Chapter 1. Neurotransmitters, Receptors, Signal Transduction

 

Chapter 1. Neurotransmitters, Receptors, Signal Transduction, and Second Messengers in Psychiatric Disorders

NEUROTRANSMITTERS, RECEPTORS, SIGNAL TRANSDUCTION, AND SECOND MESSENGERS IN PSYCHIATRIC

DISORDERS: INTRODUCTION

This chapter serves as a primer on the recent advances in our understanding of neural function both in health and in disease. It is beyond the scope of this

chapter to cover these important areas in extensive detail, and readers are referred to outstanding textbooks that are entirely devoted to the topic (Cooper et

1. 2001; Kandel et al. 2000; Nestler et al. 2001; Squire et al. 2003). Here, we focus on the principles of neurotransmission and second-messenger generation

that we believe are critical for an understanding of the biological bases of major psychiatric disorders, as well as the mechanisms by which effective

treatments may exert their beneficial effects. In particular, it is our goal to lay the groundwork for the subsequent chapters, which focus on individual

disorders and their treatments.

Although this chapter is intended to provide a general overview on neurotransmitter and second-messenger function, whenever possible we emphasize the

neuropsychiatric relevance of specific observations. In the chapter proper, we outline principles that are of utmost importance to the study and practice of

psychopharmacology; in the figure legends, we provide additional details for the interested reader.

[The work presented in this chapter was undertaken under the auspices of the National Institute of Mental Health Intramural Program. Dr. Manji is now at

Johnson & Johnson Pharmaceutical Research & Development.

The authors thank Ioline Henter for assistance in the preparation of this chapter.]

WHAT ARE NEUROTRANSMITTERS?

Several criteria have been established for a neurotransmitter, including 1) it is synthesized and released from neurons; 2) it is released from nerve terminals

in a chemically or pharmacologically identifiable form; 3) it interacts with postsynaptic receptors and brings about the same effects as are seen with

stimulation of the presynaptic neuron; 4) its interaction with the postsynaptic receptor displays a specific pharmacology; and 5) its actions are terminated by

active processes (Kandel et al. 2000; Nestler et al. 2001). However, our growing appreciation of the complexity of the central nervous system (CNS) and of

the existence of numerous molecules that exert neuromodulatory and neurohormonal effects has blurred the classical definition of neurotransmitters

somewhat, and even well-known neurotransmitters do not meet all these criteria under certain situations (Cooper et al. 2001).

Most neuroactive compounds are small polar molecules that are synthesized in the CNS via local machinery or are able to permeate the blood–brain barrier.

To date, more than 50 endogenous substances have been found to be present in the brain that appear to be capable of functioning as neurotransmitters.

There are many plausible explanations for why the brain would need so many transmitters and receptor subtypes to transmit messages. Perhaps the simplest

explanation is that the sheer complexity of the CNS results in many afferent nerve terminals impinging on a single neuron. This requires a neuron to be able

to distinguish the multiple information conveying inputs. Although this can be accomplished partially by spatial segregation, it is accomplished in large part by

chemical coding of the inputs—that is, different chemicals convey different information. Moreover, as we discuss in detail later, the evolution of multiple

receptors for a single neurotransmitter means that the same chemical can convey different messages depending on the receptor subtypes it acts on.

Additionally, the firing pattern of neurons is also a means of conveying information; thus, the firing activities of neurons in the brain differ widely, and a single

neuron firing at different frequencies can even release different neuroactive compounds depending on the firing rate (e.g., the release of peptides often

occurs at higher firing rates than that which is required to release monoamines). These multiple mechanisms to enhance the diversity of responses—chemical

coding, spatial coding, frequency coding—are undoubtedly critical in endowing the CNS with its complex repertoire of physiological and behavioral responses

(Kandel et al. 2000; Nestler et al. 2001). Finally, the existence of multiple neuroactive compounds also provides built-in safeguards to ensure that vital brain

circuits are able to partially compensate for loss of function of particular neurotransmitters.

RECEPTORS

An essential property of any living cell is its ability to recognize and respond to external stimuli. Cell surface receptors have two major functions: recognition

of specific molecules (neurotransmitters, hormones, growth factors, and even sensory signals) and activation of “effectors.” Binding of the appropriate

agonist (i.e., neurotransmitter or hormone) externally to the receptor alters the conformation (shape) of the protein. Cell surface receptors use a variety of

membrane-transducing mechanisms to transform an agonist’s message into cellular responses. In neuronal systems, the most typical responses ultimately

(in some cases rapidly, in others more slowly) involve changes in transmembrane voltage and hence neuronal changes in excitability. Collectively, the

processes are referred to as transmembrane signaling or signal transduction mechanisms. These processes are not restricted to neurons. For example,

astrocytes, which were once thought to be unrelated to neurotransmission, have recently been demonstrated to possess voltage-regulated anion channels

(VRAC), which not only transport Cl– but also allow efflux of amino acids such as taurine, glutamate, and aspartate (Mulligan and MacVicar 2006).

Interestingly, although increasing numbers of potential neuroactive compounds and receptors continue to be identified, it has become clear that translation

of the extracellular signals (into a form that can be interpreted by the complex intracellular enzymatic machinery) is achieved through a relatively small

number of cellular mechanisms. Generally speaking, these transmembrane signaling systems, and the receptors that utilize them, can be divided into four

major groups (Figure 1–1):

Those that are relatively self-contained in structure and whose message takes the form of transmembrane ion fluxes (ionotropic)

Those that are multicomponent in nature and generate intracellular second messengers (G protein–coupled)

Those that contain intrinsic enzymatic activity (receptor tyrosine kinases and phosphatases)

Those that are cytoplasmic and translocate to the nucleus to directly regulate transcription (gene expression) after they are activated by lipophilic molecules (often

hormones) that enter the cell (nuclear receptors)

FIGURE 1–1. Major receptor subtypes in the central nervous system.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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This figure depicts the four major classes of receptors in the CNS. (A) Ionotropic receptors. These receptors comprise multiple protein subunits that are combined in such a

way as to create a central membrane pore through this complex, allowing the flow of ions. This type of receptor has a very rapid response time (milliseconds). The

consequences of receptor stimulation (i.e., excitatory or inhibitory) depend on the types of ions that the receptor specifically allows to enter the cell. Thus, for example, Na+

entry through the NMDA (N-methyl-D-aspartate) receptor depolarizes the neuron and brings about an excitatory response, whereas Cl– efflux through the -aminobutyric acid

type A (GABAA) receptor hyperpolarizes the neuron and brings about an inhibitory response. Illustrated here is the NMDA receptor regulating a channel permeable to Ca2+,

Na+, and K+ ions. The NMDA receptors also have binding sites for glycine, Zn2+, phencyclidine (PCP), MK801/ketamine, and Mg2+; these molecules are able to regulate the

function of this receptor. (B) G protein–coupled receptors (GPCRs). The majority of neurotransmitters, hormones, and even sensory signals mediate their effects via seven

transmembrane domain–spanning receptors that are G protein–coupled. The amino terminus of the G protein is on the outside of the cell and plays an important role in the

recognition of specific ligands; the third intracellular loop and carboxy terminus of the receptor play an important role in coupling to G proteins and are sites of regulation of

receptor function (e.g., by phosphorylation). All G proteins are heterotrimers (consisting of , , and subunits). The G proteins are attached to the membrane by isoprenoid

moieties (fatty acid) via their subunits. Compared with the ionotropic receptors, GPCRs mediate a slower response (on the order of seconds). Detailed depiction of the

activation of G protein–coupled receptors is given in Figure 1–2. Here we depict a receptor coupled to the G protein Gs (the s stands for stimulatory to the enzyme adenylyl

cyclase [AC]). Activation of such a receptor produces activation of AC and increases in cAMP levels. G protein–coupled pathways exhibit major amplification properties, and,

for example, in model systems researchers have demonstrated a 10,000-fold amplification of the original signal. The effects of cAMP are mediated largely by activation of

protein kinase A (PKA). One major downstream target of PKA is CREB (cAMP response element–binding protein), which may be important to the mechanism of action of

antidepressants. (C) Receptor tyrosine kinases. These receptors are activated by neurotrophic factors and are able to bring about acute changes in synaptic function, as well

as long-term effects on neuronal growth and survival. These receptors contain intrinsic tyrosine kinase activity. Binding of the ligand triggers receptor dimerization and

transphosphorylation of tyrosine residues in its cytoplasmic domain, which then recruits cytoplasmic signaling and scaffolding proteins. The recruitment of effector molecules

generally occurs via interaction of proteins with modular binding domains SH2 and SH3 (named after homology to the src oncogenes–src homology domains); SH2 domains

are a stretch of about 100 amino acids that allows high-affinity interactions with certain phosphotyrosine motifs. The ability of multiple effectors to interact with

phosphotyrosines is undoubtedly one of the keys to the pleiotropic effects that neurotrophins can exert. Shown here is a tyrosine kinase receptor type B (TrkB), which upon

activation produces effects on the Raf, MEK (mitogen-activated protein kinase/ERK), extracellular response kinase (ERK), and ribosomal S6 kinase (RSK) signaling pathway.

Some major downstream effects of RSK are CREB and stimulation of factors that bind to the AP-1 site (c-Fos and c-Jun). (D) Nuclear receptors. These receptors are

transcription factors that regulate the expression of target genes in response to steroid hormones and other ligands. Many hormones (including glucocorticoids, gonadal

steroids, and thyroid hormones) are able to rapidly penetrate into the lipid bilayer membrane, because of their lipophilic composition, and thereby directly interact with these

cytoplasmic receptors inside the cell. Upon activation by a hormone, the nuclear receptor–ligand complex translocates to the nucleus, where it binds to specific DNA

sequences, referred to as hormone responsive elements (HREs), and regulates gene transcription. Nuclear receptors often interact with a variety of coregulators that promote

transcriptional activation when recruited (coactivators) and those that attenuate promoter activity (corepressors). However, nongenomic effects of neuroactive steroids have

also been documented, with the majority of evidence suggesting modulation of ionotropic receptors. This figure illustrates both the genomic and the nongenomic effects.

ATF1 = activation transcription factor 1; BDNF = brain-derived neurotrophic factor; CaMKII = Ca2+/calmodulin–dependent protein kinase II; CREM = cyclic adenosine

5′-monophosphate response element modulator; D1 = dopamine1 receptor; D5 = dopamine5 receptor; ER = estrogen receptor; GR = glucocorticoid receptor; GRK = G

protein–coupled receptor kinase; P = phosphorylation; PR = progesterone receptor.

Ionotropic Receptors

The first class of receptors contains in their molecular complex an intrinsic ion channel. Receptors of this class include those for a number of amino acids,

including glutamate (e.g., the NMDA [N-methyl-D-aspartate] receptor) and GABA ( -aminobutyric acid via the GABAA receptor), as well as the nicotinic

acetylcholine (ACh) receptor and the serotonin3 (5-HT3) receptor. Ion channels are integral membrane proteins that are directly responsible for the electrical

activity of the nervous system by virtue of their regulation of the movement of ions across membranes. Receptors containing intrinsic ion channels have been

called ionotropic and are generally composed of four or five subunits that open transiently when neurotransmitter binds, allowing ions to flow into (e.g., Na+,

Ca2+, Cl– ) or out of (e.g., K+) the neuron, thereby generating synaptic potential (see Figure 1–1).

Often, the ionotropic receptors can be composed of different compositions of the different subunits, thereby providing the system with considerable

flexibility. For example, there is extensive research into the potential development of an anxiolytic that is devoid of sedative effects by targeting GABAAPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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receptor subunits present in selected brain regions. In general, neurotransmission that is mediated by ionotropic receptors is very fast, with ion channels

opening and closing within milliseconds, and regulates much of the tonic excitatory (e.g., glutamate-mediated) and inhibitory (e.g., GABA-mediated) activity

in the CNS; as we discuss below, many of the classical neurotransmitters (e.g., monoamines) exert their effects on a slower time scale and are therefore often

considered to be modulatory in their effects.

G Protein–Coupled Receptors

Most receptors in the CNS do not have intrinsic ionic conductance channels within their structure but instead regulate cellular activity by the generation of

various “second messengers.” Receptors of this class do not generally interact directly with the various second-messenger-generating enzymes but instead

transmit information to the appropriate “effector” by the activation of interposed coupling proteins. These are the G protein–coupled receptor families. The G

protein–coupled receptors (GPCRs, which constitute more than 80% of all known receptors in the body, and number about 300) all span the plasma

membrane seven times (see Figure 1–1). GPCRs have been the focus of extensive research in psychiatry in recent years (Catapano and Manji 2007). The

amino terminus is on the outside of the cell and plays a critical role in recognition of the ligand; the carboxy terminus and third intracellular loop are inside

the cell and regulate not only coupling to different G proteins but also “cross talk” between receptors and desensitization (see Figure 1–1).

G proteins are so named because of their ability to bind the guanine nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Receptors

coupled to G proteins include those for catecholamines, serotonin, ACh, various peptides, and even sensory signals such as light and odorants (Table 1–1). As

we discuss later in the chapter, multiple subtypes of G proteins are known to exist, and they play critical roles in amplifying and integrating signals.

TABLE 1–1. Key features of G protein subunits

G protein class Members Effector(s)/Functions Examples of receptors

i

G i1–3, G o

AC (+)

2, D2, A1, , M2, 5-HT1A

Ligand-type Ca2+ channels (+)

Olfactory signals

G z, G t1–2

K + channels (+)

Ca2+ channels (–)a

GABAB

Cyclic GMP

Retinal rods, cones (rhodopsins)

Phosphodiesterase (+) (G t1–2)

q

G q, G 11, G 14, G 15, G 16 PLC- (+)

TxA2, 5-HT2C, M1, M3, M5, 1

12

G 12,G 13

RGS domain–containing rho exchange factors TxA2, thrombin

b

(x5) AC type I (–); AC types II, IV (potentiation)

PLC (+)

Receptor kinases (+)

Inactivates s

(x12)

required for interaction of subunit with receptor

Note. AC = adenylyl cyclase; A1, A2 = adenosine receptor subtypes; 1, 1, 2 = adrenergic receptor subtypes; C = cholera toxin; D1, D2 = dopamine receptor subtypes; G t

= olfactory, but also found in limbic areas; G s = stimulatory; G t = transducin; GABAB = -aminobutyric acid receptor subtype; 5-HT1A, 5-HT2C = serotonin receptor

subtypes; M1, M2, M3, M5 = muscarinic receptor subtypes; = opioid receptor; P = pertussis toxin; PLC = phospholipase C; RGS = regulators of G protein signaling; TxA2 =

thromboxane A2 receptor.

aAlthough regulation of Na+/H+ exchange and Ca2+ channels by G 1–2 and G 1–3 has been demonstrated in artificial systems in vitro, these findings await definitive

confirmation.

bEffectors are regulated by subunits as a dimer.

Autoreceptors and Heteroreceptors

Autoreceptors are receptors located on neurons that produce the endogenous ligand for that particular receptor (e.g., a serotonergic receptor on a

serotonergic neuron). By contrast, heteroreceptors are receptor subtypes that are present on neurons that do not contain an endogenous ligand for that

particular receptor subtype (e.g., a serotonergic receptor located on a dopaminergic neuron).

Two major classes of autoreceptors play very important roles in fine-tuning neuronal activity. Somatodendritic autoreceptors are present on cell bodies and

dendrites and exert critical roles in regulating the firing rate of neurons. In general, activation of somatodendritic autoreceptors (e.g., 2-adrenergic

receptors for noradrenergic neurons, serotonin1A [5-HT1A] receptors for serotonergic neurons, dopamine2 [D2] receptors for dopaminergic neurons) inhibits

the firing rate of the neurons by opening K+ channels and by reducing cyclic adenosine monophosphate (cAMP) levels, both of which may be important in

psychiatric disease. For example, TREK-1 is a background K+ channel regulator protein important in 5-HT transmission and potentially in mood-like behavior

regulation in mice (Heurteaux et al. 2006). Fundamental mechanisms of neuronal transmission—such as K+ channels, which regulate membrane

potentials—may relate to global alterations in brain functioning relevant to psychiatry.

The second major class of autoreceptors, nerve terminal autoreceptors, play an important role in regulating the amount of neurotransmitter released per

nerve impulse, generally by closing nerve terminal Ca2+ channels. Both of these types of autoreceptors are typically members of the G protein–coupled

receptor family. Neurotransmitter release is known to be triggered by influx and alterations of intracellular calcium, with the functioning of three types of

SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein [SNAP] receptor) proteins exerting critical roles. Recent advances in our

understanding of the distinct kinetics of neurotransmitter release modulators, such as botulinum and tetanus neurotoxins, suggest that these induce

prominent alterations in synaptobrevin and syntaxin, leading to calcium-independent mechanisms of neurotransmitter regulation (Sakaba et al. 2005). Most

synapses are dependent on influx of Ca2+ through voltage-gated calcium channels for presynaptic neurotransmitter release; in the retina, however, this influx

of calcium occurs via glutamatergic AMPA receptors (Chavez et al. 2006). Beyond the receptor level, presynaptic SAD, an intracellular serine threonine kinase,

is associated with the active zone cytomatrix that regulates neurotransmitter release (Inoue et al. 2006). These recent data further exemplify the dynamic

nature and ongoing advancement of our knowledge pertaining to basic processes involved in neurotransmitter regulation that may possibly aid in advancing

treatment of psychopathology.

G Protein–Coupled Receptor Regulation and Trafficking

The mechanism by which GPCRs translate extracellular signals into cellular changes was once envisioned as a simple linear model. It is now known, however,

that the activity of GPCRs is subject to at least three additional principal modes of regulation: desensitization, downregulation, and trafficking (Carman and

Benovic 1998) (Figure 1–2). Desensitization, the process by which cells rapidly adapt to stimulation by agonists, is generally believed to occur by two major

mechanisms: homologous and heterologous.

FIGURE 1–2. G protein–coupled receptors and G protein activation.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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All G proteins are heterotrimers consisting of , , and subunits. The receptor shuttles between a low-affinity form that is not coupled to a G protein and a high-affinity

form that is coupled to a G protein. (A) At rest, G proteins are largely in their inactive state, namely, as

heterotrimers, which have GDP (guanosine diphosphate) bound

to the subunit. (B) When a receptor is activated by a neurotransmitter, it undergoes a conformational (shape) change, forming a transient state referred to as a

high-affinity ternary complex, comprising the agonist, receptor in a high-affinity state, and G protein. A consequence of the receptor interaction with the G protein is that the

GDP comes off the G protein subunit, leaving a very transient empty guanine nucleotide binding domain. (C) Guanine nucleotides (generally GTP) quickly bind to this

nucleotide binding domain; thus, one of the major consequences of active receptor–G protein interaction is to facilitate guanine nucleotide exchange—this is basically the “on

switch” for the G protein cycle. (D) A family of GTPase-activating proteins for G protein–coupled receptors has been identified, and they are called regulators of G protein

signaling (RGS) proteins. Since activating GTPase activity facilitates the “turn off” reaction, these RGS proteins are involved in dampening the signal. Binding of GTP to the

subunit of G proteins results in subunit dissociation, whereby the -GTP dissociates from the

subunits. Although not covalently bound, the and subunits remain tightly

associated and generally function as dimers. The -GTP and

subunits are now able to activate multiple diverse effectors, thereby propagating the signal. While they are in

their active states, the G protein subunits can activate multiple effector molecules in a “hit and run” manner; this results in major signal amplification (i.e., one active G

protein subunit can activate multiple effector molecules; see Figure 1–11). The activated G protein subunits also dissociate from the receptor, converting the receptor to a

low-affinity conformation and facilitating the dissociation of the agonist from the receptor. The agonist can now activate another receptor, and this also results in signal

amplification. Together, these processes have been estimated to produce a 10,000-fold amplification of the signal in certain models. (E) Interestingly, the subunit has

intrinsic GTPase activity, which cleaves the third phosphate group from GTP (G-P-P-P) to GDP (G-P-P). Since -GDP is an inactive state, the GTPase activity serves as a

built-in timing mechanism, and this is the “turn off” reaction. (F) The reassociation of -GDP with

is thermodynamically favored, and the reformation of the inactive

heterotrimer (

) completes the G protein cycle.

Homologous desensitization is receptor specific; that is, only the receptor actively being stimulated becomes desensitized. This form of desensitization occurs

via a family of kinases known as G protein–coupled kinases (GRKs). When a receptor activates a G protein and causes dissociation of the subunit from the

subunits (discussed in detail later), the

subunits are able to provide an “anchoring surface” for the GRKs to allow them to come into the proximity of the

activated receptor and phosphorylate it. This phorphorylation then recruits another family of proteins known as arrestins, which physically interfere with the

coupling of the phosphorylated receptor and the G protein, thereby dampening the signal. This form of desensitization is very rapid and usually transient (i.e.,

the receptors get dephosphorylated and return to the baseline state). However, if the stimulation of the receptor is excessive and prolonged, it leads to an

internalization of the receptor, and often its degradation, a process referred to as downregulation.

Heterologous desensitization is not receptor specific and is mediated by second-messenger kinases such as protein kinase A (PKA) and protein kinase C

(PKC). Thus, when a receptor activates PKA, the activated PKA is capable of phosphorylating (and thereby desensitizing) not only that particular receptor but

also other receptors that are present in proximity and have the correct phosphorylation motif, thereby producing heterologous desensitization.

Upon prolonged or repeated activation of receptors by agonist ligands, the process of receptor downregulation is observed. Downregulation is associated

with a reduced number of receptors detected in cells or tissues, thereby leading to attenuation of cellular responses (Carman and Benovic 1998). The process

of GPCR sequestration is mediated by a well-characterized endocytic pathway involving the concentration of receptors in clathrin-coated pits and subsequent

internalization and recycling back to the plasma membrane (Tsao and von Zastrow 2000). Endocytosis can thus clearly serve as a primary mechanism to

attenuate signaling by rapidly and reversibly removing receptors from the cell surface. However, emerging evidence suggests additional functions of

endocytosis and receptor trafficking in mediating GPCR signaling by way of certain effector pathways, most notably mitogen-activated protein (MAP) kinase

cascades (discussed in greater detail later). There is also evidence that endocytosis of GPCRs may be required for certain signal transduction pathways

leading to the nucleus (Tsao and von Zastrow 2000). These diverse functions of GPCR endocytosis and trafficking are leading to unexpected insights into the

biochemical and functional properties of endocytic vesicles. Indeed, there is considerable excitement about our growing understanding of the diverse

molecular mechanisms for signaling specificity and receptor trafficking, and the possibility that this knowledge could lead to highly selective therapeutics.

Receptor Tyrosine Kinases

The receptor tyrosine kinases, as their name implies, contain intrinsic tyrosine kinase activity and are generally utilized by growth factors, such as

neurotrophic factors, and cytokines. Binding of an agonist initiates receptor dimerization and transphosphorylation of tyrosine residues in its cytoplasmic

domain (Patapoutian and Reichardt 2001) (see Figure 1–1). The phosphotyrosine residues of the receptor function as binding sites for recruiting specific

cytoplasmic signaling and scaffolding proteins. The ability of multiple effectors to interact with phosphotyrosines is undoubtedly one of the keys to the

pleiotropic effects that neurotrophins can exert. These pleiotropic and yet distinct effects of growth factors are mediated by varying degrees of activation of

three major signaling pathways: the MAP kinase pathway, the phosphoinositide-3 (PI3) kinase pathway, and the phospholipase C (PLC)– 1 pathway (see

Figure 1–9 later in this chapter).

Nuclear Receptors

Nuclear receptors are transcription factors that regulate the expression of target genes in response to steroid hormones and other ligands. Many hormones

(including glucocorticoids, gonadal steroids, and thyroid hormones) are able to rapidly penetrate into the lipid bilayer membrane, because of their lipophilic

composition, and thereby directly interact with these cytoplasmic receptors inside the cell (see Figure 1–1). Upon activation by a hormone, the nuclear

receptor–ligand complex translocates to the nucleus, where it binds to specific DNA sequences referred to as hormone-responsive elements (HREs), and

subsequently regulates gene transcription (Mangelsdorf et al. 1995; Truss and Beato 1993). Nuclear receptors often interact with a variety of coregulators

that promote transcriptional activation when recruited (coactivators) and those that attenuate promoter activity (corepressors).

With this overview of neurotransmitters and receptor subtypes, we now turn to a discussion of selected individual neurotransmitters and neuropeptides

before discussing the intricacies of cellular signal transduction systems.

NEUROTRANSMITTER AND NEUROPEPTIDE SYSTEMS

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Largely on the basis of the observation that most current effective antidepressants and antipsychotics target these systems, the monoaminergic systems

(e.g., serotonin, norepinephrine, dopamine) have been extensively studied. Serotonin (5-HT) was given that name because of its activity as an endogenous

vasoconstrictor in blood serum (Rapport et al. 1947). It was later acknowledged as being the same molecule (secretin) found in the intestinal mucosa and

that is “secreted” by chromaffin cells (Brodie 1900). Following these findings, 5-HT soon became characterized as being a neurotransmitter in the CNS

(Bogdansky et al. 1956).

5-HT-producing cell bodies in the brain are localized in the central gray, in the surrounding reticular formation, and in cell clusters located in the center, and

thus the name raphe (from Latin, meaning midline) was adopted (Figure 1–3A) (discussed more extensively in Chapter 4, “Chemical Neuroanatomy of the

Primate Brain”). The dorsal raphe (DR), the largest brain stem 5-HT nucleus, contains approximately 50% of the total 5-HT neurons in the mammalian CNS; in

contrast, the medial raphe (MR) comprises 5% (Descarries et al. 1982; Wiklund and Bjorklund 1980). Serotonergic neurons project widely throughout the

CNS rather than to discrete anatomical locations (as the dopaminergic neurons appear to do; see Figure 1–4A later in this chapter), leading to the suggestion

that 5-HT exerts a major modulatory role throughout the CNS (Reader 1980). Interestingly, evidence suggests that infralimbic and prelimbic regions of the

ventral medial prefrontal cortex (mPFCv) in rats are responsible for detecting whether a stressor is under the organism’s control. When a stressor is

controllable, stress-induced activation of the dorsal raphe nucleus is inhibited by the mPFCv, and the behavioral sequelae of the uncontrollable stress

response are blocked (Amat et al. 2005). The organism’s ability to regulate 5-HT neuron activity and function has been a major ongoing focus of psychiatric

disorder research and treatments.

FIGURE 1–3. The serotonergic system.

This figure depicts the location of the major serotonin (5-HT)–producing cells (raphe nuclei) innervating brain structures (A), and various cellular regulatory processes

involved in serotonergic neurotransmission (B). 5-HT neurons project widely throughout the CNS and innervate virtually every part of the neuroaxis. L-Tryptophan, an amino

acid actively transported into presynaptic 5-HT-containing terminals, is the precursor for 5-HT. It is converted to 5-hydroxytryptophan (5-HTP) by the rate-limiting enzyme

tryptophan hydroxylase (TrpH). This enzyme is effectively inhibited by the drug p-chlorophenylalanine (PCPA). Aromatic amino acid decarboxylase (AADC) converts 5-HTP to

5-HT. Once released from the presynaptic terminal, 5-HT can interact with a variety (15 different types) of presynaptic and postsynaptic receptors. Presynaptic regulation of

5-HT neuron firing activity and release occurs through somatodendritic 5-HT1A (not shown) and 5-HT1B,1D autoreceptors, respectively, located on nerve terminals.

Sumatriptan is a 5-HT1B,1D receptor agonist. (The antimigraine effects of this agent are likely mediated by local activation of this receptor subtype on blood vessels, which

results in their constriction.) Buspirone is a partial 5-HT1A agonist that activates both pre- and postsynaptic receptors. Cisapride is a preferential 5-HT4 receptor agonist that

is used to treat irritable bowel syndrome as well as nausea associated with antidepressants. The binding of 5-HT to G protein receptors (Go, Gi, etc.) that are coupled to

adenylyl cyclase (AC) and phospholipase C– (PLC- ) will result in the production of a cascade of second-messenger and cellular effects. Lysergic acid diethylamide (LSD)

likely interacts with numerous 5-HT receptors to mediate its effects. Pharmacologically this ligand is often used as a 5-HT2 receptor agonist in receptor binding experiments.

Ondansetron is a 5-HT3 receptor antagonist that is marketed as an antiemetic agent for chemotherapy patients but is also given to counteract side effects produced by

antidepressants in some patients. 5-HT has its action terminated in the synapse by rapidly being taken back into the presynaptic neuron through 5-HT transporters (5-HTT).

Once inside the neuron, it can either be repackaged into vesicles for reuse or undergo enzymatic catabolism. The selective 5-HT reuptake inhibitors (SSRIs) and

older-generation tricyclic antidepressants (TCAs) are able to interfere/block the reuptake of 5-HT. 5-HT is then metabolized to 5-hydroxyindoleacetic acid (5-HIAA) by

monoamine oxidase (MAO), located on the outer membrane of mitochondria or sequestered and stored in secretory vesicles by vesicle monoamine transporters (VMATs).

Reserpine causes a depletion of 5-HT in vesicles by interfering with uptake and storage mechanisms (depressive-like symptoms have been reported with this agent).

Tranylcypromine is an MAO inhibitor (MAOI) and an effective antidepressant. Fenfluramine (an anorectic agent) and MDMA (“Ecstasy”) are able to facilitate 5-HT release by

altering 5-HTT function. DAG = diacylglycerol; 5-HTT = serotonin transporter; IP3 = inositol-1,4,5-triphosphate.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,

1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc. Modified from Nestler et al. 2001.

FIGURE 1–4. The dopaminergic system.

This figure depicts the dopaminergic projections throughout the brain (A) and various regulatory processes involved in dopaminergic neurotransmission (B). The amino acid

L-tyrosine is actively transported into presynaptic dopamine (DA) nerve terminals, where it is ultimately converted into DA. The rate-limiting step is conversion of L-tyrosine

to L-dihydroxyphenylalanine (L-dopa) by the enzyme tyrosine hydroxylase (TH). -Methyl-p-tyrosine (AMPT) is a competitive inhibitor of tyrosine hydroxylase and has been

used to assess the impact of reduced catecholaminergic function in clinical studies. The production of DA requires that L-aromatic amino acid decarboxylase (AADC) act on

L-dopa. Thus, the administration of L-dopa to patients with Parkinson’s disease bypasses the rate-limiting step and is able to produce DA quite readily. DA has its action

terminated in the synapse by rapidly being taken back into the presynaptic neuron through DA transporters (DATs). DA is then metabolized to dihydroxyphenylalanine

(DOPAC) by intraneuronal monoamine oxidase (MAO; preferentially by the MAO-B subtype) located on the outer membrane of mitochondria, or is sequestered and stored in

secretory vesicles by vesicle monoamine transporters (VMATs). Reserpine causes a depletion of DA in vesicles by interfering and irreversibly damaging uptake and storage

mechanisms. -Hydroxybutyrate inhibits the release of DA by blocking impulse propagation in DA neurons. Pargyline inhibits MAO and may have efficacy in treating

parkinsonian symptoms by augmenting DA levels through inhibition of DA catabolism. Other clinically used inhibitors of MAO are nonselective and thus likely elevate the

levels of DA, norepinephrine, and serotonin. Once released from the presynaptic terminal (because of an action potential and calcium influx), DA can interact with five

different G protein–coupled receptors (D1–D5), which belong to either the D1 or D2 receptor family. Presynaptic regulation of DA neuron firing activity and release occurs

through somatodendritic (not shown) and nerve terminal D2 autoreceptors, respectively. Pramipexole is a D2/D3 receptor agonist and has been documented to have efficacy

as an augmentation strategy in cases of treatment-resistant depression and in the management of Parkinson’s disease. The binding of DA to G protein receptors (Go, Gi, etc.)

positively or negatively coupled to adenylyl cyclase (AC) results in the activation or inhibition of this enzyme, respectively, and the production of a cascade of

second-messenger and cellular effects (see diagram). Apomorphine is a D1/D2 receptor agonist that has been used clinically to aid in the treatment of Parkinson’s disease.

(SKF-82958 is a pharmacologically selective D1 receptor agonist.) SCH-23390 is a D1/D5 receptor antagonist. There are likely physiological differences between D1 and D5

receptors, but the current unavailability of selective pharmacological agents has precluded an adequate differentiation thus far. Haloperidol is a D2 receptor antagonist, and

clozapine is a nonspecific D2/D4 receptor antagonist (both are effective antipsychotic agents). Once inside the neuron, DA can either be repackaged into vesicles for reuse or

undergo enzymatic catabolism. Nomifensine is able to interfere/block the reuptake of DA. The antidepressant bupropion has affinity for the dopaminergic system, but it is not

known whether this agent mediates its effects through DA or possibly by augmenting other monoamines. DA can be degraded to homovanillic acid (HVA) through the

sequential action of catechol-O-methyltransferase (COMT) and MAO. Tropolone is an inhibitor of COMT. Evidence suggests that the COMT gene may be linked to

schizophrenia (Akil et al. 2003).

Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,

1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc.

The precursor for 5-HT synthesis is l-tryptophan, an amino acid that comes primarily from the diet and crosses the blood–brain barrier through a carrier for

large neutral amino acids. Tryptophan hydroxylase (TrpH) is the rate-limiting enzyme in serotonin biosynthesis (Figure 1–3B), and polymorphisms in this

enzyme have been extensively investigated in psychiatric disorders, with equivocal results to date. A more fruitful research strategy in humans has been

tryptophan depletion via dietary restriction to study the role of serotonin in the pathophysiology and treatment of psychiatric disorders (Bell et al. 2001).

These studies have indicated that tryptophan depletion produces a rapid depressive relapse in patients treated with selective serotonin reuptake inhibitors

(SSRIs) but not in those treated with norepinephrine reuptake inhibitors; the data suggesting induction of depressive symptoms in remitted patients orPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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individuals with family histories of mood disorders are more equivocal (Bell et al. 2001).

Serotonin Transporters

As is the case for many classical neurotransmitters, termination of the effects of 5-HT in the synaptic cleft is brought about in large part by an active reuptake

process mediated by the 5-HT transporter (5-HTT). 5-HT is taken up into the presynaptic terminals, where it is metabolized by the enzyme monoamine

oxidase (MAO) or sequestered into secretory vesicles by the vesicle monoamine transporter (see Figure 1–3B). This presumably underlies the mechanism by

which MAO inhibitors initiate their therapeutic effects; that is, the blockade of monoamine breakdown results in increasing the available pool for release when

an action potential invades the nerve terminal. It is now well established that many tricyclic antidepressants and SSRIs exert their initial primary

pharmacological effects by binding to the 5-HTT and blocking 5-HT reuptake, thereby increasing the intrasynaptic levels of 5-HT, which initiates a cascade of

downstream effects (see Figure 1–3B for details). It has been hypothesized that the first step in 5-HT transport involves the binding of 5-HT to the 5-HTT and

then a cotransport with Na+, while the second step involves the translocation of K+ across the membrane to the outside of the cell. SSRIs bind to the same

site on the transporter as 5-HT itself. Recently, elegant biochemical and mutagenesis experiments have elucidated a leucine transporter from bacterial

species, providing information that may aid in unraveling the complex process by which mammalian transporters couple ions and substrates to mediate

neurotransmitter clearance (Henry et al. 2006).

In the brain, 5-HTTs have been radiolabeled with [3H]-imipramine (Hrdina et al. 1985; Langer et al. 1980) and with SSRIs such as [3H]cyanoimipramine (Wolf

and Bobik 1988), [3H]paroxetine (Habert et al. 1985), and [3H]citalopram (D’Amato et al. 1987). The regional distribution of 5-HTT corresponds to discrete

regions of rat brain known to contain cell bodies of 5-HT neurons and synaptic axon terminals, most notably the cerebral cortex, neostriatum, thalamus, and

limbic areas (Cooper et al. 2001; Hrdina et al. 1990; Madden 2002). The specific cellular localization of 5-HTT in the CNS has also been accomplished by using

site-specific antibodies (Lawrence et al. 1995a). Immunohistochemical studies utilizing antibodies against the 5-HT carrier have revealed both neuronal and

glial staining in areas of the rat brain containing 5-HT somata and terminals (i.e., DR and hippocampus) (Lawrence et al. 1995b). Experimental alterations of

5-HTT in young mice for a brief period during early development indicate abnormal emotional behavior in the same mice later in life, similar to the phenotype

in mice where 5-HTT is deficient throughout life (Ansorge et al. 2004). This suggests the necessity of 5-HT early in emotional development and provides a

possible mechanism by which genetic changes in the 5-HTT system may lead to susceptibility to developing psychiatric diseases such as depression (Caspi et

1. 2003). Furthermore, 5-HT uptake ability has been documented in primary astrocyte cultures (Kimelberg and Katz 1985) and has been postulated to

account for considerable 5-HT uptake in the frontal cortex and periventricular region (Ravid et al. 1992). Since 5-HTT is transcribed from a single copy gene,

abnormalities in platelet 5-HTT have been postulated to reflect CNS abnormalities (Owens and Nemeroff 1998). A number of studies on platelet 5-HTT density

have been undertaken using [3H]imipramine binding or [3H]paroxetine binding in mood disorders. Although the results of these studies are not entirely

consistent, in toto the results suggest that the Bmax value for platelet 5-HT density is significantly lower in depressed subjects compared with healthy control

subjects (Owens and Nemeroff 1998).

Serotonin Receptors

In 1957, the existence of two separate 5-HT receptors was first proposed primarily because of the opposing phenomenon this neurotransmitter produces in

reference to cholinergic mediation of smooth muscle contraction (Gaddum and Picarelli 1957). Today, through the use of more precise molecular cloning and

pharmacological and biochemical studies, seven distinct 5-HT receptor families have been identified (5-HT1–7), many of which contain several subtypes. With

the exception of the 5-HT3 receptor, which is an excitatory ionotropic receptor, all the other 5-HT receptors are GPCRs. The 5-HT1A,B,D,E,F receptors are

negatively coupled to adenylyl cyclase, the 5-HT2A,B,C subtypes are positively coupled to PLC, and the 5-HT4, 5-HT5, 5-HT6, and 5-HT7 subtypes are positively

coupled to adenylyl cyclase (see Figure 1–3B) (Humphrey et al. 1993).

5-HT1 receptors

5-HT1A receptors are found in particularly high density in several limbic structures, including the hippocampus, septum, amygdala, and entorhinal cortex, as

well as on serotonergic neuron cell bodies, where they serve as autoreceptors regulating 5-HT neuronal firing rates (Blier et al. 1998; Cooper et al. 2001;

Pazos and Palacios 1985). The highest density of labeling is found in the DR, with lower densities observed in the remaining raphe nuclei (Pazos and Palacios

1985). The density and mRNA expression of 5-HT1A receptors appear insensitive to reductions in 5-HT transmission associated with lesioning the raphe or

administering the serotonin-depleting agent p-chlorophenylalanine (PCPA). Similarly, elevation of 5-HT transmission resulting from chronic administration of

an SSRI or monoamine oxidase inhibitor (MAOI) does not consistently alter 5-HT1A receptor density or mRNA in the cortex, hippocampus, amygdala, or

hypothalamus. In contrast to the insensitivity to 5-HT, 5-HT1A receptor density is downregulated by adrenal steroids. Postsynaptic 5-HT1A receptor gene

expression is under tonic inhibition by adrenal steroids in the hippocampus and some other regions. Thus, in rodents, hippocampal 5-HT1A receptor mRNA

expression is increased by adrenalectomy and decreased by corticosterone administration or chronic stress. The stress-induced downregulation of 5-HT1A

receptor expression is prevented by adrenalectomy. Mineralocorticoid receptor stimulation has the most potent effect on downregulating 5-HT1A receptors,

although glucocorticoid receptor stimulation also contributes to this effect.

In addition to being expressed on neurons, postsynaptic 5-HT1A receptors are also abundantly expressed by astrocytes and some other glia

(Whitaker-Azmitia et al. 1990) (see Figure 1–7 later in this chapter). Stimulation of astrocyte-based 5-HT1A sites causes astrocytes to acquire a more mature

morphology and to release the trophic factor S-100 , which promotes growth and arborization of serotonergic axons. Administration of 5-HT1A receptor

antagonists, antibodies to S-100 , or agents that deplete 5-HT produces similar losses of dendrites, spines, and/or synapses in adult and developing

animals—effects that are blocked by administration of 5-HT1A receptor agonists or SSRIs. These observations have led to the hypothesis that a reduction of

5-HT1A receptor function may play an important role in mood disorders that are known to be associated with glial reductions (Manji et al. 2001). The use of

conditional knockouts of the 5-HT1A receptor, in which gene expression is altered only in particular anatomical regions and/or during particular times, has

illustrated the caution necessary in attributing complex behaviors to simple “too much” or “too little” neurotransmitter/receptor hypotheses. Using a

knockout/rescue approach with regional and temporal specificity, Gross et al. (2002) demonstrated that the anxiety-related effect of the 5-HT1A receptor

knockout was actually developmental. Specifically, expression limited to the hippocampus and cortex during early postnatal development was sufficient to

counteract the anxious phenotype of the mutant, even though the receptor was still absent in adulthood (Gross et al. 2002). As is discussed in the chapters on

antidepressants and axiolytics (see Chapters 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26), there is growing interest in the observation that

antidepressants enhance hippocampal neurogenesis (Duman 2002; Malberg et al. 2000). It is noteworthy that preliminary data suggest that 5-HT1A receptor

activation is required for SSRI-induced hippocampal neurogenesis in mice (Jacobs et al. 2000). Altering 5-HT levels with the SSRI fluoxetine does not affect

division of stem cells in the dentate gyrus, but rather increases symmetric divisions of an early progenitor cell class that exists after stem cell division

(Encinas et al. 2006).

5-HT1A receptors are now known to utilize a variety of signaling mechanisms to bring about their effects in distinct brain areas. Thus, somatodendritic 5-HT1A

receptors appear to inhibit the firing of serotonergic neurons by opening a K+ channel through a pertussis toxin–sensitive G protein (likely Go, discussed later

in the section on G proteins) (Andrade et al. 1986), as well as by reducing cAMP levels. Postsynaptic 5-HT1A receptors appear to exert many of their effects by

inhibiting adenylyl cyclase via Gi (De Vivo and Maayani 1990) but have also been demonstrated to potentiate the activity of certain adenylyl cyclases (Bourne

and Nicoll 1993) and to stimulate inositol-1,4,5-triphosphate (IP3) production and activate PKC (Y. F. Liu and Albert 1991).

5-HT1D receptors are virtually absent in the rodent but have been detected in guinea pig and man (Bruinvels et al. 1993). On the basis of an approximately

74% sequence homology, it has been proposed that 5-HT1B receptors are the rodent homolog of 5-HT1D receptors (see Saxena et al. 1998). Furthermore, the

distribution of the 5-HT1D receptors in guinea pig and man is roughly equivalent to that of the 5-HT1B receptors in the rat (Bruinvels et al. 1993). Both 5-HT1B

and 5-HT1D receptors have been proposed to represent the major nerve terminal autoreceptors regulating the amount of 5-HT released per nerve impulse

(Pineyro and Blier 1999) (see Figure 1–3B). Like 5-HT1A receptors, 5-HT1B and 5-HT1D receptors inhibit cAMP formation and stimulate IP3 production and

activate PKC (Schoeffter and Bobirnac 1995). As we discuss later, this appears to be the case for many receptors coupled to Gi and Go (see Table 1–1). The

subunits of the G protein ( i and o) inhibit adenylyl cyclase and regulate ion channels, respectively, whereas the

subunits activate PLC isozymes to

stimulate IP3 production and activate PKC.

Finally, it should be noted that the 5-HT1C receptor classification has been revoked, as these receptors have structural and transductional similarities to the

5-HT2 receptor class (Hoyer et al. 1986; Saxena et al. 1998).Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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5-HT2 receptors

There are three subtypes of 5-HT2 receptors: 5-HT2A, 5-HT2B, and 5-HT2C. The highest level of 5-HT2A binding sites and mRNA for these receptors exists in the

cortex, and these receptors have been implicated in the psychotomimetic effects of agents like lysergic acid diethylamide (LSD) (for a review, see Aghajanian

and Marek 1999). In addition, lesioning of 5-HT neurons with 5,7-DHT does not reduce the 5-HT2 receptor density reported in brain regions (Hoyer et al.

1986), indicating that these receptors are primarily (if not exclusively) postsynaptic. Autoradiography performed with the potent and selective radioligand

[ 3H]MDL 100,907 has localized 5-HT2A receptors to many similar brain regions in the rat and primate brain (Lopez-Gimenez et al. 1997). Recent experiments

show that mice expressing 5-HT2A receptors only in the frontal cortex have conserved receptor signaling and behavioral responses to hallucinogenic drugs

similar to those of wild-type littermates, suggestive of cortical importance (Gonzalez-Maeso et al. 2007). Competition studies with other radioligands

(Westphal and Sanders-Bush 1994) and their mRNA distribution indicate that 5-HT2C receptors are considerably widespread throughout the CNS, with the

highest density in the choroid plexus (Hoffman and Mezey 1989). 5-HT2B receptors are detected sparingly in the brain and are more prominently located in

the fundus, gut, kidney, lungs, and heart (Hoyer et al. 1986).

Several antidepressants (e.g., mianserin, mirtazapine) and antipsychotics (e.g., clozapine) bind to 5-HT2 receptors, raising the possibility that blockade of

5-HT2 receptors may play an important role in the therapeutic efficacy of these agents. Indeed, a leading hypothesis concerning the mechanism of action of

atypical antipsychotic agents suggests that the ratio of D2/5-HT2 blockade confers “atypicality” properties on many currently available antipsychotic agents

(Meltzer 2002). Evidence from animal experiments in which cortical 5-HT2A receptors are disrupted indicates a specific role of these receptors in modulation

of conflict anxiety without affecting fear conditioning and depression-like behaviors (Weisstaub et al. 2006). Furthermore, chronic administration of many

antidepressants downregulates 5-HT2 receptors, suggesting that this effect may be important for their efficacy (J. A. Scott and Crews 1986); however,

chronic electroconvulsive shock (ECS) appears to upregulate 5-HT2 expression, precluding a simple mechanism for antidepressant efficacy. The obesity seen

in 5-HT2C knockout animals suggests that in addition to histamine receptor blockade, 5-HT2C blockade may play a role in the weight gain observed with

certain psychotropic agents; this is an area of considerable current research. In keeping, recent evidence suggests that the weight gain “orexigenic”

properties of atypical antipsychotics are likely due to potent activation of hypothalamic AMP-kinase through histamine1 (H1) receptors (Kim et al. 2007). The

regulation of 5-HT2 receptors is intriguing, as not only is it important in psychiatric disorders and therapeutic benefit, but both agonists and antagonists

appear to cause an internalization of the receptor. Moreover, emerging data suggest that mRNA editing may play an important role in regulating the levels

and activity of this receptor subtype (Niswender et al. 1998). All of the 5-HT2 receptor subtypes are linked to the phosphoinositide signaling system, and their

activation produces IP3 and diacylglycerol (DAG), via PLC activation (Conn and Sanders-Bush 1987) (see Figure 1–3B).

An exciting recent pharmacogenetic investigation searched for genetic predictors of treatment outcome in 1,953 patients with major depressive disorder who

were treated with the antidepressant citalopram in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study and prospectively assessed

(McMahon et al. 2006). In a split-sample design, a selection of 68 candidate genes was genotyped, with 768 single-nucleotide-polymorphism markers chosen

to detect common genetic variation. A significant and reproducible association was detected between treatment outcome and a marker in HTR2A (P = 1 x

10–6 to 3.7 x 10–5 in the total sample). The “A” allele (associated with better outcome) was six times more frequent in white than in black participants, for

whom treatment was also less effective in this sample (McMahon et al. 2006). The “A” allele may thus contribute to racial differences in outcomes of

antidepressant treatment. Taken together with prior neurobiological findings, these new genetic data make a compelling case for a key role of HTR2A in the

mechanism of antidepressant action.

5-HT3–7 receptors

Unlike the other 5-HT receptors, 5-HT3 receptors are ligand-gated ion channels capable of mediating fast synaptic responses (see Figure 1–3B). The cis-trans

isomerization and molecular rearrangement at proline 8 is the structural mechanism that opens the 5-HT3 receptor protein pore (Lummis et al. 2005). 5-HT3

receptors are present in multiple brain areas, including the hippocampus, dorsal motor nucleus of the solitary tract, and area postrema (Laporte et al. 1992).

The 5-HT3 receptor is effectively modulated by a variety of compounds, such as alcohols and anesthetics, and antagonists against this receptor are used as

effective antiemetics in patients who are undergoing chemotherapy (e.g., ondansetron). 5-HT4, 5-HT6, and 5-HT7 are GPCRs that are preferentially coupled to

Gs and activate adenylyl cyclases (see Figure 1–3B). 5-HT4 receptors are able to modulate the release of monoamines and GABA in the brain. 5-HT5 receptors

are located in the hypothalamus, hippocampus, corpus callosum, cerebral ventricles, and glia (Hoyer et al. 2002). The 5-HT5A receptor is negatively coupled to

adenylyl cyclase, whereas the 5-HT5B receptor does not involve cAMP accumulation or phosphoinositide turnover. 5-HT6 receptors are located in the striatum,

amygdala, nucleus accumbens, hippocampus, cortex, and olfactory tubercle (Hoyer et al. 2002). Of interest, many antipsychotic agents and antidepressants

have high affinity for 5-HT6 receptors and act as antagonists at this receptor. 5-HT7 receptors have been localized to the cerebral cortex, medial thalamic

nuclei, substantia nigra, central gray, and dorsal raphe nucleus (Hoyer et al. 2002). It appears that chronic treatment with antidepressants is able to

downregulate this receptor, whereas acute stress has been reported to alter 5-HT7 expression (Sleight et al. 1995; Yau et al. 2001).

Dopaminergic System

Dopamine (DA) was originally thought to simply be a precursor of norepinephrine (NE) and epinephrine synthesis, but the demonstration that its distribution

in the brain was quite distinct to that of NE led to extensive research demonstrating its role as a unique critical neurotransmitter. DA synthesis requires

transport of the amino acid L-tyrosine across the blood–brain barrier and into the cell. Once tyrosine enters the neuron, the rate-limiting step for DA synthesis

is conversion of L-tyrosine to L-dihydroxyphenylalanine (L-dopa) by the enzyme tyrosine hydroxylase (TH); L-dopa is readily converted to dopamine and,

hence, is used as a precursor strategy to correct a dopamine deficiency in the treatment of Parkinson’s disease (Figure 1–4B). The activity of TH can be

regulated by many factors, including the activity of catecholamine neurons; furthermore, catecholamines function as end-product inhibitors of TH by

competing with a tetrahydrobiopterin cofactor (Cooper et al. 2001).

In contrast to the widespread 5-HT and NE projections, DA neurons form more discrete circuits, with the nigrostriatal, mesolimbic, tuberoinfundibular, and

tuberohypophysial pathways comprising the major CNS dopaminergic circuits (Figure 1–4A). The nigrostriatal circuit is composed of DA neurons from the

mesencephalic reticular formation (region A8) and the pars compacta region of the substantia nigra (region A9) of the mesencephalon. These neurons give

rise to axons that travel via the medial forebrain bundle to innervate the caudate nucleus and putamen (see Anden et al. 1964; Ungerstedt 1971). The DA

neurons that make up the nigrostriatal circuit have been assumed to be critical for maintaining normal motor control, since destruction of these neurons is

associated with Parkinson’s disease; however, it is now clear that these projections subserve a variety of additional functions. For example, recent evidence

from human brain imaging studies indicates that a subject’s ability to choose rewarding actions during instrumental learning tasks can be modulated by

administration of drugs that enhance or reduce striatal DA receptor activation. This further implies that the DA reward pathway in the brain is likely

convergent on many discrete brain circuits and neurotransmitter alterations, and it shows that striatal activity can also account for how the human brain

proceeds toward making future decisions based on reward prediction (Pessiglione et al. 2006).

The mesolimbic DA circuit consists of DA neurons located in the midbrain just medial to the A9 cells in an area termed the ventral tegmental area (VTA)

(Cooper et al. 2001; Nestler et al. 2001; Squire et al. 2003). This circuit shares some similarities to the nigrostriatal circuit in that it is a parallel circuit

consisting of axons that make up the medial forebrain bundle. However, these axons ascend through the lateral hypothalamus and project to the nucleus

accumbens, olfactory tubercle, bed nucleus of the stria terminalis, lateral septum, and frontal, cingulate, and entorhinal regions of the cerebral cortex (Cooper

et al. 2001). This circuit innervates many limbic structures known to play critical roles in motivational, motor, and reward pathways and has therefore been

implicated in a variety of clinical conditions, including psychosis and drug abuse (Cooper et al. 2001). Data also suggest a potential role for dopamine—and, in

particular, mesolimbic pathways—in the pathophysiology of bipolar mania as well as bipolar and unipolar depression (Beaulieu et al. 2004; Dunlop and

Nemeroff 2007; Goodwin and Jamison 2007; Roybal et al. 2007). It is perhaps surprising that the role of the dopaminergic system in the pathophysiology of

mood disorders has not received greater study, since it represents a prime candidate on a number of theoretical grounds. The motoric changes in bipolar

disorder are perhaps the most defining characteristics of the illness, ranging from the near-catatonic immobility of depressive states to the profound

hyperactivity of manic states. Similarly, loss of motivation is one of the central features of depression, whereas anhedonia and “hyperhedonic states” are

among the most defining characteristics of bipolar depression and mania, respectively. In this context, it is noteworthy that the midbrain dopaminergic

system is known to play a critical role in regulating not only motoric activity but also motivational and reward circuits. It is clear that motivation and motor

function are closely linked and that motivational variables can influence motor output both qualitatively and quantitatively. Furthermore, there is considerable

evidence that the mesolimbic dopaminergic pathway plays a crucial role in the selection and orchestration of goal-directed behaviors, particularly thosePrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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elicited by incentive stimuli (Goodwin and Jamison 2007).

The firing pattern of mesolimbic DA neurons appears to be an important regulatory mechanism; thus, in rats, electrical or glutamatergic stimulation of medial

prefrontal cortex elicits a burst firing pattern of dopaminergic cells in the VTA and increases DA release in the nucleus accumbens (Murase et al. 1993; Taber

and Fibiger 1993). The burst firing of DA cell activity elicits more terminal DA release per action potential than the nonbursting pacemaker firing pattern

(Roth et al. 1987). The phasic burst firing of DA neurons and accompanying rise in DA release normally occur in response to primary rewards (until they

become fully predicted) and reward-predicting stimuli. Such a role has also been postulated to provide a neural mechanism by which prefrontal cortex

dysfunction could alter hedonic perceptions and motivated behavior in mood disorders (Drevets et al. 2002). Recent studies indicate that the amygdala is

important in learning new cocaine drug-seeking responses as well as the habit-forming properties of cocaine (Lee et al. 2005), expanding our knowledge of

drug addiction circuits in the brain.

Dopamine Transporters

As with serotonin, the DA signal in the synaptic cleft is terminated primarily by reuptake into the presynaptic terminal. The dopamine transporter (DAT)

comprises 12 putative transmembrane domains and is located somatodendritically as well as on DA nerve terminals (see Figure 1–4B). Like other monoamine

transporters, the DAT functions as a Na+/K+ pump to clear DA from the synaptic cleft upon its release. However, data suggest that many drugs of abuse are

capable of altering the function of these transporters. Thus, the amphetamines are thought to mediate their effects, in part, by reversing the direction of the

transporter so that it releases DA. Cocaine is capable of blocking the reuptake of DAT, leading to an increase in DA in the synaptic cleft. Of interest, altered

neuronal long-term potentiation in the VTA in response to chronic cocaine exposure has been recently linked to drug-associated memory and likely

contributes to the powerful addictive potential of this drug of abuse (Q. S. Liu et al. 2005). DA in the medial frontal cortex is taken up predominantly by the

NE transporter. Although the precise functional significance of this finding is not currently known, it goes against the dogma of transporters being able to

selectively take up only their respective neurotransmitter. Furthermore, this provides a mechanism by which NE reuptake–inhibiting antidepressants may also

increase synaptic levels of DA in the frontal cortex, effects that may be therapeutically very important.

Dopaminergic Receptors

The existence of two subtypes of DA receptors, dopamine1 (D1) and dopamine2 (D2), was initially established using classic pharmacological techniques in the

1970s (Stoof and Kebabian 1984). Subsequent molecular biological studies have shown that the D1 family contains both the D1 and dopamine5 (D5)

receptors, whereas the D2 family contains the D2, dopamine3 (D3), and dopamine4 (D4) receptors (Cooper et al. 2001). D1 receptor family members were

originally defined solely on the ability to stimulate adenylyl cyclase (AC), while the D2 family inhibited the enzyme. Interestingly, DA receptors complexed

with subunits from other subclasses of DA receptors within a receptor family are able to form distinct hetero-oligomeric receptors (also termed “kissing

cousin receptors”). Notably, hetero-oligomeric D1–D2 receptor complexes in the brain require binding to active sites of both receptor subtypes to induce

activation of the hetero-oligomeric receptor complex. These receptors have been demonstrated to use traditional D1 receptor intracellular signaling

components of Gq/11 and Ca2+/calmodulin–dependent protein kinase II (CaMKII) second-messenger activation as demonstrated in the nucleus accumbens

(Rashid et al. 2007). This work suggests possible avenues through which the brain might use different receptor subunit proportions to further fine-tune brain

neurophysiology.

D1 and D5 receptors

The D1 and D5 receptors stimulate adenylyl cyclase activity via the activation of Gs or Golf (a G protein originally thought to be present exclusively in olfactory

tissue but now known to be abundantly present in limbic areas) (see Figure 1–4B). Other second-messenger pathways have also been reported to be

activated by D1 receptors, effects that may play a role in the reported D1–D2 cross-talk (Clark and White 1987). The frontal cortex contains almost exclusively

D1 receptors (Clark and White 1987), suggesting that this receptor may play an important role in higher cognitive function and perhaps in the actions of

medications like methylphenidate. The D5 receptor is a neuron-specific receptor that is located primarily in limbic areas of the brain.

D2 receptors

Four types of D2 receptors have been identified. The two subtypes of D2 receptors (the short and long forms, D2S and D2L, respectively) are derived from

alternative splicing of the D2 gene. Although a seemingly identical pharmacological profile for these receptors exists, there are undoubtedly (yet to be

discovered) physiological differences between the two subtypes. D2 receptors mediate their cellular effects via the Gi/Go proteins and thereby several

effectors (see Figure 1–4B). In addition to the well-characterized inhibition of adenylyl cyclase, D2 receptors in different brain areas also regulate PLC, bring

changes in K+ and Ca2+ currents, and possibly regulate phospholipase A2. D2 receptors are located on cell bodies and nerve terminals of DA neurons and

function as autoreceptors. Thus, activation of somatodendritic D2 receptors reduces DA neuron firing activity, likely via opening of K+ channels, whereas

activation of nerve-terminal D2 autoreceptors reduces the amount of DA released per nerve impulse, in large part by closing voltage-gated Ca2+ channels. As

discussed extensively in Chapters 27 and 46, D2 receptors have long been implicated in the pathophysiology and treatment of schizophrenia. Recently,

transgenic mice overexpressing D2 receptors in the striatum have been found to display many phenotypic hallmarks of schizophrenia (Kellendonk et al.

2006).

D3 receptors

D3 receptors possess a different anatomical distribution than do D2 receptors and, because of their preferential limbic expression, have been postulated to

represent an important target for antipsychotic drugs. Numerous studies have investigated the position association of a polymorphism in the coding sequence

of the D3 receptor with schizophrenia, with equivocal results. It has been suggested that brain-derived neurotrophic factor (BDNF) may regulate behavioral

sensitization via its effects on D3 receptor expression (Guillin et al. 2001).

D4 receptors

The D4 receptor has received much interest in psychopharmacological research in recent years because of the fact that clozapine has a high affinity for this

receptor. Studies are currently underway that are investigating more selective D4 antagonists as adjunctive agents in the treatment of schizophrenia.

Furthermore, considerable attention has focused on the possibility that genetic D4 variants may be associated with thrill-seeking behavior (Zuckerman 1985),

attention-deficit/hyperactivity disorder (Roman et al. 2001), and responsiveness to clozapine (Van Tol et al. 1992).

Noradrenergic System

Named sympathine because it was initially encountered as being released by sympathetic nerve terminals, the molecule was later given the name

norepinephrine after meeting the criteria for a neurotransmitter in the CNS (see Cooper et al. 2001). NE is produced from the amino acid precursor L-tyrosine

found in neurons in the brain, chromaffin cells, sympathetic nerves, and ganglia. The enzyme dopamine -hydroxylase (DBH) converts DA to NE, and as is the

case for DA synthesis, tyrosine hydroxylase is the rate-limiting enzyme for NE synthesis (Figure 1–5B). The dietary depletion of tyrosine and

-methyl-p-tyrosine (a TH inhibitor) has played an important part in efforts aimed at delineating the role of catecholamines in the pathophysiology and

treatment of mood and anxiety disorders (Coupland et al. 2001; McCann et al. 1995).

FIGURE 1–5. The noradrenergic system.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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This figure depicts the noradrenergic projections throughout the brain (A) and the various regulatory processes involved in norepinephrine (NE) neurotransmission (B). NE

neurons innervate nearly all parts of the neuroaxis, with neurons in the locus coeruleus being responsible for most of the NE in the brain (90% of NE in the forebrain and 70%

of total NE in the brain). The amino acid L-tyrosine is actively transported into presynaptic NE nerve terminals, where it is ultimately converted into NE. The rate-limiting step

is conversion of L-tyrosine to L-dihydroxyphenylalanine (L-dopa) by the enzyme tyrosine hydroxylase (TH). -Methyl-p-tyrosine (AMPT) is a competitive inhibitor of tyrosine

hydroxylase and has been used to assess the impact of reduced catecholaminergic function in clinical studies. Aromatic amino acid decarboxylase (AADC) converts L-dopa to

dopamine (DA). L-dopa then becomes decarboxylated by decarboxylase to form dopamine (DA). DA is then taken up from the cytoplasm into vesicles, by vesicle monoamine

transporters (VMATs), and hydroxylated by dopamine -hydroxylase (DBH) in the presence of O2 and ascorbate to form NE. Normetanephrine (NM), which is formed by the

action of COMT (catechol-O-methyltransferase) on NE, can be further metabolized by monoamine oxidase (MAO) and aldehyde reductase to

3-methoxy-4-hydroxyphenylglycol (MHPG). Reserpine causes a depletion of NE in vesicles by interfering with uptake and storage mechanisms (depressive-like symptoms

have been reported with this hypertension). Once released from the presynaptic terminal, NE can interact with a variety of presynaptic and postsynaptic receptors.

Presynaptic regulation of NE neuron firing activity and release occurs through somatodendritic (not shown) and nerve-terminal 2 adrenoreceptors, respectively. Yohimbine

potentiates NE neuronal firing and NE release by blocking these 2 adrenoreceptors, thereby disinhibiting these neurons from a negative feedback influence. Conversely,

clonidine attenuates NE neuron firing and release by activating these receptors. Idazoxan is a relatively selective 2 adrenoreceptor antagonist primarily used for

pharmacological purposes. The binding of NE to G protein receptors (Go, Gi, etc.) that are coupled to adenylyl cyclase (AC) and phospholipase C– (PLC-b) produces a

cascade of second-messenger and cellular effects (see diagram and later sections of the text). NE has its action terminated in the synapse by rapidly being taken back into

the presynaptic neuron via NE transporters (NETs). Once inside the neuron, it can either be repackaged into vesicles for reuse or undergo enzymatic degradation. The

selective NE reuptake inhibitor and antidepressant reboxetine and older-generation tricyclic antidepressant desipramine are able to interfere/block the reuptake of NE. On the

other hand, amphetamine is able to facilitate NE release by altering NET function. Green spheres represent DA neurotransmitters; blue spheres represent NE

neurotransmitters. DAG = diacylglycerol; IP3 = inositol-1,4,5-triphosphate.

Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,

1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc. Modified from Nestler et al. 2001.

There are seven NE cell groups in the mammalian CNS, designated A1 through A7. In the brain stem, these are the lateral tegmental neurons (A5 and A7) and

the locus coeruleus (A6) (Dahlstrom 1971) (see Figure 1–5B). In general, the projections from A5 and A7 are more restricted to brain stem areas and do not

interact with those of A6. The term locus coeruleus (LC) was derived from the Greek because of its saddle shape and its “bluish color” (caeruleum). The LC is

the most widely projecting CNS nucleus known (Foote et al. 1983), responsible for approximately 90% of the NE innervation of the forebrain and 70% of the

total NE in the brain (Figure 1–5A). Indeed, the LC NE neurons, although small in number, constitute a diffuse system of projections to widespread brain

areas via highly branched axons. The extensive efferent innervation suggests that the LC plays a modulatory and integrative role, rather than a role in specific

sensory or motor processing (Foote et al. 1983).

A number of physiological roles have been ascribed to the LC, notably in the control of vigilance and the initiation of adaptive behavioral responses (Foote et

1. 1983). Considerable data support the hypothesis that NE neurons in the LC constitute a CNS response or defense system, since the neurons are activated

by “challenges” in both the behavioral/environmental and the physiological domains (Jacobs et al. 1991). Thus, while a variety of sensory stimuli are capable

of increasing LC activity, noxious or stressful stimuli are particularly potent in this regard. Moreover, considerable evidence also supports a role for LC NE

neurons in the learning of aversively motivated tasks and in the conditioned response to stressful stimuli (Rasmussen et al. 1986a, 1986b), with obvious

implications for a variety of psychiatric conditions (see Gould et al. 2003; Szabo and Blier 2001). Indeed, tonic activation of the LC appears to occur

preferentially in the response to stressful stimuli, in contrast to stimuli limited to simply evoking activation or arousal (Rasmussen et al. 1986a, 1986b).

Norepinephrine Transporter

The norepinephrine transporter (NET), the first of the monoamine transporters to be cloned in humans, transports NE from the synaptic cleft back into the

neuron (Pacholczyk et al. 1991). Like other monoamine transporters, NET comprises 12 putative transmembrane domains, and autoradiography with variousPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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NE reuptake inhibitors has been used to determine the brain distribution of NET. A high level of NET is found in the LC, with moderate to high levels found in

the dentate gyrus, raphe nuclei, and hippocampus (Tejani-Butt and Ordway 1992; Tejani-Butt et al. 1990). This pattern of expression is consistent with the

NE innervation to these structures. The NET is expressed mainly on NE terminals, as demonstrated by a drastic reduction in labeling following NE destruction

with the neurotoxin 6-hydroxydopamine or DSP-4 (Tejani-Butt and Ordway 1992; Tejani-Butt et al. 1990).

The NET is dependent on extracellular Na+ to mediate NE reuptake and the effectiveness of NE reuptake inhibitors in inhibiting NE reuptake (Bruss et al.

1997, 1999; Harder and Bonisch 1985). The uptake of NE is Cl– dependent, meaning that the electrogenic process of NE transport is Na+ and Cl– driven

(Harder and Bonisch 1985). In addition to the electrogenic process, the NET demonstrates properties of a channel-like pore, in that it can transport NE

showing an infinite stoichiometry that can be blocked by cocaine and desipramine (Galli et al. 1995, 1996). A number of studies suggest that NET can be

regulated by diverse stimuli, neuronal activity, and peptide hormones, as well as protein kinases. Indeed, studies have shown that all monoaminergic

transporters (5-HTT, DAT, and NET) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly PKC (Bauman et al. 2000).

PKC activation results in an activity-dependent transporter phosphorylation and sequestration. Protein phosphatase–1/2A (PP-1/PP-2A) inhibitors, such as

okadaic acid and calyculin A, also promote monoaminergic transporter phosphorylation and functional downregulation (Bauman et al. 2000). These

phenomena that occur beyond the receptor level may well be important in the long-term actions of psychotropic drugs known to regulate protein kinases (G.

Chen et al. 1999; Manji and Lenox 1999).

Adrenergic Receptors

The and catecholamine receptors were first discovered more than 50 years ago (Alhquist 1948) and later subdivided further into 1, 2, and 1, 2, and 3

adrenoreceptors—all of which are GPCRs—on the basis of molecular cloning and pharmacological and biochemical studies (see Figure 1–5B).

receptors

There are three subtypes of 1 receptors, denoted 1A, 1B, and 1D; they are all positively coupled to PLC and possibly phospholipase A2 (see Figure 1–5B). The

2 family comprises the 2A/D, 2B, and 2C subtypes, which couple negatively to adenylyl cyclase and regulate K+ and Ca2+ channels (see Figure 1–5B). The

2A, 2B, and 2C adrenoceptors correspond to the human genes 2-C10, 2-C2, and 2-C4, respectively (see Bylund et al. 1994). The bovine, guinea pig, rat, and

mouse a2D adrenoreceptor is thought to be a species homolog or variant of the human 2A adrenoreceptor (Bylund et al. 1994) and is often referred to as

2A/D. The 2 receptors represent autoreceptors for NE neurons, and blockade of these autoreceptors results in increased NE release—a biochemical effect that

has been postulated to play a role in the mechanisms of action of selected antidepressants (e.g., mianserin, mirtazapine) and antipsychotics (e.g., clozapine).

In the LC, 2-adrenergic receptors converge onto similar K+ channels as opioid receptors, and this convergence has been postulated to represent a

mechanism for the efficacy of clonidine (an 2 agonist) in attenuating some of the physical symptoms of opioid withdrawal. The 2 antagonist yohimbine,

which robustly increases NE neuron firing and NE release, has been used as a provocative challenge in clinical studies of anxiety disorders and as an

antidepressant-potentiating agent. Given that NE neurons colocalize and release orexins, it is of interest that this neuropeptide has been implicated in sleep

disorders and hypoglycemia through its glucose-sensing tandem-pore K+ (K2P) effects in coordinating arousal (M. M. Scott et al. 2006).

receptors

The receptor family comprises 1, 2, and 3 adrenoreceptors, which are all positively coupled to adenylyl cyclase (Bylund et al. 1994) (see Figure 1–5B). As

is discussed in greater detail in the chapters on antidepressants and anxiolytics (see Chapters 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26),

most effective antidepressants produce a downregulation/desensitization of 1 receptors in rat forebrain, leading to the suggestion that these effects may

play a role in their therapeutic efficacy. Interestingly, receptors have also been shown to play a role in regulating emotional memories, leading to the

proposal that antagonists may have utility in the treatment of posttraumatic stress disorder (PTSD) (Cahill et al. 1994; Przybyslawski et al. 1999). 3

receptors are not believed to be present in the CNS but are abundantly expressed on brown fat, where they exert lipolytic and thermogenic effects. Not

surprisingly, there is active research attempting to develop selective 3 agonists for the treatment of obesity.

Cholinergic System

ACh is the only major low-molecular-weight neurotransmitter substance that is not derived from an amino acid (Kandel et al. 2000). ACh is synthesized from

acetyl coenzyme A and choline in nerve terminals via the enzyme choline acetyltransferase (ChAT). Choline is transported into the brain by uptake from the

bloodstream and enters the neuron via both high-affinity and low-affinity transport processes (Cooper et al. 2001). In addition to the “standard” ChAT

pathway, there are several additional possible mechanisms by which ACh can be synthesized; the precise roles of these additional pathways and their

physiological relevance in the CNS remain to be fully elucidated (Cooper et al. 2001). The highest activity of ChAT is observed in the interpeduncular nucleus,

caudate nucleus, corneal epithelium, retina, and central spinal roots. In contrast to the other transmitters discussed thus far (which are most dependent on

reuptake mechanisms), ACh has its signal terminated primarily by the enzyme acetylcholine esterase, which degrades ACh (Figure 1–6B). Not surprisingly,

therapeutic strategies to increase synaptic ACh levels (e.g., for the treatment of Alzheimer’s disease) have focused on inhibiting the activity of

cholinesterases.

FIGURE 1–6. The cholinergic system.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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This figure depicts the cholinergic pathways in the brain (A) and various regulatory processes involved in cholinergic neurotransmission (B). Choline crosses the blood–brain

barrier to enter the brain and is actively transported into cholinergic presynaptic terminals by an active uptake mechanism (requiring ATP). This neurotransmitter is produced

by a single enzymatic reaction in which acetyl coenzyme A (AcCoA) donates its acetyl group to choline by means of the enzyme choline acetyltransferase (ChAT). AcCoA is

primarily synthesized in the mitochondria of neurons. Upon its formation, acetylcholine (ACh) is sequestered into secretory vesicles by vesicle ACh transporters (VATs), where

it is stored. Vesamicol effectively blocks the transport of ACh into vesicles. An agent such as -bungarotoxin or AF64A is capable of increasing synaptic concentration of ACh

by acting as a releaser and a noncompetitive reuptake inhibitor, respectively. In turn, agents such as botulinum toxin are able to attenuate ACh release from nerve terminals.

Once released from the presynaptic terminals, ACh can interact with a variety of presynaptic and postsynaptic receptors. In contrast to many other monoaminergic

neurotransmitters, the ACh signal is terminated primarily by degradation by the enzyme acetylcholinesterase (AChE) rather than by reuptake. Interestingly, AChE is present

on both presynaptic and postsynaptic membranes and can be inhibited by physostigmine (reversible) and soman (irreversible). Currently, AChE inhibitors such as donepezil

and galantamine are the only classes of agents that are FDA approved for the treatment of Alzheimer’s disease. ACh receptors are of two types: muscarinic (G

protein–coupled) and nicotinic (ionotropic). Presynaptic regulation of ACh neuron firing activity and release occurs through somatodendritic (not shown) and nerve terminal

M2 autoreceptors, respectively. The binding of ACh to G protein–coupled muscarinic receptors that are negatively coupled to adenylyl cyclase (AC) or coupled to

phosphoinositol hydrolysis produces a cascade of second-messenger and cellular effects (see diagram). ACh also activates ionotropic nicotinic receptors (nAChRs). ACh has it

action terminated in the synapse through rapid degradation by AChE, which liberates free choline to be taken back into the presynaptic neuron through choline transporters

(CTs). Once inside the neuron, it can be reused for the synthesis of ACh, can be repackaged into vesicles for reuse, or undergoes enzymatic degradation. There are some

relatively new agents that selectively antagonize the muscarinic receptors, such as CI-1017 for M1, methoctramine for M2, 4-DAMP for M3, PD-102807 for M4, and

scopolamine (hardly a new agent) for M5 (although it also has affinity for M3 receptor). nAChR or nicotine receptors are activated by nicotine and the specific

alpha(4)beta(2*) agonist metanicotine. Mecamylamine is an AChR antagonist. DAG = diacylglycerol; IP3 = inositol-1,4,5-triphosphate.

Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,

1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc. Modified from Nestler et al. 2001.

Several cholinergic pathways have been proposed, but until recently the circuits had not been worked out in the brain because of the lack of appropriate

techniques. The development of tract tracing and histochemical techniques has provided a clearer picture of the cholinergic pathways. In brief, cholinergic

neurons can act as local circuit neurons (interneurons) and are found in the caudate putamen, nucleus accumbens, olfactory tubercle, and islands of Calleja

complex (Cooper et al. 2001). They do, however, also serve to function as projection neurons that connect different brain regions; one fairly

well-characterized pathway runs from the septum to the hippocampus (Figure 1–6A). The basal forebrain cholinergic complex is composed of cholinergic

neurons originating from the medial septal nucleus, diagonal band nuclei, substantia innominata, magnocellular preoptic field, and nucleus basalis. These

nuclei project cholinergic neurons to the entire nonstriatal telencephalon, pontomesencephalotegmental cholinergic complex, thalamus, and other

diencephalic loci (see Figure 1–6A). Descending cholinergic projections from these nuclei also innervate pontine and medullary reticular formations, deep

cerebellar and vestibular nuclei, and cranial nerve nuclei (Cooper et al. 2001).

Cholinergic Receptors

There are two major distinct classes of cholinergic receptors, the muscarinic and nicotinic receptors. Five muscarinic receptors (M1 through M5) have been

cloned (Kandel et al. 2000). These receptors are G protein–coupled and act either by regulating ion channels (in particular, K+ or Ca2+) or through being

linked to second-messenger systems. Generally speaking, M1, M3, and M5 are coupled to phosphoinositol hydrolysis, whereas M2 and M4 are coupled to

inhibition of adenylyl cyclase and regulation of K+ and Ca2+ channels (Cooper et al. 2001) (see Figure 1–6B).

By contrast, the nicotinic receptors are ionotropic receptors, and at least seven different functional receptors (based on different subunit composition) have

been identified. Biochemical and biophysical data indicate that the nicotinic receptors in the muscle are formed from five protein subunits, with the

stoichiometry of 2

(Kandel et al. 2000). The binding of ACh molecules on the subunit is necessary for channel activation. By contrast, neuronal nicotinic

receptors contain only two types of subunits ( and ), with the occurring in at least seven different forms and the in three (Cooper et al. 2001). NicotinicPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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receptors may be the targets of considerable cross-talk, as a variety of kinases (including PKA, PKC, and tyrosine kinases) are able to regulate the sensitivity

of this receptor. A number of regulatory mechanisms exist. For example, the mammalian prototoxin lynx1 acts as an allosteric modulator of nicotinic

acetylcholine receptors (Miwa et al. 2006).

From a clinical standpoint, Freedman et al. (1997) demonstrated that in a cohort of patients with schizophrenia, abnormal P50 auditory evoked potentials

were linked to a susceptibility locus for this disease on chromosome 15. Notably, this is where a nicotinic receptor subunit is found, providing indirect support

for the long-standing contention that the high rates of cigarette smoking in patients with schizophrenia may represent (at least in part) an attempt to correct

an underlying nicotinic receptor defect.

Glutamatergic System

Glutamate and aspartate are the two major excitatory amino acids in the CNS and are present in high concentrations (Nestler et al. 2001; Squire et al. 2003).

As the principal mediators of excitatory synaptic transmission in the mammalian brain, they participate in wide-ranging aspects of both normal and abnormal

CNS function. Physiologically, glutamate appears to play a prominent role in synaptic plasticity, learning, and memory. However, glutamate can also be a

potent neuronal excitotoxin under a variety of experimental conditions, triggering either rapid or delayed neuronal death. Unlike the monoamines, which

require transport of amino acids through the blood–brain barrier, glutamate and aspartate cannot adequately penetrate into the brain from the periphery and

are produced locally by specialized brain machinery. The metabolic and synthetic enzymes responsible for the formation of these nonessential amino acids are

located in glial cells as well as neurons (Squire et al. 2003).

The major metabolic pathway in the production of glutamate is derived from glucose and the transamination of -ketoglutarate; however, a small proportion

of glutamate is formed directly from glutamine. The latter is actually synthesized in glia, via an active process (requiring adenosine triphosphate [ATP]), and

is then transported to neurons where glutaminase is able to convert this precursor to glutamate (Figure 1–7). Following release, the concentration of

glutamate in the extracellular space is highly regulated and controlled, primarily by a Na+-dependent reuptake mechanism involving several transporter

proteins.

FIGURE 1–7. The glutamatergic system.

This figure depicts the various regulatory processes involved in glutamatergic neurotransmission. The biosynthetic pathway for glutamate involves synthesis from glucose and

the transamination of -ketoglutarate; however, a small proportion of glutamate is formed more directly from glutamine by glutamine synthetase. The latter is actually

synthesized in glia and, via an active process (requiring ATP), is transported to neurons, where in the mitochondria glutaminase is able to convert this precursor to glutamate.

Furthermore, in astrocytes glutamine can undergo oxidation to yield -ketoglutarate, which can also be transported to neurons and participate in glutamate synthesis.

Glutamate is either metabolized or sequestered and stored in secretory vesicles by vesicle glutamate transporters (VGluTs). Glutamate can then be released by a

calcium-dependent excitotoxic process. Once released from the presynaptic terminal, glutamate is able to bind to numerous excitatory amino acid (EAA) receptors, including

both ionotropic (e.g., NMDA [N-methyl-D-aspartate]) and metabotropic (mGluR) receptors. Presynaptic regulation of glutamate release occurs through metabotropic

glutamate receptors (mGluR2 and mGluR3), which subserve the function of autoreceptors; however, these receptors are also located on the postsynaptic element. Glutamate

has its action terminated in the synapse by reuptake mechanisms utilizing distinct glutamate transporters (labeled VGT in figure) that exist on not only presynaptic nerve

terminals but also astrocytes; indeed, current data suggest that astrocytic glutamate uptake may be more important for clearing excess glutamate, raising the possibility thatPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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astrocytic loss (as has been documented in mood disorders) may contribute to deleterious glutamate signaling, but more so by astrocytes. It is now known that a number of

important intracellular proteins are able to alter the function of glutamate receptors (see diagram). Also, growth factors such as glial-derived neurotrophic factor (GDNF) and

S100 secreted from glia have been demonstrated to exert a tremendous influence on glutamatergic neurons and synapse formation. Of note, serotonin1A (5-HT1A) receptors

have been documented to be regulated by antidepressant agents; this receptor is also able to modulate the release of S100 . AKAP = A kinase anchoring protein; CaMKII =

Ca2+/calmodulin–dependent protein kinase II; ERK = extracellular response kinase; GKAP = guanylate kinase–associated protein; Glu = glutamate; Gly = glycine; GTg =

glutamate transporter glial; GTn = glutamate transporter neuronal; Hsp70 = heat shock protein 70; MEK = mitogen-activated protein kinase/ERK; mGluR = metabotropic

glutamate receptor; MyoV = myosin V; NMDAR = NMDA receptor; nNOS = neuronal nitric oxide synthase; PKA = phosphokinase A; PKC = phosphokinase C; PP-1, PP-2A,

PP-2B = protein phosphatases; RSK = ribosomal S6 kinase; SHP2 = src homology 2 domain–containing tyrosine phosphatase.

Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,

1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc. Modified from Nicholls 1994.

The major glutamate transporter proteins found in the CNS include the excitatory amino acid transporters (EAATs) EAAT1 (or GLAST-1), EAAT2 (or GLT-1),

and EAAT3 (or EAAC1), with EAAT2 being the most predominantly expressed form in the forebrain. Additionally, these transporters are differentially

expressed in specific cell types, with EAAT1 and EAAT2 being found primarily in glial cells and EAAT3 being localized in neurons. EAAT4 is mainly localized in

cerebellum. The physiological events regulating the activity of the glutamate transporters are not well understood, although there is evidence that

phosphorylation of the transporters by protein kinases may differentially regulate glutamate transporters and therefore glutamate reuptake (Casado et al.

1993; Conradt and Stoffel 1997; Pisano et al. 1996). Glutamate concentrations have been shown to rise to excitotoxic levels within minutes following

traumatic or ischemic injury, and there is evidence that the function of the glutamate transporters becomes impaired under these excitotoxic conditions

(Faden et al. 1989). It is surprising that the glutamatergic system has only recently undergone extensive investigation with regard to its possible involvement

in the pathophysiology of mood disorders, since it is the major excitatory neurotransmitter in the CNS and known to play a role in regulating the threshold for

excitation of most other neurotransmitter systems. Although direct evidence for glutamatergic excitotoxicity in bipolar disorder is lacking and the precise

mechanisms underlying the cell atrophy and death that occur in recurrent mood disorders are unknown, considerable data have shown that impairments of

the glutamatergic system play a major role in the morphometric changes observed with severe stresses (McEwen 1999; Sapolsky 2000).

It is now clear that modification of the levels of synaptic AMPA-type glutamate receptors—in particular by receptor subunit trafficking, insertion, and

internalization—is a critically important mechanism for regulating various forms of synaptic plasticity and behavior. Recent studies have identified

region-specific alterations in expression levels of AMPA and NMDA glutamate receptor subunits in subjects with mood disorders (Beneyto et al. 2007).

Supporting the suggestion that abnormalities in glutamate signaling may be involved in mood pathophysiology, AMPA receptors have been shown to regulate

affective-like behaviors in rodents. AMPA antagonists have been demonstrated to attenuate amphetamine- and cocaine-induced hyperactivity and

psychostimulant-induced sensitization and hedonic behavior (Goodwin and Jamison 2007).

Glutamatergic Receptors

The many subtypes of glutamatergic receptors in the CNS can be classified into two major subtypes: ionotropic and metabotropic receptors (see Figure 1–7).

Ionotropic glutamate receptors

The ionotropic glutamate receptor ion channels are assemblies of homo- or hetero-oligomeric subunits integrated into the neuron’s membrane. Every channel

is assembled of (most likely) four subunits associated into a dimer of dimers as has been observed in crystallographic studies (Ayalon and Stern-Bach 2001;

Madden 2002). Every subunit consists of an extracellular amino-terminal and ligand binding domain, three transmembrane domains, a reentrant pore loop

(located between the first and second transmembrane domains), and an intracellular carboxyl-terminal domain (Hollmann et al. 1994). The subunits

associate through interactions between their amino-terminal domains, forming a dimer that undergoes a second dimerization mediated by interactions

between the ligand binding domains and/or between transmembrane domains (Ayalon and Stern-Bach 2001; Madden 2002). Three different subgroups of

glutamatergic ion channels have been identified on the basis of their pharmacological ability to bind different synthetic ligands, each of which is composed of

a different set of subunits. The three subgroups are the NMDA receptors, the AMPA ( -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, and

the kainate receptor. The latter two groups are often referred to together as the “non-NMDA” receptors, but they undoubtedly subserve unique functions (see

Figure 1–7). In the adult mammalian brain, NMDA and AMPA glutamatergic receptors are collocated in approximately 70% of the synapses (Bekkers and

Stevens 1989). By contrast, at early stages of development, synapses are more likely to contain only NMDA receptors. Radioligand binding studies have

shown that NMDA and AMPA receptors are found in high density in the cerebral cortex, hippocampus, striatum, septum, and amygdala.

NMDA receptors

The NMDA receptor is activated by glutamate and requires the presence of a co-agonist, namely glycine or D-serine, to be activated, a process that likely

varies in importance according to brain region (Panatier et al. 2006). However, the binding of both glutamate and glycine is still not sufficient for the NMDA

receptor channel to open, since at resting membrane potential, the NMDA ion channel is blocked by Mg2+ ions. Only when the membrane is depolarized (e.g.,

by the activation of AMPA or kainate receptors on the same postsynaptic neuron) is the Mg2+ blockade relieved. Under these conditions, the NMDA receptor

channel will open and permit the entry of both Na+ and Ca2+ (see Figure 1–7).

The NMDA receptor channel is composed of a combination of NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B subunits (see Figure 1–7). The binding site for

glutamate has been localized to the NR2 subunit, and the site for the co-agonist glycine has been localized to the NR1 subunit, which is required for receptor

function. Two molecules of glutamate and two of glycine are thought to be necessary to activate the ion channel. Within the ion channel, two other sites have

been identified—the sigma ( ) site and the phencyclidine (PCP) site. The hallucinogenic drug PCP, ketamine, and the experimental drug dizocilpine (MK-801)

all bind at the latter site and are considered noncompetitive receptor antagonists that inhibit NMDA receptor channel function.

In clinical psychiatric studies, ketamine has been shown to transiently induce psychotic symptoms in schizophrenic patients and to produce antidepressant

effects in some depressed patients (Krystal et al. 2002). Building on these preclinical and preliminary clinical data, recent clinical trials have investigated the

clinical effects of glutamatergic agents in subjects with mood disorders. Recent clinical studies have demonstrated effective and rapid antidepressant action

of glutamatergic agents, including ketamine, an NMDA receptor antagonist, and riluzole, a glutamate release inhibitor (Sanacora et al. 2007; Zarate et al.

2006a). These and other data have led to the hypothesis that alterations in neural plasticity in critical limbic and reward circuits, mediated by increasing the

postsynaptic AMPA-to-NMDA throughput, may represent a convergent mechanism for antidepressant action (Zarate et al. 2006b). This line of research holds

considerable promise for developing new treatments for depression and bipolar disorder. The NMDA receptor agonists glycine, D-serine, and D-cycloserine

have been shown to improve cognition and decrease negative symptoms in patients with schizophrenia who are receiving antipsychotics (Coyle et al. 2002).

NMDA receptors in the amygdala may also be of critical importance in the process of transforming a fixed and consolidated fear memory to a labile state (Ben

Mamou et al. 2006).

NMDA receptors play a critical role in regulating synaptic plasticity (Malenka and Nicoll 1999). The best-studied forms of synaptic plasticity in the CNS are

long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission. The molecular mechanisms of LTP and LTD have been

extensively characterized and have been proposed to represent cellular models of learning and memory (Malenka and Nicoll 1999). Induction of LTP and LTD

in the CA1 region of the hippocampus and in many regions of the brain has now clearly been demonstrated to be dependent on NMDA receptor activation.

During NMDA receptor–dependent synaptic plasticity, Ca2+ influx through NMDA receptors can activate a wide variety of kinases and/or phosphatases that in

turn modulate synaptic strength. An important development was the finding that two of the primary molecules involved—CaMKII and the NMDA subtype of

glutamate receptor—form a tight complex with each other at the synapse (Lisman and McIntyre 2001). Interestingly, this binding appears to enhance both

the autophosphorylation of the kinase and the ability of the entire holoenzyme, which has 12 subunits, to become hyperphosphorylated (Lisman and McIntyre

2001). This hyperphosphorylated state has been postulated to represent a “memory switch” that can lead to long-term strengthening of the synapse by

multiple mechanisms. One important mechanism involves direct phosphorylation of the glutamate-activated AMPA receptors, which increases their

conductance. Furthermore, CaMKII, once bound to the NMDA receptor, may organize additional anchoring sites for AMPA receptors at the synapse. Switching

of synaptic NMDA receptor subunits, which bind CaMKII, for other NMDA receptor subunits having no affinity for this enzyme dramatically reduces LTP,

demonstrating that glutamate and calcium signaling interactions are critical for learning and memory (Barria and Malinow 2005).Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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While the NMDA receptor clearly plays important roles in plasticity, abundant evidence has shown that excessive glutamatergic signaling is also involved in

neuronal toxicity. With anoxia or hypoglycemia, the highly energy-dependent uptake mechanisms that keep glutamate compartmentalized in presynaptic

terminals fail. Within minutes, glutamate is massively released into the synaptic space, resulting in activation of excitatory amino acid receptors. This leads to

depolarization of target neurons via AMPA and kainate receptors and then to inappropriate and excessive activation of NMDA receptors. Considerable data

suggest that the large excess of Ca2+ entering cells via the NMDA receptor channel may represent an important step in the rapid cell death that occurs via

excitotoxicity.

AMPA receptors

The AMPA receptor is stimulated by the presence of glutamate and characteristically produces a fast excitatory synaptic signal that is responsible for the

initial reaction to glutamate in the synapse. In fact, as discussed above, it is generally believed that it is the activation of the AMPA receptor that results in

neuronal depolarization sufficient to liberate the Mg2+ cation from the NMDA receptor, thereby permitting its activation. The AMPA receptor channel is

composed of the combination of the GluR1, GluR2, GluR3, and GluR4 subunits and requires two molecules of glutamate to be activated (see Figure 1–7).

AMPA receptors have a lower affinity for glutamate than does the NMDA receptor, thereby allowing for more rapid dissociation of glutamate and, therefore, a

rapid deactivation of the AMPA receptor (for a review, see Dingledine et al. 1999).

Studies have indicated that AMPA receptor subunits are direct substrates of protein kinases and phosphatases. Phosphorylation of the receptor subunits

regulates not only the intrinsic channel properties of the receptor but also the interaction of the receptor with associated proteins that modulate the

membrane trafficking and synaptic targeting of the receptors (discussed in Malinow and Malenka 2002). Additionally, protein phosphorylation of other

synaptic proteins has been proposed to indirectly modulate AMPA receptor function by affecting the macromolecular complexes that are important for the

presence of AMPA receptors at the synaptic plasma membrane (Malinow and Malenka 2002; Nestler et al. 2001). Studies have been elucidating the cellular

mechanisms by which AMPA receptor subunit insertion and trafficking occur and have revealed two major mechanisms (Malinow and Malenka 2002; Nestler

et al. 2001). The first mechanism is used for GluR1-containing AMPA receptor insertion and is regulated by activity. The second mechanism is governed by

constitutive receptor recycling, mainly through GluR2/3 heteromers in response to activity-dependent signals. Data suggest that AMPA receptor subunit

trafficking may play an important role in neuropsychiatric disorders. Thus, Nestler and associates have shown that the ability of drugs of abuse to elevate

levels of the GluR1 subunit of AMPA glutamate receptors in the VTA of the midbrain is crucial for the development of sensitization (Carlezon and Nestler

2002). They have demonstrated that even transient increases in GluR1 levels within VTA neurons can trigger complex cascades of other molecular

adaptations in these neurons and, within larger neural circuits, can cause enduring changes in the responses of the brain to drugs of abuse. Chronic lithium

and valproate have been shown to reduce GluR1 expression in hippocampal synaptosomes, effects that may play a role in the delayed therapeutic effects of

these agents (Du et al. 2003; Szabo et al. 2002).

Recent studies have sought to test the hypothesis that “antidepressant anticonvulsants,” like traditional antidepressants, can enhance surface AMPA

receptors (Du et al. 2007). It was found that the predominantly antidepressant anticonvulsants lamotrigine and riluzole significantly enhanced the surface

expression of GluR1 and GluR2 in a time- and dose-dependent manner in cultured hippocampal neurons. By contrast, the predominantly antimanic

anticonvulsant valproate significantly reduced surface expression of GluR1 and GluR2. Concomitant with the GluR1 and GluR2 changes, the peak value of

depolarized membrane potential evoked by AMPA was significantly higher in lamotrigine- and riluzole-treated neurons, supporting the surface receptor

changes. In addition, lamotrigine and riluzole, as well as the traditional antidepressant imipramine, increased GluR1 phosphorylation at GluR1 (S845) in the

hippocampus after chronic in vivo treatment.

Recent clinical research has demonstrated a robust and rapid antidepressant effect of ketamine; studies were therefore undertaken to test the hypothesis

that ketamine brings about its rapid antidepressant effect by enhancing AMPA relative to NMDA throughput (Maeng et al. 2008). Although the AMPA

antagonist NBQX was without behavioral effects alone, it blocked the antidepressant-like effects of ketamine. AMPA antagonists also blocked

ketamine-induced changes in hippocampal GluR1 AMPA receptor phosphorylation. Together, these results suggest that regulating AMPA relative to NMDA

throughput in critical neuronal circuits may play an important role in antidepressant action.

Kainate receptors

The kainate receptor has pre- and postsynaptic roles, sharing some properties with AMPA receptors. It is composed of the combination of the GluR5, GluR6,

GluR7, KA1, and KA2 subunits (see Figure 1–7). The precise role of kainate receptors in the mature CNS remains to be fully elucidated, although the activity of

the receptors clearly plays a role in synaptic function in many brain areas. Increasing data suggest the involvement of aberrant synaptic plasticity in the

pathophysiology of bipolar disorder. Kainate receptors contribute to synaptic plasticity in different brain regions involved in mood regulation, including the

prefrontal cortex, hippocampus, and amygdala. GluR6 (GRIK2) is a subtype of kainate receptor whose chromosomal loci of 6q16.3–q21 have been identified

as potentially harboring genetic polymorphism(s) contributing to an increased risk of mood disorders. The role of GluR6 in modulating animal behaviors

correlated with mood symptoms was investigated using GluR6 knockout and wild-type mice (Shaltiel et al. 2008). GluR6 knockout mice appeared to attain

normal growth and showed no neurological abnormalities. GluR6 mice showed increased basal- or amphetamine-induced activity, were extremely aggressive,

took more risks, and consumed more saccharin (a measure of hedonic drive). Notably, most of these aberrant behaviors responded to chronic lithium

administration. These results suggest that abnormalities in kainate receptor throughput generated by GluR6 gene disruption may lead to the concurrent

appearance of a constellation of behaviors related to manic symptoms, including persistent hyperactivity; escalated irritability, aggression, and risk taking;

and hyperhedonia.

Metabotropic glutamate receptors

The metabotropic glutamate receptors (mGluRs) are G protein–coupled receptors. The eight types of receptors that currently have been cloned can be

organized into three different subgroups (groups I, II, and III) based largely on the signaling transduction pathways that they activate (see Figure 1–7).

These receptors have a large extracellular N-terminal consisting of two lobes forming a “venus flytrap” binding pocket involved in glutamate recognition and

a cysteine-rich extracellular domain that connects with seven transmembrane domains separated by short intra- and extracellular loops (see Figure 1–7). The

intracellular loop plays an important role in the coupling with and selectivity of the G protein. The cytoplasmic carboxyl-terminal domain is variable in length

and is involved with G protein activation and coupling efficacy (Bruno et al. 2001; Conn and Pin 1997).

The mGluR group I includes the mGluR1 (a, b, c, d), and mGluR5 (a, b) receptors (see Figure 1–7). They preferentially interact with the G q/11 subunit of G

proteins, leading to activation of the IP3/calcium and DAG/PKC cascades. The receptors are located on both pre- and postsynaptic neurons. Group II

metabotropic receptors include mGluR2 and mGluR3, which have been best characterized as inhibiting adenylyl cyclase but, like many receptors coupled to

Gi/Go, may also regulate ion channels. Group III receptors, which include mGluR4 (a, b), mGluR6, mGluR7 (a, b), and mGluR8 (a, b), are reported to produce

inhibition of adenylyl cyclase as well, but also to interact with the phosphodiesterase enzyme regulating guanosine monophosphate (cGMP) levels (Cooper et

1. 2001; Squire et al. 2003). The group II and III receptors are located in the presynaptic membrane and, because of their coupling with Gi/Go proteins,

appear to negatively modulate glutamate and GABA neurotransmission output when activated (i.e., they serve as inhibitory auto- and heteroreceptors).

Preclinical studies suggest that mGlu group II and III receptors are “extrasynaptic” in their localization; that is, they are located some distance from the

synaptic cleft and are thus activated only under conditions of excessive (pathological?) glutamate release, when there is sufficient glutamate to diffuse out of

the synapse to these receptors (Schoepp 2001). In preclinical studies, mGluR2/3 agonists have been demonstrated to exert anxiolytic, antipsychotic, and

neuroprotective properties (Schoepp 2001).

Glycine

Glycine is a nonessential amino acid that also functions as a neurotransmitter in the CNS. Although the exact metabolic pathway for glycine production has

yet to be fully elucidated, evidence suggests that glycine may be produced in the CNS by two distinct pathways. First, glycine is produced from serine by the

enzyme serine-trans-hydroxymethylase in a reversible folate-dependent reaction (Cooper et al. 2001; Squire et al. 2003). Additionally, glycine may be

produced from glyoxylate by the enzyme D-glycerate dehydrogenase. This amino acid is found in higher concentrations in the spinal cord than in the rest of

the CNS. Glycine acts as an inhibitory neurotransmitter predominantly in the brain stem and spinal cord (Nestler et al. 2001). As discussed earlier, a very

important role that glycine also plays is to augment the NMDA-mediated frequency of NMDA receptor channel opening. This effect is strychnine-insensitive

and pharmacologically suggests that the actions of glycine on NMDA receptor function are different from its effect on the spinal cord, where glycine’sPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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inhibitory effect is blocked by strychnine (Cooper et al. 2001). The allosteric modulation of NMDA receptors via a glycine-insensitive site is further

underscored by receptor binding experiments yielding an anatomic distribution similar to that of NMDA receptors. Functionally, it has been postulated that

glycine is able to augment the NMDA-mediated responses by speeding up the recovery process of the receptor (Cooper et al. 2001). Given the ability of

glycine to alter NMDA function, glycine may be beneficial in the treatment of schizophrenia (Coyle et al. 2002).

GABAergic System

-Aminobutyric acid—the major inhibitory neurotransmitter system in the CNS—is one of the most abundant neurotransmitters, and GABA-containing neurons

are located in virtually every area of the brain. Unlike the monoamines, GABA occurs in the brain in high concentrations in the order of micromoles per

milligrams (about 1,000-fold higher than concentrations of monoamines) (Cooper et al. 2001; Nestler et al. 2001; Squire et al. 2003). GABA is produced when

glucose is converted to -ketoglutarate, which is then transaminated to glutamate by GABA -oxoglutarate transaminase (GABA-T). Glutamic acid is

decarboxylated by glutamic acid decarboxylase, which leads to the formation of GABA (Figure 1–8). Indeed, the neurotransmitter and the rate-limiting

enzyme are localized together in the brain and at approximately the same concentration. Catabolism of GABA occurs via GABA-T, which is also important in

the synthesis of this transmitter.

FIGURE 1–8. The GABAergic system.

This figure depicts the various regulatory processes involved in GABAergic neurotransmission. The amino acid (and neurotransmitter) glutamate serves as the precursor for

the biosynthesis of -aminobutyric acid (GABA). The rate-limiting enzyme for the process is glutamic acid decarboxylase (GAD), which utilizes pyridoxal phosphate as an

important cofactor. Furthermore, agents such as L-glutamine- -hydrazide and allylglycine inhibit this enzyme and, thus, the production of GABA. Once released from the

presynaptic terminal, GABA can interact with a variety of presynaptic and postsynaptic receptors. Presynaptic regulation of GABA neuron firing activity and release occurs

through somatodendritic (not shown) and nerve-terminal GABAB receptors, respectively. Baclofen is a GABAB receptor agonist. The binding of GABA to ionotropic GABAA

receptors and metabotropic GABAB receptors mediates the effects of this receptor. The GABAB receptors are thought to mediate their actions by being coupled to Ca2+ or K+

channels via second-messenger systems. Many agents are able to modulate GABAA receptor function. Benzodiazepines, such as diazepam, increase Cl– permeability, and

there are numerous available antagonists directed against this site. There is also a distinctive barbiturate binding site on GABAA receptors, and many psychotropic agents are

capable of influencing the function of this receptor (see blown-up diagram). GABA is taken back into presynaptic nerve endings by a high-affinity GABA uptake transporter

(GABAT) similar to that of the monoamines. Once inside the neuron, GABA can be broken down by GABA transaminase (GABA-T), which is localized in the mitochondria;

GABA that is not degraded is sequestered and stored into secretory vesicles by vesicle GABA transporters (VGTs), which differ from VMATs in their bioenergetic dependence.

The metabolic pathway that produces GABA, mostly from glucose, is referred to as the GABA shunt. The conversion of -ketoglutarate into glutamate by the action of GABA-T

and GAD catalyzes the decarboxylation of glutamic acid to produce GABA. GABA can undergo numerous transformations, of which the simplest is the reduction of succinic

semialdehyde (SS) to -hydroxybutyrate (GHB). On the other hand, when SS is oxidized by succinic semialdehyde dehydrogenase (SSADH), the production of succinic acid

(SA) occurs. GHB has received attention because it regulates narcoleptic episodes and may produce amnestic effects. The mood stabilizer and antiepileptic drug valproic acid

is reported to inhibit SSADH and GABA-T. TBPS = t-butylbicyclophosphorothionate.

Source. Adapted from Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of Neuropharmacology, 7th Edition. New York, Oxford University Press, 2001. Copyright 1970,Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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1974, 1978, 1982, 1986, 1991, 1996, 2001 by Oxford University Press, Inc. Used by permission of Oxford University Press, Inc.

The function of this dual-role enzyme becomes apparent when placed in the context of its role in the metabolic process. GABA-T converts GABA to succinic

acid, and subsequent removal of the amino group yields -ketoglutarate. Thus, -ketoglutarate is able to be used by GABA-T in GABA biosynthesis as

mentioned above (Cooper et al. 2001). This process, called the GABA shunt, maintains a steady GABA supply in the brain. As with the monoamines, the major

mechanism by which the effects of GABA are terminated in the synaptic cleft is by reuptake through GABA transporters. The GABA transporters have a high

affinity for GABA and mediate their reuptake via a Na+ and Cl– gradient (Squire et al. 2003).

Detailed studies from the Rajkowska laboratory (Grazyna Rajkowska, The University of Mississippi Medical Center, Jackson, MS) have measured the density

and size of calbindin-immunoreactive neurons (presumed to be GABAergic) in layers II and III of the dorsolateral prefrontal cortex, revealing a 43%

reduction in the density of these neurons in patients with major depressive disorder compared with controls (discussed in Goodwin and Jamison 2007). Of

particular note, in the rostral orbitofrontal cortex, there was a trend toward a negative correlation between the duration of depression and the size of

neuronal cell bodies, suggesting changes associated with disease progression. Valproate has also been shown to have neurogenic effects in at least one

study. In cultured embryonic rat cortical cells and striatal primordial stem cells, valproate markedly increased the number and percentage of primarily

GABAergic neurons and promoted neurite outgrowth (Laeng et al. 2004).

GABA Receptors

There are two major types of well-characterized GABA receptors, GABAA and GABAB, and most neurons in the CNS possess at least one of these types. The

GABAA receptor is the more prevalent of the two in the mammalian CNS and as a result has been extensively studied and characterized. GABAA contains an

integral transmembrane chloride channel, which is opened upon receptor activation, generally resulting in hyperpolarization of the neuron (i.e., suppressing

excitability). The GABA receptor is a heteropentameric glycoprotein of approximately 275 kDa composed of a combination of multiple polypeptide subunits.

GABAA displays enormous heterogeneity, being composed of a combination of five classes of polypeptide subunits ( , , , , ), of which there are at least 18

total subtypes. The various receptors display variation in functional pharmacology, hinting at the multiple finely tuned roles that inhibitory neurotransmission

plays in brain function.

It is now well established that benzodiazepines (BZDs) function by binding to a potentiator site on the GABAA receptor, increasing the amplitude and duration

of inhibitory postsynaptic currents in response to GABA binding. Coexpression of additional subunits is believed to be necessary for the potentiation of

GABA-mediated responses by BZDs. In addition to BZDs, barbiturates and ethanol are also believed to exert many of their effects by potentiating the opening

of the GABAA receptor chloride channel (see Figure 1–8). As noted earlier, GABAA receptors have a widespread distribution in the brain, and the majority of

these receptors in the brain are targets of the currently available BZDs. For this reason, there has been considerable interest in determining if the desirable

and undesirable effects of BZDs can be differentiated on the basis of the presence of different subunit composition. Much of the work has used gene knockout

technology; thus, mutation of the BZD-binding site of the 1 subunit in mice blocks the sedative, anticonvulsive, and amnesic, but not the anxiolytic, effects of

diazepam (see Gould et al. 2003; Mohler et al. 2002). In contrast, the 2 subunit (expressed highly in the cortex and hippocampus) is necessary for diazepam

anxiolysis and myorelaxation. Thus, there is now optimism that an 2-selective ligand will soon provide effective acute treatment of anxiety disorders without

the unfavorable side-effect profile of current BZDs. A compound with this preferential affinity has already been demonstrated to exert fewer

sedative/depressant effects than diazepam in rat behavioral studies (see Gould et al. 2003; Mohler et al. 2002).

The phosphorylation of GABAA receptors is another mechanism by which this receptor complex can be regulated in function and expression. In this context, it

is noteworthy that studies have reported that knockout mice deficient in PKC isoforms show reduced anxiety and alcohol consumption and an enhanced

response to the effects of BZDs (discussed in Gould et al. 2003). Furthermore, different GABAA receptor subunit partnerships, such as 1/ , mediate tonic

inhibitory currents in the hippocampus and are highly sensitive to low concentrations of ethanol (Glykys et al. 2007).

The GABAB receptors are coupled to Gi and Go and thereby regulate adenylyl cyclase activity (generally inhibit), K+ channels (open), and Ca2+ channels

(close). GABAB receptors can function as an autoreceptor but are also found abundantly postsynaptically on non-GABAergic neurons. Of interest, there is

mounting evidence that receptor dimerization may be required for the activation of GABAB and possibly other G protein–coupled receptors; although receptor

dimerization has long been known to occur for growth factor and JAK (Janus tyrosine kinase)/STAT (signal transducers and activators of transcription)

receptors (discussed later in this chapter), this was not expected for GPCRs. However, studies have reported that coexpression of two GABAB receptor

subunits—subunit 1 (GABABR1) and subunit 2 (GABABR2)—is necessary for the formation of a functional GABAB receptor (Bouvier 2001). Some data suggest

that GABABR2 may be necessary for proper protein folding of GABABR1 (acting as a molecular chaperone) in the endoplasmic reticulum, but this remains to

be definitively established. Support for the physiological relevance of this dimerization comes from studies showing that the GABAB R1 and R2 subunits can be

co-immunoprecipitated in rat cortical membrane preparations (Kaupmann et al. 1997); thus, the dimerization is not simply an in vitro phenomenon.

PURINERGIC NEUROTRANSMISSION: FOCUS ON ADENOSINE

It has been known for quite some time that ATP is capable of exerting profound effects on the nervous system (Drury and Szent-Gyšrgyi 1929). However,

adenosine and adenosine nucleotides have gained acceptance as neuroactive substances in the CNS only relatively recently (Cooper et al. 2001). Adenosine is

released from neurons and glia, but many of the neurotransmitter criteria outlined in the beginning of this chapter are not met. Nonetheless, adenosine is

able to activate many cellular functions that are able to produce changes in neuronal and behavioral states. For example, adenosine is able to stimulate cAMP

in vitro in brain slices, and caffeine (which in addition to being a phosphodiesterase inhibitor is a well-known adenosine receptor antagonist) is able to block

this response.

Four adenosine receptors have been cloned (A1, A2A, A2B, and A3), each of which exhibits unique tissue distribution, ligand binding affinity (nanomolar

range), and signal transduction mechanisms (Cooper et al. 2001). Currently available data suggest that the high-affinity adenosine receptors (A1 and A2A)

may be activated under normal physiological conditions, whereas in pathological states such as hypoxia and inflammation (in which high adenosine

concentrations [micromolar range] are present), low-affinity A2B and A3 receptors are also activated. A2B receptors are expressed in low levels in the brain

but are ubiquitous in the rest of the body, whereas A2A receptors are found in high concentrations in areas of the brain that receive dopaminergic projections

(i.e., striatum, nucleus accumbens, and olfactory tubercle) (Nestler et al. 2001). Given this receptor’s distribution and the inverse relationship between DA

and adenosine, it has been postulated that A2A antagonists may have some utility in the treatment of Parkinson’s disease (Nestler et al. 2001). The mood

stabilizer and antiepileptic drug carbamazepine acts as an antagonist of the A1 subtype and also decreases protein levels of the receptor (for a review, see

Gould et al. 2002).

Adenosine is widely regarded as important in the homeostasis of blood flow and metabolic demands in peripheral tissue physiology. Adenosine is also able to

alter the function (both pre- and postsynaptically) of numerous neurotransmitters and their receptors, including NMDA, metabotropic glutamate receptors,

ionotropic nicotinic receptors, NE, 5-HT, DA, GABA, and various peptidergic receptors. Recent evidence implicates adenosine as a fatigue factor in the

decrease of cholinergic activity-arousal via presynaptic inhibition of glutamate release (Brambilla et al. 2005). In addition, P2X (ligand-gated ion channels)

and P2Y (G protein–coupled receptors) are purine receptors that can be activated by ATP. It has been demonstrated that ATP is released from astrocytes

(through an unknown mechanism) and that the release is accompanied by glutamate release (Ca2+-dependent) (Innocenti et al. 2000). However, more data

suggest that it may be adenosine (that is derived from ATP) that serves as the true ligand for these purinergic receptors (Fields and Stevens-Graham 2002).

The ATP/adenosine is then able to activate purine receptors (P2Y receptors) on neighboring astrocytes, and this stimulates Ca2+ influx and subsequent

release of glutamate and ATP to then impact other astrocytes and neurons. This may be a critical component in the communication process between glial

cells, as well as representing a signaling molecule from glia to neurons (Fields and Stevens-Graham 2002).

PEPTIDERGIC NEUROTRANSMISSION

Neuropeptides have garnered increasing attention as critical modulators of CNS function. In general, peptide transmitters are released from neurons when

they are stimulated at higher frequencies from those required to facilitate release of traditional neurotransmitters, but they can also be colocalized and

coreleased together with other neurotransmitters (Cooper et al. 2001; Nestler et al. 2001). Modulation of the firing rate pattern of neurons and subsequent

release of neurotransmitters and peptides in a circumscribed fashion are likely important in the basal functioning of the brain as well as response to specific

stimuli. For instance, cannabinoids, an example of a neuropeptide neurotransmitter, do not alter the firing rates of hippocampal neurons but instead changePrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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the temporal coordination of those neurons, an effect that correlates with memory deficits in individuals (Soltesz and Staley 2006). Virtually every known

mammalian bioactive peptide is synthesized first as a precursor protein in which product peptides are flanked by cleavage sites. Neuropeptides are generally

found in large dense-core vesicles, whereas other neurotransmitters, such as the monoamines, are packaged in small synaptic vesicles (approximately 50

1. nm) and are usually half the size of their peptidergic counterparts (Kandel et al. 2000; Squire et al. 2003).

Space limitations preclude an extensive discussion of the diverse array of neuropeptides known to exist in the mammalian brain. Table 1–2 highlights some of

the major neuropeptides that may be of particular psychiatric relevance. In the remainder of this section, the basic aspects of peptidergic transmission are

highlighted vis-à-vis an overview of opioidergic neurotransmission.

TABLE 1–2. Selected peptides and their presumed relevance to psychiatric disorders and treatment

Group Potential clinical reference

Opioid and related peptides

Endorphin All of these peptides may be involved in opiate dependence/drug abuse; possible antidepressant activity; chronic pain

Enkephalin

Dynorphin

Nociceptin

Gut-derived peptides

VIP

Sexual behavior

CCK Anxiety/panic

Gastrin

Secretin Autism?

Somatostatin Mood disorders and treatment

Tachykinin peptides

Substance P NK1 receptor antagonists may alleviate depression/anxiety

Substance K Regulated by antipsychotics

Neuromedin N Regulated by lithium

Pituitary peptides

Oxytocin Affiliative behavior

Vasopressin Potential novel anxiolytics?

ACTH Dysregulated in mood disorders

MSH

Hypothalamic releasing factors

CRF Strongly implicated in depressive and anxiety symptoms; potential target for novel treatments

TRF Potential antidepressant effects

GHRF

LHRF

Others

Calcitonin gene–related peptide Regulated by ECT and lithium

Angiotensin Mood disorders, bipolar disorder

Neurotensin Regulated by antipsychotics and stimulants

Leptin Satiety signal; involved in diagnosis and in treatment-induced appetite/weight changes?

CART Drug addiction, eating disorders

Galanin Potentially relevant for Alzheimer’s diagnosis and other cognitive disorders

Neuropeptide Y Potential endogenous anxiolytic; regulated by antidepressants/lithium; reduced by early maternal separation

Orexin/hypocretin Narcolepsy; sleep abnormalities in other disorders?

Note. This table summarizes selected peptides and their presumed relevance for psychiatric disorders and their treatment; it is not meant to be an exhaustive listing of

findings. It should also be noted that in some cases—for example, CRF (mood/anxiety), NPY and neurotensin (regulation by medications), oxytocin (affiliative behavior), and

orexin (narcolepsy)—the data are quite convincing. In many of the other examples noted, the evidence must be considered preliminary but is, in our opinion, quite noteworthy

and warrants further investigation. ACTH = adrenocorticotropic hormone; CART = cocaine- and amphetamine-related transcript; CCK = cholecystokinin; CRF =

corticotropin-releasing factor; ECT = electroconvulsive therapy; GHRF = growth hormone–releasing factor; LHRF = luteinizing hormone–releasing factor; MSH =

melanocyte-stimulating hormone; NPY = neuropeptide Y; TRF = thyrotropin-releasing factor; VIP = vasoactive intestinal peptide.

Opioids are a family of peptides that occur endogenously in the brain (endorphins), as botanicals, or as drugs. Pro-opiomelanocortin (POMC),

proenkephalin-derived peptides, and prodynorphin-derived peptides yield opioid peptides upon cleavage. Three opioid peptide families currently exist:

enkephalins, endorphins, and dynorphins. There are also three types of opioid receptors—namely, , , and —each of which is further subclassified. POMC

gene expression occurs in various areas of the brain as well as other tissues. POMC has tissue- and cell-specific regulatory factors at every step from gene

transcription to its posttranslational processing. Opioid peptides are stored in large dense-core vesicles and are coreleased from neurons that usually contain

a classical neurotransmitter agent (e.g., glutamate and norepinephrine). Opioids activate a variety of signal transduction processes, and different

mechanisms in their regulation are in place for different cell types. The opioid receptors are G protein–coupled receptors and exert their cellular effects by

inhibiting adenylyl cyclase and regulating K+ and Ca2+ channels, via activation of Gi/Go. Recently opiorphin, an endogenously derived enkephalin that

inactivates zinc ectopeptidase, has been described as equal to morphine in the suppression of pain (Wisner et al. 2006). Although opiates are widely

associated with and used therapeutically in pain modulation, recent evidence indicates that dynorphin can actually activate bradykinin receptors and

contribute to neuropathic pain (Altier and Zamponi 2006). The continued study of the opioid system and the second-messenger changes brought about by the

chronic administration of opioids has greatly facilitated our understanding of the molecular and cellular effects of drugs of abuse and the potential to develop

novel therapeutics (Nestler et al. 2001).

NEUROTROPHINS

Neurotrophins are a family of regulatory factors that mediate the differentiation and survival of neurons, as well as the modulation of synaptic transmission

and synaptic plasticity (Patapoutian and Reichardt 2001; Poo 2001). The neurotrophin family now includes, among others, nerve growth factor (NGF),

brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6 (NT6) (Patapoutian and Reichardt 2001). Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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These various proteins are closely related in terms of sequence homology and receptor specificity. They bind to and activate specific tyrosine receptor kinases

belonging to the Trk family of receptors, including TrkA, TrkB, and TrkC, and a pan-neurotrophin receptor p75 (Patapoutian and Reichardt 2001; Poo 2001).

Additionally, there are two isoforms of TrkB receptors: the full-length TrkB and the truncated form of TrkB, which does not contain the intracellular tyrosine

kinase domain (Fryer et al. 1996). The truncated form of TrkB can thus function as a dominant-negative inhibitor for the TrkB receptor tyrosine kinase,

thereby providing another mechanism to regulate BDNF signaling in the CNS (Eide and Virshup 2001; Gonzalez et al. 1999).

Neurotrophins can be secreted constitutively or transiently, and often in an activity-dependent manner. Observations support a model in which neurotrophins

are generally secreted from the dendrite and act retrogradely at presynaptic terminals, where they act to induce long-lasting modifications (Poo 2001).

Within the neurotrophin family, BDNF is a potent physiological survival factor that has also been implicated in a variety of pathophysiological conditions, such

as Parkinson’s disease, Alzheimer’s disease, diabetic peripheral neuropathy, and psychiatric disorders (Malberg et al. 2000; Nagatsu et al. 2000; Pierce and

Bari 2001; Salehi et al. 1998). In particular, a genetic variant of BDNF (Val66Met) has been associated with risk for development of mood disorders in

humans, as well as with mood- and anxiety-related behaviors and response to antidepressant medications in animal models (Z. Y. Chen et al. 2006;

Neves-Pereira et al. 2002; Sklar et al. 2002). Recent data also support a role for this polymorphism in human brain development and function (Frodl et al.

2007; Kleim et al. 2006). The cellular actions of BDNF are mediated through two types of receptors: a high-affinity tyrosine receptor kinase (TrkB) and a

low-affinity pan-neurotrophin receptor (p75) (see Figure 1–1 for details). TrkB is preferentially activated by BDNF and NT4/5 and appears to mediate most of

the cellular responses to these neurotrophins (Du et al. 2003; Poo 2001).

Binding of BDNF initiates TrkB dimerization and transphosphorylation of tyrosine residues in its cytoplasmic domain (Patapoutian and Reichardt 2001), a

process that involves cAMP activation (Ji et al. 2005). Binding of cytoplasmic src homology 2 (SH2) domain–containing scaffolding proteins—including Shc

and Grb-2, which recognize specific phosphotyrosine residues on the receptor—can thus result in the recruitment of a variety of effector molecules. This

recruitment of effector molecules generally occurs via interaction of proteins with modular binding domains SH2 and SH3 (named after homology to the src

oncogenes—src homology domains). The ability of multiple effectors to interact with phosphotyrosines is undoubtedly one of the keys to the pleiotropic

effects that neurotrophins can exert. The physiological effects of neurotrophins are mediated by varying degrees of activation of three major signaling

pathways—the Ras/MAP kinase pathway, the phosphoinositide-3 kinase (PI3K) pathway, and the phospholipase C– 1 (PLC- 1) pathway (Figure 1–9). Among

these pathways, the effects of the PI3K pathway and the MAP kinase pathway have traditionally been linked to the cell survival effects of neurotrophins

(Patapoutian and Reichardt 2001) (see Figure 1–9). A series of studies by Duman (2002) have shown that BDNF and TrkB are upregulated by antidepressant

treatment. The “neurotrophin hypothesis of depression” has enjoyed heuristic value in reconceptualizing mood disorders as arising from abnormalities in

neural plasticity cascades. The demonstration that decreases in hippocampal BDNF levels are correlated with stress-induced depressive behaviors and that

antidepressant treatment enhances BDNF expression has generated considerable interest. It is now accepted that the main function of BDNF in the adult

brain is regulating synaptic plasticity rather than mediating neuronal survival. Exciting results show that BDNF is first synthesized as a precursor proBDNF,

which is then proteolytically cleaved to mature BDNF (mBDNF). ProBDNF and mBDNF facilitate LTD and LTP, respectively, suggesting opposing cellular

functions. Finally, BDNF plays different and perhaps opposing roles in the brain stress versus reward system (discussed in Martinowich et al. 2007).

FIGURE 1–9. Neurotrophic cascades.

Cell survival is dependent on neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor, and the expression of these factors can be

induced by synaptic activity. Phosphorylation of tyrosine receptor kinase (Trk) receptors activates a critical signaling pathway, the Ras/MAP kinase pathway (see Figure

1–15). Phosphorylated Trk receptors also recruit the phosphoinositide-3 kinase (PI3K) pathway through at least two distinct pathways, the relative importance of which differs

between neuronal subpopulations. In many neurons, Ras-dependent activation of PI3K is the most important pathway through which neurotrophins promote cell survival (not

shown; see text). In some cells, as shown in the figure, PI3K can also be directly activated through adaptor proteins (Shc, Grb-2, and Gab-1). PI3K directly regulates certain

cytoplasmic apoptotic pathways. Akt phosphorylates the pro-apoptotic Bcl-2 family member BAD (Bcl-xl/Bcl-2–associated death promoter), thereby inhibiting BAD’s

pro-apoptotic functions (Datta et al. 1997). Akt may also promote survival in an indirect fashion by regulating another major signaling enzyme: glycogen synthase kinase–3

(GSK-3) (Woodgett 2001). Interestingly, lithium is an inhibitor of GSK-3. Phosphorylated Trk receptors also recruit phospholipase C– 1 (PLC- 1). The Trk kinase then

phosphorylates and activates PLC- 1, which acts to hydrolyze phosphatidylinositides to generate diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Antidepressant

medication and mood stabilizers increase levels of BDNF and other neurotrophic factors, suggesting a therapeutic relevance.

Retrograde Transportation of Neurotrophin Receptors as Signal to the Cell Body

Unlike most other internalized receptors, which are usually degraded after internalization, neurotrophin–Trk complexes in endocytotic vesicles function as

signal transducers and provide a mechanism for long-range signaling in the neuronal cytoplasm. Several studies have provided support for the retrograde

transportation model of neurotrophin–Trk complexes; these studies indicate that endocytotic vesicles containing neurotrophin–Trk complexes may be

functionally active and should be viewed as activated signaling complexes that spread the cytosolic signaling of neurotrophin–Trk complexes to distant parts

of the neuron via active transport mechanisms. Intriguingly, as has been shown with another tyrosine kinase (ErbB4 receptor tyrosine kinase), other hithertoPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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unappreciated mechanisms, such as cleavage of receptor fragments, may also be operative in trafficking signals from extracellular receptors to intracellular

and nuclear targets (Ni et al. 2001). Whether such novel signaling mechanisms are also utilized by neurotrophin receptors will undoubtedly be the focus of

considerable future research.

Regulation of Neurotrophin Signaling by Neuronal Activity

The neurotrophic functions of neurotrophins depend in large part on a cytoplasmic signal-transduction cascade, whose efficacy may be influenced by the

presence of electrical activity in the neuron. Seizure activity, as well as nonseizure activity of a frequency or intensity capable of inducing LTP, has been

shown to elevate BDNF mRNA levels and to facilitate the release of BDNF from hippocampal and cortical neurons (Poo 2001). Although BDNF was originally

considered to be transported only retrogradely, evidence indicates that BDNF can also act anterogradely to modulate synaptic plasticity (Poo 2001).

High-frequency neuronal activity and synaptic transmission have also been shown to elevate the number of TrkB receptors on the surface of cultured

hippocampal neurons through activation of the CaMKII pathway and may therefore facilitate the synaptic action of BDNF (Du et al. 2000). Thus, electrically

active nerve terminals may be more susceptible to synaptic potentiation by secreted neurotrophins than are inactive terminals. Neuronal or synaptic activity

is also known to promote the effects of neurotrophins on the survival of cultured retinal ganglion cells; here, neuronal or synaptic activity elevates cAMP

levels to enhance the responsiveness of the neuron to neurotrophins, apparently by recruiting extra TrkB receptors to the plasma membrane (Meyer-Franke

et al. 1998). Moreover, the internalization of BDNF receptor TrkB is also upregulated by activity as a retrograde signal to the cell body in cultured

hippocampal neurons (this regulation is discussed in some detail in Du et al. 2003). The activity-dependent regulation of BDNF signaling on BDNF synthesis

and release, TrkB insertion onto neuronal surfaces, and activated TrkB tyrosine kinase internalization are crucial for its influence on synaptic plasticity and

neuronal survival.

CYTOKINES AND JAK/STAT–COUPLED RECEPTORS

There is mounting evidence that many psychiatric disorders may be associated with altered immune function. Even more convincing is the evidence that

numerous medical disorders and treatments that regulate immune function are associated with psychiatric symptomatology (Evans et al. 2001). Thus, the

mechanism by which the immune system is able to mediate its effects through specified signaling pathways in the CNS will undoubtedly be of increasing

importance in our understanding of these complex disorders.

Numerous cytokines and growth factors are able to activate the JAK/STAT pathway; here we focus on interferons as a prototype. Interferons are cytokines

that subserve important antiviral, antigrowth, and immunomodulatory activities (Larner and Keightley 2000). The interferon/cytokine receptor family is a

group of receptors that, on binding to an extracellular site, produce dimerization or higher-order clustering. Unlike the tyrosine kinase type receptors (Trk),

these receptors associate intrinsically in a noncovalent constitutive manner with proteins of the JAK (Janus tyrosine kinase) family to mediate their effects.

Signal transducers and activators of transcription (STATs), which are SH2 domain–containing transcription factors, are required for the actions of many other

cytokines and growth factors.

There are two types of receptors for which interferons, on binding to the extracellular part of the receptor, are able to rapidly induce corresponding genes:

interferon- / (IFN- / ), or type I receptors; and IFN- , or type II receptors. The interferon-stimulated gene factors, which are more commonly known as

STATs, bind to enhancers in the promoter regions of type I and type II receptor genes to mediate transcription (Larner and Keightley 2000). It should be

mentioned that in addition to interferon, interleukin-6 and prolactin are other cytokines whose effects have been documented to be mediated by STATs.

STATs are modified through tyrosine kinases and are necessary for activation of early response genes on interferon binding to the receptor. Thus, the Janus

tyrosine kinases (JAK1–3 and TYK2) are important in the regulation of interferon-mediated cellular effects.

Evidence suggests that IFN- / cluster in receptor complexes, and upon ligand binding, these proteins are able to mediate some of the receptor–effector

responses of interferons. Type I interferon receptors consist of two subunits (IFNAR1 and IFNAR2), of which the IFNAR2 subunit has three isoforms

(IFNAR2a, IFNAR2b, and IFNAR2c). Upon binding of interferon to IFN- , the IFNAR2c subunit is necessary for the activation of the JAK/STAT pathway.

Initially, interferons bind to two sites—IFNAR1 and IFNAR2—which heterodimerize, and then the activation of TYK2 and JAK ensues to phosphorylate the

receptor (Larner and Keightley 2000). Other phosphatases and kinases are also able to interact with type I interferon receptors to produce a cascade of

intercellular effects.

NUCLEAR HORMONES: FOCUS ON STEROIDS

In contrast to the other neuroactive compounds we have discussed thus far, many hormones (including cortisol, gonadal steroids, and thyroid hormones) are

able to rapidly penetrate into the lipid bilayer membrane because of their lipophilic composition (Kandel et al. 2000). Retinoic acid (vitamin A) has recently

been shown to be involved in sleep, as well as learning and memory formation (Drager 2006). Nuclear receptors are transcription factors that regulate the

expression of target genes in response to steroid hormones and other ligands. Approximately 50 nuclear receptors are known to exist, and their structure is

defined by a number of signature functional domains. Generally, nuclear receptors comprise an amino-terminal activation function, the DNA-binding domain,

a hinge region, and a carboxy-terminal ligand-binding domain containing a second activation function (Kandel et al. 2000). Upon activation by a hormone, the

steroid receptor–ligand complex translocates to the nucleus, where it binds to specific DNA sequences referred to as hormone responsive elements (HREs),

which subsequently regulate gene transcription (Mangelsdorf et al. 1995; Truss and Beato 1993) (see Figure 1–1). It is now known that nuclear receptors are

markedly regulated by additional “accessory proteins.” Nuclear receptor coregulators are cellular factors that complement nuclear receptors’ function as

mediators of the cellular response to endocrine signals. They are generally divisible into coregulators that promote transcriptional activation when recruited

(coactivators) and those that attenuate promoter activity (corepressors).

In addition to the traditional view of steroid hormone action, it is now clear, however, that steroid hormones also have so-called nongenomic effects that

include changes in neurotransmitter receptors, other membrane receptors, and second-messenger systems. These effects are less well characterized, but

evidence for their existence includes modulation of neural activity in brain areas where there are few, if any, gonadal steroid receptors; there is also evidence

showing that estrogen directly and rapidly inhibits calcium channels in neurons (McEwen 1999; Mermelstein et al. 1996). A growing body of data is also

demonstrating bidirectional cross-talk between nuclear receptors and GPCRs. Thus, for example, gonadal steroids have long been known to modulate the

activity of monoaminergic neurons and receptors. More recently, it has been shown that -adrenergic and dopamine D1 receptors are capable of

transactivating glucocorticoid and progesterone receptors, respectively. Neuroactive steroid is the term used for a steroid that is able not only to bind to its

respective intracellular receptor and become rapidly translocated to the nucleus but also to alter neuronal excitability via interactions with certain

neurotransmitter receptors (Rupprecht 2003) (see Figure 1–1). Many of the above-mentioned neuroactive steroids are capable of altering neuronal

excitability by interacting with GABAA receptors. Studies using chimeras of GABAA/glycine receptors suggest an allosteric action of neuroactive steroids at

the N-terminal side of the middle of the second transmembrane domain of the GABA receptor 1 and/or 2 subunits (Rick et al. 1998). However, no direct

binding of the steroid to the receptor has been demonstrated. In addition to GABAA receptors, other members of the ligand-gated ion channel family

(including 5-HT3, glycine, nicotinic, ACh, and glutamate receptors) have been postulated to represent targets for neuroactive steroids (Rupprecht 2003).

In view of the GABAA-enhancing potential of 3 -reduced neuroactive steroids, these steroids have been suggested to possess sleep-modulating or -promoting

(Mendels and Chernik 1973), anticonvulsant (Frye and Scalise 2000), anxiolytic (Crawley et al. 1986), and neuroprotective (Rupprecht 2003) properties.

Finally, it has been postulated that neuroactive steroids may also contribute to psychiatric symptoms sometimes observed during pregnancy and in the

postpartum period (Pearson Murphy et al. 2001).

UNCONVENTIONAL TRANSMITTERS: FOCUS ON GASES

Many of the unconventional transmitters do not fit the well-accepted neurotransmitter criteria mentioned at the beginning of this chapter. A handful of

unconventional transmitters have been characterized and may ultimately prove to have relevance for neuropsychiatric disorders; here, we limit ourselves to a

discussion of the gases nitric oxide (NO) and carbon monoxide (CO), which have been demonstrated to exhibit neurotransmitter-like properties in the brain

(Dawson and Snyder 1994). The gases, as a result of being small and uncharged, are able to permeate the lipid bilayer and enter the neuron and directly

affect certain second-messenger generating systems directly.

Synthesis of NO is derived from arginine via an enzymatic reaction involving NO synthase, flavin adenine dinucleotide, and flavin mononucleotide enzyme

(Cooper et al. 2001). Currently, there are three different variations of NO synthase, which arise from different genes that share approximately 50% sequence

homology. The neuronal NO synthase is activated by Ca2+ and calmodulin and is also regulated by phosphorylation, which decreases its function. NO isPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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released from both neurons and glia and can activate the enzyme guanylate cyclase to augment cGMP concentrations, thereby regulating a variety of

neurotransmitter systems (Cooper et al. 2001) (Figure 1–10). These effects likely occur via the activation of protein kinase G (termed G because it is

activated by cGMP), but this remains to be definitely established. Notably, endocannabinoids, a class of fatty acid derivatives that bind to cannabinoid

receptors, exert prominent effects on NO signaling (Alger 2005).

FIGURE 1–10. Nitric oxide as a signaling molecule.

This figure depicts the various regulatory processes involved in nitric oxide (NO) signaling. Reactive oxygen species, in particular several gases, represent yet another means

by which the brain is able to transmit messages. NO is formed via NO synthase (NOS), an enzyme that is generally activated by Ca2+-calmodulin. As such, Ca2+ entry into

cells via NMDA (N-methyl-D-aspartate) receptor activation is an important means of activating NOS. NOS yields NO by converting arginine to citrulline using O2. NO then

converts GTP to cGMP, which then is able to target soluble guanylyl cyclases (GCs) (enzymes that are similar to adenylyl cyclases but are activated by cGMP rather than

cAMP). cGMP then activates the protein kinase (PKG) and, through the conversion of ATP to ADP, phosphorylates many proteins to bring about the physiological effects of NO.

Once produced, NO is then able to diffuse out of the neuron and act on other cells as a signaling molecule. Interestingly, NO is able to also diffuse back to the presynaptic

terminal, acting as a retrograde transmitter, and is thought to be important in reshaping synaptic connections (i.e., it has been linked to long-term potentiation). NO is

labeled in yellow; glutamate is labeled in purple. GTg = glial transporter for glutamate; GTn = neuronal transporter for glutamate; 5-HT1A = serotonin1A receptor; S100 =

calcium-binding protein expressed primarily by astrocytes.

Source. Adapted from Girault J-A, Greengard P: “Principles of Signal Transduction,” in Neurobiology of Mental Illness. Edited by Charney DS, Nestler EJ, Bunney BS. New

York, Oxford University Press, 1999. Copyright 1999, Oxford University Press. Used with permission.

CO appears to be formed in neurons exclusively by heme oxygenase–2 (HO-2), which cleaves the heme ring, releasing biliverdin, expelling iron from the heme

ring, and releasing a one-carbon fragment as CO. HO-2 activity occurs in neuronal populations in numerous parts of the brain and is dynamically regulated by

neuronal impulses through a kinase cascade in which PKC activates casein kinase–2, which in turn phosphorylates and activates HO-2. HO-2 activity

generates low micromolar concentrations of CO in the brain.

Similar to NO, CO augments cGMP levels to produce its effects in the brain. Additionally, protein carboxyl methylation and phospholipid methylation involve

S-adenosylmethionine acting as the methyl donor. Protein carboxyl methylation and phospholipid methylation are able to impact certain aspects of brain

function (i.e., calmodulin-linked enzymes), and indeed both NO and CO have been implicated in long-term neural alterations such as learning and memory.

Thus, it has been presumed that these gases could influence events in the nucleus, such as transcription. When released from postsynaptic neurons, these

gases have feedback potential that impacts neurotransmitter release, states of neuronal activity, and notably learning and memory.

SIGNAL TRANSDUCTION PATHWAYS

Signal transduction refers to the processes by which extracellular stimuli are transferred to—and propagated as—intracellular signals (Figure 1–11).

Multicomponent cellular-signaling pathways interact at various levels, thereby forming complex networks that allow the cell to receive, process, and respond

to information (Bhalla and Iyengar 1999; Bourne and Nicoll 1993). These networks facilitate the integration of signals across multiple time scales, the

generation of distinct outputs that depend on input strength and duration, and the regulation of intricate feed-forward and feedback loops (Bhalla and

Iyengar 1999). These properties of signaling networks suggest that they play critical roles in cellular memory; thus, cells with different histories, and

therefore expressing different repertoires of signaling molecules, interacting at different levels, may respond quite differently to the same signal over time.

Given their widespread and crucial role in the integration, regulation, amplification, and fine-tuning of physiological processes, it is not surprising that

abnormalities in signaling pathways have now been identified in a variety of human diseases (Simonds 2003; Spiegel 1998). Pertinent to the present

discussion is the observation that a variety of diseases manifest a relatively circumscribed symptomatology, despite the widespread, often ubiquitous

expression of the affected signaling proteins.

FIGURE 1–11. Principles of signal transduction.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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As described in the text, neurons regulate signaling pathways through multiple mechanisms and at multiple levels. Neuronal circuits possess a large number of extracellular

neuroactive molecules (1; labeled A, B, and C) that can interact with multiple receptors (2). Binding of neuroactive molecules to receptors can result in stimulation and/or

attenuation of multiple cellular signaling pathways (3), depending on the type of receptor, location in the central nervous system, and activity of other signaling pathways

within the cell. Thus, the potential is there to greatly amplify the signals. This signaling can then converge on one signaling pathway (4) or diverge into many signaling

pathways (5). Activation of signaling pathways alters gene transcription and activity of proteins such as ion channels and other signaling molecules (6). Additionally,

activation of signaling pathways can both positively (7) and negatively (8) regulate the function of extracellular receptors. Bcl-2 = an anti-apoptotic protein; BDNF =

brain-derived neurotrophic factor; CREB = cAMP response element–binding protein.

Although complex signaling networks are likely present in all eukaryotic cells and control various metabolic, humoral, and developmental functions, they may

be especially important in the CNS, where they serve the critical roles of first amplifying and “weighting” numerous extracellularly generated neuronal signals

and then transmitting these integrated signals to effectors, thereby forming the basis for a complex information-processing network (Bourne and Nicoll 1993;

Manji 1992). The high degree of complexity generated by these signaling networks may be one mechanism by which neurons acquire the flexibility for

generating the wide range of responses observed in the nervous system. These pathways are thus undoubtedly involved in regulating such diverse vegetative

functions as mood, appetite, and wakefulness and are therefore likely to be involved in the pathophysiology of a variety of psychiatric disorders and their

treatments.

G Proteins

As mentioned already, G proteins were originally named because of their ability to bind the guanine nucleotides guanosine triphosphate and guanosine

diphosphate. Receptors coupled to G proteins include those that respond to catecholamines, serotonin, ACh, various peptides, and even sensory signals such

as light and odorants. Gs and Gi were among the first G proteins identified and received their names because of their ability to stimulate or inhibit adenylyl

cyclase. Since that time, a multitude of G protein subunits have been identified by a combination of biochemical and molecular cloning techniques. Indeed,

genes for 16 G subunits are known and give rise via alternative splicing to at least 20 mature G subunits with differential tissue expression (Simonds 2003)

(see Table 1–1). There are four homology-based subfamilies of G subunits: the Gs subfamily, whose members stimulate adenylyl cyclase; the Gi subfamily,

which includes Gi1–3 and Gz, which inhibit adenylyl cyclase; the Gq subfamily, whose members activate PLC- ; and the G12 subfamily, whose members interact

with regulators of G protein signaling (RGS) domain–containing Rho exchange factors (see Table 1–1 and Figure 1–2) (Simonds 2003). Genes encoding 5 G

isoforms and 12 different G subunits are known in humans; effectors of G

complexes include ion channels, isoforms of adenylyl cyclase, isoforms of PLC- ,

and MAP kinase pathways (Simonds 2003) (see Table 1–1).

G Protein Function

G proteins function in the context of two interrelated cycles: a cycle of subunit association and dissociation and a cycle of GTP binding and hydrolysis

(discussed in detail in the legend to Figure 1–2). G protein heterotrimers consist of G , G , and G subunits at a 1:1:1 stoichiometry and are named according

to decreasing mass, with the subunits having an apparent mass of 40–52 kDa, subunits having an apparent mass of 35–36 kDa, and subunits having an

apparent mass of 5–20 kDa. The different types of G protein have been named on the basis of the distinct subunits they possess (i.e., Gs represents G

proteins containing G s). This classification system arose from the erroneous assumption that it was only the subunits that were responsible for the

proteins’ specific functional activity; it is now known that the and subunits exert a number of functional effects on their own (see Table 1–1) and are not

simply “anchoring proteins” for subunits. Although the and subunits are not covalently bound, they are tightly linked by noncovalent coiled-coil

interactions; thus, they are generally assumed to function as

dimers. It is very likely that different

subunits exert different effects on subunits and

effectors (e.g., 2 2 behaves differently from 2 3), but the delineation of the differential effects of the different subunit compositions is still in its infancy.

Mediation of neurotransmitter–neurotransmitter and receptor–receptor interactions

The CNS is remarkably complex, both anatomically and chemically, with a remarkable convergence of different receptors in common cortical layers and

considerable convergence of neurotransmitter action. A single neuron in the brain receives thousands of synaptic inputs on the cell body and dendrites, and

neuronal response is also modulated by a variety of hormonal and neurohormonal substances that are not dependent on synaptic organization (Kandel et al.

2000). The neuron needs to integrate all the synaptic and nonsynaptic inputs impinging on it; this integration of a multitude of signals determines the

ultimate excitability, firing pattern, and response characteristics of the neuron, which are then conveyed to succeeding targets via synaptic transmission. How

does the single neuron decipher and integrate the multitude of signals it receives and, additionally, generate unique responses to each of these signals or

combinations of signals?

Not only do G proteins amplify signals, but they also appear to form the basis of a complex information-processing network in the plasma membrane (BhallaPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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and Iyengar 1999; Manji 1992). Thus, the ability of G proteins to interact with multiple receptors provides an elegant mechanism to organize the signals from

these multiple receptors and to transmit them to a relatively much smaller number of effectors. Signals from a variety of receptors can be “weighted”

according to their intrinsic ability to activate a given G protein and integrated to stimulate a single second-messenger pathway (see Figure 1–11). Similarly,

the dual (positive and negative) regulation of adenylyl cyclase by G proteins allows for stimulatory and inhibitory signals for these pathways to be “balanced”

at the G protein level, yielding an integrated output. Thus, G proteins provide the first opportunity for signals from different receptors to be integrated. This

complex web of interactions linking receptors, G proteins, and their effectors with signals converging to shared detectors appears to be crucial for the

integrative functions performed by the CNS.

Abnormalities in a variety of human diseases have now been clearly shown to arise from primary abnormalities in G protein signaling cascades and in the G

protein subunits themselves (for an excellent discussion, see Simonds 2003; Spiegel 1998). To date, the direct evidence for the involvement of G proteins in

psychiatric disorders is more limited. Thus, although elevations in the levels of G s have been found in postmortem brain and peripheral tissue in bipolar

disorder, a mutation in the G s gene has not yet been identified (discussed in Manji and Lenox 1999). There is, however, convincing evidence that chronic

lithium administration attenuates the functioning of both Gs and Gi, resulting in an elevation of basal cAMP levels but dampened receptor-mediated effects.

The allosteric modulation of G proteins has been proposed to play a role in lithium’s long-term prophylactic efficacy in protecting susceptible individuals from

cyclic affective episodes induced spontaneously or by stress or drugs (e.g., antidepressant, stimulant) (G. Chen et al. 1999; Gould and Manji 2002).

The cAMP signaling cascade

G proteins control intracellular cAMP levels by mediating the ability of neurotransmitters to activate or inhibit adenylyl cyclase (Figure 1–12; see also Figure

1–2). The mechanism by which neurotransmitters stimulate adenylyl cyclase is well established. Activation of those neurotransmitter receptors that couple to

Gs results in the generation of free G s subunits that bind to and directly activate adenylyl cyclase. A similar mechanism appears to be the case for G olf, a

type of G protein (structurally related to G s) that is enriched in olfactory epithelium and dopamine-rich areas of the brain and mediates the ability of odorant

receptors and D1 receptors to stimulate adenylyl cyclase. The mechanism by which neurotransmitters inhibit adenylyl cyclase and decrease neuronal levels of

cAMP is somewhat less clear, and more than one mechanism may be operative. By analogy with the action of Gs, it was originally proposed that activation of

neurotransmitter receptors that couple to Gi results in the generation of free G i subunits, which could bind to, and thereby directly inhibit, adenylyl cyclase.

While this mechanism may be operative, there are also data to suggest that

subunit complexes, generated by the release of G i, might directly inhibit

certain forms of adenylyl cyclase or might bind and “tie up” free G s subunits in the membrane.

FIGURE 1–12. cAMP signaling pathway.

Receptors can be positively (e.g., -adrenergic, D1) or negatively (e.g., 5-HT1A, D2) coupled to adenylyl cyclase (AC) to regulate cAMP levels. The effects of cAMP are

mediated largely by activation of protein kinase A (PKA). One major downstream target of PKA is CREB (cAMP response element–binding protein). After activation, the

phosphorylated CREB binds to the cAMP response element (CRE), a gene sequence found in the promoter of certain genes; data suggest that antidepressants may activate

CREB, thereby bringing about increased expression of a major target gene, BDNF. Phosphodiesterase is an enzyme that breaks down cAMP to AMP. Some antidepressant

treatments have been found to upregulate phosphodiesterase. Drugs like rolipram, which inhibit phosphodiesterase, may be useful as adjunct treatments for depression.

Forskolin is an agent used in preclinical research to stimulate adenylyl cyclase.

It is now clear that there are several forms of adenylyl cyclase that make up a distinct enzyme family; these various forms are differentially regulated and

display distinct distributions in nervous and nonnervous tissues. For example, type I is found predominantly in brain, whereas types II and IV, although

abundantly expressed in the brain, have a more widespread distribution. The topographical structure of the adenylyl cyclase proteins resembles that of

membrane transporters and ion channels. However, there is currently no convincing evidence of a transporter or channel function for mammalian adenylyl

cyclases.

As would be predicted, the different forms of adenylyl cyclase are regulated by distinct mechanisms. Type I through IV enzymes differ in their ability to be

regulated by Ca2+ and calmodulin. Types I and III are stimulated by Ca2+-calmodulin complexes, whereas types II and IV are insensitive. Perhaps the most

intriguing regulation is that by the G protein and subunits. Thus, it is now clear that when type II adenylyl cyclase is concurrently stimulated by a

stimulatory receptor (e.g., -adrenergic receptor), the

subunits released from an “inhibitory receptor” (e.g., 5-HT1A, 2, GABAB) can, in fact, robustly

potentiate the cAMP response (Bourne and Nicoll 1993). Type II adenylyl cyclase thus serves as a “coincidence detector” in the CNS, capable of temporally

and spatially integrating signals to bring about dramatically different effects. An additional important mechanism by which adenylyl cyclase can be regulated

is by cross-talk with protein kinase C, thereby linking receptors linked to stimulation of adenylyl cyclase and those linked to the turnover of membrane

phosphoinositides. The physiological effects of cAMP are mediated primarily by activation of protein kinase A, an enzyme that phosphorylates and regulates

many proteins, including ion channels, cytoskeletal elements, transcription factors, and other enzymes. Indeed, one major CNS target for the actions of PKA is

the transcription factor CREB (cAMP response element–binding protein), which plays a major role in long-term neuroplasticity and is an indirect target of

antidepressants (Duman 2002) (see Figure 1–12). As we discuss in greater detail below, phosphorylation and dephosphorylation reactions play a major role

in regulating a variety of long-term neuroplastic events in the CNS.

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Phosphoinositide

Although inositol phospholipids are relatively minor components of cell membranes, they play a major role in receptor-mediated signal transduction

pathways. They are involved in a diverse range of responses, such as cell division, secretion, and neuronal excitability and responsiveness. In many cases,

Gq/11 is involved, and it is believed that G q/11 directly binds to and activates phospholipase C (Figure 1–13). In other cases, however, it is the subunits

released upon activation of receptors coupled to Gi/Go that bring about activation of the enzyme PLC to produce the intracellular second messengers

sn-1,2-diacylglycerol (DAG; an endogenous activator of PKC) and inositol-1,4,5-triphosphate (IP3). IP3 binds to the IP3 receptor and facilitates the release of

calcium from intracellular stores, in particular the endoplasmic reticulum (see Figure 1–13). The released calcium then interacts with various proteins in the

cell, including the important family of calmodulins (Ca2+-receptor protein calmodulin, or CaM) (discussed later in this chapter; Figure 1–14). Calmodulins then

activate calmodulin-dependent protein kinases (CaMKs), which affect the activity of diverse proteins, including ion channels, signaling molecules, proteins

that regulate apoptosis, scaffolding proteins, and transcription factors (Miller 1991; Soderling 2000).

FIGURE 1–13. Phosphoinositide (PI) signaling pathway.

A number of receptors in the CNS (including M1, M3, M5, 5-HT2C) are coupled, via G q/11, to activation of PI hydrolysis. Activation of these receptors induces phospholipase C

(PLC) hydrolysis of phosphoinositide-4,5-bisphosphate (PIP2) to sn-1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). DAG activates protein kinase C (PKC), an

enzyme that has many effects, including the activation of phospholipase A2 (PLA2), an activator of arachidonic acid signaling pathways. IP3 binds to the IP3 receptor, which

results in the release of intracellular calcium from intracellular stores, most notably the endoplasmic reticulum. Calcium is an important signaling molecule and initiates a

number of downstream effects such as activation of calmodulins and calmodulin-dependent protein kinases (see Figure 1–15). IP3 is recycled back to PIP2 by the enzymes

inositol monophosphatase (IMPase) and inositol polyphosphatase (IPPase; not shown), both of which are targets of lithium. Thus, lithium may initiate many of its therapeutic

effects by inhibiting these enzymes, thereby bringing about a cascade of downstream effects involving PKC and gene expression changes.

Source. Adapted from Gould TD, Chen G, Manji HK: “Mood Stabilizer Pharmacology.” Clinical Neuroscience Research 2:193–212, 2003. Copyright 2003, Elsevier. Used with

permission.

FIGURE 1–14. Calcium-mediated signaling.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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In neurons, Ca2+-dependent processes represent an intrinsic nonsynaptic feedback system that provides competence for adaptation to different functional tasks. Ca2+ is

generally mobilized in one of two ways in the cells: either by mobilization from intracellular stores (e.g., from the endoplasmic reticulum) or from outside of the cell via

plasma membrane ion channels and certain receptors (e.g., NMDA [N-methyl-D-aspartate]). The external concentration of Ca2+ is approximately 2 mM, yet resting

intracellular Ca2+ concentrations are in the range of 100 nM (2 x 104 lower). Local high levels of calcium result in activation of enzymes, signaling cascades, and, at

extremes, cell death. Release of intracellular stores of calcium is primarily regulated by inositol-1,4,5-triphosphate (IP3) receptors that are activated upon generation of IP3

by phospholipase C (PLC) activity, and the ryanodine receptor that is activated by the drug ryanodine. Many proteins bind Ca2+ and are classified as either “buffering” or

“triggering.” These include calcium pumps, calbindin, calsequestrin, calmodulin, PKC, phospholipase A2, and calcineurin. Once stability of intracellular calcium is accomplished,

transient low-magnitude changes can serve an important signaling function. Calcium action is local. Because of the high concentration of calcium-binding proteins, it is

estimated that the free Ca2+ ion diffuses only approximately 0.5 M and is free for about 50 sec before encountering a Ca2+-binding protein. Ca2+ is sequestered in the

endoplasmic reticulum (which serves as a vast web and framework for Ca2+-binding proteins to capture and sequester Ca2+). Ca2+ buffering/triggering proteins are

nonuniformly distributed, so there is considerable subcellular variation of Ca2+ concentrations (e.g., near a Ca2+ channel). The primary mechanism for Ca2+ calcium exit from

the cell is either via sodium-calcium exchange or by means of a calcium pump.

IP3 can be metabolized both by dephosphorylation to form inositol-1,4-P2 and by phosphorylation to form inositol-1,3,4,5-P4 (IP4), which has been proposed

to be involved in the entry of Ca2+ into cells from extracellular sources. Recycling of IP3 is important for continuation of phosphoinositide hydrolysis in

response to extracellular signals. This is achieved by the enzyme inositol monophosphatase (IMPase), which is the rate-limiting enzyme that converts IP3

back to phosphoinositide-4,5-bisphosphate (PIP2). Without this enzyme, PIP2 cannot be recycled adequately, potentially leading to low levels of PIP2 and

inhibition of the signaling cascades involving DAG and IP3. Lithium, at therapeutically relevant concentrations, is a noncompetitive inhibitor of IMPase (for a

review, see Gould et al. 2003). This has led to the “inositol depletion hypothesis,” which posits that lithium brings about a reduction in the levels of inositol by

inhibiting the activity of this “recycling enzyme.” Although lithium does reduce inositol levels in the areas of the brain in bipolar patients (Moore et al. 1999),

this likely represents an upstream “initiating event,” which brings about downstream changes in PKC and regulates gene expression, which may be ultimately

responsible for some of its therapeutic effects (see Figure 1–13) (Brandish et al. 2005; Gould et al. 2003; Manji and Lenox 1999).

Protein Kinase C

Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C, or PKC) is a ubiquitous enzyme, highly enriched in brain, where it plays a major role

in regulating both pre- and postsynaptic aspects of neurotransmission (Nishizuka 1992; Stabel and Parker 1991). PKC is one of the major intracellular

mediators of signals generated upon external stimulation of cells via a variety of neurotransmitter receptors (including muscarinic [M1, M3, M5],

noradrenergic [ 1], and serotonergic [5-HT2] receptors) that induce the hydrolysis of various membrane phospholipids.

Activation of PKC by DAG appears to involve the binding of the lipid to a specific regulatory site on the enzyme, resulting in an increase in the Ca2+ affinity,

and thus its stimulation at physiological ionic concentrations. Ca2+ is also believed to contribute to PKC activation by facilitating the interaction of the enzyme

with the lipid bilayer and hence with acidic phospholipids and DAG. PKC is now known to exist as a family of closely related subspecies, has a heterogeneous

distribution in brain (with particularly high levels in presynaptic nerve terminals), and, together with other kinases, appears to play a crucial role in the

regulation of synaptic plasticity and various forms of learning and memory. The multiple closely related PKC isoforms are all activated by phospholipids and

DAG, albeit with slightly different kinetics. The isoforms can be subclassified according to Ca2+ dependence: the “conventional” PKCs ( , I, II, ) are

dependent on Ca2+ for activity, whereas several others, termed “novel” ( , , , ) and “atypical” ( , , ), are calcium independent (Nishizuka 1992). The

conventional, novel, and atypical isozymes all share activation by phospholipids or DAG and an autoinhibitory pseudosubstrate region, which maintains the

enzyme in an inactive state until activated. However, the subgroups are activated by different activators. The conventional PKCs require calcium, acidic

phospholipids, and DAG for activation; the novel PKCs do not require calcium, and the atypical PKCs do not require calcium or DAG. PKC isozymes that do not

share the pseudosubstrate region ( /PKD and ) have been described, which suggests a possible different mode of action.

The differential tissue distribution of PKC isozymes, as well as the fact that several isoforms are expressed within a single cell type, suggests that eachPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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isozyme may exert distinct cellular functions. At present, it is unclear whether such putative functional specificity arises from differential in vivo activation,

differential substrate specificity, or a combination thereof. PKC has many growth-regulating properties in immature cells and has additional cell-specific

responses in individual mature cells (Kanashiro and Khalil 1998). One protein whose activity is modulated by PKC is myristoylated alanine-rich C kinase

substrate (MARCKS). This protein functions as a regulated crossbridge between actin and the plasma membrane, contributing to the cytoskeleton of the cell

and subsequently to neuronal plasticity (Aderem 1992) (see Figure 1–13). PKC is also an important activator of phospholipase A2, thus linking the

phosphoinositide cycle with arachidonic acid pathways (see Figure 1–13). Arachidonic acid functions as an important mediator of second-messenger

pathways within the brain and is regulated by chronic lithium (Axelrod et al. 1988; Rapoport 2001). The activation of phospholipase A2 by PKC (and other

pathways) results in arachidonic acid release from membrane phospholipids (Axelrod 1995). This release of arachidonic acid from cellular membrane allows

for the subsequent formation of a number of eicosanoid metabolites such as prostaglandins and thromboxanes. These metabolites mediate numerous

subsequent intracellular responses and, because of their lipid permeable nature, transsynaptic responses.

PKC also has been demonstrated to be active in many other cellular processes, including stimulation of transmembrane glucose transport, secretion,

exocytosis, smooth muscle contraction, gene expression, modulation of ion conductance, cell proliferation, and desensitization of extracellular receptors

(Nishizuka 1992). One of the best-characterized effects of PKC activation in the CNS is the facilitation of neurotransmitter release. Studies have suggested

that PKC activation may facilitate neurotransmitter release via a variety of mechanisms, including modulating several ionic conductances regulating Ca2+

influx, upstream steps regulating release of Ca2+ from intracellular stores, recruitment of vesicles to at least two distinct vesicle pools, and the Ca2+

sensitivity of the release process itself (discussed in Bown et al. 2002). Abundant data also suggest that the PKC signaling pathway may play an important

role in the pathophysiology and treatment of bipolar disorder (Manji and Lenox 1999). Thus, elevations in PKC isozymes have been reported in postmortem

brain and platelets in bipolar patients; more importantly, in animal and cell-based models, lithium and valproate exert strikingly similar effects on PKC

isozymes and substrates in a time frame mimicking their therapeutic actions.

A recent whole-genome association study of bipolar disorder has further implicated this pathway. Of the risk genes identified, the one demonstrating by far

the strongest association with bipolar disorder was diacylglycerol kinase, an immediate regulator of PKC (Baum et al. 2008). In animal models of mania,

several studies have demonstrated that both acute and chronic amphetamine exposure produces an alteration in PKC activity and its relative

cytosol-to-membrane distribution, as well as the phosphorylation of a major PKC substrate, GAP-43, which has been implicated in long-term alterations of

neurotransmitter release. Increased hedonistic drive and increased tendency to abuse drugs are well-known facets of manic behavior; notably, PKC inhibitors

attenuate these important aspects of the manic-like syndrome in rodents (Einat and Manji 2006; Einat et al. 2007). Recent preclinical studies have specifically

investigated the antimanic effects of tamoxifen (since this is the only CNS-penetrant PKC inhibitor available for humans). These studies showed that

tamoxifen significantly reduced amphetamine-induced hyperactivity and risk-taking behavior (Einat et al. 2007). Finally, with respect to cognitive dysfunction

associated with mania, Birnbaum et al. (2004) demonstrated that excessive activation of PKC dramatically impaired the cognitive functions of the prefrontal

cortex and that inhibition of PKC protected cognitive function. In summary, preclinical biochemical and behavioral data support the notion that PKC activation

may result in manic-like behaviors, whereas PKC inhibition may be antimanic. These preclinical data, along with animal studies discussed above, have

prompted clinical studies of PKC inhibitors and mood dysregulation. A number of small studies have found that tamoxifen, a nonsteroidal antiestrogen and a

PKC inhibitor at high concentrations, possesses antimanic efficacy (Bebchuk et al. 2000; Kulkarni et al. 2006). Most recently, a double-blind,

placebo-controlled trial of tamoxifen in the treatment of acute mania was undertaken (Zarate et al. 2007). Subjects showed significant improvement in mania

on tamoxifen compared with placebo as early as 5 days, and the effect size for the drug difference was very large after 3 weeks.

Calcium

Calcium is a very common signaling element and plays a critical role in the CNS by regulating the activity of diverse enzymes and facilitating neurotransmitter

release (see Figure 1–14). Importantly, excessively high levels of calcium are also a critical mediator of cell death cascades within neurons, necessitating

diverse homeostatic mechanisms to regulate intracellular calcium levels very precisely. Thus, although the external level of Ca2+ is approximately 2 mM, the

resting intracellular Ca2+ concentrations (Ca2+i) are in the range of 100 nM (that is, 2 x 104 lower) (Rasmussen 1989). Neuronal stimulation by

depolarization or receptor activation activates phosphoinositol turnover and increases Ca2+i by one to two orders of magnitude as a result of release of Ca2+

from intracellular stores and/or influx of Ca2+ through ion channels (Rink 1988). Acting via intracellular proteins such as calmodulin and enzymes such as

PKC, calcium ions influence synthesis and release of neurotransmitters (Parnas and Segel 1989), receptor signaling (Rasmussen 1986), and neuronal

periodicity (Matthews 1986).

Many proteins bind Ca2+; these are classified as either “buffering” or “triggering” and include calcium pumps, calbindin, calsequestrin, calmodulin, PKC,

phospholipase A2, and calcineurin (see Figure 1–14). Once stability of intracellular calcium is accomplished, transient low-magnitude changes can serve an

important signaling function. Importantly, calcium action is locally mediated; that is, because of the high concentration of calcium-binding proteins, it is

estimated that the free Ca2+ ion diffuses only approximately 0.5 M and is free for around 50 sec before encountering a Ca2+-binding protein. Ca2+ is

sequestered in the endoplasmic reticulum (which serves as a vast web and framework for Ca2+-binding proteins to capture and sequester Ca2+). Ca2+

buffering/triggering proteins are nonuniformly distributed, and thus there is considerable subcellular variation of Ca2+ concentrations (e.g., near a Ca2+

channel).

Calcium is generally mobilized in one of two ways in the cell, either by mobilization from intracellular stores or by selective diffusion across plasma membrane

ion channels (see Figure 1–14). Ca2+ ions pass the membrane through more or less specific channels regulated by changes of membrane potential or

transmitter binding. This Ca2+ influx lasts until Ca2+ levels reach a critical level in the submembranal compartment; a potassium current is then activated that

repolarizes the membrane. This Ca2+-dependent potassium current represents a strong inhibitory mechanism of the single neuron itself without synaptic

input. Its attractiveness for psychiatry lies in its sensitivity to modulatory influences: many amines, peptides, or drugs with relevance in the etiology and/or

treatment of these disorders (e.g., norepinephrine, dopamine, corticotrophin-releasing factor [CRF], caffeine, neuroleptics) modify (i.e., increase or decrease)

this potassium current. When activation of the potassium pump is decreased, the capacity for negative feedback after excitation becomes impaired and the

neuron switches to a state of higher activation, coincidentally with increased calcium influx. Such an overdrive in calcium currents and discharge activity

could be a functional prerequisite for states of pathological activity, possibly underlying neuropsychiatric symptoms such as epilepsy, mania, or depression.

Ca2+ released intracellularly is regulated both positively and negatively, resulting in the generation of dynamic Ca2+ waves. Once intracellular Ca2+ levels are

increased, this triggers/activates a number of proteins (e.g., adenylyl cyclase type I, CaMKs, PKC, calpain [a protease], calcineurin [a protein phosphatase]).

In neurons, Ca2+-dependent processes represent an intrinsic nonsynaptic feedback system that provides the competence for adaptation to different

functional tasks (see Figure 1–14). Regulation of intracellular Ca2+ could be of particular relevance to the study of psychiatric disorders, because the same

elevation of intracellular Ca2+ may facilitate or inhibit a given function, depending on the target enzyme, the phase of the cell cycle, the intracellular effector

protein, and the Ca2+-dependent process. In addition, higher or more sustained increases of intracellular Ca2+ may inhibit the same function that smaller

elevations facilitate (Wolff et al. 1977), so that elevated intracellular Ca2+ can produce excessive activation of some systems and inhibition of others. A

polymorphism in PPP3CC, a component of the calcium-dependent protein phosphatase calcineurin, has been associated with risk of developing schizophrenia

in at least two patient populations (Gerber et al. 2003; Y. L. Liu et al. 2007).

Signaling Cascades Generally Utilized by Neurotrophic Receptors in the CNS

The pleiotropic and often profound effects (e.g., neuronal growth, differentiation, survival) of neurotrophins and growth factors in the CNS are generally

mediated by varying degrees of activation of three major signaling pathways: MAP kinase (extracellular response kinase [ERK]) pathway, the

phosphoinositol-3 kinase pathway, and the phospholipase C– 1 pathway (see Figure 1–9). Among these pathways, the effects of the PI3K pathway and the

MAP kinase pathway have been most directly linked to the cell survival effects of neurotrophins (Patapoutian and Reichardt 2001).

MAP Kinase Cascade

MAP kinases are abundantly present in brain and have been postulated to play a major role in a variety of long-term CNS functions, in both the developing

and the mature CNS (Fukunaga and Miyamoto 1998; Kornhauser and Greenberg 1997; Matsubara et al. 1996; Robinson et al. 1998). With respect to their

actions in the mature CNS, MAP kinases have been implicated in mediating neurochemical processes associated with long-term facilitation (Martin et al.Print: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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1997), long-term potentiation (English and Sweatt 1996, 1997), associative learning (Atkins et al. 1998), one-trial and multitrial classical conditioning (Crow

et al. 1998), long-term spatial memory (Blum et al. 1999), and modulation of the addictive effects of abused drugs (Lu et al. 2005). They have also been

postulated to integrate information from multiple infrequent bursts of synaptic activity (Murphy et al. 1994). Importantly for the present discussion, MAP

kinase pathways have been demonstrated to regulate the responses to environmental stimuli and stressors in rodents (Xu et al. 1997) and to couple PKA and

PKC to CREB protein phosphorylation in area CA1 of the hippocampus (Roberson et al. 1996, 1999). These studies suggest the possibility of a broad role for

the MAP kinase cascade in regulating gene expression in long-term forms of synaptic plasticity (Roberson et al. 1999). For example, it has recently been

shown that CREB modulates excitability of neurons of the nucleus accumbens, which helps to limit behavioral sensitivity to cocaine in rodent models (Dong et

1. 2006). Thus, overall, the data suggest that MAP kinases play important physiological roles in the mature CNS and, furthermore, may be important targets

for the actions of CNS-active agents (Nestler 1998).

Growth factors acting through specific receptors (e.g., BDNF acting on TrkB) activate the Ras/MAP kinase signaling cascade (Figure 1–15). Among the targets

of the MAP kinase pathway is ribosomal S6 kinase (RSK). RSK phosphorylates CREB and other transcription factors. Studies have demonstrated that the

activation of the MAP kinase pathway can inhibit apoptosis by inducing the phosphorylation and inactivation of the pro-apoptotic protein BAD

(Bcl-xl/Bcl-2–associated death promoter) and increasing the expression of the anti-apoptotic protein Bcl-2 (the latter effect likely involves CREB) (Bonni et

1. 1999; Riccio et al. 1997). Phosphorylation of BAD occurs via activation of RSK. RSK phosphorylates BAD and thereby promotes its inactivation. Activation

of RSK also mediates the actions of the MAP kinase cascade and neurotrophic factors on the expression of Bcl-2. RSK can phosphorylate CREB, leading to the

expression of genes with neurotrophic functions, such as Bcl-2 and BDNF (see Figure 1–15). Treatment with mood-stabilizing drugs activates the ERK

(extracellular signal–related kinase) pathway in brain regions involved in mood regulation (reviewed in G. Chen and Manji 2006). Earlier work showed that

lithium and valproate induce AP-1 and CREB transcription factors and enhance expression of the bcl-2 gene. Later, it was found that chronic lithium or

valproate treatment promotes neurogenesis in the hippocampus, an effect mediated at least in part by activation of the ERK pathway (see G. Chen and Manji

2006).

FIGURE 1–15. MAP (mitogen-activated protein) kinase signaling pathway.

The influence of neurotrophic factors on cell survival is mediated by activation of the MAP kinase cascade and other neurotrophic cascades. Activation of neurotrophic factor

receptors referred to as tyrosine receptor kinases (Trks) results in activation of the MAP kinase cascade via several intermediate steps, including phosphorylation of the

adaptor protein Shc and recruitment of the guanine nucleotide exchange factor Sos. This results in activation of the small guanosine triphosphate–binding protein Ras, which

leads to activation of a cascade of serine/threonine kinases. This includes Raf, MAP kinase kinase (MEK), and MAP kinase (also referred to as extracellular response kinase, or

ERK). One target of the MAP kinase cascade is the ribosomal S6 kinases, known as RSK, which influences cell survival in at least two ways. RSK phosphorylates and

inactivates the pro-apoptotic factor BAD (Bcl-xl/Bcl-2–associated death promoter). RSK also phosphorylates cAMP response element–binding protein (CREB) and thereby

increases the expression of the anti-apoptotic factor Bcl-2 and brain-derived neurotrophic factor (BDNF). Ras also activates the phosphoinositol–3 kinase (PI3K) pathway, a

primary target of which is the enzyme glycogen synthase kinase (GSK-3). Activation of the PI3 kinase pathway deactivates GSK-3. GSK-3 has multiple targets in cells,

including transcription factors ( -catenin and c-Jun) and cytoskeletal elements such as tau. Many of the targets of GSK-3 are pro-apoptotic when activated. Thus,

deactivation of GSK-3 via activation of the PI3K pathway results in neurotrophic effects. Lithium inhibits GSK-3, an effect that may be, at least in part, responsible for

lithium’s therapeutic effects. These mechanisms underlie many of the long-term effects of neurotrophins, including neurite outgrowth, cytoskeletal remodeling, and cell

survival.

Source. Adapted from Gould TD, Chen G, Manji HK: “Mood Stabilizer Psychopharmacology.” Clinical Neuroscience Research 2:193–212, 2002. Copyright 2002, Elsevier. Used

with permission.

Inactivation of the ERK pathway in the CNS induces animal behavioral alterations reminiscent of manic symptoms, which are likely to depend on ERK’s effect

on distinct brain regions and the presence of interacting molecules (Shaltiel et al. 2007). Moreover, ERK knockout mice display behavioral abnormalities

related to manic symptoms. These data support a clinical role for the ERK pathway in therapeutic action of mood stabilizers. Nevertheless, the possible role of

this pathway in the pathophysiology of bipolar disorder has yet to be elucidated.

PI3 Kinase–Akt Pathway

The PI3K–Akt pathway is also particularly important for mediating neuronal survival in a wide variety of circumstances. Trk receptors can activate PI3KPrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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through at least two distinct pathways, the relative importance of which differs between neuronal subpopulations. In many neurons, Ras-dependent

activation of PI3K is the most important pathway through which neurotrophins promote cell survival (see Figure 1–15). In some cells, however, PI3K can also

be activated through three adaptor proteins: Shc, Grb-2, and Gab-1. Binding to phosphorylated tyrosine 490 of Shc results in recruitment of Grb-2 (see Figure

1–9). Phosphorylated Grb-2 provides a docking site for Gab-1, which in turn is bound by PI3K (Brunet et al. 2001). PI3K directly regulates certain cytoplasmic

apoptotic pathways. Akt has been proposed to act both prior to the release of cytochrome c by pro-apoptotic Bcl-2 family members and subsequent to the

release of cytochrome c by regulating components of the apoptosome. Akt phosphorylates the pro-apoptotic Bcl-2 family member BAD, thereby inhibiting

BAD’s pro-apoptotic functions (see Figure 1–15) (Datta et al. 1997).

Another important target of Akt is the enzyme glycogen synthase kinase–3 (GSK-3) (see Figures 1–11 and 1–15). GSK-3 is a serine/threonine kinase that is,

in general, constitutively active in cells. It is found in two forms— and —and currently appears to be the only kinase significantly directly inhibited by

lithium (Davies et al. 2000; Klein and Melton 1996; Stambolic et al. 1996). It may thus represent a target of the development of novel medications for the

treatment of bipolar disorder (Gould and Manji 2005). While most research has focused on the isoform, available evidence suggests that the two forms may

have very similar—though not absolutely identical—biological properties (Ali et al. 2001; Plyte et al. 1992). GSK-3 was named on the basis of its originally

described function as a kinase that inactivates glycogen synthase. Following insulin receptor stimulation, PI3K and Akt are activated, and this results in the

phosphorylation and concomitant inactivation of GSK-3. Inactivated GSK-3 no longer phosphorylates glycogen synthase, allowing the formation of glycogen

from glucose (Cohen and Frame 2001; Woodgett 2001).

In addition to regulation by Akt, other kinases, including p70 S6 kinase, RSK, and cAMP-dependent protein kinase (PKA), appear to deactivate GSK-3 by

phosphorylation (Cohen and Frame 2001; Grimes and Jope 2001). The effect of GSK-3 on transcription factors such as c-Jun, heat shock factor–1 (HSF-1),

nuclear factor of activated T-cells (NFAT), and -catenin (see below) has drawn considerable interest and is particularly noteworthy (Frame and Cohen 2001;

Grimes and Jope 2001) (see Figure 1–15). Generally, GSK-3 activity results in suppression of the activity of transcription. Conversely, inactivation of GSK-3

appears to activate these transcription factors (Grimes and Jope 2001). Thus, GSK-3 is well positioned to receive signals from multiple diverse signal

pathways, a function that is undoubtedly critical in the CNS.

GSK-3 is also a critical regulator of the Wnt pathway. Wnt is a family of secreted glycoproteins that are well known to have important roles in development.

Signaling through Wnt glycoproteins results in inactivation of GSK-3 and a subsequent increase in the transcription factor -catenin. Furthermore, some

studies suggest a role for -catenin (and Wnt) in synaptic plasticity (Salinas 1999; Salinas and Hall 1999). Additional studies have suggested that -catenin

itself may play an important role for this protein in the function of the brain. Indeed, upregulation of this protein is sufficient to cause the formation of gyri

and sulci in the mouse brain, a finding observed only in higher mammals, and is suggestive of an important role in higher mammalian cognitive functions

(Chenn and Walsh 2002).

PLC- 1 Pathway

Phosphorylated Trk receptors also recruit PLC- 1 (see Figure 1–9). The Trk kinase then phosphorylates and activates PLC- 1, which acts to hydrolyze

phosphatidylinositides to generate DAG and IP3. IP3 induces the release of Ca2+ stores, increasing levels of cytoplasmic Ca2+ and thereby activating many

pathways controlled by Ca2+. It has been shown that neurotrophins activate protein kinase C , which is required for activation of the ERK cascade and for

neurite outgrowth (Patapoutian and Reichardt 2001). As discussed previously, emerging data suggest that the regulation of hippocampal LTP by TrkB

receptors is mediated primarily through the PLC- cascade (for details, see Minichiello et al. 2002).

Phosphorylation/Dephosphorylation

For many proteins, a change in charge and conformation due to the addition or removal of phosphate groups results in alterations in their intrinsic functional

activity. Although proteins are covalently modified in many other ways—for example, by ADP ribosylation, acylation (acetylation, myristoylation),

carboxymethylation, and glycosylation—none of these mechanisms appear to be as widespread and readily subject to regulation by physiological stimuli as

phosphorylation. Indeed, protein phosphorylation/dephosphorylation represents a pathway of fundamental importance in the chemistry of biological

regulation (see Nestler et al. 2001). Virtually all types of extracellular signals are known to produce many of their diverse physiological effects by regulating

the state of phosphorylation of specific proteins in the cells that they target.

The phosphate group provides an unwieldy negative charge that often interacts with the catalytic and other regions of enzymes. The addition of a phosphate

often results in conformational changes in proteins. In the case of enzymes, this change may increase (more commonly) or decrease the affinity of an enzyme

for its substrate. Thus, phosphorylation may result in a change in kinase activity, a change in phosphatase activity, or the marking of a protein for cleavage by

proteases. The catalytic activity of an enzyme can be switched on or off, or an ion channel can be opened or closed. For many other proteins,

phosphorylation-induced changes in charge and conformation result in alterations in the affinity of the proteins for other molecules. For example,

phosphorylation alters the affinity of numerous enzymes for their cofactors and end-product inhibitors, phosphorylation of receptors can alter their affinity for

G proteins, and phosphorylation of some nuclear transcription factors alters their DNA-binding properties. Therefore, phosphorylation can produce varied

effects on cellular physiology and ultimately can have major behavioral manifestations.

Protein kinases are classified by the residues that they phosphorylate, with the two major classes being serine/threonine kinases and tyrosine kinases. Most

phosphorylation (>95%) of proteins occurs on serine residues, a small amount (about 3%–4%) on threonine residues, and very little (0.1%) on tyrosine

residues (but, as discussed earlier, the tyrosine kinases can be very important for neurotrophic signaling). In all cases, the kinases catalyze the transfer of the

terminal ( ) phosphate group of ATP to the hydroxyl moiety in the respective amino acid residue, a process that requires Mg2+. Within cells, protein kinases

often form sequential pathways, whereby one kinase phosphorylates/activates another, which phosphorylates/activates another kinase, and so forth. In this

manner, signals can be propagated within cells, allowing ample opportunity (see below) for the signal to be altered by other intracellular signals, often in a

cell type–specific manner, allowing for considerable “fine-tuning.”

Although clearly playing critical roles in modulating the function of proteins by catalyzing the cleavage of the phosphoester bond, protein phosphatases have

not been as extensively studied as kinases. In the CNS, phosphatases often function as a molecular “off switch,” thereby decreasing the activity of enzymes

and the intracellular signaling pathways they control. However, it is clear that protein phosphatases are much more than simple off switches. Thus, in an

elegant series of studies, Greengard and associates demonstrated that a major CNS phosphoprotein, known by the acronym DARPP-32 (dopamine and

cAMP-regulated phosphoprotein, 32 kDa), brings about many of its long-term neuroplastic effects by regulating the activity of a protein phosphatase (protein

phosphatase–1; PP-1) (for a review, see Greengard 2001a). Thus, they demonstrated that the DARPP-32/PP-1 pathway integrates information from a variety

of neurotransmitters and produces a coordinated response involving numerous downstream physiological effectors. DARPP-32 phosphorylation by PKA is

regulated by the actions of various neurotransmitters, principally dopamine acting at D1 receptors but also a variety of other neurotransmitters (Greengard

2001b; Nestler et al. 2001). Phospho-DARPP-32, by inhibiting the activity of PP-1, acts in a synergistic manner with different protein kinases (primarily PKA

and PKC) to increase the level of phosphorylation of various downstream effector proteins and thereby long-term neuronal adaptations that have also been

implicated in the actions of drugs of abuse and antidepressants (Greengard 2001b; Nestler et al. 2001).

While propagation of signals may be very immediate, even short-term phosphorylation of many types of proteins can have long-term effects, resulting in

“molecular and cellular memory.” Indeed, various forms of learning and memory are known to be regulated, in large part, by phosphorylation events.

Short-term memory may involve the phosphorylation of presynaptic or postsynaptic proteins in response to synaptic activity, a process that results in

transient facilitation or inhibition of synaptic transmission. Long-term memory may involve phosphorylation of proteins that play a role in the regulation of

gene expression, which would result in more permanent modifications of synaptic transmission, potentially via structural brain changes (Malenka and Nicoll

1999). As discussed, long-term potentiation, one of the most extensively studied models of learning and memory, is believed to be initiated through

short-term changes in Ca2+-dependent protein phosphorylation and maintained by long-term changes in gene expression. There is also growing appreciation

that protein phosphatases play a critical role in the extinction of memory. Thus, abundant data now suggest that rather than representing a passive process,

“forgetting” is more an active process of memory erasure (discussed in Genoux et al. 2002). In an elegant series of behavioral studies using transgenic mice,

Genoux et al. (2002) provided strong evidence that PP-1 is involved in forgetting rather than in preventing the encoding of memory. Although the precise

mechanisms by which PP-1 brings about these effects remain to be fully elucidated, these investigators postulate that CaMKII and the GluR1 subunit of the

AMPA receptor play important roles. These findings may have major implications for the ultimate development of agents that could be used to facilitate thePrint: Chapter 1. Neurotransmitters, Receptors, Signal Transduction, a… http://www.psychiatryonline.com/popup.aspx?aID=407005&print=yes…

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erasure of traumatic memories—for example, in PTSD.

CONCLUSION

In this chapter, we have attempted to provide an overview of some fundamental aspects of neurotransmitters, signal transduction pathways, and second

messengers. For most psychiatrists, molecular and cellular biology have not traditionally played a major role in day-to-day clinical practice. However, new

insights into the molecular and cellular basis of disease and drug action are being generated at an ever-increasing rate and will ultimately result in a

transformation of our understanding and management of diseases. Indeed, the last decade of the twentieth century was truly a remarkable one for

biomedical research. The “molecular medicine revolution” has utilized the power of sophisticated cellular and molecular biological methodologies to tackle

many of society’s most devastating illnesses. The rate of progress has been exciting indeed, and hundreds of G protein–coupled receptors and dozens of G

proteins and effectors have now been identified and characterized at the molecular and cellular levels. These efforts have allowed the study of a variety of

human diseases that are caused by abnormalities in cell-to-cell communication; studies of such diseases are offering unique insights into the physiological

and pathophysiological functioning of many cellular transmembrane signaling pathways.

Psychiatry, like much of the rest of medicine, has entered a new and exciting age demarcated by the rapid advances and the promise of molecular and cellular

biology and neuroimaging. There is a growing appreciation that severe psychiatric disorders arise from abnormalities in cellular plasticity cascades, leading to

aberrant information processing in synapses and circuits mediating affective, cognitive, motoric, and neurovegetative functions. Thus, these illnesses can be

best conceptualized as genetically influenced disorders of synapses and circuits rather than simply as deficits or excesses in individual neurotransmitters

(Carlson et al. 2006). Furthermore, many of these pathways play critical roles not only in synaptic (and therefore behavioral) plasticity but also in long-term

atrophic processes. Targeting these cascades in treatment may stabilize the underlying disease process by reducing the frequency and severity of the

profound mood cycling that contributes to morbidity and mortality. The role of cellular signaling cascades offers much explanatory power for understanding

the complex neurobiology of bipolar illness (Goodwin and Jamison 2007). Signaling cascades regulate the multiple neurotransmitter and neuropeptide

systems implicated in psychiatric disorders and are targets for the most effective treatments. The highly integrated monoamine and prominent neuropeptide

pathways are known to originate in and project heavily to limbic-related regions, such as the hippocampus, hypothalamus, and brain stem, which are likely

associated with neurovegetative symptoms. Abnormalities in cellular signaling cascades that regulate diverse physiological functions likely explain the

tremendous medical comorbidity associated with psychiatric disorders.

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