Chapter 14: Neurotransmission and the Central Nervous System

Cerebral Cortex. The two cerebral hemispheres constitute the largest division of the brain. Regions of the cortex are classified in several ways:

• by the modality of information processed (e.g., sensory, including somatosensory, visual, auditory, and olfactory, as well as motor and associational)

• by anatomical position (frontal, temporal, parietal, and occipital)

• by the geometric relationship between cell types in the major cortical layers ("cytoarchitectonic" classification)

The specialized functions of a cortical region arise from the interplay between connections with other regions of the cortex (corticocortical systems) and noncortical areas of the brain (subcortical systems) and a basic intracortical processing module of ~100 vertically connected cortical columns (Mountcastle, 1997). Varying numbers of adjacent columnar modules may be functionally linked into larger information-processing ensembles. The pathology of Alzheimer's disease, e.g., destroys the integrity of the columnar modules and the corticocortical connections (Chapter 22). Cortical areas termed association areas process information from primary cortical sensory regions to produce higher cortical functions such as abstract thought, memory, and consciousness. The cerebral cortices also provide supervisory integration of the autonomic nervous system and integrate somatic and vegetative functions, including those controlling the cardiovascular and gastrointestinal systems.

Limbic System. The limbic system is an archaic term for an assembly of brain regions (hippocampal formation, amygdaloid complex, septum, olfactory nuclei, basal ganglia, and selected nuclei of the diencephalon) grouped around the subcortical borders of the underlying brain core to which a variety of complex emotional and motivational functions have been attributed. Modern neuroscience tends to avoid this term because the ill-defined regions of the "limbic system" do not function consistently as a system. Parts of these limbic regions also participate individually in functions that can be more precisely defined. Thus, the basal ganglia or neostriatum (the caudate nucleus, putamen, globus pallidus, and lentiform nucleus) form an essential regulatory segment of the extrapyramidal motor system that complements the function of the pyramidal (or voluntary) motor system. Damage to the extrapyramidal system affects the ability to initiate voluntary movements and causes disorders characterized by involuntary movements, such as the tremors and rigidity of Parkinson disease or the uncontrollable limb movements of Huntington's chorea (Chapter 22). Similarly, the hippocampus may be crucial to the formation of recent memory, since this function is lost in patients with extensive bilateral damage to the hippocampus. Memory is also disrupted by Alzheimer's disease, which destroys the intrinsic structure of the hippocampus as well as parts of the frontal cortex.

Diencephalon. The thalamus lies in the center of the brain, beneath the cortex and basal ganglia and above the hypothalamus. The neurons of the thalamus are arranged in distinct clusters, or nuclei, which are either paired or midline structures. These nuclei act as relays between incoming sensory pathways and the cortex, between discrete regions of the thalamus and the hypothalamus, and between the basal ganglia and the association regions of the cerebral cortex. The thalamic nuclei and the basal ganglia also exert regulatory control over visceral functions; aphagia and adipsia, as well as general sensory neglect, follow damage to the corpus striatum or to selected circuits ending in the striatum. The hypothalamus is the principal integrating region for the autonomic nervous system and regulates body temperature, water balance, intermediary metabolism, blood pressure, sexual and circadian cycles, secretion from the adenohypophysis, sleep, and emotion.

Midbrain and Brainstem. The mesencephalon, pons, and medulla oblongata connect the cerebral hemispheres and thalamus-hypothalamus to the spinal cord. These "bridge portions" of the CNS contain most of the nuclei of the cranial nerves, as well as the major inflow and outflow tracts from the cortices and spinal cord. These regions contain the reticular activating system, an important but incompletely characterized region of gray matter linking peripheral sensory and motor events with higher levels of nervous integration. The major monoamine-containing neurons of the brain are found here. Together, these regions represent the points of central integration for coordination of essential reflexive acts, such as swallowing and vomiting, and those that involve the cardiovascular and respiratory systems; these areas also include the primary receptive regions for most visceral afferent sensory information. The reticular activating system is essential for the regulation of sleep, wakefulness, and level of arousal, as well as for coordination of eye movements. The fiber systems projecting from the reticular formation have been called nonspecific because the targets to which they project are relatively more diffuse in distribution than those of many other neuronal systems (e.g., specific thalamocortical projections). However, the chemically homogeneous components of the reticular system innervate targets in a coherent and functionally integrated manner despite their broad distribution.

Cerebellum. The cerebellum arises from the posterior pons behind the cerebral hemispheres. It is highly laminated and redundant in its detailed cytological organization. The lobules and folia of the cerebellum project onto specific deep cerebellar nuclei, which in turn make relatively selective projections to the motor cortex (by way of the thalamus) and to brainstem nuclei concerned with vestibular function (position-stabilization). In addition to maintaining the proper tone of antigravity musculature and providing continuous feedback during volitional movements of the trunk and extremities, the cerebellum also may regulate visceral functions (e.g., controlling heart rate, so as to maintain blood flow despite changes in posture).

Spinal Cord. The spinal cord extends from the caudal end of the medulla oblongata to the lower lumbar vertebrae. Within this mass of nerve cells and tracts, sensory information from skin, muscles, joints, and viscera is locally coordinated with motoneurons and with primary sensory relay cells that project to and receive signals from higher levels. The spinal cord is divided into anatomical segments (cervical, thoracic, lumbar, and sacral) that correspond to divisions of the peripheral nerves and spinal column (see Figure 8–1). Ascending and descending tracts of the spinal cord are located within the white matter at the perimeter of the cord, while intersegmental connections and synaptic contacts are concentrated within an H-shaped internal mass of gray matter. Sensory information flows into the dorsal cord, and motor commands exit via the ventral portion. The preganglionic neurons of the autonomic nervous system are found in intermediolateral columns of gray matter. Autonomic reflexes (e.g., changes in skin vasculature with alteration of temperature) can be elicited within local segments of the spinal cord, as shown by the maintenance of these reflexes after the cord has been severed.

Microanatomy of the Brain

Neurons operate either within layered structures such as the olfactory bulb, cerebral cortex, hippocampal formation, and cerebellum or in clustered groupings, defined collections of neurons that aggregate into nuclei. Specific connections between neurons within or across the macro-divisions of the brain are essential to the function of the brain. Through patterns of neuronal circuitry, individual neurons form functional ensembles to regulate the flow of information within and between the regions of the brain.

Cellular Organization of the Brain. Present understanding of the cellular organization of the CNS can be viewed from the perspective of the size, shape, location, and interconnections between neurons (Cooper et al., 2003; Shepherd, 2003).

Cell Biology of Neurons. Neurons are classified according to function (sensory, motor, or interneuron), location, the identity of the transmitter they synthesize and release or the class or classes of receptor expressed on the cell surface. Microscopic analysis of a neuron focuses on its general shape and in particular the complexity of the afferent receptive surfaces on the dendrites and cell body that receive synaptic contacts from other neurons. Neurons (Figure 14–1) exhibit the cytological characteristics of highly active secretory cells with large nuclei: large amounts of smooth and rough endoplasmic reticulum; and frequent clusters of specialized smooth endoplasmic reticulum (Golgi complex), in which secretory products of the cell are packaged into membrane-bound organelles for transport from the perikaryon to the axon or dendrites. Neurons and their cellular extensions are rich in microtubules, which support the complex cellular structure and assist in the reciprocal transport of essential macromolecules and organelles between the cell body and distant axon or dendrites. The sites of interneuronal communication in the CNS are termed synapses Although synapses are functionally analogous to "junctions" in the somatic motor and autonomic nervous systems, central junctions contain an array of specific proteins presumed to be the active zone for transmitter release and response (Husi et al., 2000). Like peripheral "junctions," central synapses are denoted by accumulations of tiny (50-150 nm) synaptic vesicles. The proteins of these vesicles have specific roles in transmitter storage, vesicle docking onto presynaptic membranes, voltage- and Ca²⁺-dependent secretion, and recycling and restorage of released transmitter (Jahn, 2004, Murthy and Camilli, 2003; Nestler et al., 2009).

Support Cells. Neurons are not the only cells in the CNS. According to most estimates, neurons are outnumbered, perhaps by an order of magnitude, by various types of support cells. These include macroglia, microglia, the cells of the vascular elements comprising the intracerebral vasculature, the cerebrospinal fluid-forming cells of the choroid plexus found within the intracerebral ventricular system, and the meninges, which cover the surface of the brain and comprise the cerebrospinal fluid-containing envelope. Macroglia are the most abundant support cells; some are categorized as astrocytes (cells interposed between the vasculature and the neurons, often surrounding individual compartments of synaptic complexes). Astrocytes play a variety of metabolic support roles including furnishing energy intermediates and supplementary removal of neurotransmitters following release (Pellerin and Magistretti, 2003). The oligodendroglia, a second prominent category of macroglia, are myelin-producing cells. Myelin, made up of multiple layers of compacted membranes, insulate segments of axons bioelectrically and permit non-decremental propagation of action potentials. Microglia are relatively uncharacterized support cells believed to be of mesodermal origin and related to the macrophage/monocyte lineage (Carson, 2002). Some microglia reside within the brain, while additional cells of this class may be recruited to the brain during periods of inflammation following either microbial infection or brain injury. The response of the brain to inflammation differs strikingly from that of other tissues (Glass et al., 2010).

Blood-Brain Barrier. Apart from exceptional instances in which drugs are introduced directly into the CNS, the concentration of an agent in the blood after oral or parenteral administration may differ substantially from its concentration in the brain. The blood-brain barrier (BBB) is an important boundary between the periphery and the CNS that forms a permeability barrier to the passive diffusion of substances from the bloodstream into the CNS. Evidence for the existence of the BBB is provided by the greatly diminished rate of access of many chemicals from plasma to the brain and the localization of several drug export systems in the cells that constitute the BBB (Chapter 5). An exception exists for lipophilic molecules, which diffuse fairly freely across the BBB and accumulate in the brain. This barrier is nonexistent in the peripheral nervous system, and is much less prominent in the hypothalamus and in several small, specialized organs (the circumventricular organs) lining the third and fourth ventricles of the brain: the median eminence, area postrema, pineal gland, subfornical organ, and subcommissural organ. While the BBB may impose limitations on the diffusion of macromolecules from the bloodstream into the brain, selective barriers to permeation into and out of the brain also exist for small, charged molecules such as neurotransmitters, their precursors and metabolites, and some drugs. These diffusional barriers are viewed as a combination of the partition of solute across the vasculature (which governs passage by definable properties such as molecular weight, charge, and lipophilicity) and the presence or absence of energy-dependent transport systems (Chapter 5). Important exceptions are the specific uptake transporters for amino acids. This accounts for the therapeutic utility of L-dopa (levodopa) used for treatment of Parkinson disease. The brain clears metabolites of transmitters into the fluid-containing lateral ventricles by excretion via the acid transport system of the choroid plexus. Substances that rarely gain access to the brain from the bloodstream often can reach the brain when injected directly into the cerebrospinal fluid. Under certain conditions, it may be possible to open the BBB, at least transiently, to permit the entry of chemotherapeutic agents. Cerebral ischemia and inflammation also modify the BBB, increasing access to substances that ordinarily would not affect the brain.

Response to Damage: Repair and Plasticity in the CNS

Because the neurons of the CNS are terminally differentiated cells, they do not undergo proliferative responses to damage, although recent evidence suggests the possibility of neural stem cell proliferation as a natural means for replacement of neurons in selected areas of the CNS and as a means of therapeutic repair (Aimone et al., 2010; Marchetto et al., 2010). In the CNS, unlike in the peripheral nervous system, glia-associated proteins are not uniformly upregulated following an injury. (Madinier A, et al., 2009). There are, in addition, proteins in myelin like Nogo-A that are potent inhibitors of axonal regeneration in the CNS (Giger et al., 2008).

Research at the cellular level identifies specific neurons and their most proximate synaptic connections that may mediate a behavior or the behavioral effects of a given drug. For example, research into the basis of emotion exploits both molecular and behavioral leads to identify the most likely brain sites at which behavioral changes pertinent to emotion can be analyzed. Such research provides clues as to the nature of the interactions in terms of interneuronal communication (i.e., excitation, inhibition, or more complex forms of synaptic interaction).

An understanding at the systems level is required to assemble the structural and functional properties of specific central transmitter systems, linking the neurons that make and release a given neurotransmitter to its behavioral effects. While many such transmitter-to-behavior linkages have been postulated, it has proven difficult to validate the essential involvement of specific transmitter-defined neurons in mediating specific mammalian behaviors. Research at the behavioral level may illuminate the integrative phenomena that link populations of neurons (often in an operationally or empirically defined manner) into extended specialized circuits or "systems" that integrate the physiological expression of a learned, reflexive, or spontaneously generated behavioral response. The entire concept of animal models of human psychiatric diseases rests on the assumption that scientists can appropriately infer from observations of behavior and physiology (heart rate, respiration, locomotion, etc.) that the states experienced by animals are equivalent to the emotional states experienced by human beings expressing similar physiological changes (Cowan et al., 2000, 2002).

CNS Transmitter Discovery Strategies. The earliest transmitters considered for central roles were acetylcholine (ACh) and norepinephrine (NE), largely because of their established roles in the somatic motor and autonomic nervous systems. In the 1960s, serotonin (5-hydroxy tryptamine, 5-HT), epinephrine, and dopamine (DA) were investigated as potential CNS transmitters; although histochemical, biochemical, and pharmacological data yielded results consistent with their roles as neurotransmitters, not all criteria were fully satisfied. In the early 1970s, the availability of selective and potent antagonists of gamma-aminobutyric acid (GABA), glycine, and glutamate, all known to be enriched in brain, led to their general acceptance as transmitters. At about the same time, a search for hypothalamic-hypophyseal factors led to an improvement in the technology to isolate, purify, sequence, and synthetically prepare a growing family of neuropeptides (Hökfelt et al., 2003). These advances, coupled with the widespread application of immunohistochemistry, strongly supported the view that neuropeptides act as transmitters. Adaptation of bioassay technology developed in studies of pituitary secretions, and later, competitive binding assays with radioactive ligands, gave rise to the discovery of endogenous peptide ligands for drugs acting at opiate receptors (Chapter 18). The search for endogenous factors whose receptors constituted drug binding sites later was extended to the benzodiazepine receptors and to a series of endogenous lipid amides as the natural ligands for cannabinoid receptors, termed the endocannabinoids (Piomelli, 2003).

Identification of Central Transmitters. An essential step in understanding the functional properties of neurotransmitters within the context of the circuitry of the brain is to identify substances that are transmitters at specific interneuronal connections. The criteria for the rigorous identification of central transmitters require the same data set used to establish the transmitters of the autonomic nervous system (Chapter 8).

1. The transmitter must be shown to be present in the presynaptic terminals of the synapse and in the neurons from which those presynaptic terminals arise.

2. The transmitter must be released from the presynaptic nerve concomitantly with presynaptic nerve activity.

3. When applied experimentally to target cells, the effects of the putative transmitter must be identical to the effects of stimulating the presynaptic pathway.

4. Specific pharmacological agonists and antagonists should mimic and antagonize, respectively, the measured functions of the putative transmitter with appropriate affinities and order of potency.

Strategies involving mutagenesis have defined how activated receptors (themselves subject to reversible phosphorylation at one or more functionally distinct sites) can interact with heterotrimeric GTP-binding protein complex. GPCRs include muscarinic cholinergic receptors, subtypes of GABA and glutamate receptors, and many other aminergic and peptidergic receptors. Transfecting cells lacking GPCRs with mRNAs for GPCRs with no known ligands has led to the identification of novel neuropeptide ligands for these "orphan" receptors (Robas et al., 2003).

Postsynaptic receptivity of CNS neurons is regulated dynamically in terms of the number of receptor sites and the threshold required to generate a response. Receptor number often depends on the concentration of agonist to which the target cell is exposed. Thus, chronic exposure to an agonist can lead to a reduction in responsiveness and in the number of receptors (desensitization and down-regulation) and consequently to subsensitivity or tolerance to the transmitter. Conversely, denervation or the administration of an antagonist can lead to supersensitive responses partially mediated by an increase in the number of receptors. For many GPCRs, down-regulation is achieved by the actions of receptor kinases (GRKs) that phosphorylate agonist occupied receptors leading in turn to internalization of the receptors (Chapter 3).

Multiple transport proteins with selectivity for various transmitters have been cloned (Figure 14–8). In contrast to the GPCRs discussed earlier, these proteins have 12 hydrophobic membrane-spanning domains. This means that both the amino and carboxy termini are on the same, intracellular, side of the membrane. Like the GPCRs, these transporters have sites for glycosylation on the cytoplasmic side, primarily on the i2 loop, and sites for phosphorylation, primarily on the amino and carboxy tails. Comparison of the sequences of the transporters for NE and DA (Figure 14–8) reveals striking conservation in the TM domains.

Neurohormones. The anterior and posterior pituitary secrete a variety of hormones and releasing factors. Hypothalamic neurons affecting the anterior pituitary release their hormones into the hypothalamic–adenohypophyseal portal blood system, which delivers them to the anterior pituitary, where they regulate the release of trophic hormones (i.e., ACTH, FSH, GH, LH, prolactin) into the blood. Other hypothalamic neurons project onto the posterior pituitary, where they release their peptide contents, oxytocin and arginine vasopression (anti-diuretic hormone, or ADH) into the systemic circulation (Figure 14–9 and Chapters 25 and 38).

Cytochemical evidence indicates that the same substances that are secreted as hormones from the posterior pituitary also mediate transmission at these sites. Thus, the designation hormone relates to the site of release at the posterior pituitary and does not necessarily describe all of the actions of the peptides.

Neuromodulators. The distinctive feature of a modulator is that it originates from non-synaptic sites, yet influences the excitability of nerve cells. Substances such as CO and ammonia, arising from active neurons or glia, are potential modulators acting through non-synaptic actions. Similarly, circulating steroid hormones, steroids produced in the nervous system (i.e., neurosteroids), locally released adenosine, and other purines, eicosanoids, and nitric oxide (NO) are regarded as modulators and/or neurotransmitters (discussed later).

Neurotrophic Factors. Neurotrophic factors are substances produced within the CNS by neurons, astrocytes, microglia, or transiently invading peripheral inflammatory or immune cells that assist neurons in their attempts to repair damage. The binding of neurotrophic factors to their receptors genarally promotes receptor dimerization and protein tyrosine kinase activity in the intracellular domains of the receptors (Figure 14–5C). Seven categories of neurotrophic peptides are recognized:

1. classic neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and the related neurotrophins)

2. neuropoietic factors, which have effects both in brain and in myeloid cells (e.g., cholinergic differentiation factor [also called leukemia inhibitory factor], ciliary neurotrophic factor, and some interleukins)

3. growth factor peptides, such as epidermal growth factor, transforming growth factors α and β, glial cell–derived neurotrophic factor, and activin A

4. fibroblast growth factors

5. insulin-like growth factors

6. platelet-derived growth factors

7. axon guidance molecules, some of which also affect cells of the immune system

Drugs designed to elicit the formation and secretion of these products or to emulate their actions may provide useful adjuncts to rehabilitative treatments (Huang and Reichardt, 2001).

GABA. Substantial data supports the idea that GABA mediates the inhibitory actions of local interneurons in the brain and may also mediate presynaptic inhibition within the spinal cord. Presumptive GABA-containing inhibitory synapses have been demonstrated most clearly between cerebellar Purkinje neurons and their targets in Deiter's nucleus; between small interneurons and the major output cells of the cerebellar cortex, olfactory bulb, cuneate nucleus, hippocampus, and lateral septal nucleus; and between the vestibular nucleus and trochlear motoneurons. GABA also mediates inhibition within the cerebral cortex and between the caudate nucleus and the substantia nigra. GABA-containing neurons and nerve terminals have been localized with immunocytochemical methods that visualize glutamic acid decarboxylase, the enzyme that catalyzes the synthesis of GABA from glutamic acid, or by in situ hybridization of the mRNA for this protein. GABA-containing neurons frequently co-express one or more neuropeptides.

The most useful compounds for confirmation of GABA-mediated effects have been bicuculline and picrotoxin (Figure 14–10); however, many convulsants whose actions previously were unexplained (including penicillin and pentylenetetrazol) are relatively selective antagonists of the action of GABA. Useful therapeutic effects have not yet been obtained through the use of agents that mimic GABA (such as muscimol), inhibit its active reuptake (such as 2,4-diaminobutyrate, nipecotic acid, and guvacine), or alter its turnover (such as aminooxyacetic acid).

GABA receptors have been divided into three main types: A, B, and C.

• The most prominent GABA-receptor subtype, the GABA_A receptor, is a ligand-gated Cl⁻ ion channel, an "ionotropic receptor."

• The GABA_B receptor is a GPCR.

• The GABA_C receptor is a transmitter-gated Cl⁻ channel.

GABA_A receptor subunit proteins have been well characterized due to their abundance. The receptor also has been extensively characterized as the site of action of many neuroactive drugs, notably benzodiazepines, barbiturates, ethanol, anesthetic steroids, and volatile anesthetics (Figure 14–11).

Based on sequence homology to the first reported GABA_A subunit cDNAs, multiple subunits have been cloned, including 6α, 4β, and 3γ subunits. They appear to be expressed in multiple multimeric, pharmacologically distinctive combinations. In addition to these subunits, which are products of separate genes, splice variants for several subunits have been described.

The GABA_A receptor is probably pentameric or tetrameric in structure with subunits that assemble together around a central pore typical for other ionotropic receptors. The major form of the GABA_A receptor contains at least three different subunits α, β, and γ, with likely stoichiometry of 2α, 2β, 1γ. All three subunits are required to interact with benzodiazepines with the profile expected of a native GABA_A receptor. The distribution of different subunit combinations of GABA_A and their functions in the mammalian brain are summarized in Table 14–2 (Fritschy and Mohler, 1995).

The GABA_B or metabotropic GABA receptor interacts with G_i to inhibit adenylyl cyclase, activate K⁺ channels, and reduce Ca²⁺ conductance. Presynaptic GABA_B receptors function as autoreceptors, inhibiting GABA release, and may play the same role on neurons releasing other transmitters. Functional GABA _B receptors are heterodimers made up of GABA_BR₁ and GABA_BR₂ subunits (Bettler et al., 2004) The GABA_C receptor is less widely distributed than the A and B subtypes. GABA is more potent by an order of magnitude at GABA_C than at GABA_A receptors, and a number of GABA_A agonists (e.g., baclofen) and modulators (e.g., benzodiazepines and barbiturates) seem not to interact with GABA_C receptors. GABA_C receptors are found in the retina, spinal cord, superior colliculus, and pituitary (Olsen and Betz, 2005).

Glycine. Many of the features described for the GABA_A receptor family also apply to the inhibitory glycine receptor, which is prominent in the brainstem and spinal cord. Multiple subunits assemble into a variety of glycine receptor subtypes. These pharmacological subtypes are detected in brain tissue with particular neuroanatomical and neurodevelopmental profiles. As with the GABA_A receptor, the complete functional significance of glycine receptor subtypes is not known. There is evidence for clustering of glycine receptors by the anchoring protein gephyrin (Sola et al., 2004). An additional role for glycine is as a co-agonist at NMDA receptors, at which both glutamate and glycine must be present for activation to occur.

Glutamate and Aspartate. Glutamate and aspartate are found in very high concentrations in brain, and both amino acids have powerful excitatory effects on neurons in virtually every region of the CNS. Their widespread distribution initially obscured their roles as transmitters, but there now is broad acceptance that glutamate and possibly aspartate are the principal fast ("classical") excitatory transmitters throughout the CNS (Bleich et al., 2003; Conn, 2003). Multiple subtypes of receptors for excitatory amino acids have been cloned, expressed, and characterized pharmacologically, based on the relative potencies of synthetic agonists and the discovery of potent and selective antagonists (Kotecha and MacDonald, 2003). Glutamate receptors are classed functionally either as ligand-gated ion channel ("ionotropic") receptors or as "metabotropic" GPCRs (Table 14–3).

Neither the precise number of subunits that assembles to generate a functional glutamate ionotropic receptor ion channel in vivo nor the intramembranous topography of each subunit has been established unequivocally. The ligand-gated ion channels are further classified according to the identity of agonists that selectively activate each receptor subtype, and are broadly divided into N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. The non-NMDA receptors include the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainic acid (KA) receptors (Table 14–3). Selective antagonists for these receptors are now available. In the case of NMDA receptors, agonists include open-channel blockers such as phencyclidine (PCP or "angel dust"), antagonists such as 5,7-dichlorokynurenic acid, which act at an allosteric glycine-binding site, and the novel antagonist ifenprodil, which may act as a closed-channel blocker. In addition, the activity of NMDA receptors is sensitive to pH and to modulation by a variety of endogenous agents including Zn²⁺, some neurosteroids, arachidonic acid, redox reagents, and polyamines such as spermine. Additional diversity of glutamate receptors arises by alternative splicing or by single-base editing of mRNAs encoding the receptors or receptor subunits. Alternative splicing has been described for metabotropic receptors and for subunits of NMDA, AMPA, and kainate receptors. For some subunits of AMPA and kainate receptors, the RNA sequence differs from the genomic sequence in a single codon of the receptor subunit that markedly affects the Ca²⁺ permeability of the receptor channel (Conn and Pin, 1997). AMPA and kainate receptors mediate fast depolarization at glutamatergic synapses in the brain and spinal cord. NMDA receptors are involved in normal synaptic transmission, but activation of NMDA receptors is usually associated more closely with the induction of various forms of synaptic plasticity rather than with fast point-to-point signaling in the brain. AMPA or kainate receptors and NMDA receptors may be co-localized at many glutamatergic synapses.

Activation of NMDA receptors is obligatory for the induction of a type of long-term potentiation (LTP) that occurs in the hippocampus. NMDA receptors normally are blocked by Mg²⁺ at resting membrane potentials. Thus, activation of NMDA receptors requires not only binding of synaptically released glutamate, but simultaneous depolarization of the postsynaptic membrane. This is achieved by activation of AMPA/kainate receptors at nearby synapses involving inputs from different neurons. AMPA receptors also are dynamically regulated to affect their sensitivity to the synergism with NMDA. Thus, NMDA receptors may function as coincidence detectors, being activated only when there is simultaneous firing of two or more neurons. A well-characterized phenomenon involving NMDA receptors is the induction of LTP. LTP refers to a prolonged (hours to days) increase in the size of a postsynaptic response to a presynaptic stimulus of given strength. NMDA receptors also can induce long-term depression (LTD; the converse of LTP) at CNS synapses. The frequency and pattern of synaptic stimulation may dictate whether a synapse undergoes LTP or LTD (Nestler et al., 2009). It is believed that NMDA-dependent LTP and LTD reflect the insertion and internalization of AMPA receptors, possibly mediated by CaM-kinase II and calcineurin/protein phosphatase 1, respectively (Figure 14–12).

Glutamate Excitotoxicity. High concentrations of glutamate lead to neuronal cell death by mechanisms that have only recently begun to be clarified (Figure 14–13). The cascade of events leading to neuronal death is thought to be triggered by excessive activation of NMDA or AMPA/kinase receptors, allowing significant influx of Ca²⁺ into neurons. Glutamate-mediated excitotoxicity may underlie the damage that occurs after ischemia or hypoglycemia in the brain, during which a massive release and impaired reuptake of glutamate in the synapse leads to excess stimulation of glutamate receptors and subsequent cell death. NMDA receptor antagonists can attenuate neuronal cell death induced by activation of these receptors (Haeberlein and Lipton, 2009). Because of their widespread distribution in the CNS, glutamate receptors have become targets for diverse therapeutic interventions. For example, a role for disordered glutamatergic transmission in the etiology of chronic neurodegenerative diseases and in schizophrenia has been postulated (Chapters 16 and 22).

Acetylcholine. Based on a non-homogeneous distribution within the CNS and the observation that peripheral cholinergic drugs could produce marked behavioral effects after central administration, many investigators addressed the possibility that ACh might also be a central neurotransmitter. In the late 1950s, Eccles and colleagues identified ACh as a neurotransmitter for the excitation of spinal cord Renshaw interneurons by the recurrent axon collaterals of spinal motoneurons. Subsequently, the capacity of ACh to elicit neuronal discharge has been replicated on scores of CNS cells (Shepherd, 2003). Eight major clusters of ACh neurons and their pathways have been characterized (Cooper et al., 2003; Nestler et al., 2009; Shepherd, 2003). For additional details, see Chapter 9.

In most regions of the CNS, the effects of ACh result from interaction with a mixture of nicotinic and muscarinic receptors. Nicotinic ACh receptors are found in autonomic ganglia, the adrenal gland, and in the CNS. Activation by ACh results in a rapid increase in the influx of Na⁺ and Ca²⁺ and subsequent depolarization. Nicotinic cholinergic receptors appear to desensitize rapidly. The receptors are composed by five heterologous subunits arranged around a central pore (see Figure 14–4). A total of 17 subunits, including 10 α, 4 β, as well as δ, ∊, and γ subunits, have been identified. The nicotinic receptor at the neuromuscular junction has the composition α₂β∊δ. However, some α subunits, including α7, α8, and α9, can form functional homo-oligomers. For additional information on neuronal nicotinic ACh receptors, see Chapter 11.

There are five subtypes of muscarinic receptors, all of which are expressed in the brain. M₁, M₃, and M₅ couple to G_q while the M₂ and M₄ receptors couple to G_i (Table 14–4). Several presumptive cholinergic pathways have been proposed in addition to that of the motoneuron-Renshaw cell.

Catecholamines. The brain contains separate neuronal systems that utilize three different catecholamines—dopamine, norepinephrine, and epinephrine. Each system is anatomically distinct and serves separate, functional roles within its field of innervation (Nestler et al., 2009)

Dopamine. Although DA was originally regarded only as a precursor of NE, assays of distinct regions of the CNS revealed that the distributions of DA and NE are markedly different. In fact, more than half the CNS content of catecholamine is DA and extremely large amounts of DA are found in the basal ganglia. There are three major DA-containing pathways in the CNS (Figure 14–14):

• the nigrostriatal pathway

• the mesocortical pathway, where neurons in the ventral tegmental nucleus project to a variety of midbrain structures and to the frontal cortex

• the tuberoinfundibular pathway, which delivers DA to cells in the anterior pituitary

Initial pharmacological studies discriminated between two subtypes of DA receptors: D₁ (which couples to G_S to stimulate adenylyl cyclase) and D₂ (which couples to G_i to inhibit adenylyl cyclase). Subsequent studies identified three additional genes encoding subtypes of DA receptors: the D₅ receptor, which is related to the D₁ receptor; and the D₃ and D₄ receptors, which are part of what is called the D2 receptor family. There are also two isoforms of the D₂ receptor that differ in the predicted length of their third intracellular loops (Nestler et al., 2009). The D₁ and D₅ receptors activate the G_s-adenylyl cyclase-cyclic AMP-PKA system. The D₂ receptors couple to multiple effector systems, including inhibition of adenylyl cyclase activity, suppression of Ca²⁺ currents, and activation of K⁺ currents. The effector systems to which the D₃ and D₄ receptors couple have not been unequivocally defined (Greengard, 2001) (Table 14–5). DA-containing pathways and receptors have been implicated in the pathophysiology of schizophrenia and Parkinson disease and in the side effects seen following pharmacotherapy of these disorders (Chapters 16 and 22).

Norepinephrine. There are relatively large amounts of NE within the hypothalamus and in certain parts of the limbic system, such as the central nucleus of the amygdala and the dentate gyrus of the hippocampus. However, this catecholamine also is present in significant, although lower, amounts in most brain regions. Detailed mapping studies indicate that noradrenergic neurons of the locus ceruleus innervate specific target cells in a large number of cortical, subcortical, and spinomedullary fields (Nestler et al., 2009). NE has been established as the transmitter at synapses between presumptive noradrenergic pathways and a wide variety of target neurons. For example, stimulation of the locus ceruleus depresses the spontaneous activity of target neurons in the cerebellum; this is associated with a slowly developing hyperpolarization and a decrease in membrane conductance (Aston-Jones et al., 2001; Nestler et al., 2009).

Multiple types and subtypes of adrenergic receptors (α₁, α₂, and β) and their subtypes have been described in the CNS; all are GPCRs (Table 14–6). As expected, β receptors couple to G_s and thence to adenylyl cyclase. α₁ Adrenergic receptors are coupled to G_q, resulting in stimulation of phospholipase C, and are associated predominantly with neurons. α₂ Adrenergic receptors are found on glial and vascular elements, as well as on neurons; they couple to G_i and thence to inhibition of adenylyl cyclase activity. α₁ Receptors on noradrenergic target neurons of the neocortex and thalamus respond to NE with prazosin-sensitive, depolarizing responses due to decreases in K⁺ conductance. However, stimulation of α₁ receptors also can augment cyclic AMP accumulation in neocortical slices in response to concurrent stimulation of the G_s pathway, possibly an example of G_q-G_S cross-talk involving Ca²⁺/calmodulin and/or PKC (Ostrom et al., 2003). α₂ Adrenergic receptors are prominent on noradrenergic neurons, where they presumably couple to G_i, inhibit adenylyl cyclase, and mediate a hyperpolarizing response due to enhancement of an inwardly rectifying K⁺ channel. As in the periphery, α₂ receptors are located presynaptically, where they function as inhibitory autoreceptors. There is also evidence for postsynaptic α₂ receptors that modulate sympathetic tone. Effects mediated through α₂ receptors on blood pressure have been reported. It is believed, e.g., that the antihypertensive effects of the α₂ selective agonist clonidine are due to stimulation of α₂ receptors in the lower brainstem.

Epinephrine. Neurons in the CNS that contain epinephrine were recognized only after the development of sensitive enzymatic assays and immunocytochemical staining techniques for phenylethanolamine-N-methyltransferase, the enzyme that converts NE into epinephrine. Epinephrine-containing neurons are found in the medullary reticular formation and make restricted connections to pontine and diencephalic nuclei, eventually coursing as far rostrally as the paraventricular nucleus of the thalamus. Their physiological properties have not been unambiguously identified.

5-Hydroxytryptamine (Serotonin). In mammals, 5-HT containing neurons are found in nine nuclei lying in or adjacent to the midline (raphe) regions of the pons and upper brainstem. Cells receiving cytochemically demonstrable 5-HT input, such as the suprachiasmatic nucleus, ventrolateral geniculate body, amygdala, and hippocampus, exhibit a uniform and dense investment of serotinergic terminals.

Molecular biological approaches have led to identification of 14 distinct mammalian 5-HT receptor subtypes (Table 14–7). These subtypes exhibit characteristic ligand-binding profiles, couple to different intracellular signaling systems, exhibit subtype-specific distributions within the CNS, and mediate distinct behavioral effects of 5-HT. Most 5-HT receptors are GPCRs coupling to a variety of G-protein α subunits. The 5-HT₃ receptor, however, is a ligand-gated ion channel with structural similarity to the α-subunit of the nicotinic acetylcholine receptor. As seen with subtypes of glutamate receptors, mRNA editing has also been observed for the 5-HT_2C receptor (Niswender et al., 2001); the resulting isoforms differ in agonist affinity and distribution in the brain.

The family of 5-HT₁ receptors is composed of at least five receptor subtypes (Table 14–7) that are linked to inhibition of adenylyl cyclase activity or to regulation of K⁺ or Ca²⁺ channels. 5-HT_1A Receptors are abundantly expressed on 5-HT neurons in the dorsal raphe nucleus, where they are thought to be involved in temperature regulation. They also are found in regions of the CNS associated with mood and anxiety such as the hippocampus and amygdala. Activation of 5-HT_1A receptors opens an inwardly rectifying K⁺ conductance, which leads to hyperpolarization and neuronal inhibition. These receptors can be activated by drugs including buspirone, which is used for the treatment of anxiety and also used off-label for panic disorders. In contrast, 5-HT_1D receptors are activated by low concentrations ofsumatriptan, which is currently prescribed for acute management of migraine headaches (Chapters 13 and 34). The 5-HT₂ receptor class has three subtypes: 5-HT_2A, 5-HT_2B, and 5-HT_2C; these receptors couple to pertussis toxin-insensitive G proteins (e.g., G_q and G₁₁) and link to activation of PLC. Based on ligand binding and mRNA in situ hybridization patterns, 5-HT_2A receptors are enriched in forebrain regions such as the neocortex and olfactory tubercle, as well as in several nuclei arising from the brainstem. The 5-HT_2C receptor, which is very similar in sequence and pharmacology to the 5-HT_2A receptor, is expressed abundantly in the choroid plexus, where it may modulate cerebrospinal fluid production. Many of the effects of antidepressants are thought to be a consequence of increased stimulation of 5-HT receptors following inhibition of 5-HT reuptake by SERT. On the other hand, inhibition of 5-HT_2c receptors may account for the increased weight gain associated with neuroleptics used to treat schizophrenia.

5-HT₃ receptors function as ligand-gated ion channels; these receptors were first recognized in the peripheral autonomic nervous system. Within the CNS, they are expressed in the area postrema and solitary tract nucleus, where they couple to potent depolarizing responses that show rapid desensitization to continued 5-HT exposure. Actions of 5-HT at central 5-HT₃ receptors can lead to emesis and anti-nociceptive actions, and 5-HT₃ antagonists are beneficial in the management of chemotherapy-induced emesis (Chapter 46).

Within the CNS, 5-HT₄ receptors occur on neurons within the inferior and superior colliculi and in the hippocampus. Activation of 5-HT₄ receptors stimulates the G_s-adenylyl cyclase-cyclic AMP pathway. Other 5-HT receptors are less well studied in the CNS. The 5-HT₆ and 5-HT₇ receptors also couple to G_s; their affinity for clozapine may contribute to its antipsychotic efficacy (Chapter 16).

The hallucinogen lysergic acid diethylamide (LSD) is a potent partial agonist at 5-HT₂ receptors. When applied iontophoretically, LSD inhibits the firing of raphe (5-HT) neurons. The inhibitory effect of LSD on raphe neurons offers a plausible explanation for its hallucinogenic effects, namely that these effects result from depression of activity in a system that tonically inhibits visual and other sensory inputs. However, typical LSD-induced behaviors are seen in animals with destroyed raphe nuclei or after blockade of the synthesis of 5-HT by p-chlorophenylalanine (Aghajanian and Marek, 1999); Nichols, 2004).

Histamine. Histamine and antihistamines have long been known to produce significant effects on animal behavior. Biochemical detection of histamine synthesis by neurons and direct cytochemical localization of these neurons have defined a histaminergic system in the CNS. Most of these neurons are located in the ventral posterior hypothalamus; they give rise to long ascending and descending tracts that are typical of the patterns characteristic of other aminergic systems. Based on the presumptive central effects of histamine antagonists, the histaminergic system is thought to affect arousal, body temperature, and vascular dynamics. Four subtypes of histamine receptors have been described; all are GPCRs (Figure 14–15). H₁ receptors, the most prominent, are located on glia and vessels as well as on neurons and act to mobilize Ca²⁺ in receptive cells through the G_q-PLC pathway.

H₂ receptors couple via G_S to the activation of adenylyl cyclase. H₃ receptors, which have the greatest sensitivity to histamine, are localized primarily in basal ganglia and olfactory regions in rat brain and act through G_i to inhibit adenylyl cyclase. Consequences of H₃ receptor activation remain unresolved but may include reduced Ca²⁺ influx and feedback inhibition of transmitter synthesis and release (Chapter 32). H₄ receptors are expressed on cells of hematopoietic origin: eosinophils, T cells, mast cells, basophils, and dendritic cells. H₄ receptors appear to couple to G_i and G_q and are postulated to play a role in inflammation and chemotaxis (Thurmond et al., 2004). Unlike the monoamines and amino acid transmitters, there does not appear to be an active process for reuptake of histamine after its release. Inhibition of H₁ receptors causes drowsiness, an effect that limits the use of H₁ antagonists to treat allergic reactions. The development of H₁ antagonists with low CNS penetration has reduced the incidence of these side effects.

Peptides. The discovery during the 1980s of numerous novel peptides in the CNS, each capable of regulating neuronal function, produced considerable excitement and an imposing catalog of substances as well as potential medications based upon interaction with peptide receptors (Darlison and Richter, 1999; Hökfelt et al., 2003). In addition, certain peptides previously thought to be restricted to the GI tract or to endocrine glands have been found in the CNS. Most of these peptides bind to GPCRs. Many of the effects are modulatory rather than causing direct excitation or inhibition. Myriad peptide neurotransmitters or neuromodulators have been described (Table 14–8). Relatively detailed neuronal maps are available that identify immunoreactivity to peptide-specific antisera. While some CNS peptides may function on their own, most are now thought to act primarily in concert with co-existing transmitters, both amines and amino acids.

Neuropeptides are processed and stored in large, dense-core vesicles (LDCVs; see Figure 14–7). The peptides may be co-localized and released together with small molecule transmitters, such as a biogenic amine. Multiple peptides may be co-localized within the same neuron. As noted, some neurons may contain two or more transmitters, including peptides, and their release can be independently regulated.

In contrast to the biogenic amines or amino acids, peptide synthesis requires transcription of DNA into mRNA and translation of mRNA into protein. This takes place primarily in perikaria and the resulting peptide is then transported to nerve terminals. Single genes can, through the post-translational action of peptidases, give rise to multiple neuropeptides. For example, proteolytic processing of proopiomelanocortin (POMC) gives rise to, among other things, ACTH, α and γ MSH, β-MSH, and β-endorphin (Figure 14–16). In addition, alternative splicing of RNA transcripts may result in distinct mRNA species. For example, calcitonin and calcitonin gene-related peptide (CGRP) are derived in specific tissues from the same primary transcript.

Organization by Function. Since most peptides were identified initially on the basis of bioassays, their names reflect these biologically assayed functions (e.g., thyrotropin-releasing hormone and vasoactive intestinal polypeptide). These names become trivial when more ubiquitous distributions and additional functions are discovered. Some general integrative role might be hypothesized for widely separated neurons (and other cells) that make the same peptide. However, a more parsimonious view is that each peptide has unique messenger roles at the cellular level that are used repeatedly in biologically similar pathways within functionally distinct systems.

Most neuropeptide receptors are GPCRs. In comparison to GPCRs for smaller ligands such as biogenic amines and amino acids, the extracellular domains of neuropeptide receptors play a larger role in ligand binding. As seen with other transmitter systems, there are often multiple subtypes of receptor for the same peptide transmitter (Table 14–9). For example, there are five subtypes of receptor for somatostatin and all inhibit adenylyl cyclase through an interaction with G_i; they differ in their interaction with various somatostatin analogs. The cloning of the major members of the opioid-peptide receptors has revealed unexpected, and as yet unexplained, homologies with receptors for somatostatin, angiotensin, and other peptides. Multiple melanocortin receptors exist that respond to various peptides derived from POMC. Not surprisingly, the receptor on adrenal cortical cells responds to ACTH, while that on melanocytes responds to α-MSH. Further evidence for complexity comes from the realization that agonists at subtypes of melanocortin receptors are associated with a variety of biological effects including skin darkening (MCR1), decreased appetite (MCR3 and/or MCR4), and sexual arousal (MCR4).

Although most peptide receptors are GPCRs, exceptions do exist. The amiloride-sensitive FMRF amide (phe-met-arg-phe-amide) receptor is a peptide-gated ion channel. The large number of neuropeptides and peptide receptors has lead to increased interest in identifying therapeutic agents. Examples include experimental substance P antagonists as antidepressants or to treat anxiety, and antagonists of CRH for stress related disorders.

Comparison with Other Transmitters. Peptides differ in several important respects from the monoamine and amino acid transmitters. Peptide synthesis takes place in the rough endoplasmic reticulum. The propeptide is cleaved (processed) to the secreted form as secretory vesicles are transported from the perinuclear cytoplasm to the nerve terminal. Active mechanisms for the local synthesis of peptides have not been described; thus, peptidergic nerve terminals depend on distant sites of synthesis. As for structure-activity relationships for peptide transmitters, linear chains of amino acids can assume many conformations at their receptors, making it difficult to define the sequences and their steric relationships that are critical for activity. Until recently, it was difficult to develop non-peptidic synthetic agonists or antagonists that interact with specific peptide receptors. Such agents now are being developed for many neuropeptides (Hökfelt et al., 2003). Natural products have not been good sources of drugs that affect peptidergic transmission. Only one plant alkaloid, morphine, has been found to act selectively at peptidergic synapses. Fortunately for pharmacologists, morphine was discovered before the endorphins, or rigid molecules capable of acting at peptide receptors might have been deemed impossible to develop.

Other Regulatory Substances.

Cannnabinoids. Delta-9-tetrahydrocannabinol (THC) is one of several active substances in marijuana (Figure 14–17). It has dramatic short-term effects, including causing feelings of euphoria and altered sensory perception. After chronic or long-term use, withdrawal symptoms include irritability and sleep disturbances. The primary pharmacologic effects of THC follow its interaction with CB₁ receptors in the CNS and CB₂ receptors in the periphery. CB₁ receptors are found primarily in the basal ganglia, hippocampus, cerebellum, and cerebral cortex. They are also expressed in some non-neuronal cells and tissues, including leukocytes and testis. CB₂ receptors are expressed in the spleen, tonsils, bone marrow, and on peripheral blood leukocytes. The natural endogenous ligands for these receptors are arachidonic acid derivatives including anandamide and 2-arachidonyl glycerol (Figure 14–17). Both CB₁ and CB₂ receptors are linked to G_i and inhibition of adenylyl cyclase activity. Activation of CB₁ receptors results in inhibition of glutamate release. Efforts to develop CB₁ antagonists like rimonabant have focused on possible treatments for drug addiction and obesity. Efforts are also underway to develop agonists that interact with CB₁ and CB₂ receptors for the relief of pain. THC (dronabinol, MARINOL) is sometimes used in the control of nausea and moderate pain (Chapter 46).

Purines. Adenosine, ATP, UDP, and UTP have roles as extracellular signaling molecules (Robertson et al., 2001; Siegel et al., 2006). High concentrations of ATP are found in adrenergic storage vesicles and ATP is released along with catecholamines and the other contents of adrenergic storage granules. Intracellular nucleotides may also reach the cell surface by other means (Lazarowski et al., 2003), including as a consequence of cellular hypoxia or cell death, and extracellular adenosine can result from cellular release and metabolism of ATP (Jackson and Raghvendra, 2004). The concentration of ATP may greatly exceed the concentration of adenosine and, given the presence of nucleotidases, it can be difficult to unambiguously distinguish between effects of, e.g., adenosine and ATP.

Extracellular nucleotides and adenosine act on a family of purinergic receptors that is divided into two classes, P1 and P2 (Table 14–10). P1 receptors are GPCRs that interact with adenosine; two of these receptors (A₁ and A₃) couple to G_i and two (A_2a and A_2b) couple to G_s; methylxanthines antagonize A₁ and A₃ receptors. Activation of A₁ receptors is associated with inhibition of adenylyl cyclase, activation of K⁺ currents, and in some instances, with activation of PLC; stimulation of A₂ receptors activates adenylyl cyclase. The P2 class includes a large number of P2X receptors that are ligand-gated ion channels and the P2Y receptors, a similarly large subclass of GPCRs that couple to G_q or G_i and their associated effectors. The P2Y₁₄ receptor is expressed in the CNS; it interacts with UDP-glucose and may couple to G_q. The P2Y₁₂ receptor is important clinically, since inhibition of this receptor in platelets inhibits platelet aggregation.

Although many of these receptors have been detected in brain, most of the current interest stems from pharmacological rather than physiological observations. Adenosine can act presynaptically throughout the cortex and hippocampal formation to inhibit the release of amine and amino acid transmitters. ATP-regulated responses have been linked pharmacologically to a variety of pathophysiological functions, including anxiety, stroke, and epilepsy. A₁ antagonists are being investigated as potential therapeutic agents to enhance awareness and learning. A₂ receptors and dopamine D₂ receptors appear to be functionally antagonistic, leading to investigation of A_2a antagonists as adjunctive therapy for Parkinson disease (Jacobson and Gao, 2006).

Lipid Mediators. Substances identified as physiological regulators in systems throughout the body have been examined for their roles within the CNS. Arachidonic acid, normally stored within the cell membrane as a glycerol ester, can be liberated during phospholipid hydrolysis (by pathways involving phospholipases A₂, C, and D). Phospholipases are activated via a variety of receptors (Chapter 3). Arachidonic acid can be converted to highly reactive regulators by three major enzymatic pathways (Chapter 33): cyclooxygenases (leading to prostaglandins and thromboxane), lipoxygenases (leading to the leukotrienes and other transient catabolites of eicosatetraenoic acid), and CYPs (which are inducible and also expressed at low levels in brain). Arachidonic acid metabolites have been implicated as diffusible modulators in the CNS, possibly involved with the formation of LTP and other forms of neuronal plasticity.

Nitric Oxide and Carbon Monoxide. Nitric Oxide (NO), an important regulator of vascular and inflammatory mediation, came into focus with respect to roles in the CNS after the characterization of brain nitric oxide synthase (NOS) activities (see Chapter 3 and Boehning and Snyder, 2003). Both constitutive and inducible forms of NOS are expressed in the brain. The application of inhibitors of NOS (e.g., methylarginine and nitroarginine) and of NO donors (such as nitroprusside) suggests the involvement of NO in a host of CNS phenomena, including neurotransmitter release and enhancement of glutamate (NMDA)-mediated neurotoxicity and LTP. Neuronal NOS (nNOS) is activated following NMDA-mediated receptor mediated increases in intracellular Ca²⁺. NO diffuses freely across membranes, acting primarily to stimulate soluble guanylyl cyclase, which catalyzes the formation of cyclic GMP. Formation of NO can also result in S-nitrosylation of cysteine residues in a variety of proteins including G proteins and ion channels. Other gases may also act as intracellular messengers. One candidate is carbon monoxide (CO), which is generated in neurons by three isoforms of hemeoxygenase (HO), isoform 2 predominating in neurons. Like NO, CO stimulates soluble guanylyl cyclase.

Cytokines. Cytokines are a large and diverse family of polypeptide regulators that are produced widely throughout the body by cells of diverse embryological origin. The effects of cytokines are regulated by the conditions imposed by other cytokines, interacting as a network with variable effects leading to synergistic, additive, or opposing actions. Tissue-produced peptides, termed chemokines, serve to attract immune and inflammatory cells into interstitial spaces. These special cytokines have received attention as potential regulators of nervous system inflammation (as in early stages of dementia, following infection with human immunodeficiency virus, and during recovery from traumatic injury). Some of the more conventional neuronal- and glial-derived growth-enhancing and growth-retarding factors have been identified earlier. The fact that neurons and astrocytes may be induced under some pathophysiological conditions to express cytokines or other growth factors further blurs the dividing line between neurons and glia (Campbell, 2004; Wang et al., 2002).

Specificity and Nonspecificity of CNS Drug Actions

The effect of a drug in the CNS is considered to be specific when it affects an identifiable molecular mechanism unique to target cells that bear receptors for that drug. Conversely, a drug is regarded as being nonspecific when it produces effects at a variety of different target cells, thus affecting a diverse set of neurobiological systems. This distinction is often affected by the dose-response relationship of the drug and the cell or mechanisms under scrutiny. Even a drug that is highly specific when tested at low concentrations may exhibit nonspecific actions at higher doses. In general, the more potent a drug at its desired target, the lower the probability that it will have off-target effects. Conversely, even drugs that have a broad spectrum of activity may not act identically at all levels of the CNS. For example, sedatives, hypnotics, and general anesthetics would have very limited utility if central neurons that control the respiratory and cardiovascular systems were especially sensitive to their actions. Although relief of pain is the goal when administering an opiate, one must also contend with potential off-target effects including respiratory depression and constipation.

General (Nonspecific) CNS Depressants

This category includes the anesthetic gases and vapors, the aliphatic alcohols, and some hypnotic-sedative drugs. These agents share the capacity to depress excitable tissue at all levels of the CNS, leading to a decrease in the amount of transmitter released by the nerve impulse, as well as to general depression of postsynaptic responsiveness and ion flux. At sub-anesthetic concentrations, these agents (e.g., ethanol) can exert relatively specific effects on certain groups of neurons, which may account for differences in their behavioral effects, especially the propensity to produce dependence.

General (Nonspecific) CNS Stimulants

The drugs in this category include pentylenetetrazol and related agents that are capable of powerful excitation of the CNS, and the methylxanthines, which have a much weaker stimulant action. Stimulation may be accomplished by one of two general mechanisms: (1) blockade of inhibition or (2) direct neuronal excitation that may involve increased transmitter release or more prolonged transmitter action, as occurs when the reuptake of a released transmitter is inhibited.

Drugs That Selectively Modify CNS Function. The agents in this group may cause either depression or excitation. In some instances, a drug may produce both effects simultaneously on different systems. Some agents in this category have little effect on the level of excitability in doses that are used therapeutically. The principal classes of these CNS drugs are anticonvulsants, drugs used in treating Parkinson disease, opioid and non-opioid analgesics, appetite suppressants, anti-emetics, analgesic-antipyretics, certain stimulants, antidepressants, anti-manic and anti-psychotic agents, tranquilizers, sedatives and hypnotics, and medications employed in the treatment of Alzheimer's disease (cholinesterase inhibitors and anti-glutamate neuroprotectants). Although selectivity of action may be remarkable, a drug usually affects several CNS functions to varying degrees. When only one constellation of effects is wanted in a therapeutic situation, the remaining effects of the drug are regarded as limitations in selectivity (i.e., unwanted side effects or off-target effects). The specificity of a drug's action is frequently overestimated. This is partly due to the fact that drugs are often identified with the effect that is implied by the class name.

General Characteristics of CNS Drugs

Combinations of centrally acting drugs frequently are administered to therapeutic advantage (e.g., an anticholinergic drug and levodopa for Parkinson disease). However, other combinations of drugs may be detrimental because of potentially dangerous additive or mutually antagonistic effects. The effects of a CNS drug may be additive with the physiological state and the effects of other depressant and stimulant drugs. For example, anesthetics are less effective in a hyperexcitable subject than in a normal patient; the converse is true for stimulants. In general, the depressant effects of drugs from different categories are additive (e.g., the potentially fatal combination of barbiturates or benzodiazepines with ethanol), as are the effects of stimulants. Therefore, respiration depressed by morphine is further impaired by depressant drugs, while stimulant drugs can augment the excitatory effects of morphine to produce vomiting and convulsions.

Antagonism between depressants and stimulants is variable. Some instances of true pharmacological antagonism among CNS drugs are known; for example, opioid antagonists can selectively antagonize the effects of opioid analgesics. However, the antagonism exhibited between two CNS drugs is most often physiological in nature. For example, an individual whose CNS is depressed by an opiate cannot be returned entirely to normal by stimulation with caffeine.

The selective effects of drugs on specific neurotransmitter systems may be additive or competitive. The potential for drug interactions must be considered whenever such drugs are concurrently administered. To minimize such interactions, a drug-free period may be required when modifying therapy; in fact, development of desensitized and supersensitive states with prolonged therapy may limit the speed with which one drug may be halted and another started. An excitatory effect is commonly observed with low concentrations of certain depressant drugs due either to depression of inhibitory systems or to a transient increase in the release of excitatory transmitters. Examples include the stage of excitement seen during induction of general anesthesia. The excitatory phase typically occurs with low concentrations of the depressant; uniform depression ensues with increasing drug concentration. The excitatory effects can be minimized, when appropriate, by pretreatment with a depressant drug that is devoid of such effects (e.g., benzodiazepines in preanesthetic medication). Acute, excessive stimulation of the cerebrospinal axis normally is followed by depression, which is in part a consequence of neuronal fatigue and exhaustion of stores of transmitters. Postictal depression is additive with the effects of depressant drugs. Acute, drug-induced depression generally is not followed by stimulation. However, chronic drug-induced sedation or depression may be followed by prolonged hyperexcitability upon abrupt withdrawal of the medication (barbiturates or alcohol). This type of hyperexcitability can be controlled effectively by the same or another depressant drug (Chapters 17, 23, 24).

Organization of CNS-Drug Interactions. The structural and functional properties of neurons provide a means to specify the possible sites at which drugs could interact, specifically or generally, in the CNS. In this scheme, drugs that affect neuronal energy metabolism, membrane integrity, or transmembrane ionic equilibria would be generally acting compounds. Similarly general in action would be drugs that affect molecular motors and thereby affect the transport of materials from cell bodies to nerve terminals and back. These general effects may exhibit different dose-response or time-response relationships based, e.g., on neuronal properties such as rate of firing, dependence of discharge on external stimuli or internal pacemakers, resting ionic fluxes, or axon length. In contrast, when the actions of a drug can be related to specific aspects of the metabolism, release, or function of a neurotransmitter, then the site, specificity, and mechanism of action of the drug can be defined by systematic studies of dose-response and time-response relationships.

Transmitter-dependent actions of drugs can be grouped into presynaptic and postsynaptic categories. The presynaptic category includes the events in the perikaryon and nerve terminal that regulate transmitter synthesis (including the acquisition of adequate substrates and co-factors), storage, release, and metabolism. Transmitter concentrations can be lowered by blockade of synthesis, inhibition of storage, or both. The amount of transmitter released per impulse generally is stable but may be subject to regulation. The effective concentration of transmitter may be increased by inhibition of metabolic enzymes or by blockade of re-uptake transporters. The transmitter that is released at a synapse also can exert actions on the terminal from which it was released by interacting with receptors at these sites (autoreceptors). Activation of presynaptic autoreceptors can inhibit or stimulate the rate of release of transmitter and thereby provide a feedback mechanism that controls the concentration of transmitter in the synaptic cleft.

The postsynaptic category includes all the events that follow release of the transmitter in the vicinity of the postsynaptic receptor. Examples include the molecular mechanisms by which receptor occupancy alters the properties of the membrane of the postsynaptic cell (shifts in membrane potential), as well as more enduring biochemical actions (e.g., changes in second messenger concentrations, protein kinase and phosphoprotein phosphatase activities, and phosphoprotein formation). Direct postsynaptic effects of drugs generally require relatively high affinity for the receptors or resistance to metabolic degradation. Each of these presynaptic or postsynaptic actions is potentially highly specific and can be envisioned as restricted to a single, chemically defined subset of CNS cells.

Convergence, Synergism, and Antagonism Result from Transmitter Interactions. Although the power of the reductionist approach to clone cDNAs for receptors or receptor subunits and to determine their properties by expression in cells that do not normally express the receptor or subunit cannot be underestimated, the simplicity of cell culture models may not reproduce the nuances of receptor function in vivo and may divert attention from the complexity of the intact CNS. A given neurotransmitter may interact simultaneously with all of the various isoforms of its receptor on neurons that also are under the influence of multiple other afferent pathways and their transmitters. Thus, the use of model systems to predict the behavioral or therapeutic consequences of drugs in humans may fail as a consequence of the complexity of the interactions possible, including differences between normal and diseased tissue.

CNS Drug Discovery. As is evident from the chapters to follow, a large number of agents have been developed to treat neuropsychiatric diseases. With few exceptions these agents offer primarily symptomatic improvement; few are truly disease modifying. For example, the use of L-dopa to treat Parkinson disease alleviates the symptoms effectively but the disease continues to progress. Similarly, although antipsychotics and antidepressants are often efficacious, the symptoms tend to recur. Moreover, many drugs developed to treat CNS diseases are not uniformly effective: Approximately one-third of patients with severe depression are "treatment-resistant." CNS diseases are complex, with multiple symptoms, some of which, like the negative symptoms of schizophrenia, tend to be resistant to treatment. Furthermore, the complexity of the brain and its neuronal pathways results in significant risk of side effects, even when the most biochemically selective agent is administered. Particularly disquieting side effects include tardive dyskinesias that can result from prolonged treatment with antipsychotics and the hypnotic effects of agents used for the treatment of seizure disorders.

The lack of disease-modifying treatments for CNS diseases represents a very significant unmet medical need and makes the discovery of such agents a high priority. Drug discovery is generally a high-risk/high-reward effort (Chapter 1) and CNS-active agents are no exception. Overall, the probability of success in developing a drug from the time a compound enters into clinical trials is ~10%; the success rate for CNS drugs is somewhat lower. Myriad factors contribute to the increased difficulty and reduced probability of success in efforts to develop drugs to treat CNS disease. Factors that reduce the probability of success include the complexity of neural pathways governing behavior and its pathology, and the permeability barriers that restrict access of drugs to CNS sites (including the blood-brain barrier and drug export systems; see Chapters 2 and 5). For example, a drug that affects serotinergic transmission may affect 14 5-HT receptor subtypes that are involved in a plethora of biological systems. Moreover, animal models of CNS diseases are often incompletely validated and an effect in an animal model may not be predictive of efficacy in human disease. For instance, a number of agents reduce the size of the infarct when given to animals following occlusion of the middle cerebral artery, yet none of these agents has shown positive results in human clinical trials. A number of agents that inhibit the reuptake of 5-HT and NE are effective in treating depression. In a number of animal models of depression, these agents produce an effect after a single dose, which corresponds nicely with the time course for the inhibition of neurotransmitter reuptake in experimental animals, whereas, a period of several weeks is typically required to see a therapeutic effect in humans. Given this discrepancy, one can understand the reluctance of pharmaceutical companies to invest in clinical trials of new antidepressants on the basis of effects in animal models.

Clinical trials represent another area of challenge in developing new therapies for CNS diseases. For example, testing an agent to treat depression likely requires a trial lasting 6 or more weeks and the placebo response rate may exceed 50%. Such conditions necessitate large, prolonged, and therefore expensive trials. Studies of treatments for neurodegenerative disease are even more difficult. With current diagnostic capabilities, it is difficult to detect a significant change in the rate of progression of cognitive decline in patients with Alzheimer's disease in less than a year. One way to circumvent the long time necessary to detect a biological meaningful result is through the use of surrogate markers (e.g., a decrease in serum cholesterol for improved cardiovascular morbidity and mortality). Regrettably, there are relatively few useful surrogate markers for CNS diseases.

Impact of Genomics on CNS Drug Discovery. The sequencing of the human genome has the potential to significantly change drug discovery in the CNS. Thus, genetic testing may predict the likelihood that a given individual will develop a particular disease, will respond to a particular therapy, or will suffer side effects from a particular treatment paradigm. Genetic testing may be particularly important in the case of CNS diseases, where the etiology is likely to be multigenic. Molecular approaches will likely speed development of more and improved animal models that better mimic human disease.