Veterinary Psychopharmacology

Contributors

Sharon L. Crowell‐Davis, DVM, PhD, DACVB

Professor of Behavioral Medicine

Department of Veterinary Biosciences and Diagnostic Imaging

College of Veterinary Medicine

University of Georgia

Athens, GA, USA

Leticia Mattos de Souza Dantas DVM, MS, PhD, DACVB

Clinical Assistant Professor of Behavioral Medicine

University of Georgia Veterinary Teaching Hospital

Department of Veterinary Biosciences and Diagnostic Imaging

College of Veterinary Medicine

University of Georgia,

Athens, GA, USA

Mami Irimajiri BVSc, PhD, DACVB

Synergy General Animal Hospital

Animal Behavior Service

Saitama, Japan

Adjunct Professor

Kitasato University

College of Veterinary Medicine

Towada, Aomori, Japan

Thomas F. Murray, PhD

Provost,

Creighton University

Department of Pharmacology

Omaha, NE, USA

Niwako Ogata BVSc, PhD, DACVB

Associate Professor of Veterinary Behavior Medicine Purdue University

College of Veterinary Medicine

West Lafayette, IN, USA

Lynne Seibert DVM, MS, PhD, DACVB

Veterinary Behavior Consultants

Roswell,

GA, USA

Preface

The first edition of this book grew out of a series of phone calls that Dr. Crowell‐Davis received over the years from various veterinarians wanting information about their patients’ behavior problems and the psychoactive medications that might help them. What were appropriate drugs for given problems? What were appropriate doses? What side effects should be watched for? The first answer to this steadily accumulating set of questions was a continuing education course in psychopharmacology specifically organized for veterinarians. The course was first presented at the University of Georgia in November of 2001 and is now part of UGA’s Outpatient Medicine annual Continuing Education, as Behavioral Medicine has become integrated with all other specialties of our teaching hospital. From the original courses, taught by Dr. Murray and Dr. Crowell‐Davis and the assistance from the clinical residents at the time (Dr. Lynne Seibert and Dr. Terry Curtis), the next logical step was a textbook so that practicing veterinarians would have a resource to turn to for the answers to their various questions. Years later, Dr. Crowell‐Davis and Dr. Dantas felt an urgent need to update the book and add several new drugs that more recently are used by diplomates of the American College of Veterinary Behaviorists, so this knowledge could be available to general practitioners. Where studies were available, we tried to make this edition purely evidence‐based and avoided including personal communications and short publications as much as possible. As this edition goes to print, we are already planning for the third as new information and protocols in veterinary mental health care keep being tested and developed.

Information on the effects of various psychoactive drugs in dogs, cats, and other veterinary patients comes from two major sources. First, animals were often used to test and study the actions of various drugs during their initial development. Thus, the reader who peruses the references will find papers published as early as the 1950s, when major breakthroughs in psychopharmacology were being made to much newer publications in human and veterinary neuroscience. With the establishment of the American College of Veterinary Behaviorists in 1993 and the overall rapid development of the field of Clinical Behavioral Medicine, there has been increasing research on the efficacy of various medications on the treatment of various mental health and behavioral/psychiatry disorders of companion animals, zoo animals, and other nonhuman animals.

There are often huge gaps in our knowledge, and the reader may note them throughout the book. While we can glean bits and pieces of pharmacokinetic and other data from studies done on dogs and cats during early drug development, the quality and quantity of the information are highly variable. Studies of teratology and carcinogenicity are typically done on rats, mice, and rabbits, while comprehensive studies of all aspects of pharmacological activity in the body are done only in humans, the species that has historically been of interest. It is hoped that, as interest in this field continues to evolve, more comprehensive data will become available; new data will be supplied in future editions.

Sharon L. Crowell‐Davis, DVM, PhD, DACVB

Professor of Behavioral Medicine

Department of Veterinary Biosciences and Diagnostic Imaging

College of Veterinary Medicine

University of Georgia

Athens, GA, USA

Leticia Mattos de Souza Dantas, DVM, MS, PhD, DACVB

Clinical Assistant Professor of Behavioral Medicine

University of Georgia Veterinary Teaching Hospital

Department of Veterinary Biosciences and Diagnostic Imaging

College of Veterinary Medicine

University of Georgia

Athens, GA, USA

Acknowledgments

There is so much to be thankful for on this second edition of Veterinary Psychopharmacology. From all the veterinarians who request consults and always ask questions about psychoactive medications; reminding us of how important this resource is, to all of the students who push us to be updated, creative and enthusiastic about practicing and teaching. We dream of a time where mental health and psychiatry care will be fully integrated into the standard of care in veterinary medicine across the globe and part of the curriculum of every veterinary medicine school. To all of you that are eager to learn and provide the best care for your patients, we thank you. You are leading the way in our profession and this book is for you.

We wanted to keep the acknowledgments from the first edition to the many people who, besides the authors, contributed to the work involved in bringing together the information presented at that time. Of particular assistance were Linda Tumlin, Wendy Simmons, and Lucy Rowland. In their capacity as librarians and reference librarians they were invaluable in locating and obtaining much of the information provided in our first edition.

We also could not have developed and run the Behavioral Medicine Service and the didactic program at the University of Georgia to this date without the continuing support of various administrators over the years. In the first edition, Dr. Royce Roberts, Dr. Crowell‐Davis’ department head of many years was acknowledged. On this edition, we would like to thank Dr. Stephen Holladay for all his encouragement and support to both of us. Dr. David Anderson, Dr. Keith Prasse, Dr. Bob Lewis, and Dr. Jack Munnell have also facilitated Dr. Crowell‐Davis’ continuing work in this field previously. In the past 10 years, our service has had major support from our hospital director, Dr. Gary Baxter, to whom Dr. Dantas is incredibly grateful as he has supported and allowed for the service’s revitalization, allowing for a more competitive and business‐oriented approach to her practice.

Finally, this book is for all animals who co‐exist with humankind, providing us with so much affection, companionship and even health benefits, but who have to adapt to our lifestyle and often undergo significant mental suffering that can remain ignored, undiagnosed, and untreated. Our mission is to heal and to improve the quality of life of all patients we have the privilege to treat; and increase the awareness in our society that the mental and emotional suffering of animals matters.

Part I

Principles of Veterinary Psychopharmacology

1

General Principles of Psychopharmacology

Thomas F. Murray

Creighton University, Omaha, NE, USA

Drug Action

Pharmacology is the science of drug action, and a drug is defined as any agent (chemical, hormone, peptide, antibody, etc.) that, because of its chemical properties, alters the structure and/or function of a biological system. Psychopharmacology is a sub‐discipline of pharmacology focused on the study of the use of drugs (medications) in treating mental disorders. Most drugs used in animals are relatively selective. However, selectivity of drugs is not absolute inasmuch as they may be highly selective but never completely specific. Thus, most drugs exert a multiplicity of effects.

Drug action is typically defined as the initial change in a biological system that results from interaction with a drug molecule. This change occurs at the molecular level through drug interaction with molecular target in the biologic system (e.g. tissue, organ). The molecular target for a drug typically is a macromolecular component of a cell (e.g. protein, DNA). These cellular macromolecules that serve as drug targets are often described as drug receptors, and drug binding to these receptors mediates the initial cellular response. Drug binding to receptors either enhances or inhibits a biological process or signaling system. Of relevance to the field of psychopharmacology, the largest group of receptors are proteins. These include receptors for endogenous hormones, growth factors, and neurotransmitters; metabolic enzymes or signaling pathways; transporters and pumps; and structural proteins. Usually the drug effect is measured at a much more complex level than a cellular response, such as the organism level (e.g. sedation or change in behavior).

Drugs often act at receptors for endogenous (physiologic) hormones and neurotransmitters, and these receptors have evolved to recognize their cognate signaling molecules. Drugs that mimic physiologic signaling molecules at receptors are agonists, that is, they activate these receptors. Partial agonist drugs produce less than maximal activation of activation of receptors, while a drug that binds to the receptor without the capacity to activate the receptor may function as a receptor antagonist. Antagonists that bind to the receptor at the same site as agonists are able to reduce the ability of agonists to activate the receptor. This mutually exclusive binding of agonists and antagonists at a receptor is the basis for competitive antagonism as a mechanism of drug action. One additional class of drugs acting at physiologic receptors are inverse agonists. At physiologic receptors that exhibit constitutive activity in the absence of activation by an endogenous agonist, inverse agonists stabilize an inactive conformation and therefore reduce the activation of the receptor. Thus, inverse agonists produce responses that are the inverse of the response to an agonist at a given receptor. Theoretical log concentration‐response curves for these four classes of drugs are depicted in Figure 1.1.

Figure 1.1 Theoretical logarithmic concentration‐response relationships for agonist, partial agonist, antagonist, and inverse agonist drugs acting at a common receptor. In this theoretical set of concentration‐response curves, the agonist produces a maximum response while the partial agonist is only capable of evoking a partial response. The antagonist binds to the receptor but is not capable of activating the receptor and therefore does not produce a response. Inverse agonists bind to an inactive form of the receptor and produce an effect which is in the inverse direction of that produced by the agonist.

Dose Dependence of Drug Interaction with Receptors

Receptor occupancy theory assumes that drug action is dependent on concentration (dose) and the attendant quantitative relationships are plotted as dose‐ or concentration‐response curves. Dose–response analysis is typically reserved to describe whole animal drug effects, whereas concentration‐response curves describe in vitro drug action where the actual concentration of the drug interacting with a receptor is known. Inspection of dose–response relationships reveals that for any drug, there is a threshold dose below which no effect is observed, and at the opposite end of the curve there is typically a ceiling response beyond which higher doses do not further increase the response. As shown in Figure 1.2, these dose‐ or concentration‐response curves are typically plotted as a function of the log of the drug dose or concentration. This produces an S‐shaped curve that pulls the curve away from the ordinate and allows comparison of drugs over a wide range of doses or concentrations.

Figure 1.2 Theoretical logarithmic concentration‐response relationships for three agonists which differ in relative potency. Drug A is more potent than Drug B, which in turn is more potent than Drug C.

A drug‐receptor interaction is typically reversible and governed by the affinity of the drug for the receptor. The affinity essentially describes the tightness of the binding of the drug to the receptor. The position of the theoretical S‐shaped concentration‐response curves depicted in Figure 1.2 reveals the potency of these drugs. The potency of a drug is a function of its affinity for a receptor, the number of receptors, and the fraction of receptors that must be occupied to produce a maximum response in a given tissue. In Figure 1.2, Drug A is the most potent and Drug C is least potent. The efficacy of all three drugs in Figure 1.2, however, is identical in that they all act as full agonists and produce 100% of the maximal effect. As a general principle in medicine, for drugs with similar margins of safety, we care more about efficacy than potency. The comparison of potencies of agonists is accomplished by determining the concentration (or dose) that produces 50% of the maximum response (Effective Concentration, 50% = EC50). In Figure 1.2, the EC50 values are 10−8, 10−7, and 10−6 M, respectively, for Drugs A, B and C; hence, the rank order of potency is Drug A > Drug B > Drug C, with Drug A being the most potent since its EC50 value is the lowest. Figure 1.3 depicts three additional theoretical concentration‐response curves for drugs with identical potencies but different efficacies. In this example, Drug A is a full agonist, producing a maximum response, whereas Drugs B and C are partial agonists, producing responses, respectively, of 50% and 25% of the maximum. Similar to receptor antagonist drugs, partial agonists can compete with a full agonist for binding to the receptor. Increasing concentrations of a partial agonist will inhibit the full agonist response to a level equivalent to its efficacy, whereas a competitive antagonist will completely eliminate the response of the full agonist.

Figure 1.3 Theoretical logarithmic concentration‐response relationships for three agonists with similar potency but different efficacies. Drug A is an agonist that produces a maximum response while Drugs B and C are partial agonists only capable of evoking a partial response. Drug A is therefore more efficacious than Drug B, which in turn is more efficacious than Drug C.

Structural Features of the Central Nervous System (CNS) and Neurotransmission

The cellular organization of the mammalian brain is more complex than any other biologic tissue or organ. To illustrate this complexity, consider that the human brain contains 10¹² neurons, 10¹³ glia, and 10¹⁵ synapses. Understanding how this complex information processor represents mental content and directs behavior remains a daunting biomedical mystery. Recent reconstruction of a volume of the rat neocortex found at least 55 distinct morphological types of neurons (Makram et al. 2015). The excitatory to inhibitory neuron ratio was estimated to be 87:13, with each cortical neuron innervating 255 other neurons, forming on average more than 1100 synapses per neuron. This remarkable connectivity reveals the complexity of microcircuits within even a small volume of cerebral cortex.

Most neuron‐to‐neuron communication in the CNS involves chemical neurotransmission at up to a quadrillion of synapses. The amino acid and biogenic amine neurotransmitters must be synthesized in the presynaptic terminal, taken up, and stored in synaptic vesicles, and then released by exocytosis, when an action potential invades the terminal to trigger calcium influx. Once released into the synaptic cleft, transmitters can diffuse to postsynaptic sites where they are able to bind their receptors and trigger signal transduction to alter the physiology of the postsynaptic neuron. Just as exocytotic release of neurotransmitters is the on‐switch for cell‐to‐cell communication in the CNS, the off‐switch is typically a transport pump that mediates the reuptake of the transmitter into the presynaptic terminal or uptake into glia surrounding the synapse. A schematic of a presynaptic terminal depicted in Figure 1.4 illustrates the molecular sites that regulate neurotransmission. Once synthesized or provided by reuptake, the neurotransmitter is transported into the synaptic vesicle for subsequent exocytosis. The pH gradient across the vesicular membrane is established by the vacuolar H+‐ATPase, which uses ATP hydrolysis to generate the energy required to move H+ ions into the vesicle (Lohr et al. 2017). This movement of H+ ions creates the vesicular proton gradient and establishes an acidic environment inside the vesicle (pH of ~5.5). Specific reuptake transporters are localized on the plasma membrane where they recognize transmitters and transport them from the synaptic cleft into the cytoplasm of the terminal (Torres et al. 2003). These transporters have evolved to recognize specific transmitters such as dopamine, serotonin, norepinephrine, glutamate, and gamma‐aminobutyric acid (GABA). In all cases, these presynaptic transporters regulate the extracellular concentration of transmitters and therefore a mechanism for termination of their respective synaptic actions. The monoamine transporters (dopamine, norepinephrine, and 5‐hydroxytryptamine) are the pharmacological targets for antidepressants and psychostimulants.

Figure 1.4 Presynaptic terminal of monoaminergic neuron, depicting sites of vesicular release, reuptake transport, and vesicular transport and storage. Monoamine transmitters are synthesized in the cytoplasm or vesicle. Transport from the cytoplasm to the vesicular compartment is mediated by the reserpine sensitive vesicular membrane transporter (VMAT2). Release into the synapse occurs by exocytosis triggered by an action of potential invasion of the terminal. Neurotransmitters are rapidly transported from the synaptic cleft back into the cytoplasm of neuron by a process termed reuptake, which involves a selective, high‐affinity, Na+‐dependent plasma membrane transporter.

Presynaptic terminals also express neurotransmitter autoreceptors that function as local circuit negative feedback inhibitor mechanisms to inhibit further exocytotic release of the transmitter when its synaptic concentration is elevated.

Figure 1.5 illustrates the comparison of presynaptic terminals for the biogenic amine neurons: dopamine, norepinephrine, and 5‐hydroxytryptamine (serotonin). The biosynthesis of each biogenic amine transmitter is indicated with uptake and storage in synaptic vesicles. The vesicular uptake of all three biogenic amines depicted is mediated by a common transporter, vesicular monoamine transporter 2 (VMAT2). VMAT2 is the vesicular monoamine transporter that transports dopamine, norepinephrine, and 5‐hydroxytryptamine into neuronal synaptic vesicles. VMAT2 is an H+‐ATPase antiporter, which uses the vesicular electrochemical gradient to drive the transport of biogenic amines into the vesicle (Lohr et al. 2017). In contrast to VMAT2 being expressed in all three biogenic amine neurons, each neurotransmitter neuron expresses a distinct plasma membrane transporter. These transporters are members of the SLC6 symporter family that actively translocate amino acids or amine neurotransmitters into cells against their concentration gradient using, as a driving force, the energetically favorable coupled movement of ions down their transmembrane electrochemical gradients. The dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SERT) are all uniquely expressed in their respective neurotransmitter neurons and couple the active transport of biogenic amines with the movement of one Cl− and two Na+ ions along their concentration gradient. The ionic concentration gradient is created by the plasma membrane Na+/K+ ATPase and serves as the driving force for transmitter uptake. Examples of drugs that act as selective inhibitors for all three biogenic transporters are listed. The three monoamine transporters, DAT, NET, and SERT, represent important pharmacological targets for many behavioral disorders including depressive, compulsive and appetite‐related behavioral problems. The three neurotransmitter terminals also express unique presynaptic autoreceptors that regulate exocytotic release.

Figure 1.5 Schematic comparison of dopamine, norepinephrine, and 5‐hydroxytryptamine (serotonin) synapses. Each neuron expresses a monoamine transporter selective for its neurotransmitter. These transporters function as reuptake pumps that terminate the synaptic actions of the transmitters and promote uptake and eventual storage of the transmitter in vesicles. Selective drug inhibitors of each monoamine transporter are shown. Abbreviations: DA, dopamine; DAT, dopamine transporter; NE, norepinephrine; NET, norepinephrine transporter; 5‐HT, 5‐hydroxytryptamine; SERT, serotonin transporter.

Biogenic Amine Neurotransmitters and Affective Disorders

The role of biogenic amines in affective disorders has a long history, beginning in the 1950s. The biogenic amine theory for affective disorders emerged as pharmacologists and psychiatrists began to explore the biologic basis for mental disorders. Initially, insights were gained from better understanding of the cellular actions of drugs and correlation of this knowledge of drug action with the therapeutic and behavioral responses to the same drugs in the clinic. In its original formulation, the biogenic amine theory for affective disorders stated that depression was due to a deficiency of biogenic amines in the brain, while mania was due to an excess of these transmitters. In the 1950s, iproniazid was used in the treatment of tuberculosis, and it was observed that in some patients with depressive symptoms, their mood improved over the course of a chronic regimen with iproniazid. Concurrently, preclinical research showed that iproniazid was an inhibitor of the enzyme monoamine oxidase (MAO). MAO catalyzes the degradation of dopamine (DA), norepinephrine (NE), and serotonin (5‐HT), and inhibition of MAO was found to elevate the levels of these transmitters in animal brains. Also, in the 1950s, reserpine was being used as an antihypertensive. Some patients treated with reserpine developed depressive symptoms severe enough in some cases to produce suicide ideation. Animals given reserpine also developed depression‐like symptoms consisting of marked sedation. Reserpine was shown to deplete the CNS of DA, NE, and 5‐HT by virtue of its ability to block the vesicular uptake of these monoamines. Blocking the vesicular uptake of monoamines leads to a depletion of the transmitters due to degradation by the mitochondrial enzyme MAO. Therefore, vesicular storage of monoamines is not only a prerequisite for exocytosis but also a means of preventing degradation of the transmitters in the cytosolic compartment. One other observation in the 1950s was that imipramine, developed initially as an antipsychotic drug candidate, elevated mood in a subpopulation of schizophrenic patients with comorbid depressive illness. Preclinical research revealed that imipramine, and other tricyclic antidepressants, were able to block monoamine transport into presynaptic terminals. This action would therefore produce an elevation of synaptic levels of biogenic amines. All these observations with iproniazid, reserpine, and imipramine were therefore consistent with the original formulation of the biogenic amine hypothesis for affective disorders.

Although today we continue to recognize the role of biogenic amines in depression, several discrepancies in the original hypothesis are appreciated. As an example, some clinically effective antidepressants do not block the presynaptic transport of monoamines and are not MAO inhibitors. However, importantly for a hypothesis that attempts to correlate synaptic levels of monoamines with mood, while synaptic levels of monoamines are elevated within a time domain of a few hours after antidepressant administration, the symptoms of depression do not resolve until several weeks of chronic therapy with antidepressant drugs. Contemporary hypotheses to explain the mechanism of action of antidepressant drugs therefore seek an appropriate temporal correlation between neurochemical drug action and the mitigation of the symptoms of depression. Rather than a focus on the synaptic levels of biogenic amines, contemporary views of the mechanism of action of antidepressants are focused on the regulation of receptor signaling.

References

Lohr, K.M., Masoud, S.T., Salahpour, A., and Miller, G.W. (2017). Membrane transporters as mediators of synaptic dopamine dynamics: implications for disease. European Journal of Neuroscience 45 (1): 20–33.

Makram, H., Muller, E., Ramaswamy, S. et al. (2015). Reconstruction and stimulation of neocortical microcircuitry. Cell 163: 456–492.

Torres, G.E., Gainetdinov, R.R., and Caron, M.G. (2003). Plasma membrane monoamine transporters: structure, regulation and function. Nature Reviews Neuroscience 4 (1): 13–25.

2

Amino Acid Neurotransmitters: Glutamate, GABA, and the Pharmacology of Benzodiazepines

Thomas F. Murray

Creighton University, Omaha, NE, USA

Introduction

In addition to their role in intermediary metabolism, certain amino acids function as small molecule neurotransmitters in the central and peripheral nervous systems. These specific amino acids are classified as excitatory or inhibitory, based on the characteristic responses evoked in neural preparations. Application of excitatory amino acids such as glutamic acid and aspartic acid typically depolarize mammalian neurons, while inhibitory amino acids such as gamma aminobutyric acid (GABA) and glycine characteristically hyperpolarize neurons. Glutamate, aspartate, and GABA all represent amino acids that occur in high concentrations in the brain. The brain levels of these amino acid transmitters are high (μmol g−1) relative to biogenic amine transmitters (nanomol g−1) such as dopamine, serotonin, norepinephrine, and acetylcholine. In mammals, GABA is found in high concentrations in the brain and spinal cord, but is present in only trace amounts in peripheral nerve tissue, liver, spleen, or heart (Cooper et al. 2003). These observations reveal the enrichment of this amino acid in the brain and suggest an important functional role in the central nervous system (CNS).

Glutamatergic Synapses

Glutamate and aspartate produce powerful excitation in neural preparations and glutamate is generally accepted as the most prominent neurotransmitter and major excitatory transmitter in the brain. The establishment of glutamate as a neurotransmitter in the brain was historically difficult due to its role in general intermediary metabolism. Glutamate is also involved in the synthesis of proteins and peptides, and also serves as the immediate precursor for GABA in GABAergic neurons. The enzyme glutamic acid decarboxylase (GAD) converts glutamate to GABA in these GABAergic neurons. In contrast to GABA, the glutamate content of brain outside of glutamatergic neurons is high as a consequence of its role in intermediary metabolism and protein synthesis. An array of neurochemical methods accordingly has demonstrated that all cells contain some glutamate, and in the brain all neurons contain measurable amounts of glutamate.

In neurons, glutamate is primarily synthesized from glucose through the pyruvate→acetyl‐CoA → 2‐oxoglutarate pathway, and from glutamine that is synthesized in glial cells, transported into nerve terminals, and converted by neuronal glutaminase into glutamate. In terminals of glutamatergic neurons, glutamate is stored in synaptic vesicles from which Ca²+‐dependent release in response to depolarization occurs. This synaptically released glutamate is taken up, in part, by glial cells and converted to glutamine by the enzyme glutamine synthetase. This glutamine is then transported back to neurons where glutamate is regenerated through the action of glutaminase (Figure 2.1).

Figure 2.1 Schematic representation of a glutamatergic synapse. Glutamine (Gln) is converted to glutamate (Glu) by mitochondrial glutaminase in glutamatergic neurons. Glutamate is released into the synaptic cleft where it may activate both pre‐ and postsynaptic receptors. Glutamate in the synaptic cleft is recaptured by neuronal and glial plasma membrane transporters that terminate the synaptic actions of the excitatory transmitter. Glial glutamate is converted to glutamine by the enzyme glutamine synthetase. This glutamine is then shuttled back to glutamatergic neurons to replenish the glutamate. Glutamate receptors include both G‐protein coupled (mGluRs) and ligand‐gated ion channel (AMPA, NMDA and kainite) receptors.

Extracellular glutamate concentrations are maintained within physiological levels by a family of transmembrane proteins known as excitatory amino acid transporters (EAATs). At least five EAATs have been identified with individual subtypes, differing with respect to their pharmacology and distribution within the CNS. Two of these EAATs are localized primarily on glial cells in the CNS with the other three EAAT subtypes being localized to neurons. The two glial glutamate transporters have been shown to be the primary regulators of extracellular glutamate in the CNS (Amara and Fontana 2002). EAATs accomplish glutamate influx driven by the cotransport of 3 Na+ and 1 H+ ions with the countertransport of 1 K+ ion. EAAT‐mediated glutamate uptake is therefore electrogenic and dependent on the Na+ gradient. The 3:1 ratio of Na+ to glutamate molecules transported causes a significant Na+ influx into the glial cells when the glutamate uptake is stimulated (Kirischuk et al. 2016). This elevation of intracellular Na+ can trigger a reverse mode of action of the Na+/Ca²+ exchanger (NCX), leading to a stimulation of Ca²+ signaling pathways in glia or neurons.

There is some evidence that neuronal EAATs are localized predominantly outside the synapse where they control extrasynaptic, rather than synaptic, glutamate concentration. This extrasynaptic glutamate may function to activate pre‐ and postsynaptic metabotropic glutamate receptors (mGluRs). Presynaptic mGluRs are involved in the feedback regulation of synaptic glutamate release.

In the normal brain, the prominent glutamatergic pathways are: (i) the cortico‐cortical pathways; (ii) the pathways between the thalamus and the cortex; and (iii) the extrapyramidal pathway (the projections between the cortex and striatum). Other glutamate projections exist between the cortex, the substantia nigra, the subthalmic nucleus, and the pallidum. Glutamate‐containing neuronal terminals are ubiquitous in the CNS and their importance in brain function and neurotransmission is therefore considerable. Estimates of the fraction of neurons in the brain that use glutamate as a neurotransmitter range from 70% to 85%.

Glutamate receptors are categorized into two main classes, namely, ionotropic glutamate receptors (iGluRs) and metabotropic receptors (mGluRs). The iGluRs were originally classified using a pharmacologic approach that led to identification of three subtypes bearing the names of selective agonists: (i) the AMPA; (ii) kainate; and (iii) NMDA receptors. These glutamate receptor subtypes are often described as being either NMDA (N‐methyl‐D‐asparate) or non‐NMDA (AMPA and kainate) receptors based on their sensitivity to the synthetic aspartate analog NMDA. All of these iGluRs represent ligand‐gated cation channels permeable to Na+ and K+ with differing permeabilities to Ca²+. Activation of these receptors by glutamate or selective agonists at normal membrane potentials allows Na+ to enter the cell with attendant membrane depolarization; this is the underlying mechanism for the rapid excitatory response of most neurons to glutamate. In addition to Na+ permeability NMDA receptors also have a high permeability to Ca²+ and display a voltage‐dependence of inward currents carried by Na+ and Ca²+ ions. The voltage dependence of the inward ionic current through the NMDA receptor arises from Mg²+ blockade of this channel at normal resting membrane potentials. This channel‐blocking action of extracellular Mg²+ is relieved when the cell is depolarized. Thus, the NMDA receptor signaling requires depolarization of the cell through the excitatory actions of non‐NMDA receptors before this ligand‐gated ion channel can produce an inward current. This property of NMDA receptors has led to the channel being termed a coincident detector due to the requirement for simultaneous activation of NMDA receptors and excitatory input to a cell as a precondition for the passage of ionic current through NMDA receptor ion channels.

A molecular classification of glutamate receptors has confirmed the subdivision based on pharmacological profiles of receptor subtypes. Molecular cloning techniques have identified gene families corresponding to each functional subtype of glutamate receptor. NMDA receptors are formed by assemblies of three gene families including NR1, NR2A‐D, and NR3A/3B (Mayer and Armstrong 2004). Functional NMDA receptors exist as heteromers containing two NR1 and two NR2 subunits (Erreger et al. 2004). NMDA receptors can also contain NR3A subunits that modulate the channel function. AMPA receptors are comprised of assemblies from the GluR1–GluR4 gene family, whereas kainate receptors are assemblies of GluR5–GluR7 and KA1 and KA2 subunits.

In addition to the iGluRs, there are metabotropic glutamate receptors (mGluRs) that are members of the large family of G‐protein coupled receptors. These mGluRs are therefore not ligand‐gated ion channels, but, rather, change cell physiology through an interaction with G‐proteins that in turn regulate the activity of enzymes and/or ion channels involved in cell signaling cascades. These mGluRs are widely distributed in the brain where they mediate a variety of effects including the modulation of glutamate release from glutamatergic neurons. These presynaptic mGluRs therefore function as autoreceptors.

Inasmuch as glutamate receptors mediate most of the excitatory transmission in the brain, they represent important potential targets for therapeutic intervention in a number of behavioral disorders.

Pharmacology of Ketamine and Tiletamine

Ketamine is an anesthetic agent that was first introduced in clinical trials in the 1960s. It is a dissociative anesthetic, which is a term originally introduced by Domino and collaborators to describe the unique state of anesthesia produced by ketamine in which the subject is profoundly analgesic while appearing disconnected from the surrounding environment (Miyasaka and Domino 1968). Domino's laboratory attributed this unique anesthetic state to a drug‐induced dissociation of the EEG activity between the thalamocortical and limbic systems. It was demonstrated that the cataleptic anesthetic state induced by intravenous ketamine (4 mg kg−1) in cats was associated with an alternating pattern of hypersynchronous δ wave bursts and low voltage, fast wave activity in the neocortex and thalamus. Subcortically, the δ wave bursts were observed prominently in the thalamus and caudate nucleus, and the EEG patterns of thalamic nuclei were closely related phasically to the δ waves of the neocortex. In contrast to the marked δ wave bursts in the neocortex, thalamus, and caudate nucleus, prominent δ waves were not observed in the cat hippocampus, hypothalamus, or midbrain reticular formation. The hippocampus showed θ arousal waves in spite of the appearance of high voltage, hypersynchronous δ wave bursts in the thalamus and neocortex. Thus, ketamine was demonstrated to produce a functional dissociation of the EEG activity between the hippocampus and thalamocortical systems.

Ketamine and the newer dissociative anesthetic tiletamine act as noncompetitive antagonists of NMDA receptors in the CNS (Figure 2.2). The discovery by Lodge et al. (1983) of the ability of ketamine and related arylcyclohexylamines to antagonize specifically the neuronal excitation mediated by the synthetic aspartate analog, NMDA, provided a pivotal advance in our understanding of the mechanism of action of dissociative anesthetics. Based on the earlier observation that ketamine selectively reduced polysynaptic reflexes in which excitatory amino acids were the transmitter, Lodge and coworkers investigated the action of ketamine on the excitation of cat dorsal horn interneurons by amino acids used in the classification of excitatory amino acid receptors, namely, NMDA, quisqualate, and KA (Lodge and Mercier 2015). The microionotophoretic or intravenous administration of ketamine selectively reduced the increased firing rate of dorsal horn neurons evoked by focal application of NMDA. The excitatory responses elicited by quisqualate and KA remained little affected. The selective NMDA‐blocking effect was not restricted to ketamine inasmuch as the dissociative anesthetics, phencyclidine (PCP) and tiletamine, had similar actions that paralleled their relative anesthetic potencies. The primary molecular target for ketamine‐induced analgesia and anesthesia therefore appears to be brain NMDA receptors. The inhibitory concentration for ketamine antagonism of NMDA responses in rat cortical preparations range from 6 μM to 12 μM. These values are comparable to the plasma concentration (20–40 μM) obtained in rats following intravenous anesthetic doses. It therefore appears likely that a large fractional occupancy of NMDA receptors may be required for ketamine induction of anesthesia (Murray 1994).

Figure 2.2 Schematic of the NMDA subtype of glutamate receptors. NMDA receptors possess binding sites for the transmitter glutamate and the co‐agonist, glycine. Competitive antagonists bind to the glutamate site, whereas noncompetitive antagonists such as ketamine and tiletamine bind to a site in the ion channel domain. Mg++ exerts a voltage‐dependent block of the ion channel.

Subanesthetic doses of ketamine produce a spectrum of psychoactive actions in humans including mood elevation, distortions in body image, hallucinations, delusions, and paranoid ideation. These effects resemble those of PCP (phencyclidine) and are responsible for the illicit use of ketamine. The availability of ketamine in veterinary medicine has resulted in numerous case reports of ketamine abuse by veterinarians. Similar to the anesthetic and analgesic actions of ketamine, the psychoactive properties appear to be related to the noncompetitive antagonism of NMDA receptors.

Great interest in ketamine as an antidepressant has emerged due to human clinical data that has demonstrated the rapid and sustained antidepressant effects of ketamine (Murrough et al. 2017). Ketamine has therefore been repurposed as a rapidly acting antidepressant, triggering great interest in glutamate signaling mechanisms underlying depressive disorders. Ketamine, its metabolites, and other NMDA receptor antagonists produce rapid antidepressant‐like effects in mouse behavioral models that are dependent on rapid protein synthesis of brain‐derived neurotrophic factor (BDNF). Experiments in animal models show that ketamine‐mediated blockade of NMDA receptors at rest deactivates eEF2 kinase, resulting in a desuppression of BDNF translation (Kavalali and Monteggia 2015). A key role for BDNF in mediating antidepressant efficacy has previously been established (Bjorkholm and Monteggia 2016).

GABAergic Synapses

Of all the putative neurotransmitters in the brain, γ‐aminobutyric acid (GABA) is perhaps the one whose candidacy rests on the longest history of investigation. Glutamic acid decarboxylase (GAD), the enzyme that catalyzes the formation of GABA, appears to be largely restricted to GABAergic neurons and therefore affords a suitable marker for this population of neurons. In brain regions such as the hippocampus, histochemical studies have demonstrated that GAD is distributed in the neuropil with highest concentrations between cell bodies reflecting the presence of GABAergic neuron terminals. The abundance of GABAergic interneurons and projection neurons in the brain has been estimated to represent 17–20% of the neurons in the brain (Somogyi et al. 1998). Upon activation, these GABAergic neurons release GABA from presynaptic terminals into the synaptic cleft. The concentration of GABA in the synaptic cleft is controlled by the high affinity uptake into presynaptic terminals and glial cells. In contrast to glutamate which is predominantly taken up by astrocytes, GABA represents the primary inhibitory neurotransmitter in the mammalian brain and is removed from the synaptic cleft mainly by the neuronal GABA transporter (GAT) subtype GAT1. Because of the high capacity and abundance of synaptic GAT1, GABA rarely escapes the synapse (Kirischuk et al. 2016).

GABA represents the major inhibitory transmitter in the brain and this inhibition is mediated by GABA binding to postsynaptic receptors. GABAergic systems serve important regulatory functions in the brain such as vigilance, anxiety, muscle tension, epileptogenic activity, and memory. In brain areas, such as the cerebral cortex and the hippocampus, GABAergic neurons are predominantly interneurons that function as primary regulators of the activity of the projecting glutamatergic pyramidal neurons. The activity of these GABAergic interneurons is largely driven by glutamatergic afferents arising from either projecting afferents or recurrent glutamatergic collaterals (Figure 2.3).

Figure 2.3 Schematic representation of a GABAergic synapse. Glutamate is the immediate precursor of GABA in these neurons where it is metabolized by the enzyme glutamic acid decarboxylase (GAD). The GABA is stored in and released from vesicles into the synaptic cleft. Synaptic GABA activates both pre‐ and postsynaptic receptors. The latter are primarily ligand‐gated ion channels (GABAA receptors), whereas presynaptic GABAB receptors are G‐protein coupled receptors involved in the regulation of neurotransmitter release. GABA in the synaptic cleft is recaptured by an active transporter (GAT) in the plasma membrane of both neurons and glia. GABA is metabolized by the mitochondrial enzyme GABA‐transaminase (GABA‐T) to succinic semialdehyde which in turn is converted to succinic acid by the enzyme succinic semialdehyde dehydrogenase. Succinic acid exerts a negative feedback inhibition on GAD. Succinic semialdehyde dehydrogenase is inhibited by the anticonvulsant sodium valproate.

It is now generally recognized that GABAergic‐mediated inhibition results from GABA activation of GABAA receptors (GABAARs). GABAARs are heteropentameric Cl−‐selective ligand‐gated ion channels that mediate fast inhibition within the CNS. When activated by GABA, these GABAARs promote the diffusion of this ion according to its concentration gradient. Thus, GABAA‐receptor activation may depolarize or hyperpolarize membranes depending on the difference in Cl− concentration of the postsynaptic neuron and extracellular milieu (Figure 2.4). Although excitatory responses to GABA have been described in embryonic cells that maintain high intracellular Cl− concentrations, the typical response of an activated GABAA receptor in the mature CNS is hyperpolarization mediated by Cl− influx. Functional GABAA receptors are pentameric ligand‐gated ion channels assembled from members of seven different subunit classes, some of which have multiple isoforms: α (1–6), β (1–3), γ (1–3), δ, Е, θ, and π. A pentameric assembly could theoretically be composed of over 50 distinct combinations of these subunits; however, GABAA receptor subunits appear to form preferred assemblies resulting in possibly dozens of distinct receptor complexes in the brain. Most GABAA receptor subtypes are presumed to be composed of α‐, β‐, γ‐subunits. Molecular studies have demonstrated that distinct GABAA receptor assemblies often have different physiologic and pharmacologic profiles, suggesting that subunit composition is an important determinant of pharmacological diversity in GABAA receptor populations. Deficits in GABAAR function have been demonstrated in a range of behavioral and CNS disorders, including anxiety, psychosis, and epilepsy.

Figure 2.4 Schematic structure of the GABAA receptor pentamer composed of two α‐subunits, two β‐subunits and one γ‐ subunit. The neurotransmitter GABA binds to a site at the interface between the α‐ and β‐subunits (●) causing the Cl− channel to open. Benzodiazepines such as clonazepam and diazepam bind to a site at the interface of the α‐ and γ‐subunits and act as positive allosteric modulators to augment the actions of GABA.

GABAA receptors represent the molecular target for all of the characteristic pharmacological actions of benzodiazepines, including sedation, muscle relaxation, seizure suppression, and anxiety reduction (anxiolysis). Benzodiazepines allosterically enhance the GABAA receptor opening frequency, producing a potentiation of the GABA‐induced inhibitory response (Rudolph et al. 1999). The benzodiazepine binding site is believed to be located at the interface between the α‐ and γ2‐subunits of a pentameric GABAA receptor complex. There are six types of α subunit, α1–α6, and GABAA receptors containing the α1‐, α2‐, α3‐, or α5‐subunits in combination with any β‐subunit and the γ2‐subunit are sensitive to benzodiazepines, such as diazepam, alprazolam, and clonazepam (Möhler et al. 2002). GABAA receptors containing these four α‐subunits are most abundant in the brain and the most prevalent receptor complex is comprised of α1β2γ2 subunits. GABAA receptors that do not respond to benzodiazepines such as diazepam and clonazepam are less abundant in the brain and are characterized by the presence of the α4‐ and α 6‐subunits (Möhler et al. 2002). The use of transgenic mice with mutated GABAA receptors has recently demonstrated that it may be possible to develop benzodiazepine‐like drugs that are anxioselective, meaning that they may reduce anxiety in the absence of sedation and muscle relaxation (leading to incoordination). The anxiolytic action of diazepam is selectively mediated by potentiation of GABAergic inhibition in a population of neurons expressing α2‐subunit containing GABAA receptors, which constitute only 15% of all diazepam‐sensitive GABAA receptors (Möhler et al. 2001). The α2‐GABAA receptor expressing neurons in the cerebral cortex and the hippocampus are therefore specific targets for the future development of selective anxiolytic drugs. The sedative actions of benzodiazepines, in contrast, appear to be mediated by α1‐subunit containing GABAA receptors. Changes in sleep pattern and EEG frequency associated with classical benzodiazepines are attributable to most GABAARs (other than α1‐containing receptors), with α2 receptors having the most profound influence. These studies indicate that future drug development of more subunit‐specific benzodiazepine ligands may have more selective pharmacologic profiles.

References

Amara, S.G. and Fontana, A.C.K. (2002). Excitatory amino acid transporters: keeping up with glutamate. Neurochemistry International 41: 313–318.

Bjorkholm, C. and Monteggia, L.M. (2016). BDNF – a key transducer of antidepressant effects. Neuropharmacology 102: 72–79.

Cooper, J.R., Bloom, F.E., and Roth, R.H. (2003). The Biochemical Basis of Neuropharmacology, 7e. New York: Oxford University Press.

Erreger, E., Chen, P.E., Wyllie, D.J.A., and Traynelis, S.F. (2004). Glutamate receptor gating. Critical Reviews in Neurobiology 16: 187–224.

Kavalali, E.T. and Monteggia, L.M. (2015). How does ketamine elicit a rapid antidepressant response? Current Opinion in Pharmacology 20: 35–39.

Kirischuk, S., Héja, L., Kardos, J., and Billups, B. (2016). Astrocyte sodium signaling and the regulation of neurotransmission. Glia 64: 1655–1656.

Lodge, D., Anis, N.A., Berry, S.C., and Burton, N.R. (1983). Arylcyclohexylamines selectively reduce excitation of mammalian neurons by aspartate like amino acids. In: Phencyclidine and Related Arylcyclohexylamines: Present and Future Applications (ed. J.‐M. Kamenka, E.F. Domino and P. Geneste). Ann Arbor, MI: NPP Brooks.

Lodge, D. and Mercier, M.S. (2015). Ketamine and phencyclidine: the good, bad and the unexpected. British Journal of Pharmacology 172: 4254–4276.

Mayer, M.L. and Armstrong, N.