The Neurobiology of Addiction

Transcription

1 The Neurobiology of Addiction Name ASAM Disclosure of Relevant Financial Relationships Content of Activity: ASAM Review Course 2014 Commercial Interests Relevant Financial Relationships: What Was Received Relevant Financial Relationships: For What Role No Relevant Financial Relationships with Any Commercial Interests Dr. Eliot Gardner X Eliot Gardner, MD Chief, Neuropsychopharmacology Section, Intramural Research Program National Institute on Drug Abuse, NIH Animal Models of Addiction Neuropharmacology Cellular and Molecular Mechanisms in Addiction Neuroimaging of Addiction and Related Phenomena Electrical Brain Stimulation Reward (BSR; ICSS) Conditioned Place Preference (CPP) Drug Self Administration Drug Seeking Maintained by Second Order Reinforcement Progressive Ratio Break Point Shifts Resistance to Extinction of Drug Seeking Behavior Relapse to Drug Seeking Behavior (Reinstatement Model) Relapse to Drug Seeking Behavior (Reactivation Model) Incubation of Drug Craving Stimulator 1

2 The Pleasure/Reward Circuitry of the Brain The Pleasure/Reward Circuitry of the Brain Acc VTA FCX GLU HIPP AMYG GLU CRF 5HT OPIOID OPIOID DYN 5HT DA BNST ENK VP OFT Opiates Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine NE ABN HYPOTHAL ICSS NE Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids LC LAT TEG PAG END 5HT Raphé To dorsal horn RETIC 2

3 Representative BSR Stimulation Response Curves Conditioned Place Preference (CPP) Conditioned Place Preference The Highly Selective Dopamine D3 Receptor Antagonist SB A Blocks Expression of Nicotine Enhanced CPP P< 0.01 P< 0.01 P< P< 0.01 P<

4 Drug Self Administration Pump Drug Self Administration Cocaine? DRUG SELF ADMINISTRATION SCHEDULES OF REINFORCEMENT Fixed Ratio (FR) Variable Ratio (VR) Fixed Interval (FI) Variable Interval (VI) Others Drug Seeking Maintained by Second Order Reinforcement 4

5 Drug Self Administration Under Second Order Reinforcement Pump Cocaine? Drug Self Administration under progressiveratio reinforcement Drug Self Administration Under Progressive Ratio Reinforcement Looking at Shifts in Break Point Pump PR Schedule Reward Work Demand (# Infusion) (# Lever Press) Cocaine? SB A Lowers Progressive Ratio Break Point for Cocaine (0.5mg/kg/inf) Self Administration 5

6 Resistance of Extinction of Drug Seeking Behavior Can be Done With Conditioned Place Preference, Drug Self Administration Resistance to CPP Extinction is Dose Orderly Animals with the Genetic Trait of Resistance to Addiction Show More Rapid Extinction of Opiate Induced CPP Highly Selective Dopamine D3 Receptor Antagonism Produces More Rapid Extinction of Cocaine Self Administration Relapse to Drug Seeking Behavior Reinstatement Model (Based on Drug Self Administration) Relapse to Drug Seeking Behavior Using the Reinstatement Model Pump Cocaine? 6

7 Triggers to Relapse Re exposure to DRUG Cross triggering between drug classes is very real Exposure to STRESS Mild stress extremely effective Exposure to environmental CUES Sights, sounds, smells associated with drug use People, places, things Alcoholics Anonymous The Reinstatement Model of Relapse to Drug Seeking Behavior Cocaine + Cues Saline No Cues Cocaine Selective Cannabinoid CB1 Receptor Antagonism (by AM251) Dose Dependently Inhibits Cocaine Triggered Relapse to Cocaine Seeking Behavior (Reinstatement Model) Selective Dopamine D3 Receptor Antagonism (by NGB 2904) Dose Dependently Attenuates Cocaine Triggered Relapse to Cocaine Seeking Behavior (Reinstatement Model) Selective Dopamine D3 Receptor Antagonism (by SB277011A Micro Injected into the Nucleus Accumbens) Attenuates Stress Triggered Relapse to Cocaine Seeking Behavior (Reinstatement Model) Selective Dopamine D3 Receptor Antagonism (by SB277011A) Dose Dependently Attenuates Cue Triggered Relapse to Cocaine Seeking Behavior (Reinstatement Model) 7

8 Relapse to Drug Seeking Behavior Using the Reactivation Model (Based on CPP) Relapse to Drug Seeking Behavior Reactivation Model (Based on Drug Induced Conditioned Place Preference) TRIGGERS TO RELAPSE Selective Dopamine D3 Receptor Antagonism (by SB277011A) Dose Dependently Attenuates Morphine Triggered Relapse to Cocaine Seeking Behavior (Cross Triggering) (Reactivation Model) Re exposure to DRUG Cross triggering between drug classes is very real Exposure to STRESS Mild stress extremely effective Exposure to environmental CUES Sights, sounds, smells associated with drug use People, places, things Alcoholics Anonymous Incubation of Drug Craving (Based on Intravenous Drug Self Administration) Incubation of Drug Craving (Incubation of Relapse Vulnerability with the Mere Passage of Time) Pump Cocaine? 8

9 Incubation of Relapse Vulnerability Over Time Selective Dopamine D3 Receptor Antagonism (by SB A) inhibits incubation of cocaine craving Selective Dopamine D3 Receptor Antagonism (by SB A) dose dependently inhibits incubation of cocaine craving Neuropharmacology Related to Addiction Neurotransmitters Receptors Second Messenger Transduction Cascades Relevance to the Neurobiology of Addiction Neurotransmitters More than 300 known to exist Presynaptic versus postsynaptic Extrasynaptic Metabotropic versus Ionotropic Acetylcholine First chemical confirmed as a synaptic transmitter Two receptors well identified and studied Muscarinic Nicotinic Receptor structure and function Relevance to addiction 9

10 Acetylcholine Structure Synthesis and Degradation Acetylcholine (ACh) is synthesized in neurons by the enzyme choline acetyltransferase from the compounds choline and acetyl CoA. The enzyme acetylcholinesterase converts acetylcholine into the inactive metabolites choline and acetate. Acetylcholinesterase is abundant in the synaptic cleft, and its role in rapidly clearing free ACh from the synapse is essential for proper synaptic function (or neuromuscular function in the case of ACh transmission at the neuromuscular junction in the periphery). NICOTINIC ACH RECEPTOR nachrs are ligand gated ion channels, and, like other members of the cys loop ligand gated ion channel superfamily, are composed of five protein subunits symmetrically arranged like staves around a barrel. Subunit composition is highly variable across different tissues. Each subunit contains four regions which span the membrane and consist of approximately 20 amino acids. Region II, which sits closest to the pore lumen, forms the pore lining. Binding of acetylcholine to the N termini of each of the two alpha subunits results in the 15 rotation of all M2 helices, opening voltage gated sodium channels in the postsynaptic membrane. Three subtypes of nachr are found in the CNS: α 4 β 2 α 3 β 4 α 7 Muscarinic ACh Receptor machrs do not conformationally alter themselves to form ion channels when activated. Instead, machrs belong instead to the superfamily of G protein coupled receptors that activate ion channels via a second messenger cascade. When bound by ACh, the machr activates a G protein. The alpha subunit of the G protein deactivates adenylate cyclase while the beta gamma subunit activates K channels resulting in hyperpolarization of the membrane in which the receptor resides. G proteins contain an alpha subunit that is critical to receptor function. These subunits can take a number of forms. There are four broad classes of G protein: G s, G i, G q, and G 12/13. The various G protein subunits act differently upon second messengers camp, phospholipases, etc. Five subtypes of machr are known: M 1 primarily linked to G q upregulates phospholipase C M 2 linked to G i decreases camp M 3 linked to G q upregulates phospholipase C M 4 linked to G i decreases camp the most common machr in CNS M 5 linked to G q upregulates phospholipase C The addictive substance nicotine acts primarily on the α 4 β 2 and α 7 subtypes. Neuroanatomic ACh Pathways Three primary ACh pathways in the CNS are known: Brainstem cholinergic system (pons to thalamus and cortex) Basal forebrain cholinergic system (Nucleus Basalis of Meynert to cortex and hippocampus) Septohippocampal pathway Principal ACh Neuroanatomic Pathways in the Brain ACh interneurons are also widely distributed throughout the CNS 10

11 Relevance to Addiction The muscarinic ACh neuronal systems of the brain appear to play virtually no role in addiction. They are critically involved in learning and in memory storage and retrieval, and degeneration of the ACh projections from the Nucleus Basalis of Meynert to the cortex and hippocampus is the primary neuropathology in Alzheimer s Disease. Nicotinic ACh neuronal modulation of the core dopaminergic reward/relapse pathway originating in the ventral tegmental area and projecting to the nucleus accumbens appears to be the fundamental CNS substrate for nicotine addiction. The anti nicotine addiction medication varenicline (Chantix ) is an α 4 β 2 and α 7 nicotinic agonist, although the α 7 agonist activity appears irrelevant to the therapeutic efficacy of varenicline. It is widely accepted that addiction involves some form(s) of aberrant and/or pathological learning attributing higher value to artificial rewards (drugs) than to natural rewards. Recently, it has been reported that machr and nachr activation in the nucleus accumbens is necessary for the acquisition of an appetitive task. Thus, future research may reveal a role for machr function(s) in addiction. Norepinephrine NE is a catecholamine with multiple roles including those as a hormone and a CNS synaptic transmitter Two receptors well identified and studied Alpha Beta Receptor structure and function Relevance to addiction Norepinephrine Structure Synthesis and Degradation Norepinephrine (NE) is synthesized from dopamine by dopamine β hydroxylase. NE s actions within the synaptic cleft are primarily terminated by NE being taken back up into the presynaptic neuron or other cells via the presynaptic NE reuptake transporter (NET). Two forms of such uptake have been identified Uptake 1 and Uptake 2. Uptake 1 mediates NE uptake into the presynaptic cytosol. Uptake 2 mediates NE uptake into non neuronal cells in the vicinity. Additionally, there is vesicular uptake from the presynaptic cytosol into synaptic vesicles mediated by the vesicular monoamine transporter (VMAT). NE is also enzymatically inactivated by catechol O methyltransferase (COMT), which converts NE into normetanephrine. In the brain, COMT dependent NE degradation is of particular importance in brain regions with low expression of NET. This process takes place in postsynaptic neurons, as, in general, COMT is located intracellularly in the CNS. The Alpha (α)receptor Two types of alpha (α) receptors exist: α 1 G q coupled producing phospholipase C (PLC) activation, which in turn produces cleavage of phosphatidylinositol 4,5 bisphosphate (PIP 2 ) into inositol trisphosphate (IP 3 ) and diacylglycerol (DAG), producing a rise in calcium. α 2 G i coupled producing inactivation of adenylate cyclase and decrease in cyclic adenosine monophosphate (cyclic AMP, camp). The Beta (β)receptor Three types of beta (β) receptors exist: β 1 G s coupled producing activation of adenylate cyclase and increase in cyclic adenosine monophosphate (cyclic AMP, camp). β 2 G s coupled producing activation of adenylate cyclase and increase in camp (there is also a degree of Gi coupling; producing opposite effects on adenylate cyclase and camp). β 3 G s coupled producing activation of adenylate cyclase and increase in cyclic adenosine monophosphate (cyclic AMP, camp). 11

12 Neuroanatomic NE Pathways The Locus Coeruleus/Dorsal NE Bundle System Two primary NE pathways in the CNS are known: The locus coeruleus/dorsal NE bundle system The brain stem/ventral NE bundle system The locus coeruleus/dorsal NE bundle system originates in the locus coeruleus (LC) a very small nucleus in the dorsal part of the pons. In humans, the LC contains only approximately 10,000 neurons, and therefore has only modest computational capacity. Furthermore, the LC/dorsal NE system projects diffusely to virtually the entire brain. The wiring of this system is rather like a fire alarm system in an office building. And the LC/dorsal NE system is known to be involved in diffusely activating the brain during dangerous or stressful situations. Electrical stimulation of the LC/dorsal NE system produces severe anxiety like and panic like behaviors in nonhuman primates (monkeys). The brain stem/ventral NE bundle system is much more anatomically discrete, and one specific pathway within that system plays an important role in addiction (see below) The Locus Coeruleus/Dorsal NE Bundle System (LC and projections) The Brain Stem/Ventral NE Bundle System (Brain Stem nuclei A1 A7 and projections) Relevance to Addiction The lateral tegmental NE neuronal projections originating in brainstem NE nucleus A2 and projecting to the hypothalamus, bed nucleus of the stria terminalis, nucleus accumbens, and amygdala is one of two brain circuits mediating stress triggered relapse to drug seeking behavior in animals behaviorally extinguished from (and pharmacologically detoxified from) prior drug taking behavior(s). See next slide. That makes the A2 lateral tegmental NE neuronal projection system a rational and logical target for pharmacotherapies targeting stress triggered relapse to drug seeking and drug taking behavior(s). Anatomic targeting of such pharmacotherapies to the A2 lateral tegmental NE neuronal projection system is possible in research animals (via direct intracerebral microinjection), but not yet possible in humans. Yet, precise targeting at the human level will be necessary to avoid nonspecific inhibition of the 8 other NE neuronal projection systems in the brain, i.e., to avoid unwanted side effects. At the human level, use of viral vectors may be one way of selectively targeting the A2 NE system for anti relapse therapeutic purposes. Acc VTA ENK FCX VP Opiates OFT GLU ABN ICSS HIPP AMYG GLU CRF OPIOID DYN DA BNST HYPOTHAL 5HT OPIOID 5HT Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids NE Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine LC NE LAT TEG PAG END 5HT Raphé RETIC To dorsal horn Relevance to Addiction Continued In addition, the LC/Dorsal NE neuronal system is highly activated during withdrawal from addictive drugs. This is believed to mediate some of the subjectively unpleasant aspects of withdrawal. Clonidine (trade names Catapres, Kapvay, Nexiclon and others) is a centrally acting α 2 adrenergic agonist which acts to inhibit the LC/Dorsal NE neuronal system, as well as acting as a sympatholytic medication. These CNS actions make clonidine useful in easing withdrawal symptoms associated with the longterm use of narcotics, alcohol, and nicotine. Although some of clonidine s therapeutic efficacy in treating withdrawal is attributable to its alleviation of sympathetic nervous system responses such as tachycardia, hypertension, hot and cold flashes, sweating, and general restlessness, some of clonidine s therapeutic efficacy in treating withdrawal is attributable to its dampening of hyperactivity in the LC/Dorsal NE projections. 12

13 Dopamine DA is a catecholamine with multiple roles including those as a hormone and a CNS synaptic transmitter Five receptors are well identified and studied D1 like group D1, D5 D2 like group D2, D3, D4 Receptor structure and function Relevance to addiction Dopamine Structure Synthesis and Degradation Dopamine (DA) is synthesized from levodopa (L dopa) by decarboxylation. After synthesis, DA is transported from the cytosol into synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2). DA remains in these vesicles until an action potential occurs and causes the contents of the vesicles to be ejected into the synaptic cleft. Once in the synapse, DA binds to and activates DA receptors either on postsynaptic target cells or on the membrane of the DA releasing cell itself (i.e., autoreceptors). DA quickly unbinds from its receptors and is absorbed back into the presynaptic cell, via reuptake mediated either by the high affinity DA transporter (DAT) or the low affinity plasma membrane monoamine transporter (PMAT). Once back in the cytosol, DA is repackaged into vesicles by VMAT2, making it available for future release. Excess DA is subjected to enzymatic breakdown by monoamine oxidase (MAO) into 3,4 dihydroxyphenylacetic acid (DOPAC). DA is also broken down into inactive metabolites by a set of enzymes acting in sequence MAO, aldehyde dehydrogenase (ALDH), and catechol O methyl transferase (COMT). Both isoforms of MAO, MAO A and MAO B, are equally effective. The end product of this enzymatic degradation is homovanillic acid (HVA). In prefrontal cortex, there are very few DATs, and DA is inactivated instead by reuptake via the NET, presumably on neighboring NE neurons, then enzymatic breakdown by COMT into 3 Methoxytyramine (3 MT). Dopamine Receptors D 1 like family Activation of D 1 like receptors is coupled to the G protein G sα, which activates adenylyl cyclase, increasing intracellular concentrations of the second messenger camp. D 1 D 5 D 2 like family Activation of D 2 like receptors is coupled to the G protein G iα, which directly inhibits formation of camp by inhibiting adenylyl cyclase. D 2 there are two forms: D 2 Sh (short) and D 2 Lh (long): D 2 Sh is pre synaptically situated; an autoreceptor regulating neurotransmission by feed back mechanisms, affecting synthesis, storage, and release of DA D 2 Lh functions as a classical post synaptic receptor D 3 Maximum expression in the islands of Calleja and nucleus accumbens D 4 Neuroanatomic DA Pathways The Pleasure/Reward Circuitry of the Brain Seven DA pathways in the CNS are known: A8 The retrorubal DA cell field A9 The substantia nigra; axonal projections to striatum, globus pallidus, and subthalamic nucleus (the nigrostriatal system) A10 The ventral tegmental area; projections to nucleus accumbens and the prefrontal cortex as well as several other areas (the mesolimbic system) A11 Cell field in posterior hypothalamus, projecting to spinal cord A12 Cell field in arcuate nucleus of hypothalamus; projections to median eminence and pituitary gland A13 Cell field in the zona incerta; projections to several areas of the hypothalamus; participate in the control of gonadotropin releasing hormone A14 Cell field in the periventricular nucleus of hypothalamus; important projections to the pituitary gland 13

14 Acc ENK FCX VP Opiates OFT VTA GLU ABN ICSS HIPP AMYG CRF GLU BNST OPIOID DYN DA HYPOTHAL 5HT OPIOID 5HT Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids NE Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine LC NE LAT TEG PAG END 5HT Raphé RETIC To dorsal horn The Crucial Reward Neurotransmitter is Dopamine (DA) How Do We Know This? Virtually all addictive drugs are DA agonists (directly or indirectly) The one common feature they share Microinjections of DA agonists support: Conditioned place preference Intracranial self administration Effects of DA antagonists Negative reinforcers in animals Subjective effects in humans (neuroleptic induced anhedonia) Effects of DA antagonists on drug self administration: Initially, a compensatory increase in drug intake Followed by behavioral extinction of drug taking Nucleus Accumbens (NAcc) neurochemistry during self administration In vivo brain microdialysis (animals self administer addictive drugs to maintain an elevated DA level within reward related synapses of the nucleus accumbens) Pump Cocaine? IV heroin self administration 14

15 Relevance to Addiction DA (in the A10 mesolimbic projections from ventral tegmental area to nucleus accumbens) is the crucial reward/pleasure neurotransmitter in the brain. Animals selfadminister addictive drugs to maintain an elevated DA level within the reward related synapses of the nucleus accumbens. This is the neuronal substrate of the drug induced high. DA deficiency within the A10 VTA accumbens system produces a reward deficiency syndrome, which disposes to drug taking behavior. This DA deficiency can be produced genetically, by environmental events (stress; social defeat in an animal dominance hierarchy), and/or by chronic exposure to addictive drugs. Addictive drugs derive their addictive potential by acting on the A10 VTA accumbens DA system more powerfully than natural biologically essential rewards (food, sex, etc). The A10 VTA accumbens DA system is also involved in reward prediction, prediction error, and incentive salience. In addition, the A10 VTA acumbens DA system is the neural substrate for drug triggered relapse to drug seeking and drug taking behavior(s). Relevance to Addiction Continued The DA D 3 receptor is an interesting target for anti addiction, anti craving, anti relapse pharmacotherapeutic compounds. The D 3 receptor is remarkably limited in its neuroanatomic distribution found only in the DA mediated reward/pleasure circuitry and the DA mediated drug triggered relapse circuitry. Also, dramatic increases in D 3 receptor density are seen in the brain reward circuits of human cocaine users and in laboratory animals exposed to chronic cocaine treatment regimens. The development of highly selective D 3 antagonists is a drug design chemist s nightmare, due to the extraordinary structural homology between DA D 2 and D 3 receptors. DA D 2 antagonists are non starters as potential anti addiction therapeutic agents, due to their dysphorigenic actions. To date, approximately a half dozen D 3 antagonists with satisfactory selectivity (at least 100:1) for D 3 versus D 2 receptors have been designed, synthesized, and tested in animal models relevant to addiction. These compounds show extraordinary promise in these animal models as anti addiction, anti craving, anti relapse medications. Serotonin (5HT) Serotonin or 5 hydroxytryptamine (5HT) is a monoamine neurotransmitter derived from tryptophan. 5HT is found in the GI tract, platelets, and the CNS) Thirteen 5HT receptors have been identified Receptor structure and function Relevance to addiction 5HT Structure Synthesis and degradation 5HT is synthesized from the amino acid L tryptophan by a short metabolic pathway consisting of two enzymes: tryptophan hydroxylase (TPH) and amino acid decarboxylase. The TPH mediated reaction is the rate limiting step in the pathway. TPH has been shown to exist in two isoforms: TPH1, found in several tissues, and TPH2, which is neuron specific. 5HT synthesis from tryptophan; and principal degradation pathway 5HT is metabolized by MAO and aldehyde dehydrogenase to form 5 hydroxyindoleacetic acid (5 HIAA). (See next slide). 5HT synaptic action is terminated primarily via uptake of 5HT from the synapse. This is accomplished through a specific monoamine transporter for 5HT on the presynaptic neuron the serotonin transporter (SERT). A newly discovered monoamine transporter, known as the plasma membrane monoamine transporter (PMAT; a low affinity monoamine transporter protein) may account for a significant percentage of 5HT synaptic clearance. 15

16 5HT Receptors of the 5HT 1 Receptor Family 5HT 1 receptor family signals via G i/o inhibition of adenylyl cyclase: 5HT 1A receptor Mediates inhibitory neurotransmission. Implicated in the following functions: learning; memory; anxiety; depression; positive, negative, cognitive symptoms of schizophrenia; aggression; analgesia; DA release in prefrontal cortex; 5HT synthesis and release. 5HT 1B receptor An autoreceptor. Implicated in: vasoconstriction; aggression; bone mass. 5HT 1D receptor Implicated in vasoconstriction. 5HT 1E receptor Function(s) unknown. 5HT 1F receptor Function(s) unknown. 5HT Receptors of the 5HT 2 Receptor Family 5HT 2 receptor family signals via G q activation of phospholipase C: 5HT 2A receptor Implicated in the following functions: anxiety; depression; positive and negative symptoms of schizophrenia; effects of psychedelic drugs; NE release from LC; glutamate release in prefrontal cortex 5HT 2B receptor Implicated in cardiovascular functions 5HT 2C receptor Implicated in appetite regulation; antipsychotic effects; antidepressant effects; ACh release in prefrontal cortex; DA release in mesocorticolimbic pathway Other 5 HT receptors 5HT 3 receptor An ionotropic receptor (i.e., a ligand gated ion channel). Implicated in the following functions: emesis; anxiolysis 5HT 4 receptor Signals via G αq activation of adenylyl cyclase. Implicated in GI tract function(s); learning; memory; antidepressant effects 5HT 5A receptor Signals via G i/o inhibition of adenylyl cyclase. Implicated in memory consolidation 5HT 6 receptor Signals via G s activation of adenylyl cyclase. Implicated in cognition; antidepressant effects 5HT 7 receptor Signals via G s activation of adenylyl cyclase. Implicated in cognition; antidepressant effects Neuroanatomic 5HT Pathways The 5HT anatomic projection pathways in the brain arise from the raphé nuclei, which are considered to be part of the reticular formation, appearing as cell clusters in the central and medial portions of the brain stem. From caudal to rostral, the raphé nuclei are: Raphé nuclei of the medulla oblongata Nucleus raphé obscurus Nucleus raphé magnus Nucleus raphé pallidus Raphé nuclei of the pontine reticular formation Nucleus raphé pontis Nucleus centralis inferior Raphé nuclei of the midbrain reticular formation Median raphénucleus Dorsal raphé nucleus Neuroanatomic 5HT Pathways Neuroanatomic 5HT Pathways The caudal raphé nuclei, including the nucleus raphé magnus, nucleus raphé pallidus, and nucleus raphé obscurus, all project dorsally or caudally towards the cerebellum and spinal cord. The more rostral nuclei, including the nucleus raphé pontis, median raphé nucleus, and nucleus raphé dorsalis project rostrally towards the brain areas of higher function. 16

17 Relevance to Addiction Historically, the 5HT systems of the CNS have been thought to be not involved in addiction. Pharmacological treatment with 5HT antagonists either direct or indirect has proven to be not successful in addiction treatment. Pharmacological treatment with 5HT agonists has proven similarly not successful. However, recent animal studies suggest the possibility that adaptations in 5HT function(s) may serve as a neuronal switch from controlled to compulsive drug use. A reduction in basal 5HT levels and specific down regulation of 5HT 2C receptor function may be involved in increasing impulsive choice a classical aspect of drug addiction. Gamma Aminobutyric Acid () ɣ Aminobutyric acid () is an amino acid neurotransmitter that is the principal inhibitory neurotransmitter in the mammalian CNS, regulating neuronal excitability throughout the nervous system. Two principal receptor complexes are known. Receptor structure and function Relevance to addiction Structure Synthesis and degradation is synthesized in the brain from glutamate using the enzyme L glutamic acid decarboxylase, with pyridoxal phosphate (the active form of vitamin B6) as a cofactor. is converted back to glutamate by a metabolic pathway called the shunt. The enzyme transaminase catalyzes the conversion of 4 aminobutanoic acid and 2 oxoglutarate into succinic semialdehyde and glutamate. Succinic semialdehyde is then oxidized into succinic acid by succinic semialdehyde dehydrogenase and as such enters the citric acid cycle as a usable source of energy. Receptors acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in both presynaptic and postsynaptic neuronal membranes. This binding causes the opening of ion channels to allow the flow of either negatively charged chloride ions into the cell or positively charged potassium ions out of the cell. This action results in a negative change in the transmembrane potential, usually causing hyperpolarization. Two general classes of receptor are known: A ionotropic receptors in which the receptor is part of a ligand gated ion channel complex, and B metabotropic receptors, which are G protein coupled receptors that open or close ion channels via G proteins and secondmessenger cascades. The A Receptor The A receptor is an ionotropic receptor and ligand gated ion channel. Its endogenous ligand is, the principal inhibitory neurotransmitter in the CNS. Upon activation, the A receptor selectively conducts Cl through its pore, resulting in hyperpolarization of the neuron causing neuronal inhibition. The active site of the A receptor is the binding site for and several drugs such as muscimol and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, Z drugs (Zolpidem [Ambien], Zaleplon [Sonata], Eszopiclone [Lunesta], etc), barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin. A receptors occur in all organisms that have a nervous system. Due to their wide distribution within the CNS, A receptors play a role in virtually all brain functions. 17

18 A Receptor Structure Top: side view of the receptor imbedded in a cell membrane. Bottom: view of the receptor from the extracellular face of the membrane. The subunits are labeled according to standard A nomenclature and the approximate locations of the and benzodiazepine (BZ) binding sites are noted (between the α and β subunits and between the α and γ subunits respectively). A Receptor Structural Schematic Schematic diagram of a A receptor protein ((α1) 2 (β2) 2 (γ2)) which illustrates the five combined subunits that form the protein, the chloride (Cl ) ion channel pore, the two active binding sites at the α1 and β2 interfaces, and the benzodiazepine (BDZ) allosteric binding site. The A Receptor The ionotropic A receptor protein complex is the target of the benzodiazepines and barbiturates. Benzodiazepines do not bind to the same receptor site on the protein complex as (whose binding site is located between α and β subunits), but to distinct benzodiazepine binding sites situated at the interface between the α and γ subunits. In order for A receptors to be sensitive to the action of benzodiazepines they need to contain an α and a γ subunit, between which the benzodiazepine binds. Once bound, the benzodiazepine locks the A receptor into a conformation which increases s affinity for the A receptor, increasing the frequency of opening of the associated chloride ion channel and hyperpolarizing the membrane. This potentiates the inhibitory effect of the available leading to sedative and anxiolytic effects. Barbiturates bind to a site distinct from the benzodiazepine binding site. Barbiturates also produce different effects on binding. Whereas benzodiazepines cause chloride channel opening to occur more often, barbiturates cause the duration of chloride channel opening to become longer. As these are separate modulatory effects, they can both take place at the same time. Thus, the combination of benzodiazepines with barbiturates is strongly synergistic, and can be dangerous if dosage is not strictly controlled. Barbiturate action at this complex is less dependent upon endogenous levels; this may explain why some benzodiazepineresistant patients obtain therapeutic effect from barbiturates. The B Receptor B receptors are metabotropic transmembrane receptors that are linked via G proteins to potassium channels. Receptor activation stimulates the opening of K + channels hyperpolarizing the neuron. This prevents sodium channels from opening, action potentials from firing, and voltage dependent calcium channels from opening, and so stops neurotransmitter release. Thus B receptors are inhibitory receptors. B receptors also reduce the activity of adenylyl cyclase and decrease the cell s Ca 2+ conductance. B Receptors are similar in structure to (and in the same receptor family with) metabotropic glutamate receptors. There are two subtypes of the receptor, B1 and B2. These assemble as heterodimers in neuronal membranes by linking up via their intracellular C termini. B receptors are involved in the behavioral actions of ethanol and gammahydroxybutyric acid (GHB). Baclofen is a direct B agonist; gamma vinyl is an indirect B agonist. Neuroanatomic Pathways ergic neurons are profusely distributed throughout the CNS. is the most widespread inhibitory neurotransmitter known. Relevance to Addiction The A receptor is the site of action of numerous addictive drugs including benzodiazepines, barbiturates, alcohol, the Z drugs, and others. Desensitization of the A receptor by chronic exposure to addictive drugs may be involved in addictive drug withdrawal symptoms. ergic neurons synapse upon and modulate all neuronal systems known to be involved in addiction including the DA mediated reward/pleasure circuits, the DA mediated drug triggered relapse circuits, the NE mediated stress triggered relapse circuits, the CRF mediated stress triggered relapse circuits, and the glutamate mediated environmental cue triggered relapse circuits. These facts, on their face, would appear to make the ergic system a logical target for development of anti addiction, anti craving, antirelapse pharmacotherapies. Indeed, promising preliminary anti addiction effects have been reported for baclofen and gamma vinyl. However, the ubiquitous anatomic distribution of ergic neurons in the CNS makes it highly likely that ergically active pharmacotherapeutic compounds would have unacceptable side effects. 18

19 Glutamate Glutamate is an amino acid neurotransmitter that is the principal excitatory neurotransmitter in the mammalian CNS, regulating neuronal excitability throughout the nervous system. Two principal glutamate receptor classes are known. Ionotropic Metabotropic Receptor structure and function Relevance to addiction Glutamate Structure Synthesis and degradation Synthesis and degradation Synthesis and degradation Glutamate Receptors Glutamate receptors are synaptic receptors located primarily on the membranes of neurons. Glutamate (glutamic acid) is abundant in the human body, but particularly in the nervous system and especially in the brain where it is the most prominent neurotransmitter and the brain s main excitatory neurotransmitter. Glutamate receptors are responsible for glutamate mediated postsynaptic excitation of neural cells, and are especially implicated in learning, memory, synaptic plasticity, and excitotoxic neuronal damage. Two general classes of glutamate receptor are known: ionotropic and metabotropic. Three glutamate ionotropic receptors are known: the NMDA receptor, the kainate receptor, and the AMPA receptor. Subtypes of these receptors are known. The metabotropic glutamate receptor (mglur) consists of three families of receptors: Group I, Group II, and Group III. 19

20 Ionotropic Glutamate Receptors 1a NMDA receptor: The N methyl D aspartate receptor (NMDA receptor) is an ionotropic glutamate receptor, important in controlling synaptic plasticity and memory. Ionotropic Glutamate Receptors 1b Depiction of an activated NMDAR. Glutamate is in the glutamate binding site and glycine is in the glycine binding site. Allosteric sites that would cause inhibition of the receptor are not occupied. NMDARs require the binding of two molecules of glutamate or aspartate and two of glycine. Activation of the NMDA receptor results in the opening of an ion channel that is nonselective to cations with an equilibrium potential near 0 mv. A property of the NMDA receptor is its voltage dependent activation, a result of ion channel block by extracellular Mg 2+ and Zn 2+ ions. This allows the flow of Na + and small amounts of Ca 2+ ions into the cell and K + out of the cell to be voltagedependent. The NMDA receptor is distinct in two ways: first, it is both ligandgated and voltage dependent; second, it requires co activation by two ligands: glutamate and either D serine or glycine. The NMDA receptor forms a heterotetramer between two GluN1 and two GluN2 subunits. Multiple receptor isoforms with distinct brain distributions and functional properties arise by selective splicing of GluN1 transcripts and differential expression of GluN2 subunits. Ionotropic Glutamate Receptors 1c GluN1: There are eight variants of the GluN1 subunit produced by alternative splicing of the GRIN1 gene: Ionotropic Glutamate Receptors 1d GluN2 subunit in vertebrates GluN1 1a, GluN1 1b; GluN1 1a is the most abundantly expressed form. GluN1 2a, GluN1 2b; GluN1 3a, GluN1 3b; GluN1 4a, GluN1 4b; GluN2: Four distinct isoforms of the GluN2 subunit are expressed in vertebrates GluN2A, GluN2B, GluN2C, GluN2D. They contain the bindingsite for the neurotransmitter glutamate. More importantly, each GluN2 subunit has a different intracellular C terminal domain that can interact with different sets of signaling molecules. Unlike GluN1 subunits, GluN2 subunits are expressed differentially across various cell types and control the electrophysiological properties of the NMDA receptor. One particular subunit, GluN2B, is mainly present in immature neurons and in extrasynaptic locations. Ionotropic Glutamate Receptors 1e Known NMDA receptor agonists include: D Cycloserine, L aspartate, Quinolinate, D serine, L alanine Known NMDA partial agonists include: N Methyl D aspartic acid (NMDA), 3,5 dibromo L phenylalanine Known NMDA receptor agonists include: Amantadine, Ketamine, Phencyclidine (PCP), Nitrous oxide, Dextromethorphan, Dextrorphan, Memantine, AP5, Ethanol, Atomoxetine Known dual opioid and NMDA receptor antagonists include: Methadone, Tramadol, Dextropropoxyphene, Ibogaine Ionotropic Glutamate Receptors 1f NMDA receptor functions: Calcium flux through NMDARs is thought to be critical in synaptic plasticity, a cellular mechanism for learning and memory. NMDAR mediated currents are directly related to membrane depolarization. NMDA agonists therefore exhibit fast Mg 2+ unbinding kinetics, increasing channel open probability with depolarization. This property is fundamental to the role of the NMDA receptor in memory and learning, and it has been suggested that this channel is a biochemical substrate of Hebbian learning, where it can act as a coincidence detector for membrane depolarization and synaptic transmission. NMDA receptors are also believed to play a key role in a wide range of pathological processes (e.g., neuronal excitotoxicity). 20

21 Ionotropic Glutamate Receptors 2a Kainate receptor: Kainate receptors (KARs) are ionotropic receptors for the neurotransmitter glutamate. They were first identified as a distinct receptor type through their selective activation by the agonist kainate. KARs are less understood than NMDA and AMPA receptors, the other ionotropic glutamate receptors. Postsynaptic kainate receptors are involved in excitatory neurotransmission. Presynaptic kainate receptors have been implicated in inhibitory neurotransmission by modulating release of the inhibitory neurotransmitter through a presynaptic mechanism. There are five types of kainate receptor subunits: GluK1, GluK2, GluK3, GluK4, and GluK5, which are similar to NMDA and AMPA receptor subunits and can be arranged in different ways to form a tetramer, a four subunit receptor. GluR5 7 can form homomers (except a receptor composed entirely of GluR5) and heteromers (except a receptor composed of both GluR5 and GluR6). GluK1 and GluK2 can only form functional receptors by combining with one of the GluK5 7 subunits. Ionotropic Glutamate Receptors 2b Kainate receptor: The ion channel formed by kainate receptors is permeable to sodium and potassium ions. The single channel conductance of kainate receptor channels is similar to that of AMPA channels. However, rise and decay times for postsynaptic potentials generated by KARs are slower than for AMPA postsynaptic potentials. Their permeability to Ca 2+ is usually very slight but varies with subunits. Kainate receptors have both presynaptic and postsynaptic actions. They have a somewhat more limited distribution in the brain than NMDA and AMPA receptors, and their function is less known. The convulsant kainic acid induces seizures, in part, by activation of kainate receptors containing the GluK2 subunit and also probably via AMPA receptors. KARs play only a minor role in synaptic signaling. Rather, KARs may have a more subtle role in synaptic plasticity, affecting the likelihood that the postsynaptic cell will fire in response to future stimulation. Activating KARs in the presynaptic cell can affect the amount of neurotransmitters that are released. This effect appears to occur quickly and last for a long time. Ionotropic Glutamate Receptors 2c Kainate receptor: Ethanol is a KAR antagonist. So too is Tezampanel, which suppresses withdrawal symptoms from morphine and other opioids, and suppresses the development of opioid tolerance. Ionotropic Glutamate Receptors 3a AMPA receptor: The α amino 3 hydroxy 5 methyl 4 isoxazolepropionic acid receptor (AMPA receptor, AMPAR) is a non NMDA type ionotropic transmembrane receptor for glutamate that mediates fast synaptic transmission in the CNS. AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system. AMPARs are composed of four subunits GluR1, GluR2, GluR3, and GluR4, which combine to form tetramers. Most AMPARs are heterotetrameric, consisting of symmetric dimer of dimers of GluR2 and either GluR1, GluR3 or GluR4. Each AMPAR has four sites to which an agonist (e.g., glutamate) can bind, one for each subunit. The binding site is believed to be formed by the N tail, and the extracellular loop between transmembrane domains three and four. When an agonist binds, these two loops move towards each other, opening the ion pore. The channel opens when two sites are occupied, and increases its current as more binding sites are occupied. AMPARs open and close quickly; they are responsible for most of the fast excitatory synaptic transmission in the central nervous system. Ionotropic Glutamate Receptors 3b AMPA receptor: The AMPAR s permeability to calcium and other cations, such as sodium and potassium, is governed by the GluR2 subunit. If an AMPAR lacks a GluR2 subunit, it will be permeable to sodium, potassium, and calcium. The presence of a GluR2 subunit almost always renders the channel impermeable to calcium. Almost all AMPARs in the CNS contain the GluR2 subunit. This means that the principal ions gated by AMPARs are sodium and potassium, distinguishing AMPARs from NMDA receptors (the other main ionotropic glutamate receptors in the brain), which permit calcium influx. Ionotropic Glutamate Receptors 3c AMPA receptor: AMPA receptors (AMPAR) are both glutamate receptors and cation channels that are integral to synaptic transmission and plasticity at many postsynaptic membranes. One of the most widely and thoroughly investigated forms of plasticity in the nervous system is long term potentiation (LTP). There are two necessary components of LTP: presynaptic glutamate release and postsynaptic depolarization. LTP produces a sustained increase in the amplitude of the excitatory postsynaptic potential (EPSP), and is thought to be an important physiological substrate of learning and memory. AMPARs play an integral role in LTP. The underlying physiological substrate for the increase in EPSP size is postsynaptic upregulation of AMPARs in the membrane, which is accomplished through the interactions of AMPARs with many cellular proteins. The simplest explanation for LTP is as follows. Glutamate binds to postsynaptic AMPARs and another glutamate receptor, the NMDA receptor (NMDAR). Ligand binding causes the AMPARs to open, and Na + flows into the postsynaptic cell, resulting in depolarization. NMDARs, on the other hand, do not open directly because their pores are occluded at resting membrane potential by Mg 2+ ions. NMDARs can open only when a depolarization from the AMPAR activation leads to repulsion of the Mg 2+ cation out into the extracellular space, allowing the pore to pass current. Unlike AMPARs, however, NMDARs are permeable to both Na + and Ca 2+. The Ca 2+ that enters the cell triggers AMPAR upregulation, which results in the long lasting increase in EPSP size underlying LTP. 21

22 Ionotropic Glutamate Receptors 3d AMPA receptor: One of the key indicators of LTP induction is the increase in the ratio of AMPARs to NMDARs following high frequency stimulation. AMPARs appear to be trafficked from the dendrite into the synapse and incorporated into the perisynaptic membrane through a series of signaling cascades. Once AMPA receptors are transported to the perisynaptic region, they are then trafficked to the postsynaptic density (PSD). At present levels of understanding, this is believed to constitute an important substrate for morphological synaptic remodeling in learning and memory. Metabotropic Glutamate Receptors 1a Group I metabotropic glutamate receptors: Metabotropic glutamate receptors (mglurs) are a type of glutamate receptor that are members of the group C family of G protein coupled receptors, or GPCRs. Their activation sets in motion a cascade of second messenger events that ultimately result in ion fluxes that result in neuronal excitation. mglurs appear to be involved in a wide variety of CNS functions including learning, memory, anxiety, and the perception of pain. They are found on pre and postsynaptic neurons in synapses throughout the CNS. Like other metabotropic receptors, mglurs have seven transmembrane domains that span the neuronal membrane. Eight different types of mglurs, labeled mglur 1 to mglur 8, are divided into groups I, II, and III based on receptor structure and physiological activity. Metabotropic Glutamate Receptors 1b Group I metabotropic glutamate receptors: Group I mglurs consist of two types mglur 1 and mglur 5. mglur 1 and mglur 5 receptors are G q coupled. Their activation leads via a complex cascade of second messengers to increased Na + and K + conductances. Their activation leads to neuronal excitation. Their activation can cause more glutamate to be released from the presynaptic cell, but they also increase inhibitory postsynaptic potentials (IPSPs). They can also inhibit glutamate release and can modulate voltage dependent calcium channels. They are primarily located postsynaptically. Their activation increases NMDA receptor activity. mglur 1 and mglur 5 receptors are distinguished from each other primarily on the basis of the agonists that they respond to. Metabotropic Glutamate Receptors 1c Group II metabotropic glutamate receptors: Group II mglurs consist of two types mglur 2 and mglur 3. mglur 2 and mglur 3 receptors are G i /G o coupled. Their activation leads to inhibition of the enzyme adenylyl cyclase, which forms camp from ATP. This prevents the formation of camp. These receptors are involved in presynaptic inhibition, and do not appear to affect postsynaptic membrane potentials by themselves. Neuronal excitation is the end result. They are primarily located presynaptically. Their activation decreases NMDA receptor activity. mglur 2 and mglur 3 receptors are distinguished from each other primarily on the basis of the agonists that they respond to. Like other glutamate receptors, Group II mglurs have been shown to be involved in synaptic plasticity and in neurotoxicity and neuroprotection. They are involved in long term potentiation and long term depression. Metabotropic Glutamate Receptors 1d Group III metabotropic glutamate receptors: Group III mglurs consist of four types mglur 4, mglur 6, mglur 7, and mglur 8. Like Group II mglurs, Group III mglurs are G i /G o coupled. Their activation leads to inhibition of the enzyme adenylyl cyclase, and prevention of the camp formation. These receptors are involved in presynaptic inhibition, and do not appear to affect postsynaptic membrane potentials by themselves. Neuronal excitation is the end result. They are primarily located presynaptically. Their activation decreases NMDA receptor activity. mglur 4, mglur 6, mglur 7, and mglur 8 receptors are distinguished from each other primarily on the basis of the agonists that they respond to. Like other glutamate receptors, Group III mglurs have been shown to be involved in synaptic plasticity and in neurotoxicity and neuroprotection. They are involved in long term potentiation and long term depression. Neuroanatomic Glutamate Pathways Glutamatergic neurons are profusely distributed throughout the CNS. Glutamate is the most wide spread excitatory neurotransmitter known. Of particular relevance to addiction are glutamatergic neuronal pathways originating in the frontal and cingulate cortices, and projecting to the core DA reward/pleasure/relapse circuitry running from ventral tegmental area to nucleus accumbens. Also of relevance to addiction are glutamatergic neuronal pathways originating in the ventral subiculum of the hippocampus and in the basolateral complex of the amygdala, and projecting to the core DA reward/pleasure/relapse circuitry running from ventral tegmental area to nucleus accumbens. (see next slide for schematic). 22

23 Acc ENK FCX VP Opiates OFT VTA GLU ABN ICSS HIPP AMYG GLU CRF OPIOID DYN DA BNST HYPOTHAL 5HT OPIOID 5HT Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids NE Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine LC NE LAT TEG PAG END 5HT Raphé RETIC To dorsal horn Relevance to Addiction Glutamatergic neurons synapse upon and modulate many neuronal systems known to be involved in addiction. Of special relevance are: 1) The glutamatergic pathways running from the frontal cortices to the core DA rewardrelated circuitry originating in the ventral tegmental area and projecting to the nucleus accumbens. These pathways are believed to convey information relating to decisionmaking and impulsivity. 2) The glutamatergic pathways running from the hippocampus and basolateral amygdala to the core DA reward related circuitry originating in the ventral tegmental area and projecting to the nucleus accumbens. These pathways are known to constitute the environmental cue triggered relapse circuits of the brain. These facts make the glutamatergic system a logical target for development of anti addiction, anti craving, anti relapse pharmacotherapies. In fact, mglur antagonists and negative allosteric modulators show promise in animal models. Neuropeptides Corticotropin releasing factor (CRF) is a peptide hormone and neurotransmitter involved in the stress response. It is also involved in stresstriggered relapse to drug seeking and drug taking behavior(s). Two receptors are known. CRF1 CRF2 Receptor structure and function Relevance to addiction CRF Structure The 41 amino acid sequence of CRF is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII CRF Corticotropin releasing factor (also known as corticotropin releasing hormone CRH) is a peptide hormone and neurotransmitter involved in the stress response. It is a 41 amino acid peptide derived from a 196 amino acid preprohormone. As a hormone, its main function is the stimulation of the pituitary synthesis of ACTH, as part of the HPA Axis. In this role, CRF is secreted by the paraventricular nucleus of the hypothalamus in response to stress. It is released at the median eminence from neurosecretory terminals of these neurons into the primary capillary plexus of the hypothalamo hypophyseal portal system. This system carries CRF to the anterior lobe of the pituitary, where it stimulates corticotropes to secrete adrenocorticotropic hormone (ACTH) and other biologically active substances (β endorphin). ACTH stimulates the synthesis of cortisol, glucocorticoids, mineralocorticoids and DHEA. As a neurotransmitter, it is used by one of the two stress triggered relapse circuits in the brain. CRF Receptors Corticotropin releasing factor receptors (CRFRs) are a G protein coupled receptor family that binds corticotropinreleasing factor (CRF). Two CRF receptors are known: 1) CRF 1 2) CRF 2 23

24 CRF 1 Protein Structure Neuroanatomic CRF Pathways Of relevance to addiction is the CRF circuit originating in the central nucleus of the amygdala and projecting to the bed nucleus of the stria terminalis (see next slide for schematic). This neuroanatomic circuit is known to mediate stress triggered relapse to drug seeking and drug taking behavior(s). Acc ENK FCX VP Opiates OFT VTA GLU ABN ICSS HIPP AMYG GLU CRF OPIOID DYN DA BNST HYPOTHAL 5HT OPIOID 5HT Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids NE Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine LC NE LAT TEG PAG END 5HT Raphé RETIC To dorsal horn Relevance to Addiction Recent research has linked the activation of the CRF 1 receptor with the euphoric feelings that accompany alcohol consumption. A CRF 1 receptor antagonist developed by Pfizer (CP 154,526) is under investigation for the potential treatment of alcoholism. Other CRF 1 receptor antagonists (α helical CRF, antalarmin, pexacerfont) have been extensively studied in animal models of stress triggered relapse to drug seeking and drug taking behavior(s), with extremely promising findings. However, no human trials have been carried out. Endogenous Opioids Opioid peptides are short sequences of amino acids that are produced within the body and bind to opioid receptors in the brain; opiate drugs mimic the effects of these peptides. Three classes of receptors for these endogenous opioids are known. Receptors in these classes have subtypes. Receptor structure and function Relevance to addiction Endogenous Opioids The endogenous opioid peptides are produced by the body itself, by proteolytic cleavage of precursor proteins. The effects of these opioid peptides vary, but they all resemble those of opiates. Brain opioid peptide systems are known to play an important role in motivation, emotion, attachment behavior, response to stress and pain, and food intake. There are four classes of endogenous opioid peptides: 1) Endorphins 2) Enkephalins 3) Dynorphins 4) Endomorphins Nociceptin is sometimes listed as an endogenous opioid. But it is not strictly speaking an opioid, as it does not act at classical opioid receptors (mu, kappa, delta), and its actions are not antagonized by the opioid antagonist naloxone. Nociceptin does have opioid like actions (especially potent analgesia) and is very widely distributed in the CNS (both brain and spinal cord). There is evidence that it may be involved in opioid induced hyperalgesia. Nociceptin is probably best described as an opioid related neuropeptide neurotransmitter. 24

25 Endorphins 1 Endorphins ( endogenous morphine ) are endogenous opioid inhibitory neuropeptides. They are produced in the CNS and pituitary gland. Three endorphins are currently known: 1) Beta Endorphin (β Endorphin) 2) Alpha Neoendorphin (α Neoendorphin) This is a is an endogenous opioid peptide with a decapeptide structure and the amino acid sequence Tyr Gly Gly Phe Leu Arg Lys Tyr Pro Lys. Extremely little is known about the functions of this opioid peptide. 3) Beta Neoendorphin (β Neoendorphin) This is an endogenous opioid peptide with a nonapeptide structure and the amino acid sequence Tyr Gly Gly Phe Leu Arg Lys Tyr Pro. Extremely little is known about the functions of this opioid peptide. Endorphins 2 β Endorphin is released into blood from the pituitary gland and into the spinal cord and brain from hypothalamic neurons (although other parts of the brain may well contain neurons that similarly synthesize and use β endorphin as a neurotransmitter. β endorphin is a cleavage product of pro opiomelanocortin (POMC), which is also the precursor hormone for adrenocorticotrophic hormone (ACTH). The behavioural effects of β endorphin are exerted by its actions in the brain and spinal cord. β endorphin has highest affinity for mu 1 (μ 1 ) opioid receptors, slightly lower affinity for μ 2 and delta (δ) opioid receptors, and very low affinity for kappa 1 (κ 1 ) opioid receptors. μ Opioid receptors are the main receptor through which morphine acts. μ Opioid receptors are often presynaptic, and inhibit neurotransmitter release. Via that mechanism, they inhibit the release of the inhibitory neurotransmitter, and disinhibit DA pathways, causing more DA to be released. By hijacking this process, exogenous opioids (e.g., heroin) cause inappropriate DA release, leading to the drug induced high and to aberrant synaptic plasticity, which likely plays an important role in addiction. Opioid receptors mediate many other important functions in brain and periphery e.g., modulating pain, cardiac, gastric, and vascular functions, and possibly panic and satiety. Also, opioid receptors are found at postsynaptic locations as well as at presynaptic locations. Enkephalins 1 Enkephalins are pentapeptides importantly involved in regulating nociception in the body. Enkephalins are internally derived and bind to the body s opioid receptors. Two enkephalins are currently known: Enkephalins 2 Structural correlation between met enkephalin (left) and morphine (right) 1) Methionine enkephalin (Met enkephalin) Tyr Gly Gly Phe Met. 2) Leucine enkephalin (Leu enkephalin) Tyr Gly Gly Phe Leu. The met enkephalin peptide sequence is coded for by the enkephalin gene; the leu enkephalin peptide sequence is coded for by both the enkephalin gene and the dynorphin gene. Enkephalins 3 The receptors for the enkephalins are the delta (δ) opioid receptors. All endogenous opioid (μ, δ, and κ) receptors are G protein coupled receptors. Dynorphins 1 Dynorphins (Dyn) are a class of opioid peptides that arise from the precursor protein prodynorphin. Prodynorphin is cleaved by proprotein convertase 2 (PC2) to produce multiple opioid peptides: dynorphin A, dynorphin B, α neoendorphin, and β neo endorphin. Depolarization of a neuron containing prodynorphin stimulates PC2 processing, which occurs within synaptic vesicles in the presynaptic terminal. Occasionally, prodynorphin is not fully processed, leading to the release of big dynorphin. This 32 amino acid molecule consists of both dynorphin A and dynorphin B. Dynorphins are stored in large dense core vesicles that are considerably larger than vesicles storing other neurotransmitters. These large dense core vesicles differ from small synaptic vesicles in that a more intense and prolonged stimulus is needed to cause the large vesicles to release their contents into the synaptic cleft. Dense core vesicle storage is characteristic of opioid peptides storage. Although dynorphins are found widely distributed in the CNS, they have the highest concentrations in the hypothalamus, medulla, pons, midbrain, and spinal cord. 25

26 Dynorphins 2 The receptors for the dynorphins are the kappa (κ) opioid receptors. Endomorphins 1 The endomorphins are the most recently discovered endogenous opioid peptides. At present levels of knowledge, two endomorphin opioid peptides are known: endomorphin 1 (Tyr Pro Trp Phe NH2) and endomorphin 2 (Tyr Pro Phe Phe NH2). Both are tetrapeptides, and have the highest known affinity and specificity for the μ opioid receptor. Endomorphin 1 is located in the nucleus of the solitary tract, the periventricular hypothalamus, and the dorsomedial hypothalamus, where it is found within histaminergic neurons and may regulate sedation and arousal. It is assumed that endomorphins are the cleavage products of a larger precursor, but this polypeptide or protein has not yet been identified. Endomorphins 1 Endormorphin 1 (left); Endomorphin 2 (right) Endogenous Opioids Receptors 1 μ Opioid receptors 1 μ Opioid receptors (MORs) are a class of opioid receptors with high affinity for enkephalins and β endorphin but low affinity for dynorphins. The prototypical μ receptor agonist is morphine. Three variants of the μ opioid receptor are well characterized, though reverse transcriptase PCR has identified up to 10 total splice variants in humans. The three well characterized variants are: μ 1 More is known about the μ 1 opioid receptor than the other variants. μ 2 μ 3 First described in 2003; responsive to opiate alkaloids but not opioid peptides. MORs can exist either presynaptically or postsynaptically depending upon cell type. MORs exist mostly presynaptically in the periaqueductal gray region and in the dorsal horn of the spinal cord (the substantia gelatinosa); in both regions MORs play a crucial role in synaptic pain gating (hence, analgesia). Other areas where MORs have been located include the olfactory bulb external plexiform layer, nucleus accumbens, in several layers of cerebral cortex, in some amygdala nuclei, thalamus, rostral ventromedial medulla, and the nucleus of the solitary tract. MORs mediate acute changes in neuronal excitability via disinhibition of presynaptic release of. Endogenous Opioids Receptors 1 μ Opioid receptors 2 MOR activation by an agonist such as morphine causes analgesia, sedation, slightly reduced blood pressure, itching, nausea, euphoria, decreased respiration, miosis (constricted pupils), and decreased bowel motility often leading to constipation. Some of these effects, such as analgesia, sedation, euphoria, and decreased respiration, tend to lessen with continued use as tolerance develops. Miosis and reduced bowel motility tend to persist; little tolerance develops to these effects. As with other G protein coupled receptors, MOR signaling is terminated through several different mechanisms, which are upregulated with chronic use, leading to rapid tachyphylaxis. The most important regulatory proteins for the MOR are the β arrestins Arrestin β 1 and Arrestin β 2, and the RGS (Regulators of G protein signaling) proteins RGS4, RGS9 2, RGS14 and RGSZ2. Long term or high dose opioid use may also lead to additional mechanisms of tolerance. This includes downregulation of MOR gene expression, so the number of receptors on the cell surface is reduced, as opposed to the more short term desensitization induced by β arrestins or RGS proteins. Another long term adaptation to opioid use can be upregulation of glutamatergic and other brain pathways which can exert an opioid opposing effect and so reduce the effects of opioid drugs by altering downstream pathways, despite MOR activation. Endogenous Opioids Receptors 1 μ Opioid receptors 3 Functions mediated by the three well characterized MOR variants are: μ 1 analgesia, physical dependence. μ 2 euphoria, physical dependence, respiratory depression, miosis, reduced GI motility. μ 3 vasodilatation. 26

27 Endogenous Opioids Receptors 2 δ Opioid receptor 1 Endogenous Opioids Receptors 3 κ Opioid receptor 1 δ opioid receptors (DORs) are a class of opioid receptors that has enkephalins as endogenous ligands. The two well characterized variants are: δ 1 δ 2 DORs are found in both the brain and in peripheral sensory neurons. In the brain, they are found in pontine nuclei, amygdala, olfactory bulb, and deep layers of cortex. They are also found as co neurotransmitters in neuronal tracts within or synapsing upon the core DA reward/pleasure/relapse circuitry. Activation of DORs produces some analgesia, although less than that of MOR agonists. DOR activation also may be involved in the opiate induced high and thus in addiction. DOR activation produces substantially less respiratory depression than MOR activation. Until recently, there were few pharmacological tools for the study of DORs. As a consequence, our understanding of their function is much more limited than those of other opioid receptors for which selective ligands have long been available. However there are now several selective DOR agonists available, including peptides such as DPDPE and deltorphin II. Selective DOR antagonists are also available, with the best known being the opiate derivative naltrindole. κ opioid receptors (KORs) are a class of opioid receptors that has dynorphins as endogenous ligands. The three well characterized variants are: κ 1 κ 2 κ 3 KORs are found in both the brain and in peripheral sensory neurons. In the brain, they are found in hypothalamus, periaqueductal gray, and claustrum. They are also found as co neurotransmitters in neuronal tracts within or synapsing upon the core DA reward/pleasure/relapse circuitry. They are also found in the substantia gelatinosa of the dorsal spinal cord. Activation of DORs produces some analgesia, although very much less than that of MOR agonists. DOR activation also may be involved in the opiate induced physical dependence. DOR activation also produces convulsant effects. Of high relevance to addiction, DOR activation produces strong dysphoria. This may provide a natural addiction control mechanism. Endogenous Opioids Receptors 3 κ Opioid receptor 2 Only one KOR cdna clone has been identified. Hence, these receptor subtypes likely arise from interaction of one KOR protein with other membrane associated proteins. KOR activation is coupled to the G i/o protein, which activates phosphodiesterase. Phosphodiesterases break down camp, producing an inhibitory effect in neurons. KORs also couple to inwardly rectifying K + and to N type calcium ion channels. Recent studies also show that agonist induced KOR stimulation, like other G protein coupled receptors, can result in the activation of mitogen activated protein kinases (MAPK). The synthetic alkaloid ketazocine and the terpenoid natural product salvinorin A are potent and selective KOR agonists. The KOR also mediates the hallucinogenic side effects of opioids such as pentazocine. It is widely accepted that KOR agonists (and partial agonists) have dissociative and delirium inducing effects, as exemplified by salvinorin A. Such effects are undesirable in medicinal drugs. Thus, the KOR is likely not a promising target for medication development. The hallucinogenic effects of drugs such as butorphanol, nalbuphine, and pentazocine serve to limit their abuse potential. In the case of salvinorin A, a structurally novel KOR agonist, these deliriant and dissociative effects are sought after, even though the experience is often termed dysphoric by the user. While salvinorin A is considered a hallucinogen, its effects are qualitatively different than those produced by classical psychedelic hallucinogens such as LSD or mescaline. Endogenous Opioids Receptors 3 κ Opioid receptor 3 Crystallographic structure of the human κ 1 homodimer imbedded in a cartoon of a lipid bilayer. Each monomer is individually color coded (blue = N terminus; red = C terminus). The receptor is bound to the selective KOR ligand JDTic. Endogenous Opioids Receptors 4 Endomorphin receptor 1 As noted above, endomorphins appear to exert their effects by acting upon the MOR. Relevance to Addiction The relevance of the endogenous opioid systems of the CNS to addiction is enormous. First, they are obviously the neural substrates upon which opiate drugs of abuse act to exert their addictive effects. Second and perhaps more importantly the endogenous opioid systems of the CNS appear to be involved in the CNS actions of virtually all drugs of abuse. Thus, the actions of addictive drugs on brain reward circuits and neural substrates are in almost all cases naloxone blockable even for such unexpected and unlikely addictive compounds as Δ 9 tetrahydrocannabinol (THC; the psychoactive and addictive constituent of marijuana and hashish), phencyclidine (PCP), and ethanol. This strongly implies that there are endogenous opioid neuronal substrates and mechanisms at play in the addictive process for virtually all addictive substances. In addition, neuroadaptive mechanisms such as long term potentiation (LTP) and long term depression (LTD) are believed to play a significant role in the addiction process. Addiction alters prefrontal cortical regulation of the nucleus accumbens, including reduced ability to induce LTP and LTD. This important potential mechanism of impaired prefrontal regulation of behavior has recently been shown to apply to opiates as well as cocaine. Heroin self administration results in impaired LTP and LTD in nucleus accumbens following in vivo stimulation of the prefrontal cortex. Such data suggest that compromised synaptic plasticity in prefrontal to accumbens projections may be a common feature of addiction, with possible endogenous opioid system involvement. 27

28 Endocannabinoids Endocannabinoids are fatty acid neurotransmitters that are produced within the body and bind to specific receptors in the brain; endocannabinoid function appears to be crucially involved in addiction. Two receptors for these endogenous lipid signaling systems are known. Receptor structure and function Relevance to addiction Endocannabinoids The endocannabinoids are lipid signaling molecules produced in the body by specific synthetic pathways. They are involved in many physiological processes including appetite, pain sensation, motor learning, mood, memory, synaptic plasticity, and addiction. Endocannabinoids mediate the psychoactive effects of cannabis. At present levels of knowledge, two major endocannabinoids are known: Anandamide (N arachidonoylethanolamide, AEA) 2 Arachidonoylglycerol (2 AG) Endocannabinoids are all eicosanoids. AEA and 2 AG are physiological ligands for the cannabinoid receptors. The enzymes that synthesize and degrade AEA and 2 AG are known, and the metabolic pathways well described. At present levels of knowledge, two G protein coupled cannabinoid receptors (CB1 and CB2) are known. AEA and 2 AG are located and known to function in the central and peripheral nervous systems. Endocannabinoids are reverse signaling molecules within the nervous system. They are released from dendrite and/or cell body, flow backwards across the synapse, and activate receptors located on axon terminals. Endocannabinoids In addition to AEA and 2 AG, the following compounds are currently believed to also function as endocannabinoids: Noladin ether N Arachidonoyldopamine Viradhamine Endocannabinoids 1 Anandamide (AEA) AEA is synthesized on site and on demand. Synthesis appears to be exclusive; both endocannabinoids are not co synthesized. This exclusion is based on synthesis specific channel activation: a recent study found that in the bed nucleus of stria terminalis, activation of mglur 1/5 receptors triggered synthesis of AEA, while calcium entry via voltage sensitive calcium channels produced an L type current resulting in 2 AG production. Enzymatic activation within the postsynaptic neuron catalyzes the first step of AEA synthesis by converting phosphatidylethanolamine, a membrane resident phospholipid, into N acylphosphatidylethanolamine (NAPE). NAPE selective phospholipase D cleaves NAPE to yield AEA. After interacting with cannabinoid receptors on the pre synaptic neuron, AEA is taken up by a transporter on the postsynaptic neuron and degraded by fatty acid amide hydrolase (FAAH), which cleaves AEA into arachidonic acid and ethanolamine or monoacylglycerol lipase (MAGL). Glial uptake of AEA and subsequent FAAH mediated inactivation has also been proposed. Endocannabinoids 2 2 Arachidonoylglycerol (2 AG) 2 AG is similarly synthesized on site and on demand. Unlike AEA, formation of 2 AG is calcium dependent and is mediated by the activities of phospholipase C (PLC) and diacylglycerol lipase (DAGL). 2 AG is synthesized from arachidonic acid containing diacylglycerol (DAG) derived from increased inositol phospholipid metabolism by the action of diacylglycerol lipase (DAG lipase). 2 AG can can also be formed via other pathways such as the hydrolysis of the diaclygly derived from phosphatidylcholine (PC) PC and phosphatidic acid by the action of DAG lipase and the hydrolysis of arachidonic acid containing lysophosphatidic acid by the action of a phosphatase. The relative importance of these pathways may depend on the types of cells and stimuli. After interacting with cannabinoid receptors on the pre synaptic neuron, 2 AG is taken up by a transporter on the postsynaptic neuron and degraded by monoacylglycerol lipase (MAG Lipase), fatty acid amide hydrolase (FAAH), and the uncharacterized serine hydrolase enzymes ABHD6 and ABHD12. The exact contribution of each of these enzymes to the degradation of 2 AG in vivo is unknown, although it is estimated that MAG Lipase is responsible for approximately 85% of this activity in the brain. 28

29 AEA NAPE PLD? NAPE Presynaptic neuron NAT 2 AG MGL Neurotransmitter vesicles CB 1 ET Ca 2+ DAGL ET AA COX 2 AG DAG PG PLC AEA Phospholipid NAPE PLD? NAPE NAT? Postsynaptic neuron Endocannabinoids 3 Additional possible endocannabinoids Endocannabinoid research is currently in an enormous state of flux with additional discoveries, insights, and hypotheses on virtually a monthly basis. In addition to AEA and 2 AG, a number of additional structurally similar compounds have been suggested to be additional endocannabinoids. These include Noladine ether, Virodhamine, and N Arachidonoyldopamine. Guindon et al., (2009) Pharmacology of the cannabinoid system, IASP Press HO HO HO Endocannabinoids O O HO HN O HO Anandamide 2 Arachidonoylglycerol HO O O HN HO Endocannabinoids 4a Endocannabinoid Receptors At present levels of knowledge, two endocannabinoid receptors in the body are known. However, cannabinoid and endocannabinoid research is currently in an enormous state of flux with additional discoveries, insights, and hypotheses on virtually a monthly basis. It is believed that additional endocannabinoid receptors may well be discovered. The two that are currently known are the: Noladin ether N Arachidonoyldopamine CB 1 CB 2 NH 2 O O Virodhamine Both receptors are G protein coupled, which means that second messenger cascades intermediate between receptor activation and post receptor ion membrane flows (which, of course, means that second messenger cascades intermediate between receptor activation and post receptor neuronal excitation/inhibition. Endocannabinoids 4b The Endocannabinoid CB 1 Receptor 1 The CB 1 receptor is located primarily in the central and peripheral nervous system. It is activated by the endocannabinoid neurotransmitters AEA and 2 AG; by plant cannabinoids, such as THC (an active ingredient of cannabis); and by synthetic analogues of THC. The CB 1 receptor shares the structure characteristic of all G protein coupled receptors, possessing 7 transmembrane domains connected by 3 extracellular and 3 intracellular loops, an extracellular N terminal tail, and an intracellular C terminal tail. CB 1 receptors may exist as homodimers or form heterodimers or oligomers when coexpressed with other G protein coupled receptors. Known heterodimers include CB 1 A 2 A, CB 1 D 2, and CB 1 orexin 1 ; many more may exist. CB 1 receptors may also possess an allosteric binding site, a target for modulating cannabinoid effects. The CB 1 receptor is a pre synaptic heteroreceptor that modulates neurotransmitter release when activated in a dose dependent, stereoselective and pertussis toxin sensitive manner. Most CB 1 receptors are coupled through G i/o proteins. The CB 1 receptor exerts its effects mainly through activation of G i, which decreases intracellular camp concentration by inhibiting adenylate cyclase, and increases mitogen activated protein kinase (MAP kinase) concentration. In rare cases CB 1 receptor activation may be coupled to G s proteins, which stimulate adenylate cyclase. Endocannabinoids 4b The Endocannabinoid CB 1 Receptor 2 camp serves as a second messenger coupled to a variety of ion channels, including positively influenced inwardly rectifying K + channels (IRK), and calcium channels, which are activated by camp dependent interaction with such molecules as protein kinase A (PKA), protein kinase C (PKC), Raf 1, ERK, JNK, p38, c fos, c jun, and others. In terms of function, the inhibition of intracellular camp shortens the duration of pre synaptic action potentials by prolonging the rectifying potassium A type currents. This inhibition grows more pronounced due to the fact that activated CB 1 receptors also limit calcium entry into the cell, which does not occur through camp but by a direct G proteinmediated inhibition. As presynaptic Ca 2+ entry is a requirement for vesicle release, this CB 1 action decreases the transmitter that enters the synapse upon release. The relative contribution of each of these two inhibitory mechanisms depends on the variance of ion channel expression by cell type. The CB 1 receptor is also allosterically modulated in either a positive or negative manner. In summary, CB 1 receptor activity is coupled to membrane ion channels, in the following manners: Positively to inwardly rectifying and A type outward K + channels. Negatively to D type outward K + channels Negatively to N type and P/Q type Ca 2+ channels 29

30 Cannabinoid CB1 and CB2 Receptors CB1 Mediated Signal Transduction ATP AC AMPc PKA MAPK Gene expression NA+/H+ exchanger K + AA CB 1 Ca 2+ Guindon, Beaulieu and Hohmann (2009) Pharmacology of the cannabinoid system, IASP Press Endocannabinoids 4b The Endocannabinoid CB 1 Receptor 3 The CB 1 receptor is one of the most prolifically distributed neurotransmitter receptors in the CNS (see next slide). The CB 1 receptor is expressed presynaptically at both glutaminergic and ergic interneurons and acts to inhibit release of glutamate and. Repeated administration of CB 1 receptor agonists may result in receptor internalization and/ or a reduction in receptor protein signaling. Limiting glutamate release causes reduced excitation, while limiting release suppresses inhibition, a common form of short term plasticity in which the depolarization of a single neuron induces a reduction in mediated inhibition, so exciting the postsynaptic neuron. Endocannabinoids 4b The Endocannabinoid CB 1 Receptor 4 The CB 1 receptor is widely expressed in all major brain regions (a mouse brain is used below to illustrate). The receptor is relatively absent in much of the thalamus. The CB 1 receptor is extremely widespread throughout the CNS in the olfactory bulb, cortical regions (neocortex, pyriform cortex, hippocampus, and amygdala), several parts of basal ganglia, hypothalamic nuclei, septal area, cerebellar cortex, and many brainstem nuclei (e.g., the periaqueductal gray, where it is involved in pain gating only one of the anatomic sites for cannabinoid induced analgesia). Endocannabinoids 4b The Endocannabinoid CB 1 Receptor 5 The CB 1 receptor is intimately involved in many forms of synaptic plasticity. This has been most extensively studied in the hippocampus. CB 1 receptors are abundant on ergic interneurons of the hippocampus, and densely located on hippocampal pyramidal cells, which release glutamate. Cannabinoids suppress the induction of LTP and LTD in the hippocampus by inhibiting these glutamatergic neurons. By reducing glutamate below the threshold necessary to depolarize NMDA receptors known to be directly related to induction of LTP and LTD cannabinoids play a crucial role in neuroplasticity and in memory. From these hippocampal mechanisms, an as yet unknown complex (presumably fractal like) feedforward network allows the brain to weaken specific synapses while others are enhanced, allowing long term memory to be formed. Endocannabinoids 4b The Endocannabinoid CB 2 Receptor 1 The CB 2 receptor is a G protein coupled receptor structurally related to the CB 1 (the CB 1 receptor being largely responsible for endocannabinoid mediated presynaptic inhibition, the psychoactive properties of THC and other natural cannabinoids). The principal endogenous ligand for the CB 2 receptor is 2 AG. The CB 2 receptor was cloned in 1993 by a research group from Cambridge University looking for a second cannabinoid receptor that could explain the many pharmacological properties of THC not satisfactorily explained by action at the CB 1 receptor. The CB 2 receptor was identified among cdnas based on its similarity in amino acid sequence to the CB 1 receptor. The discovery of the CB 2 receptor helped provide a molecular explanation for the established effects of cannabinoids on the immune system. As commonly seen in G protein coupled receptors, the CB 2 receptor has 7 transmembrane spanning domains, a glycosylated N terminus, and an intracellular C terminus. The C terminus appears to play a critical role in the regulation of ligand induced receptor desensitization and downregulation following repeated agonist application, perhaps causing the receptor to become less responsive to particular ligands. 30

31 Endocannabinoids 4b The Endocannabinoid CB 2 Receptor 2 Like CB 1 receptors, CB 2 receptors inhibit the activity of adenylyl cyclase via their G i /G oα subunits. Via their G βγ subunits, CB 2 receptors are also coupled to the MAPK ERK pathway, a complex and highly conserved signal transduction pathway, which regulates a number of important cellular processes. Activation of the MAPK ERK pathway by CB 2 receptor agonists ultimately results in changes in cell migration as well as induction of the growth related gene Zif268. The Zifi268 gene encodes a transcriptional regulator implicated in neuroplasticity and long term memory formation. At present, there are 5 recognized cannabinoids produced endogenously in the body: AEA, 2 AG), noladin ether (2 arachidonyl glyceryl ether), N arachidonoyl dopamine, and virodhamine. Some of these ligands appear to exhibit functional selectivity at the CB 2 receptor: 2 AG preferentially activates the MAPK ERK pathway, while noladin ether preferentially inhibits adenylyl cyclase. Like noladin ether, the synthetic cannabinoid ligand CP 55,940 (widely used in cannabinoid research) has also been shown to preferentially inhibit adenylyl cyclase in CB 2 receptors. Together, these results support the emerging concept of agonist directed trafficking at cannabinoid receptors. Endocannabinoids 4b The Endocannabinoid CB 2 Receptor 3 CB 2 receptors are widely and prolifically expressed in peripheral tissues of the immune system throughout spleen, tonsils, and thymus gland. In immune tissues, CB 2 Rs are responsible for mediating cytokine release, and are localized on monocytes, B cells, T cells, and macrophages. Indeed, the CB 2 R was initially referred to as being entirely a peripheral immune system receptor. However, CB 2 Rs are also found throughout the GI system, where they modulate intestinal inflammatory responses. Thus, CB 2 receptor agonists are a potential therapeutic target for inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. In addition, CB 2 Rs are found in the peripheral nervous system and in the CNS. In the brain CB 2 Rs are found directly on neurons and glial cells, and CB 2 agonists have recently been found to strongly modulate certain behaviors in laboratory animals. CB 2 R density in the brain is very low roughly on the same order of magnitude as the μ opioid receptor. Endocannabinoids 4b Summary Characteristics of CB 1 and CB 2 Receptors Both densely distributed throughout the body CB 1 Rs highly enriched in central nervous system Located on axon terminals Mediate retrograde signaling (Dendrite Axon) G protein coupled CB 2 Rs highly enriched in periphery Especially in immune system CB 2 Rs also in brain and CNS Fewer than CB 1 Rs; ~ Same density as μ opioid receptors CB 2 s modulate neural signaling Endocannabinoids 5 Cannabinoid Ligands A very large number of ligands have been discovered for the CB 1 and CB 2 receptors. See following slides. AM630 Natural cannabinoids Synthetic Cannabinoids 31

32 Cannabinoid antagonists Cannabinoid antagonists CB 1 antagonists CB 2 antagonist AM630 Relevance to Addiction 1 The relevance of the endocannbinoid systems of the CNS to addiction is enormous. First, they are obviously the neural substrates upon which cannabinoid drugs of abuse act to exert their addictive effects. Second and perhaps more importantly the endocannabinoid systems of the CNS appear to be involved in the CNS actions of virtually all drugs of abuse. Thus, the actions of addictive drugs on brain reward circuits and neural substrates are in most cases blockable by CB 1 antagonists/inverse agonists) or by CB 2 agonists even for such unexpected and unlikely addictive compounds as cocaine, heroin, or alcohol. This strongly implies that there are endocannabinoid neuronal substrates and mechanisms at play in the addictive process for virtually all addictive substances. In addition, neuroadaptive mechanisms such as long term potentiation (LTP) and long term depression (LTD) are believed to play a significant role in the addiction process. Endocannabinoid mechanisms in the nucleus accumbens, hippocampus, and prefrontal cortex have been shown to play an important role in both LTP and LTD. Cannabidiol a naturally occurring cannabinoid found in cannabis may have anti addiction efficacy. Relevance to Addiction 2 Nabiximols (trade name Sativex) is a patented cannabinoid oromucosal mouth spray developed by the UK company GW Pharmaceuticals for multiple sclerosis patients, who use it to alleviate neuropathic pain, spasticity, overactive bladder, and other MS symptoms. Nabiximols is distinct from other pharmaceutically produced cannabinoids currently available because it is a mixture of compounds derived from Cannabis plants, rather than a synthetic product. Its principal active cannabinoid components are THC and cannabidiol (CBD) in a near 1:1 ratio of CBD to THC. Nabiximols is currently approved for prescription use in the UK, Canada, and a large number of European countries. It is currently under review by the FDA for approval in the United States. Highly credible reports from physician prescribers in the UK and Canada (where it has been available for prescription use for the longest periods of time) indicate that it is a highly effective analgesic (especially for neuropathic pain) and an effective anti addiction pharmacotherapy. Cellular and Molecular Mechanisms in Addiction Neuroadaptation Epigenetic phenomena Relevance to addiction Neuroadaptation 1 An utterly enormous number of types of neuroadaptation occur within the CNS. These range from such simple things as receptor internalization following ligand binding to much more complex forms of neuroadaptation such as LTP and LTD. Their relevance to addiction is in some cases evident, in many cases conjectural, and in some cases delusional or self serving on the part of proponents. All of the following (and many other neuronal phenomena) can be considered to be types of neuroadaptation: Synaptic enhancement Short term synaptic enhancement results from an increased probability of synaptic terminals releasing transmitters in response to pre synaptic action potentials. Synapses will strengthen for a short time because of either an increase in size of the readily releasable pool of packaged transmitter or an increase in the amount of packaged transmitter released in response to each action potential. Depending on the time scales over which it acts synaptic enhancement is classified as: Neural facilitation Synaptic augmentation Post tetanic potentiation Synaptic depression Usually attributed to depletion readily releasable vesicles. Can also arise from post synaptic processes and from feedback activation of presynaptic receptors. 32

33 Neuroadaptation 2 Heterosynaptic depression believed to be linked to the release of astrocytic ATP. Homosynaptic plasticity A type of synaptic plasticity. This plasticity is input specific, meaning changes in synapse strength occur only at post synaptic targets specifically stimulated by a pre synaptic input. Therefore, the spread of signal from the presynaptic cell is localized. Synaptic strengthening via homosynaptic plasticity is associative dependent on the firing of a presynaptic and postsynaptic neuron closely in time. These mechanisms appear to underlie at least some forms of learning and short term memory. This is often termed Hebbian neuroplasticity in honor of Donald Olding Hebb, the towering neuropsychologist of the 20th century, who proposed in 1949 that strengthening of synaptic connections occurred because of coordinated activity between the pre synaptic terminal and post synaptic dendrite. According to Hebb, these two cells are strengthened because their signaling occurs together in space and/or time, also known as coincident activity. This postulate is often summarized as Cells that fire together, wire together, synapses that have coincident firing are strengthened, while other synapses on the same neurons remain unchanged. Hebb's postulate has provided a conceptual framework for how synaptic plasticity underlies long term information of storage in the brain. Neuroadaptation 3 Heterosynaptic plasticity Another type of synaptic plasticity. In the case of heterosynaptic plasticity, the activity of a particular neuron leads to changes in the strength of synaptic connections between another pair of neurons. A number of distinct forms of heterosynaptic plasticity have been found in a variety of brain regions and organisms. These different forms of heterosynaptic plasticity contribute to a variety of neural processes including associative learning, the development of neural circuits, and homeostasis of synaptic input. Modulatory input dependent plasticity (a well confirmed type of heterosynaptic plasticity) One well studied example of heterosynaptic plasticity is modulatory inputdependent plasticity. This is dependent upon the action of neuromodulators, synaptic signaling molecules that do not directly generate electrical responses in target neurons. Rather, the release of neuromodulators alters the efficacy of neurotransmission in nearby chemical synapses, often in quite long lasting fashion. A number of neurotransmitters act as neuromodulators, particularly biogenic amines such as DA and 5 HT. These neuromodulators use G protein coupled receptors which mediate slower modulatory effects and neither hyperpolarize nor depolarize cells. Due to these qualities, GPCRs can initiate long lasting changes in heterosynaptic strength. Neuroadaptation 3 In heterosynaptic plasticity (left), neurons that are not specifically innervated undergo changes in synaptic plasticity in addition to those that are specifically innervated. In modulatory input dependent plasticity (right), neuron C acts as an interneuron, releasing neuromodulators, which changes synaptic strength between Neuron A and Neuron B Neuroadaptation 5 Homeostatic plasticity Homeostatic plasticity refers to the capacity of neurons to regulate their own excitability relative to network activity, a compensatory adjustment that occurs over the timescale of days. Synaptic scaling has been proposed as a mechanism of homeostatic plasticity. Homeostatic plasticity is thought to balance Hebbian plasticity. Spike timing dependent plasticity Spike timing dependent plasticity (STDP) adjusts the strength of connections between neurons, based on the relative timing of a particular neuron s output and input action potentials (or spikes). If an input to a neuron tends, on average, to occur immediately before that neuron s output spike, then that particular input is strengthened. If an input spike tends, on average, to occur immediately after an output spike, then that particular input is weakened. Thus, inputs that cause a post synaptic neuron s excitation are made even more likely to contribute in the future, whereas inputs that are not causal become less likely to contribute in the future. The process continues until a subset of the initial set of connections remain, while the influence of all others is reduced to zero. Since a neuron produces an output spike when many of its inputs occur within a brief period the subset of inputs that remain are those that tended to be correlated in time. In addition, since the inputs that occur before the output are strengthened, the inputs that provide the earliest indication of correlation will eventually become the final input to the neuron. Neuroadaptation 6 Neuroadaptation 7 Long term depression (LTD) LTD is an activity dependent reduction in efficacy of neuronal synapses lasting hours or longer following a long patterned stimulus. LTD occurs in many areas of the CNS with varying mechanisms depending upon brain region. LTD in the hippocampus and cerebellum are the best characterized. LTD occurs in different types of neurons using various neurotransmitters. However, the most common neurotransmitter involved is glutamate. Glutamate acts on NMDARs, KARs, AMPARs, and mglurs during LTD. LTD can result from strong synaptic stimulation or persistent weak synaptic stimulation. In conjunction, LTD and LTP are the major factors affecting synaptic plasticity. LTD is thought to result mainly from a decrease in postsynaptic receptor density, although a decrease in presynaptic neurotransmitter release may also play a role. Hippocampal LTD may be important for the clearing of old memory traces. Hippocampal/cortical LTD can be dependent on NMDARs, mglurs, or endocannabinoids. LTD results in the phosphorylation of AMPARs and their elimination from the synaptic surface. LTD is one of several processes that serve to selectively weaken specific synapses in order to make constructive use of synaptic strengthening caused by LTP (below). This is necessary because, if allowed to continue increasing in strength, synapses would ultimately reach a ceiling level of efficiency, which would inhibit the encoding of new information. Long term depression (LTD) Continued LTD often occurs via the internalization of AMPA receptors (AMPARs) into the postsynaptic membrane of the synapse undergoing a change in connective strength. Ca 2+ is one signaling ion that causes this AMPA receptor density change by inducing a cascade of biological changes within the cell. LTD occurs via Ca 2+ activation of protein phosphatases, which dephosphorylate and cause AMPAR internalization. In order to create input specific changes in synaptic strength, the Ca 2+ signal must be restricted to specific dendritic spines. Dendritic restriction of Ca 2+ is mediated by several mechanisms. Extracellular Ca 2+ can enter the spine through NMDARs and voltage gated Ca 2+ channels (VGCCs). Both NMDARs and VGCCs are concentrated on dendritic spines, mediating spine specific Ca 2+ influx. Intracellular stores of Ca 2+ in the endoplasmic reticulum and mitochondria may also contribute to spine restricted signaling. Clearance of Ca 2+ is controlled by buffer proteins, which bind to Ca 2+ and keep it from trickling out to other spines. Restricted diffusion of Ca 2+ across the neck of the dendritic spine also helps isolate it to specific dendrites. 33

34 Neuroadaptation 8 Neuroadaptation 9 Long term potentiation (LTP) LTP is a form of long lasting increase in synaptic strength between two neurons that results from stimulating them synchronously. It is one of several phenomena underlying synaptic plasticity, the ability of chemical synapses to change their strength. As memories are thought to be encoded by modification of synaptic strength, LTP is widely considered one of the major cellular mechanisms that underlies learning and memory. LTP is the opposing process to LTD, and many of the underlying mechanisms are similar. To induce LTP, Ca 2+ activates Ca 2+ /calmodulindependent protein kinase II (CAMKII) and protein kinase C (PKC), causing phosphorylation and insertion of AMPARs into the neuronal membrane. Many of the mechanisms seen in LTD for keeping the Ca 2+ signal restricted to specific dendritic spines also apply in LTP. Another mechanism for input specific LTP is temporal. NMDARs require both depolarization, to remove their magnesium block, and glutamate activation, to open their channels, to allow Ca 2+ influx. LTP is thus localized at sites where NMDA channels are opened by active synaptic inputs that are releasing glutamate and causing depolarization of the postsynaptic cell, and will not affect nearby inactive synapses. In order to stabilize LTP and make it last longer, new proteins are synthesized in response to stimulation at a potentiating synapse. The challenge that arises is how to get specific, newly synthesized proteins to the correct input specific synapses they are needed at. Two solutions to this problem include synaptic tagging and local protein synthesis: Synaptic tags mark where synaptic plasticity has occurred and provide information on synaptic strength and potential for long term plastic changes. The tag is temporary and involves a large number of proteins, activated by influx of Ca 2+ into the postsynaptic cell. In addition, depending on the type and magnitude of synaptic change, different proteins are used for tagging. During LTD, calcineurin is used. During LTP, CaMKII is used. In order for synaptic plasticity to be input specific, these synaptic tags are essential on post synaptic targets, to ensure that synaptic plasticity is localized. These tags later initiate protein synthesis that in turn cause changes in synaptic strength at the specific synapse. Local protein synthesis at dendrites is also apparently involved. The depolarization and resulting activation of AMPA and NMDA receptors in the postsynaptic cell causes endocytosis of these receptors. Local protein synthesis is required to maintain the number of surface receptors at the synapse. These new proteins help stabilize the structural changes. There is evidence that dendritic ribosomes manufacture these proteins. There is also evidence of RNA granules in dendrites, indicating the presence of newly made proteins. LTP can be induced from dendrites severed from the soma of the post synaptic target neuron. Contrarily, LTP can be blocked in these dendrites by protein synthesis inhibitors, which further suggests local protein synthesis. In sum, evidence shows local protein synthesis is necessary for L LTP to be stabilized and maintained. Neuroadaptation 10 As important as these neuroadaptations are to addiction especially for pathological adaptations in reward and memory systems another type of neuroadaptation is compelling as a substrate for addiction. This additional type consists of an adaptive change in locus of control over drug seeking and drug taking behavior(s) specifically, a change from reward driven drug taking to habit driven drug taking. See following slides. Progression of drug seeking behavior from reward driven to habit driven Long history of involvement of dorsal striatum in habit formation Pavlovian to Instrumental transfer (PIT) Animals trained to associate CS with a reward (Pavlovian learning) Animals then trained to lever press for same reward (Instrumental) Test: Ability of CS to enhance lever pressing in extinction (models addiction) Lesions of CeA and NAc core abolish PIT Lesions of BLA or NAc shell have no effect on PIT Dopamine D2/D3 receptor antagonism abolishes PIT Amphetamine potentiates PIT Robbins and Everitt, Neurobiology of Learning and Memory 78: , 2002 Ascending spiral of striato nigral striato loop pathways from NAc shell to dorsolateral striatum Haber et al, Journal of Neuroscience 20: , 2000 Compulsive drug seeking behavior is inflexible, since it persists despite considerable cost to the addict, becomes dissociated from subjective measures of drug value, becomes elicited by specific environmental stimuli, and involves complex goal directed behaviors for procurement and self administration of drugs. Limbic cortical ventral striatopallidal circuits that underlie goal directed drugseeking actions may eventually consolidate habitual, S R drug seeking through engagement of corticostriatal loops operating through the dorsal striatum. This progression from action to habit may have its neural basis within the spiraling loop circuitry of the striatum, by which each striatal domain regulates its own DA innervation and that of its adjacent domain in a ventral to dorsal progression (Haber et al, 2000). Thus, the NAc shell regulates its own DA innervation via projections to the VTA and also that of the NAc core. The NAc core in turn regulates its own DA innervation via projections to the VTA and also that of the next, more dorsal tier of the dorsal striatum via projections to the substantia nigra pars compacta and so on. Chronically self administered drugs, through their ability to increase striatal DA, may consolidate this ventral to dorsal striatal progression of control over drug seeking as an habitual form of responding. Robbins TW and Everitt BJ. Limbic striatal memory systems and drug addiction. Neurobiology of Learning and Memory 78: ,

35 Epigenetic Phenomena in Addiction 1 Epigenetic Phenomena in Addiction 2 Histone acetylation. Most of our knowledge on epigenetic regulation of addiction has focused on the effects of psychostimulants such as cocaine and amphetamine on histone modifications. Among these, the most common modifications studied involve histone acetylation and methylation. Cocaine exposure alters acetylated H3 and H4 levels in the nucleus accumbens. Acute cocaine exposure increases H4 acetylation at the promoter of c Fos, an immediate early gene and a marker of neuronal activation, while chronic exposure results in no such a change. Nevertheless, chronic cocaine exposure can also result in gene activation that is not induced by acute treatment. One example is the acetylation of H3 at the brain derived neurotrophic factor (BDNF) and cyclin dependent kinase 5 (CDK5) promoter regions. While cocaine administration can alter histone acetylation at many gene promoters, it does not necessarily result in altered transcription in the nucleus accumbens. It is worth mentioning that the lack of correlation does not imply that a similar changes in BDNF promoter acetylation have been detected following cocaine exposure, but rather highlights the complexity of transcriptional output resulting from changes in histone acetylation. Consistent with the above studies, behavioral tests measuring the effect of histone deacetylase (HDAC) deletion on cocaine sensitivity and reward have also resulted in mixed outcomes. For instance, while deletion of HDAC1 in the nucleus accumbens attenuates behavioral responses to cocaine, deletion of HDAC2 or HDAC3 in the accumbens does not. Histone acetylation Continued. Interestingly, inhibition of HDAC3, the most highly expressed HDAC in the brain, enhances extinction and prevents reinstatement of cocaine seeking in a conditioned place preference animal model. To date, most behavioral studies have investigated the effects of psychostimulants on drug seeking and locomotor sensitization. However, to obtain a more complete picture on the role of epigenetic modifications in drug addiction, additional behavioral models of addiction, such as intravenous drug self administration, will be necessary. Epigenetic Phenomena in Addiction 3 Epigenetic Phenomena in Addiction 4 Histone methylation. Several recent studies have investigated the effects of drugs of abuse on histone methylation. While drug exposure fails to have a general effect on histone methyltransferases (HMTs) and histone demethylases (HDMs), chronic cocaine treatment represses G9a in the nucleus accumbens, as evidenced by decreases in histone methyltranferase H3K9 (H3K9) dimethylation. Additionally, histone methyltransferase G9a (G9a) inhibition in the nucleus accumbens, either genetically or pharmacologically, increases behavioral responses to cocaine and opiates, and overexpressing G9a can reverse these effects. Furthermore, Cre dependent knockout of G9a in the nucleus accumbens increases dendritic arborization, suggesting that H3K9 dimethylation by G9a may play a role in drug dependent synaptic plasticity. Mechanistically, G9a appears to play a central role in a negative feedback loop with ΔFosB, a long lasting transcription factor activated in drug addiction. G9a inhibits induction of ΔFosB, and in turn, ΔFosB inhibits expression of G9a. Additionally, prolonged HDAC inhibition not only inhibits behavioral responses to cocaine, but also induces G9a expression, a finding consistent with the ability of G9a overexpression to inhibit such behavioral responses to psychostimulants. Histone methylation Continued. While these findings support the involvement of epigenetic regulation in drug reward, one cannot undermine the role of transcription factors in the recruitment and modulation of epigenetic modifying enzymes. Indeed, transcription factors such as ΔFosB, myocyte enhancer factor 2 (MEF2), and CREB are all known to recruit epigenetic modifying enzymes. ΔFosB can drive CDK5 transcription by recruiting CBP and, conversely, can inhibit c Fos transcription by recruiting HDAC1. MEF2 can recruit the class II HDAC, p300, while CREB also binds CBP. It is therefore likely that transcription factors and epigenetic enzymes work in concert to mediate the transcriptional regulation of drug reward. Neuroimaging in Addiction The most compelling neuroimaging work in addiction research comes from animal models, and are seen in the context of the Reward Deficiency understanding of addiction See following slides REWARD DEFICIENCY AS A DRIVING FORCE IN ADDICTION 35

36 Is it possible, then, that some substance abusers have a defect in their ability to capture reward and pleasure from everyday experience, as postulated by some clinicians and as postulated by Blum and colleagues in the context of their formulation of reward deficiency syndrome? Interestingly, this very concept of a basal hypofunctionality in brain mechanisms subserving normal reward and pleasure functions was originally postulated by Dole and colleagues nearly 40 years ago during the development of methadone maintenance for heroin addiction. If these conceptions have merit, they have profound implications: (a) our goals are not only to rescue addicts from the clutches of their addictions, but also, more importantly, (b) to restore their reward functions to a level of func tionality that enables them to get off on the real world; and (c) pharmaco therapeutic interventions for treatment of substance abuse that are based on simple blockade of brain reward functions are doomed to failure. Gardner, EL. Brain reward mechanisms. In Lowinson JH, Ruiz P, Millman RB, Langrod JG (Eds), Substance Abuse: A Compre hensive Textbook, 4 th edn. Philadelphia: Lippincott Williams and Wilkins, 2005, pp

37 Behavioral measures of trait impulsivity in high impulsive and low impulsive rats Reduced D2/D3 receptor binding in nucleus accumbens of drug naïve trait impulsive rats Black circles High impulsive rats White circles Non impulsive rats 37