Opioid and CB Notes PDF

Peptides - Opioids Opioids: endogenous ligands à enkephalins, beta-endorphin, dynorphin Opiate drugs: exogenous ligands; (e.g. opium, morphine, heroin) heroin  Peptides - Opioids...

Peptides - Opioids Opioids: endogenous ligands à enkephalins, beta-endorphin, dynorphin Opiate drugs: exogenous ligands; (e.g. opium, morphine, heroin) heroin  Peptides - Opioids Opioids: endogenous ligands à enkephalins, beta-endorphin, dynorphin Opiate drugs: exogenous ligands; (e.g. opium, morphine, heroin) The receptors… mu µ – delta d – kappa k (and a few others) Periaqueductal gray, hypothalamus, other brain areas  Peptides - Opioids Opioids: pain perception and reward/reinforcement Morphine: µ opioid agonist Naloxone: µ opioid antagonist Rats stimulated through electrodes in the central periaqueductal gray (PAG) show a stimulation-induced analgesia that is reversed by naloxone. Measure latency (in seconds) to tail flick. Akil et al. 1976 (Science)  Peptides - Opioids Self-administration: Opiate reinforcement  Endocannabinoids Discovered in the early 1990s Highly lipid soluble. (AEA; “Ananda” = “joy, bliss”) Act as retrograde messengers. Receptors CB1 (1988) – Brain (lots!), lungs, liver, kidneys CB2 (1993) – immune system, brain Often inhibit release of other neurotransmitters Cannabis sativa △ 9 THC  Cannabinoids Common behavioural effects of THC: Analgesia Motor impairment Hyperphagia Learning and memory deficits Sample Test/Choice Delay  Cannabinoids Common behavioural effects of THC: Analgesia Motor impairment Hyperphagia Learning and memory deficits Kosiorek et al. (2003) Synapses and synaptic transmission Pharmacological and genetic manipulation of behaviour (Neurotransmitters Supplement) Chapter 4  NEUROTRANSMITTERS Chemicals that act as messengers between cells in the nervous system; they transmit impulses across the synaptic cleft from a neuron to another neuron, a muscle, a gland…  Behavioural Genetics Random Mutations Population-level Statistical Assessments Selective Breeding Deliberate Genetic Manipulations (gene knockout and gene knockin) Global gene expression Spatially limited gene expression Temporally limited gene expression  Behavioural Pharmacology Agonist: a drug that facilitates the effects of a particular neurotransmitter Antagonist: a drug that inhibits the effects of a neurotransmitter Review DRUG EFFECTS ON RECEPTORS Competitive Binding Noncompetitive Binding DRUG EFFECTS ON RECEPTORS Competitive Binding Direct Agonist Receptor Blocker or Direct Antagonist  DRUG EFFECTS ON RECEPTORS Noncompetitive Binding Neuromodulators Orthosteric binding site Allosteric binding site Pharmacological and genetic manipulation of behaviour Specific transmitter systems Chapter 4 Copyright © 2014 Pearson Education, Inc.  AMINO ACID NEUROTRANSMITTERS Fast acting; directed synapses  AMINO ACID NEUROTRANSMITTERS Fast acting; directed synapses Glutamate: excitatory neurotransmitter. Gamma-aminobutyric Acid (GABA): inhibitory. Glycine: inhibitory. Aspartate: excitatory.  Glutamate Excitatory amino acid neurotransmitter  Neuron-glia interactions: synthesis of glutamate “Glutamine cycle” VGLUT = Vesicular Glutamate Transporter EAAT = Excitatory Amino Acid Transporter  Glutamate Excitatory amino acid neurotransmitter Produced in many neurons throughout the brain Four Receptors 1- NMDA receptor 2- AMPA receptor Ionotropic (Na+/Ca2+) Receptors 3- Kainate receptor 4- Metabotropic Glutamate Receptor (mGluR - 8 subtypes): post-synaptic (second messengers) and pre-synaptic. NMDA receptor Formation of Memories Schizophrenia ?? ADHD U of T researcher earns world’s richest prize for brain research IVAN SEMENIUK - SCIENCE REPORTER The Globe and Mail Published Tuesday, Mar. 01, 2016 9:34PM EST Last updated Tuesday, Mar. 01, 2016 9:37PM EST “Dr. Collingridge, who was born in Britain, began working on his prize-winning discovery while a postdoctoral fellow at the University of British Columbia in the 1980s. His contribution was working out the key role that the protein N-methyl-D- aspartate (NMDA) [receptor] plays in changing the level of connectivity between neurons.” FAST excitatory transmission Obligatory co- agonist Voltage – dependent At rest Depolarized  1 2 Obligatory co- agonist 3 Voltage – dependent 4 NMDA receptor = At rest Depolarized “coincidence detector”  Hebb’s Postulate “When an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased” OR… “Cells that fire together wire together.” “Synaptic Plasticity” Basis for Long-term Memory? The Organization of Behavior (1949) **See section 11.8 in Pinel** Long-Term Potentiation (LTP) An enduring change in communication between pre and post synaptic cells in response to salient stimulation (incl behavioural)… Requires NMDAR. Bliss & Lomo, 1973  (Unconditioned (Conditioned Stimulus) Stimulus) (Unconditioned Response) (Conditioned Stimulus) (Conditioned Response)  Hebb’s Postulate “When an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased” OR… “Cells that fire together wire together.” “Synaptic Plasticity” Basis for Long-term Memory? The Organization of Behavior (1949) Figure 8.3 NMDA receptors are composed of four subunits  Behavioural Genetics Random Mutations Population-level Statistical Assessments Selective Breeding Deliberate Genetic Manipulations (gene knockout and gene knockin) Global gene expression Spatially limited gene expression Temporally limited gene expression Copyright © 2009 Allyn & Bacon NR1 subunit CA1-KO mice Tsien et al., 1996 Tsien et al., 1996. Cell 87, 1327–1338 The NR1 subunit in CA1 is needed to produce LTP (LTP = Long-Term Potentiation) High frequency stimulation High frequency stimulation Therefore, NMDA receptor is necessary for LTP…  Morris Water Maze Training Phase Testing Phase “Allocentric” spatial memory The NR1 subunit in CA1 is needed for Place Learning Figure 8.3 NMDA receptors are composed of four subunits  NR2B overexpression: The ‘Doogie’ Mouse Throughout the forebrain. Tang et al., 1999. Nature 401, 63-69 NR2B overexpression: Enhanced LTP  Morris Water Maze Training Phase Testing Phase NR2B overexpression: Enhanced Spatial Learning Tang et al., 1999. Nature 401, 63-69 Object Recognition Spontaneous Object Recognition Sample Test/Choice Delay NR2B overexpression: Enhanced Object Memory Training: two identical objects Testing: two objects; one novel, one familiar Tang et al., 1999. Nature 401, 63-69  GABA (Gamma-Aminobutyric Acid) Inhibitory amino acid neurotransmitter Most are interneurons.  GABA (Gamma-Aminobutyric Acid) Inhibitory amino acid neurotransmitter The inhibitory effects of GABA are crucial for normal brain functions. Delicate balance between excitation and inhibition… Reduced GABAergic inhibition à ??? GABA is synthesized from glutamate GABA (Gamma-Aminobutyric Acid) Two receptors: GABAA à ionotropic (Cl-) GABAB à metabotropic (less common)  All others are ??? (???) Benzodiazepines: (valium, librium). Anxiolytic; muscle relaxing; anti-convulsant; impair learning and memory Barbiturates: calming (low doses) à anesthesia (higher doses); impair learning and memory Steroids: e.g. progesterone and its metabolites. Calming. Picrotoxin: a poison found in an East Indian shrub Opposite effects, it inhibits GABAA receptors (indirect antagonist) At high doses à ?? Alcohol: regulates GABAA function (benzodiazepine site? Barbiturate site?)  MONOAMINE NEUROTRANSMITTERS For each: synthesis from one amino acid  MONOAMINE NEUROTRANSMITTERS For each: synthesis from one amino acid Tyrosine L-DOPA Catecholamines: Dopamine – Dopamine Norepinephrine - Epinephrine Norepinephrine (or Noradrenaline) Epinephrine (or Adrenaline)  MONOAMINE NEUROTRANSMITTERS For each: synthesis from one amino acid Tyrosine L-DOPA Catecholamines: Dopamine – Dopamine Epinephrine – Norepinephrine Norepinephrine (or Noradrenaline) Tryptophan Epinephrine (or Adrenaline) Indolamines: e.g., Serotonin Serotonin  Monoamines Catecholamines: dopamine, norepinephrine, epinephrine Indolamine: serotonin Localized synthesis (brain stem nuclei) Widespread functions  Dopamine Tyrosine Synthesis in the mesencephalon: L-DOPA Dopamine Substantia nigra Norepinephrine (or Noradrenaline) Ventral Tegmental Area Epinephrine (or Adrenaline) Substantia nigra à striatum (nigrostriatal pathway) Limbic System (mesolimbic pathway) Ventral Tegmental area Prefrontal cortex (mesocortical pathway)  Dopamine Tyrosine L-DOPA Dopamine Substantia nigra à striatum (nigrostriatal system) Degeneration à Parkinson’s Disease If Dopamine is insufficient, why don’t we just administer it to Parkinson’s patients? L-DOPA used instead à but limited efficacy because of side effects (cognitive: confusion, anxiety, hallucinations). Inhibitory feedback also reduces its effectiveness… ???.  Dopamine Tyrosine 5 dopamine receptors identified L-DOPA D1 – D2 – D3 – D4 - D5 Dopamine D1–like: D1 and D5 Two subfamilies: D2–like: D2, D3 and D4 All are metabotropic.  Dopamine Tyrosine 5 dopamine receptors identified L-DOPA D1 – D2 – D3 – D4 - D5 Dopamine D1–like: D1 and D5 Two subfamilies: D2–like: D2, D3 and D4 Movement (NS) Dopamine is involved in many functions: Attention (MC) Learning (ML/MC) major component of the Various forms of reward system reinforcement - mesolimbic pathway (food, sex, drugs of abuse) (nucleus accumbens)  DOPAMINERGIC DRUGS L-DOPA: agonist. Parkinson’s disease. Amphetamines: agonist. Disorders of excessive sleep. Antischizophrenic (Antipsychotic) drugs (e.g., Haloperidol): D2 receptor antagonists… problem?  MONOAMINE NEUROTRANSMITTERS For each: synthesis from one amino acid Tyrosine L-DOPA Catecholamines: Dopamine – Dopamine Norepinephrine - Epinephrine Norepinephrine (or Noradrenaline) Epinephrine (or Adrenaline)  Norepinephrine Tyrosine L-DOPA Or noradrenaline… Dopamine Synthesis mainly in the pons Norepinephrine (metencephalon): (or Noradrenaline) Locus ceruleus Epinephrine (or Adrenaline) Released mainly diffusely by Non Directed Synapses  Norepinephrine Tyrosine L-DOPA Or noradrenaline… Dopamine Synthesis mainly in the pons Norepinephrine (metencephalon): (or Noradrenaline) Locus ceruleus Epinephrine (or Adrenaline) Released mainly diffusely by Non Directed Synapses Most actions are similar to those of Epinephrine Also synthesized and released by post-ganglionic Neurons in the sympathetic (autonomic) nervous system Lots of cortical innervation: arousal, attention, learning and memory  Epinephrine Tyrosine L-DOPA Or adrenaline… Dopamine Synthesis less prevalent in the brain… Norepinephrine (or Noradrenaline) In the medulla of the adrenal glands Epinephrine (or Adrenaline) kidney Periphery: autonomic nervous system CNS: neurotransmitter/neuroactive molecule – limited role Same receptors as norepinephrine  Adrenergic Receptors a1 – a2 – b1 – b2; metabotropic They bind both epinephrine (adrenaline) and norepinephrine (noradrenaline) Responses to arousing, activating, stressful events. Help to prepare the body for action. Adrenergic Receptors a1 – a2 – b1 – b2; metabotropic They bind both epinephrine (adrenaline) and norepinephrine (noradrenaline) Responses to arousing, activating, stressful events. Epinephrineà mostly in the periphery Norepinephrine à mostly in the CNS  Adrenergic Receptors a1 – a2 – b1 – b2; metabotropic a2 receptors implicated in ADHD common treatment: atomoxetine NE transporter inhibitor Monoamine oxidase inhibitors: formerly used to treat depression; side effects Beta-blockers (e.g. propranolol): used for anxiety disorders (e.g., GAD); reduce peripheral aspects of anxiety (sympathetic activation), not anxiety per se.  COCAINE, A CATECHOLAMINE AGONIST It increases activity of both Dopamine and Norepinephrine It blocks their reuptake à the synapses remain active  COCAINE, A CATECHOLAMINE AGONIST It increases activity of both Dopamine and Norepinephrine It blocks their reuptake à the synapses remain active Analgesic properties - ???  Cocaine hydrochloride Crack Coca bush Coca paste  COCAINE PSYCHOSIS Excitability, anxiety, talkativeness, increased pulse rate and blood pressure, dilation of pupils, faster breathing, temperature, sweating, loss of appetite, insomnia. Bizarre, erratic, sometimes violent behaviour and paranoid psychosis that disappears if discontinued. Sometimes misdiagnosed as Paranoid Schizophrenia.  Serotonin / 5-hydroxytryptamine (5-HT) Tryptophan Serotonin Very important neurotransmitter, for its implication in: Mood disorders Aggression Feeding Sleep  Serotonin / 5-hydroxytryptamine (5-HT) Tryptophan Synthesis in the midbrain, pons and medulla: Serotonin Mainly in neurons of the raphe nuclei Median raphe à Cerebral cortex/HPC (cognitive functions) Dorsal raphe à basal ganglia/cerebellum (motor control) Very complex system, at least 18 different receptors - most are metabotropic  Major Noradrenergic, Dopaminergic and Serotonergic Pathways in the CNS SIGMA-ALDRICH Serotonin Dopamine Norepinephrine  Serotonergic Drugs Fluoxetine: selective serotonin reuptake inhibitor (SSRI). Active component of Prozac, used to treat: Depression Some forms of Anxiety Disorders Obsessive Compulsive Disorder Fenfluramine: reuptake inhibitor AND stimulates release. Appetite suppressant à formerly used to treat obesity discontinued: heart disease  ACETYLCHOLINE (ACh) Cholinergic neurons First neurotransmitter discovered: abundant in brain AND periphery… Neuromuscular junctions à contraction Nicotinic receptors  Acetylcholine (ACh) Cholinergic neurons Neuromuscular junctions à contraction Ganglia of the autonomic nervous system In the brain: basal forebrain à various regions Brain functions: regulation of sleep, learning, memory, etc.  Brain stem nuclei – in pons and midbrain Involved in sleep/arousal. Involved in learning and memory/attention.  Acetylcholine (ACh) – Antagonism Botulinum Toxin (Botox): an anaerobic bacterium. US: 200 infections/yr - mortality rate: 9% 8 cases/yr in Canada Source: foodborne (often improperly canned vegetables), wound, intestinal AND Inhalation  EXOCYTOSIS Docking: SNARE proteins It blocks the release of Acetylcholine à paralysis Death from respiratory depression. JAMA. 2001;285:1059-1070  Therapeutic use of Botox Before Before After A patient with blepharospasm From, US food and drug administration, web site Also: - Migraines (muscles in neck) - Wrinkles…  Acetylcholine (ACh) - Agonism Black Widow Spider Venom: it stimulates the release of ACh Several bites needed to kill a healthy adult. Muscle spasms/convulsions.  Acetylcholine (ACh) Two types of Acetylcholine Receptors Muscarinic receptors = metabotropic Amanita muscaria Nicotinic receptors = ionotropic (Na+)  Loss of cholinergic basal forebrain neurons in aging and Alzheimer’s disease… Involved in learning and memory/attention. Learning and memory in rats: Spontaneous Object Recognition Sample Test/Choice Delay  Scopolamine – competitive muscarinic receptor antagonist Drug injection Sample Test/Choice Delay Memory acquisition/encoding Memory retrieval Scopolamine – competitive muscarinic receptor antagonist Memory impairment Winters et al. (2006) Journal of Neuroscience Scopolamine – competitive muscarinic receptor antagonist Drug injection Sample Test/Choice Delay Memory acquisition Memory retrieval Scopolamine – competitive muscarinic receptor antagonist Memory impairment Winters et al. (2006) Journal of Neuroscience  Acetylcholine (ACh) Two types of Acetylcholine Receptors Muscarinic receptors = metabotropic Amanita muscaria Nicotinic receptors = ionotropic (Na+)  Acetylcholine (ACh) Nicotinic receptors Curare: extract of certain woody vines in South America Nicotinic ACh receptor antagonist (blocker) Trivia: South American natives used to coat the tips of their hunting arrows and darts with curare.  Acetylcholine (ACh) Nicotinic receptors Curare: extract of certain woody vines in South America Nicotinic ACh receptor antagonist (blocker) Trivia: South American natives used to coat the tips of their hunting arrows and darts with curare. ???  Acetylcholine (ACh) Nicotinic receptors Curare: extract of certain woody vines in South America Nicotinic ACh receptor antagonist (blocker) ACh and agonists (e.g., nicotine) acting on nicotinic receptors in brain can enhance memory and attention. E.g., Smoking can make you feel more alert; Schizophrenia/ADHD. Rats: object recognition, etc.  PEPTIDE NEUROTRANSMITTERS Larger Molecule Neuroactive Peptides bradykinin beta-endorphin bombesin calcitonin cholecystokinin enkephalins dynorphin insulin gastrin substance P neurotensin glucagon Secretin somatostatin motilin vasopressin Oxytocin prolactin thyrotropin angiotensin II thyrotropin- sleep peptides galanin neuropeptide Y releasing hormone gonadotropnin- growth hormone- vasoactive luteinizing releasing releasing intestinal hormone hormone hormone peptide Highlighted in blue: endogenous opioid Peptides à pain perception/analgesia Synapses and synaptic transmission Chapter 4 Synaptic transmission: chemical transmission of signal from one neuron to another  Presynaptic Membrane Axon Postsynaptic Membrane Synaptic Cleft  TYPES OF SYNAPSES Axodendritic Synapses: Dendrodendritic Synapses: Axon - Dendrites Dendrite - Dendrite Axosomatic Synapses: Axoaxonal Synapses: Axon – Cell body (soma) Axon - Axon Chemical synapses – 5 msec Electrical synapses - faster Electrical synapses: – membrane coupling – direct transmission Invertebrates (e.g. crayfish) Mammals ELECTRICAL SYNAPSES - PROPERTIES Gap junctions à membrane coupling Free transit of small molecules and ions Passive spread of depolarization ELECTRICAL SYNAPSES – GAP JUNCTIONS Gap junctions: sets of channels. 6 connexins à 1 connexon 2 connexons à 1 gap junction. ELECTRICAL SYNAPSES – GAP JUNCTIONS ELECTRICAL SYNAPSES – GAP JUNCTIONS Why not more electrical synapses? Synaptic transmission: chemical transmission of signal from one neuron to another Typical chemical Synapse Many neurons synthesize and release one neurotransmitter (e.g., “dopaminergic”, “cholinergic”, “serotonergic”, etc) …but there is often Colocalisation between multiple transmitters  NEUROTRANSMITTERS Synthesis: in the cell body (soma) then… Bigger peptides/proteins: Somal vesicle packaging Then active axoplasmatic transport (~40 cm/day) Small molecules: Axonal travel – usually passive (concentration gradient) Button vesicle packaging Exocytosis Directed Synapses Non-Directed Synapses  RELEASE OF NEUROTRANSMITTERS The action potential reaches the synaptic terminal Voltage-activated Calcium (Ca2+) channels open Ca2+ ion influx Presynaptic Vesicles fuse (dock) with the cell membrane Release of neurotransmitter in the synaptic cleft Exocytosis Copyright © 2014 Pearson Education, Inc.  EXOCYTOSIS Docking: SNARE proteins  RECEPTOR ACTIVATION - 1 Receptor: a protein that contains the binding site for a specific neurotransmitter (ligand)  RECEPTOR ACTIVATION - 1 Receptor: a protein that contains the binding site for a specific neurotransmitter (ligand) Receptor Subtypes: e.g. dopamine has 5 receptor subtypes; D1, D2….D5 Differential brain distribution à different functions  CELL MEMBRANE - PROTEINS Transmembrane or integral proteins  RECEPTOR ACTIVATION - 2 IONOTROPIC RECEPTOR: ligand-activated ion channel. Fast effect. Open - Close  RECEPTOR ACTIVATION - 2 IONOTROPIC RECEPTOR: ligand-activated ion channel. Fast effect. Open - Close ?  CELL MEMBRANE - PROTEINS Transmembrane or integral proteins Peripheral membrane proteins  RECEPTOR ACTIVATION - 3 METABOTROPIC RECEPTOR: Associated with signal proteins and G proteins. Slower effect. Longer lasting. Varied responses. G proteins: Guanosine-tri- phosphate (GTP)-sensitive.  CELL MEMBRANE - PROTEINS Transmembrane or integral proteins Peripheral membrane proteins e.g., G-proteins  b g G protein a neurotransmitter ions b g a = “G-protein activated” ion channel  neurotransmitter ions b g a enzyme = Intracellular biochemical cascade Second messenger AUTORECEPTORS  AUTORECEPTORS Metabotropic receptors on the PRE-synaptic neuron terminal region, soma, or dendrites Bind to own neurotransmitter Often affect intracellular processes (e.g., in terminal – neurotransmitter release) Self-regulation through inhibitory (negative) feedback  INTRACELLULAR RECEPTORS Receptors for liposoluble neurotransmitters Nuclear receptors: known also as nuclear factors. à gene transcription. Cytosolic receptors: (1) interact with intracellular reactions (2) gene transcription TERMINATION OF SYNAPTIC ACTIVITY Reuptake: cells recycle. Enzymatic degradation: e.g., Acetylcholinesterase (AChE).  TERMINATION OF SYNAPTIC ACTIVITY active transporter systems on e.g., Acetylcholinesterase (AChE) the presynaptic terminal Pinel -Figure 4.12 Excitable Cell Membranes and Action Potentials Chapter 4  CELL BODY Mitochondria (energy) Nucleus (DNA) Cytoplasm Endoplasmatic Reticulum Rough Smooth (With (Without ribosomes) ribosomes Protein Fat Synthesis Synthesis Microtubules Golgi Complex (transport within the cell) (vesicle packaging)  CELL MEMBRANE Semi-permeable barrier ??? polar head (glycerol – phosphate) hydrophilic: Water-loving Phospholipid two nonpolar fatty acid tails hydrophobic: Water-fearing  CELL MEMBRANE - LIPIDS Extracellular Space (OUT) Aqueous Phospholipid bilayer Nonpolar areas form a hydrophobic region between the hydrophilic heads Intracellular Space (IN) Aqueous (Cytosol) At body temperatures the interior of the bilayer is fluid.  CELL MEMBRANE - PROTEINS Transmembrane or integral proteins Peripheral membrane proteins  TRANSMEMBRANE OR INTEGRAL PROTEINS They have hydrophobic and hydrophilic regions Signal proteins Channel proteins PERIPHERAL, NON-TRANSMEMBRANE PROTEINS Extracellular or intracellular side of transmembrane proteins They facilitate chemical reactions (usually enzymes) Can facilitate the functions of transmembrane proteins  TRANSMEMBRANE TRAFFIC Lipids of membrane form a barrier to hydrophilic (water soluble) molecules. Substances do go through Diffusion Facilitated Diffusion Active Transport Active Transport: requires energy consumption (mitochondria) Passive Processes: no energy consumption  DIFFUSION Movement of substance (liquid or gas) along a concentration gradient ??? Lipid soluble molecules Example: steroid hormones (estrogens, progesterone, testosterone), gases  ELECTROSTATIC PRESSURE Charged particles = ions (+ or -) Same charge à particles repel each other Opposite charge à particles attract each other Concentration gradient and electrostatic pressure act together to determine distribution of ions… …BUT, ions are NOT lipid soluble…  ION CHANNELS à Facilitated Diffusion Specialized e.g. Na+, Cl-, Ca2+, etc Open or closed à differential membrane permeability Regulated by different mechanisms: chemical, mechanical, electrical…  CHARGES NEAR THE CELL MEMBRANE The electrical properties of the cell membrane are determine by the uneven distribution of charges (ions) on either side. Sodium: Na+ Potassium: K+ Chloride: Cl- Proteins - Sodium: Na+ More concentrated OUTSIDE the cell Chloride: Cl- (“physiological saline”) Potassium: K+ More concentrated INSIDE the cell Proteins - Overall: INSIDE is more negative than OUTSIDE  Oscilloscope Axon in NaCl solution (Squid Giant Axon)  Oscilloscope Axon in NaCl solution (Squid Giant Axon) Membrane potential: difference in electrical charge between the inside and the outside of a cell (related to uneven distribution of ions and proteins across the membrane) Neuronal Resting Potential -70 mV (= “polarized”) How is the differential concentration of ions across the membrane maintained? Cell membrane à not equally permeable to all ions Impermeable to the electrically charged proteins (-) Not very permeable to sodium (Na+) ions Relatively permeable to potassium (K+) ions Very permeable to chloride (Cl-) ions Experiments by Alan Hodgkin and Andrew Huxley (1950) They calculated the electrostatic charge needed to keep the various ions in the location where, at rest, they are in greater concentration: Cl-: 70 mV à 70 mV - 70 mV = 0 (Resting potential: - 70 mV) Equilibrium K+: 90 mV à 90 mV - 70 mV = 20 mV Na+: 50 mV à 50 mV + 70 mV = 120 mV Force of diffusion: driven by concentration gradient  Cl-: 70 mV à 70 mV - 70 mV = 0 K+: 90 mV à 90 mV - 70 mV = 20 mV Na+: 50 mV à 50 mV + 70 mV = 120 mV Hodgkin and Huxley also showed that: K+ ions “leak out” Na+ ions get into the cell Yet, at rest... the distribution remains relatively stable…  SODIUM-POTASSIUM PUMP It is an active co-transport mechanism It uses energy provided by the mitochondria (ATP: Adenosine Triphosphate) 3 Na+ ions OUT 2 K+ ions IN ATP ADP + P P= Phosphate group  Outside Outside Outside Outside Na+ K+ K+ Na+ K+ K+ K+ K+ Na+ Na+ P P P Na+ K+ Na+ P K+ Inside Inside Inside Inside Synapses on dendritic spines or the cell body Synaptic neurotransmitters à changes in the potential of post-synaptic neurons Neuron’s resting potential: -70 mV = “Polarized”  Neuron’s resting potential: -70 mV 0 mV Depolarization -70 mV Hyperpolarization Depolarization à excitatory postsynaptic potentials Hyperpolarization à inhibitory postsynaptic potentials ??? excitatory Depolarization à excitatory postsynaptic potentials (EPSP) inhibitory Hyperpolarization à inhibitory postsynaptic potentials (IPSP) Small changes – increase or decrease the chance of cell “firing” an action potential… POST SYNAPTIC POTENTIALS - 1 Graded responses Travel very rapidly Decremental conduction Decremental Conduction  NEURON Axon (emerges from the Axon Hillock)  POST SYNAPTIC POTENTIALS - 2 Post synaptic potentials add together (integration) Spatial summation Temporal summation At the same time In rapid succession in different locations at the same synapse SPATIAL SUMMATION TEMPORAL SUMMATION  Integration Neurons constantly integrate signals from many synapses over both time and space.  ACTION POTENTIAL Summation à threshold of excitation à neuron fires (about -65 mV “Action Potential Threshold”) All or none  ACTION POTENTIAL Summation à threshold of excitation à neuron fires (about -65 mV) In the axon’s membrane adjacent to the axon hillock  ACTION POTENTIAL Summation à threshold of excitation à neuron fires (about -65 mV) In the axon’s membrane adjacent to the axon hillock Action potential: a massive, rapid (1 millisecond) reversal of the membrane potential from -70 mV to +50 mV PRODUCTION OF ACTION POTENTIALS - 1 1) Threshold is reached (about -65 mV) PRODUCTION OF ACTION POTENTIALS - 2 2) Na+ channels open 3) Massive influx of Na+ ions 4) The cell membrane starts depolarizing 5) Depolarization opens K+ channels (“voltage gated”) 6) K+ ions exit the cell following their concentration gradient PRODUCTION OF ACTION POTENTIALS - 3 7) Full depolarization of the cell membrane (+50 mV) 8) Na+ channels close 9) K+ ions still exit the cell because of electrostatic pressure (inside of cell has become positive) 10) cell membrane starts REpolarizing 11) K+ channels close gradually PRODUCTION OF ACTION POTENTIALS - 4 12) Temporary hyperpolarization 13) Refractory period 14) Ready to start all over  REFRACTORY PERIODS Absolute Refractory Period Relative Refractory Period  REFRACTORY PERIODS Example (1): the eye can perceive high or low intensity light stimulations. Graded manner. Example (2): a muscle can be contracted at different strengths. Graded manner. Example (3): the secretion from a gland can be stimulated at high or low levels. Graded manner. All or none  REFRACTORY PERIODS Example (1): the eye can perceive high or low intensity light stimulations. Graded manner. Example (2): a muscle can be contracted at different strengths. Graded manner. Example (3): the secretion from a gland can be stimulated at high or low levels. Graded manner. Firing rate of neurons Number of neurons  REFRACTORY PERIODS - 2 Thanks to the Refractory Periods… Neuronal Firing Rate is related to Stimulus Intensity High intensity à firing rate set by absolute refractory period Low intensity à firing rate set by relative refractory period Also, action potentials cannot travel backwards within the axon under natural conditions…  REFRACTORY PERIODS - 2 Thanks to the Refractory Periods… Action potentials cannot travel backwards within the axon under natural conditions… Orthodromic conduction: normal direction, from axon hillock towards axon terminal. BUT: Antidromic conduction: opposite direction, from axon terminal towards axon hillock. CONDUCTION OF ACTION POTENTIALS - 1 Post Synaptic Potentials Action Potentials Close to the Synapses Along the axon Very fast Slower Decremental “Non-decremental” Passive Active CONDUCTION OF ACTION POTENTIALS - 2 Non-decremental CONDUCTION OF ACTION POTENTIALS - 3 Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+  Na+ It is a step process Active Step Passive Step Axonal Conduction: single wave, actively spreading CONDUCTION IN MYELINATED AXONS Can you guess? Will myelination make axonal conduction faster or slower? Why?  CONDUCTION IN MYELINATED AXONS Saltatory Conduction (Saltare = to jump) Slow Very fast Slow Very fast  CONDUCTION IN MYELINATED AXONS Slow Very fast Slow Very fast CONDUCTION IN NON-MYELINATED AXONS Slow Slow Slow Slow Slow Slow Slow  Cats: 100 m/sec Human motor neurons: 60 m/sec IN VIVO MEASUREMENTS OF NEURONAL ACTIVITY Normal Electrode: Activity of an area Micro Electrode: Activity of a single neuron Acute studies with anesthetized animals Chronic studies with “behaving” animals (e.g., place cells) Measurement or stimulation (e.g., Jose Delgado’s bull) e.g., Working memory in prefrontal cortex; face recognition neurons; mirror neurons Neurotransmitter Systems and Behavior B.D. Winters Department of Psychology University of Guelph 1 Table of Contents Introduction Amino acid neurotransmitters Glutamate Glutamate synthesis and the glutamine cycle Glutamate receptors The NDMA receptor and its many binding sites The NMDA Receptor, Hebb’s Postulate, and Long-Term Potentiation The NMDA Receptor and Long-term Memory GABA GABA Synthesis and Degradation GABAergic receptors Monoamine neurotransmitters Catecholamines Dopamine Norepinephrine Epinephrine Adrenergic receptors Catecholamine drugs Serotonin and the indolamines Acetylcholine Peripheral acetylcholine Central acetylcholine Acetylcholine receptors Peptide Neurotransmitters Unconventional Neurotransmitters 2 Introduction Neurons can be referred to as ‘electro-chemical transducers’ because they convert electrical signals (i.e., action potentials) into chemical signals through the release of neurotransmitters. These neurotransmitters typically travel across the synaptic cleft from the pre-synaptic to the post- synaptic neuron, where they bind to receptor proteins to initiate new cellular events. In the following sections, we will consider many of the best understood neurotransmitter systems. Most of these are referred to as ‘conventional’ or ‘classical’ neurotransmitters because they were the substances first identified to play signalling roles at synapses in the brain. The classical neurotransmitters are relatively small molecules that operate predominantly according to the fundamental features of synapses that we have already discussed. As we will see, there are also several ‘large molecule’ neurotransmitters (e.g., peptide neurotransmitters such as endogenous opioids) and other ‘unconventional’ neurotransmitters (e.g., gaseous neurotransmitters), many of which violate the original ‘rules’ of neurotransmission. Indeed, as the study of synaptic structure and function has progressed, so too has our understanding of the complex interplay of mechanisms1 regulating neuronal communication. As always, we will place these mechanisms in the context of their behavioural consequences. To do so, we will focus on two primary sources of evidence: behavioural pharmacology and behavioural genetics. Behavioural pharmacology deals with the effects of drugs on neurotransmitter systems and behaviours. Behavioural genetics concerns the study of the relationship between genetic factors and behaviour; specifically, we will cover studies that have used modern molecular biology techniques to produce genetic manipulations affecting neurotransmitter functions and related behaviours. Together, findings from studies using behavioural pharmacological and/or behavioural genetic techniques have shed significant light on the workings of synapses and the specific roles that different neurotransmitter systems play in behaviour. Table 1 provides an overview of the various neurotransmitter systems we will consider in the following sections, as well as their general classifications. We will begin with a discussion of the classical, small molecule neurotransmitters, starting with the amino acids. 3 Table 1. Classes of neurotransmitters. Small-Molecule Neurotransmitters Glutamate Aspartate Glycine GABA Catecholamines Dopamine Norepinephrine Epinephrine Indolamines Serotonin (5-HT) Acetylcholine Acetylcholine Large-Molecule Neurotransmitters Neuroactive Peptides Endogenous Opioids Endorphins Enkephalins Dynorphins Hypothalamic Peptides e.g., Corticotrophin-releasing hormone; gonadotropin- releasing hormone Pituitary Peptides e.g., oxytocin; adrenocorticotropic hormone Gut Peptides e.g., cholesystokinin Unconventional Neurotransmitters Steroid Hormones Estrogen Testosterone Cortisol Soluble Gases Nitric oxide Carbon monoxide Endocannabinoids Anandamide 2-AG Amino Acid Neurotransmitters The amino acid neurotransmitters, being amino acids, are some of the smallest of small molecule neurotransmitters. All of these, except for GABA, also play important roles as building blocks for proteins and are readily available in the proteins we consume as part of our diet. GABA, however, appears to play an exclusive role in neurotransmission and is derived from the modification of glutamate. Amino acid neurotransmitters are generally classified in terms of whether they predominantly produce excitatory2 or inhibitory3 effects when acting at their receptors. Glutamate is by far the most common excitatory neurotransmitter in the brain, whereas GABA is the most prevalent inhibitory neurotransmitter. Aspartate and glycine are two additional examples of excitatory and inhibitory amino acid neurotransmitters, respectively, but they are far less common in the brain. The amino acid neurotransmitters, perhaps more than any other neurotransmitters that we will discuss, are released primarily at directed synapses4 and produce 4 relatively fast-acting effects on post-synaptic neurons. Because of their prevalence, we will focus our discussion on glutamate and GABA in turn. GLUTAMATE Glutamate is the primary excitatory neurotransmitter in the central nervous system. It is abundant throughout the brain, and, unlike many of the other conventional neurotransmitters we will discuss, its synthesis is not particularly localised; many neurons throughout the cortex and subcortical brain regions produce and release it (Fig. 1). Figure 1. Simplified illustration of glutamate cell body distribution and axon projections in the brain. Glutamatergic neurons are localized to many cortical and subcortical sites, and their projections can travel long distances (both ascending and descending) to facilitate communication between brain areas. Glutamate Synthesis and the Glutamine Cycle The synthesis and degradation of glutamate – as part of the glutamine cycle – is an excellent example of neuron-glia interactions. The glutamine cycle is so called because glutamine serves as both a precursor to glutamate synthesis and a metabolite (or by-product) of glutamate degradation. Glutamine contained within the nerve terminals of a glutamatergic (i.e., glutamate producing) 5 neuron is converted into glutamate by the synthesizing enzyme glutaminase. Once synthesized, glutamate can be packaged into synaptic vesicles for release (exocytosis); this packaging is accomplished by the actions of small transporter proteins called VGLUTs (Vesicular GLUtamate Transporter proteins), which are embedded in the membranes of the synaptic vesicles. After release into the synaptic cleft, glutamate can produce excitatory responses at post-synaptic receptors (see next section). It is imperative, however, that the glutamate molecules are eventually removed from the synapse, as prolonged stimulation can have neurotoxic5 effects. This synaptic clean-up is accomplished through two main mechanisms, both of which involve the actions of Excitatory Amino Acid Transporters (EAATs). The axon terminals of glutamatergic neurons contain EAATs that bind glutamate molecules and transport them back into the neuron, where they will be repackaged into vesicles for subsequent re-release. Alternatively, EAATs embedded in the membranes of nearby astrocytes can also bind glutamate; however, in this case, the molecules are taken up by the astrocyte and converted to glutamine by the degrading enzyme glutamine synthetase. Thus, the glial cells and neurons form a valuable metabolic partnership in this scenario, because astrocytes can help to reduce the potential for detrimental glutamate build-up in the synapse and subsequently transfer the resultant glutamine back to the neuron terminals for future glutamate synthesis. Glutamate Receptors There are several sub-types of glutamate receptors, which can be classified into the two main receptor superfamilies: ionotropic and metabotropic (Table 2). There are at least eight sub-types of G-protein coupled (i.e., metabotropic) glutamate receptors (mGluRs). Although we will not cover them in detail in this course, it is worth noting that the mGluRs are involved in many of the same kinds of functions we will discuss for the ionotropic receptors. Furthermore, mGluRs are also found on pre-synaptic terminals and can play a role in autoreceptor-mediated negative feedback. Of the ionotropic glutamate receptors, there are three main families: AMPA (α-amino-3-hydroxyl- 5-methyl-4-isoxazole proprionic acid), kainate, and NMDA (N-methyl-D-aspartic acid), which are all named after prominent agonists. All three contain ligand-gated ion channels selective for positively-charged ions, hence their relatively fast excitatory effects when bound by glutamate. However, the specific functions of these receptors – and their behavioural roles – depend on the ion selectivity of their channels. In particular, whereas AMPA and kainate receptors are relatively typical ionotropic receptors, allowing sodium ions to flow into post-synaptic neurons when stimulated by glutamate, NMDA receptors have a more complicated mechanism of action and also conduct calcium ions (see next section). 6 Table 2. Glutamate receptors subtypes. Subtype Superfamily Ion selectivity Second messengers AMPA Ionotropic Sodium, potassium --------------------------- Kainate Ionotropic Sodium, potassium --------------------------- NMDA Ionotropic Sodium, potassium, --------------------------- calcium Metabotropic Metabotropic ------------------------- cAMP, IP3, DAG, Given its aforementioned prevalence in the nervous system, it makes sense that glutamate and glutamatergic receptors would be involved in many aspects of behaviour. Indeed, dysfunctional glutamate transmission has been implicated in cognitive and other behavioural deficits related to such human disorders as Alzheimer’s disease, schizophrenia, and attention deficit hyperactivity disorder (ADHD), to name a few, and novel therapies targeting glutamate receptors have been proposed in recent years (Arnsten and Wang, 2016). Many of these disorders are characterized by significant impairments in learning and memory, and it is this aspect of behaviour to which we now turn for a deeper consideration of one of the most well established functions of glutamatergic transmission. The NMDA Receptor and its Many Binding Sites Activation of ionotropic glutamate receptors causes depolarization of the post-synaptic neuron by entry of positively-charged ions. For the AMPA and kainate receptors, this occurs primarily because of the influx of sodium ions. The NMDA receptor conducts sodium, but it also allows calcium to enter the neuron. However, unlike the AMPA and kainate receptors, several conditions must be met before NMDA receptors will allow passage of these ions into the cell. These conditions are related to the fact that the NMDA receptor contains a number of binding sites that can influence the functioning of the receptor (Fig. 2). First, two of the subunits that comprise the NMDA receptor protein contain a binding site for glutamate (i.e., the othosteric binding sites); both of these sites must be bound by glutamate in order for the ion channel to open (Fig. 2, 1). However, this is not sufficient, as one molecule of either glycine or D-serine must also be bound to a separate co-agonist site on the NMDA receptor (Fig. 2, 2); this co-agonist site is thought to be occupied under most physiological conditions. Two other very influential binding sites are located not on the surface but within the ion channel of the NMDA receptor protein. The PCP binding site (Fig. 2, 3) has no known endogenous ligand, but is well-established to bind to certain drugs of abuse, including PCP (phencyclidine; “angel dust”) and ketamine. These drugs, like MK-801 (an experimental compound often used in the laboratory setting), are non-competitive antagonists of the NMDA receptor because they occupy the PCP binding site, thereby blocking the passage of ions through the channel of the NDMA receptor even when the glutamate and co-agonist sites are bound. In small doses, drugs like PCP or ketamine produce feelings of intoxication and numbness due to their blockade of excitatory signalling. Moderate doses produce analgesia and anaesthesia, and high doses can cause convulsions. 7 Finally, the magnesium binding site is also located within the ion channel of the NMDA receptor (Fig. 2, 4). Under resting membrane potential conditions, this site is bound by magnesium ions that block the NMDA receptor channel from conducting sodium or calcium. A key feature of this ‘magnesium block’ is that it is voltage dependent. This means that the magnesium ions are less likely to bind as the membrane potential becomes increasingly depolarized (i.e., the affinity of the magnesium ions for the magnesium binding site changes as the membrane voltage changes). So, as more pre-synaptic glutamate is released, the greater the stimulation of AMPA and/or kainate receptors, and the greater the depolarization of the post-synaptic neuron from sodium entry through these ionotropic receptors. As this membrane depolarization increases, the post-synaptic neuron will approach action potential threshold, and the magnesium block will be removed, enabling NMDA receptors to conduct sodium (which further depolarizes the cell) and calcium (which can affect signalling pathways within the neuron). Thus, the NMDA receptor only conducts ions when two things are occurring together or in close temporal contiguity: 1. Glutamate must be released pre-synaptically to stimulate the post-synaptic glutamate receptors; and 2. The post-synaptic cell Figure 2. The many binding sites of the NMDA receptor. The receptor protein, like many ionotropic receptors, consists of several subunits that come together to create a pore through which ions (in this case sodium and calcium) can pass into the neuron. However, regulation of pore opening is under the influence of several potential ligands. Glutamate must bind to two orthosteric binding sites on the NR1 subunits. There is also an obligatory co-agonist site that must bind either glycine of D-serine. Two other sites within the channel also can affect the passage of ions even if the agonist sites are activated. PCP and other drugs of abuse bind to the PCP site to prevent ion passage. Finally, the magnesium site binds magnesium in a voltage-dependent manner. As the neuronal membrane becomes less polarized, magnesium binds with less affinity to this site, such that this ‘block’ is totally removed when the neuron is fully depolarized and calcium and sodium can enter the cell. 8 membrane is sufficiently depolarized to remove the magnesium block. That is, the pre- and post- synaptic neurons are firing action potentials at the same time, and because the NMDA receptor responds specifically to this scenario, it is often referred to as a biological ‘coincidence detector’, signalling the coincident firing of the pre- and post-synaptic neurons. It is this characteristic of the NMDA receptor that determines its central role in synaptic plasticity and long-term memory. The NMDA Receptor, Hebb’s Postulate, and Long-Term Potentiation How is the foregoing discussion relevant to behaviour? Well, one of the leading theories describing how memories are stored in the brain for long periods of time, which was articulated by Donald Hebb6 over 60 years ago (Hebb, 1949), proposed a process for learning-induced synaptic plasticity7 that is eerily similar to the mechanism of NMDA receptor ion conduction that we just described. In what has become known as Hebb’s rule or Hebb’s postulate, Donald Hebb stated: “When an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased” (Hebb, 1949: 62). Hebb’s postulate is often paraphrased as, “Cells that fire together wire together”. We can now see that, should Donald Hebb’s theory prove to be accurate, the coincidence detector role of the NMDA receptor, as discussed in the preceding section, could be central to such a mechanism of synaptic plasticity. That is, the passage of calcium8 into the post-synaptic neuron through the NMDA receptor channel, which occurs only when the pre- and post-synaptic neurons are firing together, would seem to serve a potentially important signaling role, and the resultant changes to the synapse (what Hebb called a “growth process”) might provide the physiological basis for information storage. But, does such a mechanism for synaptic change even exist? When Hebb’s book was first published in 1949, nobody knew about the magnesium block or the potential for NMDA receptors as “coincidence detectors”. Nor was this information available some 24 years later when Bliss and Lomo published their findings on long-term potential (LTP), a phenomenon which appears to represent a physiological instantiation of Hebb’s ideas (indeed, LTP is now commonly referred to as ‘Hebbian plasticity’). In their seminal paper, for which they were studying the rabbit hippocampus, Bliss and Lomo (1973) described an enduring enhancement of communication between pre- and post-synaptic neurons in response to salient stimulation. In their preparation, Bliss and Lomo were able to stimulate electrically pre-synaptic neurons and record the responses in post-synaptic neurons. When they delivered a relatively weak electrical stimulus to the pre-synaptic neuron, they recorded a relatively weak post-synaptic response. As expected, when they increased the strength of the pre-synaptic stimulus, the post-synaptic response increased as well. What they did not expect to see was that, when they later delivered the original weak stimulus, the post-synaptic response was greater than before; something about the intervening strong stimulus induced a change at the synapse such that the communication between pre- and post-synaptic neurons was now strengthened or potentiated. That is, as Hebb had proposed, Cell A’s efficiency in firing Cell B had increased. An important aspect of the Bliss and Lomo study was the high frequency strong stimulus; by stimulating the pre-synaptic neuron at a high frequency (i.e., many action potentials in a short amount of time), they guaranteed that stimulation of the post-synaptic cell would be maximized, 9 causing it to fire at the same time. The well-established ability of such high frequency stimuli to induce LTP at synapses is consistent both with Hebb’s postulate and the proposal that NMDA receptors might be involved in this form of synaptic plasticity. Indeed, numerous subsequent studies revealed that active NMDA receptors are required for LTP to occur (Bliss and Collingridge, 1993), consistent with their proposed role in detecting the coincident firing of pre- and post- synaptic neurons. Later research demonstrated that there are many forms of LTP, but a key feature of all of these is the lasting or persistent enhancement in neuronal communication. The fact that these synaptic changes can last for days or even weeks after the inducing stimulus is presented, as well as the finding that LTP can also be produced by behavioural stimulation (Martin et al., 2000), strongly suggests that this form of synaptic plasticity provides a mechanism by which the brain stores information about behavioural experiences. We now turn our attention first to a simple example to explain how such a process might actually store behaviourally-relevant information and subsequently to two studies that provide empirical evidence for a role of NDMA receptor- dependent LTP in learning and memory. The NMDA Receptor and Long-term Memory While we have established that LTP resembles Hebb’s proposed mechanism for synaptic change and that it requires NMDA receptors, we have yet to discuss actual evidence for the involvement of either in behavioural memory. Suffice to say, there is ample evidence that both LTP and NMDA receptors are essential to many forms of long-term memory (i.e., memory lasting several hours or more). But before we describe some of this evidence, a simplified example of how synaptic plasticity might form the basis for associations underlying learning and memory. Imagine you are at the beach. Most people would find this to be a rather pleasant event. Now consider that whenever we experience a specific sensory stimulus or combination of stimuli, this activates a certain sub-set of neurons9 in our brains. To really simplify this example, let’s say that being at the beach activates your brain’s beach neuron (Fig. 3A). You really like the beach and have never had a particularly bad experience there, so your beach neuron has no significant synaptic connections with any neuronal circuits responsible for negative emotions such as fear (although the potential for such connections exist). However, imagine that one day you encounter a great white shark while in the water swimming. This activates your shark neuron, which is strongly connected to your fear neuron (Fig. 3B), which consequently also fires. Consider, however, that this event takes place at the beach and, as such, your beach neuron also remains active throughout the encounter. This means that your beach neuron was firing at the same time as your shark and fear neurons and, according to the theory outlined above, any connection between these cells, however weak, might be strengthened as a result of this coincident firing. Consequently, assuming you survived the encounter, the next time you are at the beach (or maybe even when you just think about the beach), your fear neuron will be much more likely to fire because of the enhanced communication at the synapse between the beach and fear neurons (Fig. 3C). As a result, you may now have a fearful response to the beach. This admittedly simplified account demonstrates how ‘Hebbian plasticity’ could strengthen connections in the brain in response to behavioural experiences and how these synaptic changes could influence future 10 behavioural responses. This is the basic definition of learning and memory, and the empirical literature strongly supports the idea that NMDA receptor-dependent plasticity (i.e., LTP) plays a central role in the acquisition and maintenance of many forms of long-term memory. Figure 3. A simplified example of how synaptic plasticity could underlie learning and memory functions that produce significant behavioral changes. Sensory neurons activated at the beach have the potential to activate fear-related neurons, but normally do not do so to any significant degree. However, if you encounter a shark at the beach, the neurons associated with the shark will strongly activate the fear neurons, resulting in simultaneous firing of these along with the beach neurons. Because the beach neurons fired together with the fear neurons, their connections are now strengthened as a result of LTP-like synaptic plasticity. Next time you go to the beach, you will likely experience more fear than before. One of the earliest studies to use behavioural genetics to investigate this question was conducted by Tsien et al. (1996). These researchers knew that NMDA receptors are made up of combinations of different sub-units (see Fig. 2). Although the specific sub-unit composition varies between NMDA receptor sub-types, all NMDA receptor sub-types contain NR1 sub-units, and these are essential for the receptor to function properly. Tsien and colleagues decided to use genetic 11 manipulation techniques to ‘knockout’ the NR1 sub-unit in the CA1 region of the hippocampus of mice (Fig. 4). CA1 CA3 DG Figure 4. The general organization of the rodent hippocampus. Many cortical inputs enter the hippocampus in the dentate gyrus (DG). Granule cells from the DG project to the CA3 region, where many neurons have axons projecting to area CA1. Tsien and colleagues (1996) knocked out the NR1 subunit in the CA1 region, which is known to be important for many of the cognitive functions of the hippocampus. This spatially limited gene knockout (KO) technique is analogous to a brain lesion, only it is exponentially more selective in its effect, as only the gene responsible for a particular protein (in this case, the NR1 sub-unit) is targeted. No other aspects of cell function are intentionally altered, and no neurons are killed. So these researchers were looking for effects of a very specific neuronal manipulation. Knocking out the gene responsible for the NR1 sub-unit should render these mice unable to produce functional NMDA receptors; thus, if the NMDA receptor is important for LTP and long-term memory, these KO mice should have impairments in both. This is exactly what Tsien and colleagues reported. First, they confirmed that the knockout was successful, demonstrating with immunocytochemistry10 that NR1 sub-unit protein was indeed significantly reduced in the CA1 region of the hippocampus in KO mice compared with wild-type control mice who were not subject to the genetic knockout. This NR1 reduction was also spatially restricted, as there was no such decrease in other hippocampal regions (e.g., dentate gyrus; CA3) of the KO mice. Similarly, LTP induction remained intact in the dentate gyrus, but was severely reduced in the CA1 region of KO mice compared with controls. Consistent with these results, the KO mice were also significantly impaired in tests of hippocampus-dependent long-term spatial memory, including the Morris Water Maze (MWM)11. Despite slower learning in the MWM and a lower final peak level of acquisition, KO mice performed the same as control mice when tested in the pool with a visible escape platform12; this suggests that the impairment in KO mice was indeed related to learning and memory and not explicable by disrupted basic motivational, motor, or sensory processes. Thus, Tsien and colleagues, consistent with many pharmacological and genetic studies that followed (Morris, 2013), concluded that NMDA receptors are essential for hippocampal plasticity and spatial memory. 12 A complementary follow-up study demonstrated that NMDA receptors can be targeted to improve synaptic plasticity and long-term memory. In this study, Tang and colleagues used genetic techniques to knock in (i.e., overexpress) the gene responsible for the NR2B sub-unit. Some NMDA receptors have NR2A sub-units, while others have NR2B. Those with NR2B sub-units appear to be more sensitive, so Tang et al. (1999) predicted that mice with an overexpression of NR2B-containing NMDA receptors would have enhanced LTP and better long-term memory. This time, the genetic manipulation was not quite as spatially restricted; mice had NR2B overexpression throughout the entire forebrain rather than just within the hippocampus. Accordingly, these mice performed significantly better on a variety of learning and memory tasks, including the MWM, fear conditioning, and object recognition, demonstrating improvements particularly with relatively long memory retention delays at which control mice could not perform. As predicted, the NR2B knock-in mice13 also had significantly enhanced LTP in the hippocampus. These studies provide just a couple of examples of evidence for the important role of NMDA receptors and synaptic plasticity in the acquisition and retention of information in long-term memory. Although exceptions can be seen in the literature, these are typical findings, and short- term memory (i.e., retaining information for a few seconds or a few minutes) does not tend to require NMDA receptors or LTP-like plasticity; other mechanisms are involved in this type of information storage. But we will leave those for another day. GABA GABA (gamma-aminobutyric acid) is the primary inhibitory neurotransmitter in the central nervous system. Like glutamate, it is found throughout the brain and is synthesized and released by many neurons throughout the telencephalon, brainstem, and spinal cord (Fig. 5). The vast majority of these GABAergic cells are interneurons, relatively small neurons with short axons that are specialized for coordinating neuronal activity within relatively local brain regions. For example, many GABAergic interneurons are found in the hippocampus, and they help to synchronize activity among the primary pyramidal cells in the various hippocampal layers before signals are sent elsewhere in the brain. This typical relationship between inhibitory GABAergic interneurons and excitatory pyramidal cells highlights a critical aspect of brain function: the important balance between inhibitory and excitatory transmission. As we noted when discussing glutamate, too much excitation can be toxic to neurons and other brain cells. Similarly, unnaturally low GABAergic inhibition can lead to brain hyperactivity and seizures. Conversely, too much inhibition can also be bad, resulting in drowsiness, intoxication, coma, and even death. 13 Figure 5. Simplified illustration of the distribution of GABAergic cell bodies and axon projections in the brain. GABAergic neurons are found throughout the brain and spinal cord. Many of these are interneurons with relatively short axons to facilitate local communication within an anatomically defined brain region. GABA Synthesis and Degradation Unlike the excitatory amino acid neurotransmitters glutamate and aspartate, GABA appears to function only as a neurotransmitter and does not participate in other typical amino acid roles such as protein synthesis and metabolism. Thus, GABA is only found in neurons that use it as a neurotransmitter. Interestingly, glutamate can also be found within GABAergic neurons because glutamate is the precursor to GABA. Indeed, glutamate is converted to GABA in a single step by the synthesizing enzyme glutamic acid decarboxylase (GAD). After release, GABA can be removed from the synapse by either GABAergic neurons or astrocytes, where it is either recycled or degraded by the enzyme GABA aminotransferase (GABA-T). GABAergic Receptors Two main GABAergic receptors have been identified, one from each of the receptor superfamilies. The GABAA receptor is ionotropic and the GABAB receptor is metabotropic. Both produce inhibitory effects, but via different mechanisms. For example, GABAB receptors can activate second messenger cascades linked to the opening of potassium channels, which can cause inhibition by allowing potassium to flow out of the neuron. Much more is understood about the 14 actions and behavioural effects of the ionotropic GABAA receptor, so we will focus on it for the remainder of this section. The GABAA receptor is found widely throughout the brain and spinal cord. It is very complex because of the variety of binding sites it possesses and the number of ligands – both endogenous and exogenous – that can influence its function. The typical GABAA receptor possesses an orthosteric binding site for GABA and at least four additional separate allosteric binding sites. When GABA binds to the orthosteric site it can cause a chloride channel to open on the receptor protein; this allows chloride to flow into the neuron, hyperpolarizing the membrane. Several other ligands can bind to the receptor protein at the allosteric sites, often indirectly influencing this inhibitory mechanism. The benzodiazepine site, as its name implies, is activated by benzodiazepine drugs such as valium and librium. These drugs are allosteric or indirect agonists because they can amplify the inhibitory effect of GABA binding to the orthosteric site. Notably, these drugs typically have no effect on their own; rather, if they are bound to the benzodiazepine site at the same time that GABA binds the orthosteric site, more chloride will enter the neuron to produce a greater hyperpolarization. Barbiturates, acting at the separate barbiturate site, have very similar effects at low doses, but can actually directly open the chloride channel at higher doses. Both benzodiazepines and barbiturates are highly effective anti-anxiety drugs (benzodiazepines are commonly prescribed as anxiolytics14) and also have anti-convulsant and muscle relaxing properties. However, both classes of drug produce undesired side effects (e.g., respiratory depression) and have high abuse potential so their medical use should be closely monitored. A third allosteric site on the GABAA receptor appears to favour endogenous steroids and their metabolites. For example, progesterone and certain stress hormones such as cortisol produce metabolites that bind here and can enhance GABAergic inhibition, producing calming and sedative effects. One function of this process could be to return the body and brain to a relaxed state after a stressful event. The picrotoxin site is named after an exogeneous ligand, a derivative of a poisonous plant. Picrotoxin is an indirect antagonist, inducing effects opposite to those discussed for the benzodiazepines and barbiturates. When bound, it can reduce the inhibitory effect of GABA by decreasing the amount of chloride that enters the neuron. Consequently, in large doses, picrotoxin can cause seizures and convulsions. Finally, a comment about the effects of alcohol is warranted. Evidence suggests that, among many other things, alcohol influences the GABAA receptor. Indeed, alcohol shows cross-tolerance15 with both benzodiazepines and barbiturates, suggesting a common mechanism of action for these drugs. However, alcohol is a very small and simple molecule that has numerous non-specific effects as well, and its exact actions at the GABAA receptor are not fully understood. Nonetheless, a GABAergic influence is certainly consistent with the established relaxing and depressant effects of alcohol. It is interesting to note that, apart from the steroid binding site, no definitive endogenous ligands have yet been discovered for the many allosteric sites of the GABAA receptor. It is reasonable to 15 suggest that such ligands exist, considering the fact that these sites did not likely evolve to respond to substances from outside of the body. In the years ahead, researchers will almost certainly identify endogenously produced ligands for these sites, the existence of which is testament not only to the complexity of the GABAA receptor but also the elaborate arrangement of signalling mechanisms that have developed to ensure fine control of inhibitory transmission in the nervous system. Monoamine Neurotransmitters Next within the broad category of small molecule, classical neurotransmitters are the monoamines, which can be further divided into the catecholamines and indolamines (Table 1). These distinctions are based on the chemical precursors and structures of the specific neurotransmitters within each class. Specifically, the monoamines are so-called because each is derived from a single amino acid precursor (Fig. 6). The three catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine) all derive from the amino acid tyrosine. Each step in the sequence of catecholamine synthesis shown in Figure 6 requires a different enzyme. Thus, the specific enzymes produced within a given neuron will determine where the synthesis pathway ends and which specific catecholamine neurotransmitter that neuron releases. Similarly, tryptophan is converted into serotonin, which can serve as a precursor for other indoleamines like melatonin. Figure 6. (A) The catecholamines are monoamine neurotransmitters that derive from the amino acid tyrosine. Each step in the pathway from tyrosine to epinephrine is catalyzed by a specific enzyme (not shown). (B) Serotonin is an indolamine that is derived from the amino acid tryptophan. Unlike the amino acid neurotransmitters, the monoamines are synthesized in a relatively localized manner. That is, there are relatively small numbers of neurons, typically within defined brain stem 16 regions, that synthesize and release specific monoamines. We will see the specific regions as we go through the different monoamine neurotransmitter systems. Despite this localized synthesis, however, many monoamine synthesizing neurons have extensive connections throughout the forebrain and hindbrain, so their influence is widespread. Consequently, the monoamines, as we will see, regulate a wide array of behaviours. We will now consider the most well studied monoamine neurotransmitters in turn, beginning with the catecholamines. CATECHOLAMINES The catecholamine neurotransmitters include dopamine, norepinephrine (also known as noradrenaline), and epinephrine (also know as adrenaline). They are monoamines because they are derived from the single amino acid tyrosine. As indicated in Figure 6A, dopamine is a direct precursor for norepinephrine, and norepinephrine is converted directly into epinephrine. Each of these chemical conversions is catalysed by a different enzyme, and therefore the specific gene expression for specific enzymes in each neuron will determine whether that neuron is dopaminergic, noradrenergic, or adrenergic. Let’s consider these catecholamine neurotransmitters in sequence. Dopamine Most of the dopamine synthesizing neurons in the brain are found in the mesencephalon (midbrain). There are two primary dopaminergic nuclei16 in the midbrain: the substantia nigra and the ventral tegmental area (VTA). Neurons in each of these nuclei have unique patterns of innervation that define distinct dopaminergic pathways or systems in the brain. Specifically, the substantia nigra neurons project their axons to the striatum (part of the basal ganglia) to form the nigrostriatal17 pathway. The VTA neurons, on the other hand, project to a variety of limbic system structures (including the nucleus accumbens and amygdala), as well as the prefrontal cortex; these pathways are referred to, respectively, as the mesolimbic and mesocortical dopaminergic systems. Figure 7 illustrates the three main dopaminergic pathways and their main targets in the forebrain. 17 Figure 7. The three main dopaminergic projection pathways in the brain. The nigrostriatal pathway begins in the substantia nigra and terminates in the striatum. The mesolimbic pathway projects from the VTA to the nucleus accumbens and other limbic structures like the amygdala. The mesocortical pathway begins in the VTA and terminates in the frontal cortex. Considering the widespread projection patterns of the midbrain dopaminergic neurons, it should come as no surprise that these pathways are implicated in a variety of behavioural functions. For example, dopamine released by the mesolimbic projections can influence emotional behaviour and motivation. Indeed, one major function of the mesolimbic projections to the nucleus accumbens is in reward processing and reinforcement. Dopamine released in the nucleus accumbens interacts with other neurotransmitters there to determine responses to typically pleasurable or rewarding stimuli, such as food, social reward, sex, and drugs of abuse. The reason we find many of these things pleasurable is because of the effects of dopamine in the nucleus accumbens and other limbic areas, and depression is associated with reduced dopaminergic effects at these sites. The mesocortical dopaminergic pathways, conversely, are implicated in many cognitive functions, including attention and working memory. Working memory is a form of short-term memory that involves the online maintenance of information used to guide ongoing behaviours (so, for example, keeping the directions active in short-term memory while you navigate a new environment). Working memory is one of the executive functions, which are broadly defined as cognitive abilities that help one to execute goal-directed behaviours. These include forms of planning and decision making, and are very reliant on the prefrontal cortex. The nigrostriatal system is strongly implicated in motor behaviour, specifically generation of voluntary movements. Much of what we know about the role of this pathway in motor behaviour comes from research on Parkinson’s disease, which is characterized by muscle rigidity, tremors, and an inability to initiate voluntary movements. Indeed, Parkinson’s disease is caused by 18 degeneration of the nigrostriatal pathway when the dopaminergic neurons in the substantia nigra die. The most common form of pharmaceutical treatment for Parkinson’s disease is L-Dopa, which is the direct precursor to dopamine (see Fig. 6A). L-Dopa is prescribed rather than dopamine because the latter does not cross the blood-brain barrier. L-Dopa is quite effective for treating the motor disturbances in Parkinson’s disease because it enables the remaining substantia nigra neurons to produce dopamine more efficiently, but it also produces various side effects, including cognitive (e.g., confusion, anxiety, hallucinations) and autonomic (e.g., low blood pressure, abnormal heart beat) effects. Five dopamine receptor sub-types have been identified (D1-D5). All of these are metabotropic receptors. The two most common sub-types are D1 and D2 receptors, and the others are categorized into two families (D1-like and D2-like) depending on their similarity to one or the other. The D1-like receptor family includes D1 and D5 receptors, and these generally produce excitatory responses. The D2-like family includes D2, D3, and D4, which all produce generally inhibitory responses. Many clinically-relevant drugs influence the dopaminergic system. For example, L-Dopa, as discussed above, is prescribed for motor impairment in Parkinson’s disease. Amphetamines, which prevent catecholamine re-uptake, have been prescribed for sleep disorders as they are powerful stimulants; they also are highly addictive. Finally, many antipsychotic drugs (e.g., haloperidol) prescribed for schizophrenia are D2 receptor antagonists. A leading theory of schizophrenia states that there is too much dopamine in certain parts of the brain. Indeed, dopamine antagonists are very effective for reducing many symptoms of the disease. However, they can also produce substantial motor side effects. Recall that L-Dopa treatment for Parkinson’s disease is associated with cognitive side effects, including confusion and hallucinations. The varied behavioural effects of bidirectional dopamine system modulation provide a clear demonstration of the fact that one neurotransmitter can be involved in many functions, and these behavioural roles are related to the widespread release of dopamine throughout the brain. It is imperative that drugs be as selective as possible to target a particular therapeutic function while limiting effects on other systems that are not disrupted in a given disorder. Too much or too little activity can both produce undesired effects. Norepinephrine Like dopamine, norepinephrine (or noradrenaline; the terms are often use interchangeably) is synthesized by a relatively localized collection of neurons in the brain stem, but the main noradrenergic nucleus is slightly lower than the dopaminergic regions discussed above. The locus ceruleus contains the majority of norepinephrine synthesizing neurons in the brain and is located in the pons within the metencephalon. Like the dopamine pathways, the axons originating in the locus ceruleus project widely throughout the brain, exerting modulatory control over many brain areas and behavioural functions. Norepinephrine is released primarily by these axons in a diffuse manner at non-directed synapses18, which enables a single axon to influence the activity of many post-synaptic neurons simultaneously. Norepinephrine is also synthesized and released by post- ganglionic neurons in the sympathetic branch of the autonomic nervous system. Similarly, both norepinephrine and epinephrine are produced in the adrenal glands and released into the bloodstream during stressful events. The release of these two transmitters in the periphery by both 19 sympathetic neurons and the adrenal glands relates to the stimulatory effects that they produce, and similar effects are observed in the brain. Indeed, noradrenergic projections from the locus ceruleus innervate many cortical and subcortical regions, generally producing excitatory responses to enhance arousal, attention, and aspects of learning and memory. Epinephrine As the names imply, epinephrine (or adrenaline) is closely related – both structurally and functionally – to norepinephrine. Although for years it was thought not to be synthesized in the brain, a few epinephrine-producing brain stem nuclei have been identified in more recent years. However, the vast majority of epinephrine is made in the periphery, within the medulla of the adrenal glands. Indeed, although it is synthesized and acts on receptors in the brain, producing the same kinds of effects as norepinephrine, the primary role of epinephrine is restricted to the periphery, where, along with norepinephrine and other adrenal stress hormones, it helps to coordinate fight-or-flight responses to stressful events. Adrenergic Receptors Epinephrine and norepinephrine act on the same receptors. The peripheral effects of epinephrine and norepinephrine in response to arousing stressful events are mediated by actions at adrenergic receptors located on organs such as the heart and lungs. Similarly, the stimulant effects of these transmitters in the brain are accomplished by action at adrenergic receptors there. There are four main types of adrenergic receptors, all metabotropic: α1, α2, β1, and β2. Depending on the specific receptor type, epinephrine or norepinephrine will produce different effects in the same or different parts of the body. Consistent with the established stimulant properties of these transmitters, α2 adrenergic receptors are implicated in attention deficit disorder (ADD). Indeed, atomoxetine, a common ADD treatment, is a norepinephrine reuptake inhibitor; by preventing removal of norepinephrine from the synapse, this drug can prolong the stimulant and attention-promoting effects of adrenergic receptor activation. Beta-blockers, such as propranolol, are a class of drug commonly prescribed for heart disorders (e.g., hypertension or high blood pressure). By blocking β adrenergic receptors, these drugs can inhibit many of the sympathetic activational effects of epinephrine and norepinephrine. Interestingly, this action of β-blockers has been exploited to treat severe anxiety disorders, as a reduction in the peripheral sympathetic effects of stress (e.g., racing heart, palpitations, sweaty palms, etc.) can have secondary effects on the central aspects of anxiety, helping patients to cope more effectively. Catecholamine drugs Considering the substantial chemical similarities between the catecholamine neurotransmitters, it is perhaps not surprising that many drugs actually affect transmission in more than one of these systems. Monoamine oxidase (MAO) is an enzyme that degrades all of the catecholamines. A popular class of anti-depressant drugs called monoamine oxidase inhibitors (MAOIs) prevents this metabolism, thereby increasing the synaptic availability of dopamine and norepinephrine. While this improves mood, MAOIs are also associated with many undesired side-effects, which is 20 unsurprising given the variety of behavioural functions in which the catecholamines are implicated. Cocaine is another example of a well-known drug that influences the catecholamines broadly. By inhibiting the reuptake pumps for dopamine and norepinephrine, cocaine prolongs their activity at the synapse. This helps to explain the many effects of cocaine, which include analgesia19, increased pleasure, and stimulant effects. Cocaine is highly addictive, and this is likely because of enhanced dopamine transmission in the aforementioned mesolimbic pathway. As with other drugs of abuse, animals will learn operant responses (i.e., pressing a lever in a skinner box) to self- administer cocaine, and these behaviours can be used to study the neurobiological bases of addiction and drug taking behaviour. In humans, prolonged cocaine consumption can lead to a syndrome called cocaine psychosis, the symptoms of which include excitability, anxiety, increased blood pressure and other sympathetic effects, bizarre, erratic behaviour, and paranoid psychosis. Clearly, cocaine is a complex drug with a multitude of consequences for the user. It also provides an excellent example of how a better understanding of the systems influenced by a particular drug can help us to explain often diverse effects. SEROTONIN AND THE INDOLAMINES The other major class of monoamine neurotransmitters are the indolamines, which include histamine, melatonin, and serotonin. However, serotonin is by far the most prevalent and influential of these, so we will focus our attention on it. Serotonin (or 5-hydroxytryptamine; 5- HT) is derived from the amino acid tryptophan. Like the catecholamines, 5-HT synthesis is mostly restricted to brain stem nuclei, although these are slightly more spread out within the midbrain, pons, and medulla. The predominant regions of 5-HT synthesis in the brain stem are the raphe nuclei, which are clusters of neurons centred around the reticular formation. Two main divisions of the raphe nuclei are the dorsal and median raphe. Serotonergic axons of the dorsal raphe target the cerebellum and basal ganglia and are implicated in motor coordination and control. Many more of the projections from the median raphe innervate neocortex and hippocampus and are involved in cognitive functions like learning and memory. Like the other transmitter systems discussed, however, serotonergic projections are widespread, and 5-HT is implicated in myriad behavioural functions ranging from mood and aggression to feeding and sleep. The serotonergic system is very complex, and it contains at least 18 different receptors, almost all of which are metabotropic. Because of the widespread influence of this system, there has been intense interest from pharmaceutical companies to understand the roles of these many receptors and their potential application to treat various human disorders. Two examples of clinically- relevant serotonergic drugs are fluoxetine and fenfluramine. Fluoxetine, the active ingredient in Prozac, is used to treat various mood and anxiety disorders, including depression and obsessive compulsive disorder. It is a selective serotonin reuptake inhibitor (SSRI), which prolongs the action of 5-HT in the synapse by preventing its reuptake. Although SSRIs can improve mood in many patients, their mechanism of action is actually poorly understood, positive effects can take 21 weeks to become apparent, and in some cases depressed and even suicidal thoughts can become worse. In addition to reducing aggressive behaviour, 5-HT appears to decrease appetite. Fenfluramine is a reuptake inhibitor that also stimulates 5-HT release. This is a highly effective appetite suppressing drug, which previously was widely prescribed to treat obesity. However, fenfluramine is also associated with severe side effects, including heart disease; when these effects became apparent, fenfluramine was removed from obesity treatments. Acetylcholine Acetylcholine (ACh), which was the first substance identified by researchers as a neurotransmitter, is in a class of its own due to its unique chemical structure. Acetylcholine is synthesized by the combination of choline with an acetyl group, a chemical reaction that is catalyzed by the enzyme choline acetyltransferase (ChAT). PERIPHERAL ACETYLCHOLINE It is perhaps not surprising that ACh was discovered first because it is highly abundant in both the peripheral and central nervous systems. In the periphery, ACh is the main neurotransmitter at neuromuscular junctions, where motor neurons interface with muscles to cause contractions based on signals coming from the brain (Fig. 8A). The motor neurons release ACh, which binds to nicotinic receptors (see below) embedded in the post-synaptic membrane of muscle cells. Acetylcholine stimulates the receptors to induce muscle contraction. Botulinum toxin (Botox) is an example of an exogenous substance that can disrupt this process of cholinergic transmission at the neuromuscular junction (Fig. 8B). Remember that when neurotransmitter is released from axon terminals by exocytosis synaptic vesicles must dock to the pre-synaptic membrane with the help of SNARE proteins. The different strains of botulinum toxin are taken up into the terminals of motor neurons at the neuromuscular junction and damage SNARE proteins such that ACh can no longer be released via exocytosis. When ACh is not released, the muscles cannot contract and will be paralysed. When this happens at the neuromuscular junctions of vital organs it can be life- threatening. Indeed, botulinum toxin is a highly potent poison, which, if not treated immediately, often kills the infected organism by respiratory paralysis. Despite its significant toxicity, Botox is used medically and cosmetically in very small doses. It can be used to treat individuals who suffer from abnormal muscle contractions. For example, some migraines result from overactive neck muscles. Blepharospasm is a condition in which the patient cannot open her eyes because of excessive muscle contraction. In these cases, minute doses of locally administered Botox can be very helpful in restoring function by temporarily paralysing the overactive muscles. Some forms of wrinkles, of course, can also be counteracted with the same approach. Also in the peripheral nervous system, ACh is found in both the sympathetic and parasympathetic branches of the autonomic nervous system. It is released by pre-ganglionic neurons in both branches, as well as post-ganglionic neurons in the parasympathetic branch. 22 Figure 8. Botulinum toxin poisoning of neuromuscular junctions. (A) At a healthy neuromuscular junction, a motor neuron releases acetylcholine, which binds to nicotinic receptors to cause the post-synaptic muscle to contract. (B) Botulinum toxin prevents this acetylcholine release by damaging SNARE proteins within the axon terminal of the motor neuron; SNARE proteins are essential for exocytosis. When less acetylcholine is released, the muscle does not contract sufficiently, resulting in paralysis. CENTRAL ACETYLCHOLINE Acetylcholine is synthesized by neurons in several nuclei within the c