Academic Half-Day The Chemical Basis for Neuronal Communication Marie-Pierre Thibeault-Eybalin, R4 November 5th, 2008

Introduction 100 billion (1011) neurons in the brain Up to 100,000 terminal contacts / neuron 1016 connections between neurons / brain Connections = Synapses Chemical messenger is released at pre-synaptic membrane of axon or dendrite terminal It travels across synaptic cleft It binds onto its receptor on post-synaptic membrane of other neuron It activates effector system Chemical messenger may be released at non-synaptic locations to influence distant neurons

Criteria to define chemical messenger as neurotransmitter Localization: A putative neurotransmitter must be localized to the presynaptic elements of an identified synapse and must be present also within the neuron from which the presynaptic terminal arises. Release: The substance must be shown to be released from the presynaptic element upon activation of that terminal and simultaneously with depolarization of the parent neuron. Identity: Application of the putative neurotransmitter to the target cells must be shown to produce the same effects as those produced by stimulation of the neurons in question.

Examples of neurotransmitters

Synaptic transmission Variable synaptic delay from pre-synaptic neurotransmitter release to excitation or inhibition of post-synaptic neuron Synaptic delay depends on complexity of transduction mechanisms at post-synaptic membrane Synaptic delay Receptor Neurotransmitter Effector system Fast Few msec Ligand-gated ion channel Small molecule Flux of ions to generate transmembrane electrical potential (EPSP, IPSP ± AP if reach threshold) Slow Hundreds of msec G-protein-coupled Neuropeptide Indirect effect on ion channel Enzyme activation to produce 2nd chemical messenger (intracellular)

Sequence of events

Regulatory mechanisms To regulate amount of neurotransmitter release Pre-synaptic receptor-mediated autoregulation Neurotransmitter in synaptic cleft binds to pre-synaptic receptor Inhibitory feedback mechanism Retrograde transmission 2nd chemical messenger diffuses from post-synaptic to pre-synaptic membranes, e.g. NO

Secretory vesicles Small = Synaptic vesicles For small molecules Synthesized within vesicles, e.g. NE or uploaded by high-affinity ATP-proton-coupled transporters in terminals, e.g. ACh Recycled 50 nm diameter Cluster in active zones Large dense-cored vesicles For neuropeptides, "built-in" in neuronal soma ± co-stored small molecule Not-recycled 75-150 nm diameter Found in intraneuronal locations + terminals, less numerous Neurosecretory vesicles Hypothalamic neuron terminals in neurohypophysis For neurohormones 150-200 nm diameter

Secretory vesicles

Exocytic release Fusion pore Docking complex

Signal transduction Most receptors are transmembrane glycoproteins Binding of neurotransmitter to receptor induces conformational change 4 transduction mechanisms Ligand-gated ion channels G-protein-coupled receptors Enzymes e.g. tyrosine kinase Ligand-dependent regulators of nuclear transcription e.g. testosterone Receptors often named after family of neurotransmitters they bind e.g. cholinergic and adrenergic receptors Multiple subtypes based on response Nicotinic ACh receptors usually excitatory Muscarinic ACh receptors usually inhibitory Individual neurotransmitter family members of have different potency Rank order of potency according to EC50% Concentration of individual neurotransmitter required to reach 50% of maximal response expected The same neurotransmitter may have excitatory or inhibitory responses depending on receptor type

Structure of neurotransmitter receptors Ligand-gated ion channels Multiple subunits = transmembrane glycoproteins connected via intra-and extra-cellular loops Cylindrical Binding site in transmembrane portion Conformation changes opens gate inside channel Selectively pass small ions 2 genetic families based on AA sequence homology Nicotinic ACh, serotonin, GABA, glycine Glutamate

G-protein-coupled receptors Glycoprotein chains with multiple transmembrane loops -helices β-pleated sheets Binding site in transmembrane or extra-cellular portion 3 components Receptor GTP-binding heterotrimer Effector protein (enzyme or ion channel) Examples Rhodopsin Odorants Biogenic amines Bioactive peptides β2-adrenergic receptor                         

G-protein action

Examples of effector proteins Enzyme 2nd intracellular messenger Adenylyl cyclase cAMP Guanylate cyclase cGMP Phospholipase C IP3 and DAG Phospholipase A2 Members of the eicosanoid family

Receptor regulation Desensitization Reduction in receptor agonist-induced response after seconds to minutes of stimulation mediated by conformational changes Homologous Heterologous Phosphorylation of intracellular portion of receptor altering its binding affinity Downregulation of receptor number at post-synaptic membrane Internalization of receptor by invagination of post-synaptic membrane

Maintenance of synaptic environment To reduce or eliminate neurotransmitters in synaptic cleft Enzymatic degradation ACh cleaved by acetylcholinesterase Neuropeptides degraded by peptidases Transporter-mediated reuptake of small molecules (not neuropeptides) by pre-and post-synaptic neuron or glia (extraneuronal monoamine transport; EMT) NET for norepinephrine DAT for dopamine SERT for serotonin After reuptake, neurotrasmitter either recycled or degraded by mitochondria (MAO) COMT for norepinephrine

Pharmacologic modification of synaptic transmission Drugs may affect: Neurotransmitter synthesis Vesicular uptake and storage Depolarization-induced exocytosis Neurotransmitter receptor binding Termination of neurotransmitter action Post-synaptic effector system

Metyrosine for pheochromocytoma -Methyldopa VAMT Reserpine MAO inhibitors Yohimbine Guanethidine Cocaine Propranolol

Synopsis of clinical points Many drugs function by altering chemical transmission at the synaptic cleft. Neuropeptides play a role in the body's response to stress. Some drugs must traverse the plasma membrane to access receptors. Epinephrine is used in cardiopulmonary resuscitation and to treat anaphylactic reactions. The excess production of catecholamines, seen in tumors such as pheochromocytoma, can be treated by the drug metyrosine. Reserpine is sometimes used to treat hypertension. Reserpine may precipitate Parkinson-like symptoms or galactorrhea, or worsen clinical depression. α-Methyldopa is effective for managing hypertension during pregnancy. The side effects of guanethidine include reduced heart rate, nasal congestion, and orthostatic hypotension. Propranolol is used in the management of angina pectoris, hypertension, and congestive heart failure. Yohimbine may be effective in treating male impotence of vascular or psychogenic origin. Amphetamines enhance motor performance and relieve fatigue; these are habit-forming if used inappropriately.