NEUROTRANSMITTER

1. NEUROTRANSMITTER Sadiah Achmad : Department of Biochemistry FACULTY OF MEDICINE UNIVERSITY OF PADJADJARAN BANDUNG

2. NEUROTRANSMITTER : • Chemical substances that mediate signaling from neuron to another neuron or a muscle or gland cells through chemical synapses SYNAPSES : • Synapses : junctions where neurons pass signals to a postsynaptic target cell • Two types of synapses : Electrical and Chemical

3. Chemical synapses • The majority of nerve to nerve signaling • All nerve to muscle and nerve to gland signaling • Impulses are transmitted by NTs which are released from the axon terminal of the presynaptic cell into the synaptic cleft and subsequently bound to specific receptors on the postsynaptic cell • Impulse transmission occurs with a small time delay

4. Chemical synapses

5. Electrical synapses • Much less common than chemical synapses • Ions pass directly from the presynaptic cells to the postsynaptic cells through 2 nm gap junctions • An action potential in one cell generates a local current that causes an action potential in an adjacent cell • Impulse transmission is nearly instantaneous • Found in cardiac muscle and in many types of smooth muscle

6. Electrical synapses

7. TYPES OF NTs : • Acetylcholine • Monoamines: - catecholamines:dopamine, epinephrine norepinephrine - serotonin (5-hydroxytryptamin, 5-HT) - histamine • Amino acids : - aspartate, glutamate, glycine, taurine GABA • Neuropeptides:- enkephalins, endorphins, substance P vasopressin, oxytocin, etc

8. The classic NTs: • Small molecules NTs • Amino acids or derivatives, except acetylcholine • Synthesized in the cytosol of axon terminals • Stored in the synaptic vesicles and released by exocytosis • Each neuron produces one type of classic NTs • Neuropeptides are stored in a different type of vesicle.

9. Small molecules neurotransmitters

10. NTs maybe excitatory or inhibitory • Excitatory NTs: - acetylcholine, catecholamines, serotonin, histamine, aspartate, glutamate, substance P • Inhibitory NTs: - GABA, glycine, taurine • Excitatory NT: increasing membrane permeability to Na+, causes depolarization of the membrane • Inhibitory NT: increasing membrane permeability to Cl- or K+, causing hyperpolarization

11. PROCESS OF TRANSMISSION STEPS : • Arrival of an action potential opens voltage-gated Ca2+ channels cytosolic Ca2+ levels • The rise in Ca2+ triggers exocytosis of the synaptic vesicles and release of NTs • The NTs diffuse across the synaptic cleft, bind to receptors on the postsynaptic membrane  change membrane potential • Synaptic vesicles are endocytosed and recycled

12. PROCESS OF TRANSMISSION • Several inactivation mechanisms terminate the process : - enzymatic degradation of NTs - reuptake of NTs by presynaptic neuron - diffusion of NTs from the synaptic cleft • Signaling by most of the classic NTs is terminated by reuptake • Signaling by acetylcholine and neuropeptides is terminated by enzymatic degradation

13. SYNTHESIS OF NTs • Nonpeptide NTs are synthesized in the cytosol of axon terminals • Monoamine NTs are synthesized in a series of enzymatic steps from the precursor amino acids  packaged into storage granules (synaptic vesicles).

14. STORAGE OF NTs • Small molecules NTs are imported from the cytosol into synaptic vesicles by a proton-coupled antiporters in the vesicle membrane and stored • Synaptic vesicles : 40-50 nm in diameter • Synaptic vesicles membrane contains V-type ATPase (proton pumps) which maintains low intravesicular pH • Synaptic vesicles membrane consist of at least eight types of membrane proteins that function in vesicle docking and fusion

15. RELEASE OF NTs • On stimulation of the nerve cells, NTs are released by exocytosis • Exocytosis involves vesicle - targeting and fusion • Synaptic vesicle fuse with axonal membrane releasing their contents into the synaptic cleft • Synaptic vesicle are recycled locally to the axon terminus after fusion with the plasma membrane.

17. Cocaine : inhibits the transporters for norepinephrine, serotonin, and dopamine. Binding of cocaine to dopamine transporter inhibits reuptake of dopamine  prolonging signaling at key brain synapses. Dopamine transporter : principal brain “cocaine receptor”. Antidepressant drugs : • fluoxetine (prozac) & imipramine : block serotonin uptake • tricyclic desipramine blocks norepinephrine uptake

18. Fig. from Devlin 23.6a

19. Proteins of the synaptic vesicle membrane • Synapsin:- a fibrous P-protein that links synaptic vesicles - phosphorylated by c-AMP-dependent protein kinase and Ca-calmodulin (CaM) kinase  regulates number of free or bound vesicles - bind to cytoskeletal proteins actin and spectrin • VAMP (vesicle- associated membrane protein, synaptobrevin) : - involved in vesicle transport and exocytosis • Rab 3 (GTP-binding proteins) : - involved in docking & fusion of exocytosis. • Synaptotagmin : - contains four Ca2+ binding sites - Ca2+-sensing protein  triggers vesicle exocytosis

20. Fig 23.8 Devlin

21. VAMP  mechanism of action of botulinum-B toxin Botulinum –B toxin : - a bacterial protein - composed of 2 polypeptides : * one peptide binds to motor neuron that release acetylcholine at neuromuscular synapse * the other, a protease, enter into the cytosol and destroy VAMP  prevents acetylcholine release  causing paralysis

22. RECEPTORS OF NEUROTRANSMITTER Two classes of NT receptors : • Ligand-gated ion channels receptors • mediate rapid postsynaptic responses (msec) • contain 5 subunits, each has a transmembrane M2 α- helix that lines the channel • NT binding triggers a conformational change leading to channel opening and permit ion passage • G-protein coupled receptors • linked to a separate ion channel  regulate ion channel indirectly • mediate slow postsynaptic responses (seconds or more)

23. RECEPTORS OF NEUROTRANSMITTER • Depend on the specific R, the same NT can induce either excitatory or inhibitory response • Stimulation of excitatory Rs causes depolarization of postsynaptic membrane  generates an action potential • Stimulation of inhibitory Rs causes hyperpolarization of postsynaptic membrane  represses an action potential

24. ACETYLCHOLINE • Cholinergic neurons: - projection neurons : 2 clusters - basal forebrain complex - mesopontine complex • interneurons : - several brain regions including the striatum • Periphery : - preganglionic autonomic neurons - postganglionic parasympathetic neurons - neuromuscular junction

25. ACETYLCHOLINE • Cholinergic pathway in the brain : memory formation. • Alzheimer’s disease : majority of nucleus basalis neurons in the basal forebrain are lost leading to impairments in the cortical cholinergic innervation  correlate with the severity of dementia • Dementia in Parkinson’s disease : due to degenerative process in the basal ganglia and other parts of the brain

26. ACETYLCHOLINE • SYNTHESIS, STORAGE AND RELEASE : • Synthesis : transfer of acetyl group from acetyl CoA to choline, catalyzed by choline acetyltransferase • Choline is derived from diet, transported from blood by high affinity transport mechanism. Choline availability is a rate limiting factor • Stored into synaptic vesicles through vesicular H+-acetylcholine antiporter • Released from the vesicles into the synaptic cleft, reacts with the nicotinic-acetylcholine receptor on the postsynaptic membrane

27. ACETYLCHOLINE • The action of acetylcholine is terminated by acetylcholinesterase which hydrolyzes acetylcholine to choline and acetate. Choline is transported back into the nerve terminal by a H+-choline symporter  reuse Acetate is reabsorbed into the blood. • Acetylcholinesterase inhibitors is used to treat dementia of Alzheimer (augment cholinergic transmission) • Neurotoxins and nerve gasses inhibit acetylcholinesterase  prolong the action of acetylcholine  extending the period of membrane depolarization. Such inhibitors can be lethal if they prevent relaxation of the respiratory muscles

28. FIG.

29. ACETYLCHOLINE RECEPTORS • Two major classes : - Muscarinic receptors : G-protein coupled - Nicotinic acetylcholine receptors : ligand-gated ion channels • Muscarinic : - implicated in learning & memory, sleep regulation, pain perception and regulation of seizure susceptibility - 5 subtypes which are heterogenous - M1, M3, M5 : stimulate phosphoinositides - M2 & M4 : inhibit adenylate cyclase - M1 : implicated in learning & memory processes - M4 : putative targets for anticholinergics used as antiparkinson

30. ACETYLCHOLINE RECEPTORS • Muscarinic : - in peripher : - M2 regulate heart rate & contractility - M3 mediate smooth muscle contraction & glandular secretion - binding of acetylcholine to muscarinic Rs in heart muscle causes dissociation of G protein open K+ channels. Influx of K+ ions hyperpolarizes the cell membrane  slowing heart contraction (fig. page )

31. ACETYLCHOLINE RECEPTORS • Nicotinic acetylcholine Receptor : NAChR • In the brain, NAChR are found at highest densities in the area implicated in cognitive function : hippocampus, neocortex, substantia nigra, basal forebrain • Admits both K+ & Na+ • Composed of pentameric protein radially arranged around a central ion pore : subunits α2βγδ which are heterogenous • The channel opens when R cooperatively binds two ACh molecules at the interfaces of the αδ and αγ subunits • Its role is best known in synapses between motor neurons and skeletal muscle cells  neuromuscular junction

32. ACETYLCHOLINE RECEPTORS • NAChR • Binding of ACh to NAChR at neuromuscular junction triggers a rapid increase in permiability of the membrane to Na+ & K+ ions, depolarization  action potential  contraction of the muscle • Cortical NAChR : - diminished in Alzheimer’s, Nicotine administration : -improves attention defects in some Alzheimer patients - improves measures of sensory gating in some schizophrenia - Some rare familial epilepsy syndromes are associated with mutation of NAChR

33. NICOTINIC ACETYLCHOLINE RECEPTOR

34. NICOTINIC ACETYLCHOLINE RECEPTOR

35. ACETYLCHOLINE RECEPTOR

36. SEROTONIN • Serotonin systems influence CNS activity at all levels of neuraxis • Serotonergic neurons are clustered in midbrain, pons and medulla, project extensively throughout the brain and descend to the spinal cord • Two types of fibres: • Fine with small varicosities : dorsal raphe axons • Beaded with large spherical varicosities : median raphe axons • Both type of fibres are found in the neocortex, which receive serotonergic innervation from both nuclei • Caudal raphe serotonergic neurons project to medulla, cerebellum and spinal cord • MDMA (methylene-dioxy-methamphetamine, ecstasy) produces selective loss of fine axons

37. SEROTONIN • SYNTHESIS : • Serotonin is synthesized from tryptophan. • Brain uptake of tryptophan (active carrier mechanism) is determined by circulatory tryptophan & by ratio of tryptophan to other large neutral amino acids • Steps : - hydroxylation of tryp by tryp hydroxylase  5-hydroxytryp (rate limiting) - decarboxylation of 5-hydroxytryp by aromatic amino acid decarboxylase  5-hydroxytryptamine (serotonin)

38. SEROTONIN • DEGRADATION : - Mediated by monoamine oxidase type A (MAOA) which oxidizes amino group to form aldehyde - Further oxidation by aldehyde dehydrogenase  5-hydroxy- indolacetic acid (5-HIAA) • Kidney, liver tissue, fecal bacteria : convert tryptophan to tryptamine  indole 3-acetate. Urinary catabolites : 5-HIAA & indole 3-acetate - MAO inhibitors : antidepressant effect  elevation of serotonin

40. SEROTONIN RECEPTOR • Great diversity : a single NT produce a wide variety of cellular effects in multiple neuronal systems • Two types : - G protein-coupled : cAMP & inositol-3P as 2nd messengers - ligand-gated ion channels • G-protein-coupled • 5-HT1 : - the largest subfamily with subtypes - inhibits adenylate cyclase  ↓ cAMP - 5-HT1A : postsynaptic & presynaptic(autoR) Stimulation of autoR suppresses activity of serotonergic neuron

41. SEROTONIN RECEPTOR • 5-HT2 : - stimulates phosphoinositide turnover  IP3 - antidepressant, antipsychotic : antagonize 5-HT2C R - hallucinogen (LSD) : agonist activity at 5-HT2 R • 5-HT4, 5-HT6, 5-HT7 : stimulate adenylate cyclase   cAMP  indirectly modulate K+ channel • Ligand-gated ion channel • 5-HT3: - passage Na+ & K+  rapid excitatory effects in postsynaptic neurons

42. Serotonin modulatory synapse

43. CATECHOLAMINES • DOPAMINE • Dopamine neurons : more widely distributed • Three dopamine systems : 1. nigrostriatal 2. mesocorticolimbic 3. tuberohypophyseal • Nigrostriatal: - cell bodies : in the pars compacta substantia nigra - ascending projection to dorsal striatum : modulate motor function - motor disturbances in Parkinson’s disease : degenerative disorder of nigrostriatal system - extrapyramidal adverse effects of antipsychotic drugs result from blockade of striatal receptors

44. DOPAMINE • 2. Mesocorticolimbic: - Ascending projection  innervating limbic structures & associated cortical structures - Regulate a wide variety of stimuli, including drugs of abuse - Target of antipsychotic drugs (dopamine R antagonist) • 3. Tuberohypophyseal : - dopaminergic neurons in hypothalamic arcuate & periventricular nuclei. - projections to pituitary : inhibitory regulation of prolactin release Administration of antipsychotic drugs → galactorhea

45. NOREPINEPHRINE & EPINEPHRINE • Function as both systemic hormones & NTs • NE : at synapses in CNS and peripheral neurons (synapses with smooth muscles innervated by sympathetic motor neurons) • SYNTHESIS • In adrenal medulla & the brain • Hydroxylation of tyrosine to dopa by tyrosine hydroxylase • Decarboxylation of dopa to dopamine by dopa decarboxylase • Hydroxylation of dopamine to NE by dopamine β-hydroxylase, in catecholaminergic vesicles within adrenergic and noradrenergic neurons • Conversion of NE to E by phenylethanolamin-N-methyl transferase (PNMT) in adrenergic neurons

47. CATECHOLAMINES • DEGRADATION • The action of CA is terminated by reuptake into the presynaptic neuron  repackaged into synaptic vesicles or metabolized • Metabolism by : - catechol-O-methyl transferase (COMT) & - monoamine oxidase (MAO) • COMT: catalyzes transfer of a methyl group from S-adenosyl- methionine to a phenolic -OH group • MAO : catalyzes oxidative deamination of amines to aldehydes • End product - dopamine : homovanillic acid (HVA) - NE & E : 3-methoxy-4-hydroxymandelic acid (MHMA) • CA metabolites : indicators of the activity of catecholaminergic systems

48. COMT : - distributed throughout the brain & peripheral tissues - wide substrate specifity - catalyzing transfer of methyl groups from S-adenosyl- methionine to hydroxyl group of catechol compouds • MAO : - located on the outer membrane of mitochondria - oxidatively deaminates catecholamines to aldehydes - two isoenzymes : MAOA & MAOB - MAOA: preferentially deaminates serotonin & NE - MAOB: deaminates histamine, dopamine & phenylethylamine - in peripheral tissues (GI & liver) : prevent accumulation of toxic amines • MAO inhibitor : - block MA catabolism  monoamines in the brain - adverse effects :  peripheral amines

49. CATECHOLAMINES RECEPTORS • All known CA Rs are coupled to G proteins • Different Rs are linked to different G pr  different 2nd messengers • DOPAMINE Rs : D1-5 • D1 : - stimulates - adenylate cyclase   cAMP - phosphoinositide turnover   IP3 - not found on dopaminergic neuron ( not an autoR) - contribute to the effects of cocain in CNS - low affinity for antipsychotic (butyrophenones, halloperidol) • D5 , D1-like : - structural similarity - stimulates adenylate cyclase

50. DOPAMINE RECEPTOR • D2 : - interacts with a variety of G pr diverse 2nd messenger  - ↓ cAMP - modulates Ca2+ & K+ channel - alters phosphoinositide production - functions as postsynaptic or autoR on dopaminergic terminals, cell bodies & dendrites of dopaminergic neuron - high affinity for antipsychotic drugs - in ant. pituitary : inhibition of prolactin & MSH release - in schizophrenia :  D2R - extrapyr adverse effects of antipsychotic: block of striatal D2R • D3,D4 , D2-like :- similar in structure & pharmacology - D4R : more abundant in heart than in the brain - in schizophrenia :  D4R