1. Neurotransmitters
    • Two general types:
    • Small, nitrogen-containing molecules
    • Neuroactive peptides
  2. Synthesis of small molecule NTs
    • Molecules and enzymes necessary for synthesizing small molecule NTs are contained in the presynaptic terminal
    • This ensures that the supply of NT can keep up with electrical activity
    • NT synthesis is regulated by neuronal activity levels: synthesis occurs in the cytosol, but then NTs are packaged in vesicles to protect them from degradation and to prepare them for release
  3. Cofactors in NT synthesis
    • Folic acid
    • SAM(S-adenosylmethionine)
    • O2
    • Cu2
    • Vitamins: C, B6 and B12
  4. Storage in vesicles
    • NTs are concentrated into presynaptic vesicles, which are assembled in the terminal through a process of endocytosis that provides a mechanism for recycling material
    • NTs enter vesicles using transporter proteins in the vesicular membrane
    • Transport depends on a vesicular ATPase that pumps protons into the lumen
    • The transporter exchanges H+ in the lument with NT in the cytoplasm
    • This efficient mechanism allows vesicle sot concentrate NTs to 50-100mM levels
  5. Reserpine – action
    • Blocks the vesicular transporter
    • Prevents refilling of vesicles and inhibits synaptic transmission
  6. Neurotransmitter Release
    • Depends on Ca influx
    • Fate of NT once released:
    • 1. binding to presynaptic receptors
    • 2. binding to postsynaptic receptors
    • 3. diffusion out of synaptic cleft
    • 4. enzymatic degradation
    • 5. reuptake across the plasma membrane
  7. Inotropic receptors
    • Composed of 4-5 subunits that form a pore in the membrane for passage of ions
    • Diversity comes from the variety of forms each subunit may have
    • So different receptor sybtypes can exist in different locations with different physiological/pharmacological properties and functional role
    • Subunit composition can change developmentally
  8. Metabotropic receptors
    • Transmitter binding is coupled to G protein activation and second messenter pathways
    • Diversity comes form the different typs of G proteins that are coupled to receptors and the specific subunits associated with them
  9. Reuptake by Plasma Membrane (pm) Transporters
    • Plasma membrane transporters efficiently allow nts and other molecules to cross the cell membrane
    • Pm transporters depend oncotransport of Na+ and other ions to move transmitters into the terminal against their concentration gradients
    • They can produce a 10,000 x increase in presynaptic nT concentration compared to extracellular space
    • This is a diverse group f molecules that is expressed within and outside the nervous system. Some transmitters have several transporter subtypes, which vary in location, specificity, and pharmacology
    • ImplicationL the CNS changes molecular structure to match specific needs at specific locations – most drugs canno take advantage of these differences and consequently affect the entire class of molecules
  10. Function of NT reuptake
    • 1. terminate action of NT at receptor
    • 2. prevent NT diffusion to other synapses
    • 3. recycle supply of NT in presynaptic terminal
  11. pm transporters can run in reverse
    when NT levels are high intracellularly!
  12. Some molecules: “false NTs”
    • Tyramine, guanethidine, ephedrine, amphetamine
    • Mimic NTs and bind to pm transporter to enter teminals and then bind to the vesicular transporter to enter vesicles
    • They displace the real NTs, which accumulate in the terminal cytosol
    • This can result in a large, nonvesicular leak of real NT out of the terminal and massive stimulation of receptors.
    • In addition, the false NTs may be released from synaptic vesicles to have reduced effects on post synaptic receptors (inhibitor or partial agonist)
  13. Criteria for NTs
    • Synthesized in neuron
    • Stored in nerve terminal
    • Released in quantities sufficient to affect postsynaptic cell
    • Exogenous application mimics action
    • Mechanism for removal
  14. Amino Acid Neurotransmitters
    • Glutamate, aspartate, GABA, glycine
    • These AAs are common to all cells/neurons
    • To be a transmitter, must be taken up into synaptic vesicles
    • Essential AAs cross the blood brain barrier (BBB) via transporters to enter brain
    • However, AA neurotransmitters DO NOT CROSS BBB!: ! They are restricted from entry
    • NTs must be synthesized by neurons and glia from TCA intermediates and other AAs
  15. Glutamate Synthesis
    • 70% synthesized from glutamine by glutaminase: .
    • Glucose converted to alpha-ketoglutarate to glutamate
    • The major excitatory NT in the CNS
  16. Glutamate receptors
    • Ionotropic – 14 possible subunits arranged in groups of 4 to 3 types: AMPA, Kainate, NMDA
    • Metabotropic – 8 types: mGluR1-R8
    • SIGNIFICANCE: potentially, many types of tetra/penta-meric ionotropic receptors can be made from different combinations of subunits, all responding to the same neurotransmitter
  17. Reuptake by plasma membrane Glu transporter
    • The primary mechanism for inactivation of Glu in the synapse
    • PmGlu transporter is found primarily on astrocytes (few on neurons)
    • Glia: big role in Glu inactivation and recycling
    • Astrocytes take up glutamate, convert it to glutamine via glutamine synthetase and transport it out to extracellular environment
    • Neurons take up glutamine via a glutamine transporter and convert it to glutamate
    • 5 subtypes differing in affinity, specificity, location
    • Highly effective at lowering extracellular Glu concentration
    • Elevated Glu levels are neurotoxic: !!! Glu transporter is important in buffering Glu especially if released in excessive amounts by neurons in pathological conditions
  18. GABA Synthesis
    • Gamma-amino butyric acid
    • Glutamate is converted to GABA by glutamic acid decarboxylase: (and from glutamine)
    • The major inhibitory neurotransmitter in CNS; major importance in controlling potential for seizures, anxiety, sedation. Drugs facilitate receptor function
  19. GABA vesicular transporter
    Concentrates GABA in vesicles
  20. GABA receptors
    Ionotropic – GABA-A, GABA-C; metabotropic – GABA-B
  21. GABA inactivation
    • Reuptake by neurons and glia
    • 4 different pmGABA transporters identified that differ in structure, type of cell found (neuron/glia/other), pharmacology
  22. Glycine synthesis
    • Synthesized from glucose via glycolytic intermediates
    • 2- or 3-phosphoglycerate converted to Serine
    • and Serine converted to glycine by addition of FH4
    • Inhibitory transmitter in the brainstem and spinal cord
  23. Glycine transported by
    GABA receptors also transport glycine
  24. Glycine receptor
    One type ionotropic
  25. Glycine Inactivation
    Via pmGlycine transporter (several types) on neurons and glia
  26. Monoamine Neurotransmitters
    Dopamine, norepinephrine, epinephrine, serotonin
  27. Catecholamines – 3 NTs and general characteristics
    • Dopamine, Norepinephrine, Epinephrine
    • Synthesized by a small percentage of neurons but terminals have wide distribution to large areas of brain
    • Act as excitatory and inhibitory neurotransmitters, but they also have powerful, modulatory effect (ingluence release of other transmitters) that influence motor activity, emotion, mood, attention, and arousal
    • All based on structure of catechol
  28. Synthesis of catecholamines
    • All synthesized from tyrosine: or indirectly from phenylalanine
    • Remember: phenylalanine (essential AA) converted to Tyrosine (nonessential AA) via PAH
    • The disorder PKU (PAH defect): results in low catecholamine levels
  29. Transporter for catecholamines
    • One type of vesicular transporter in brain for ALL monoamines: but
    • Second type of vesicular monoamine transporter in adrenal medulla
  30. Reserpine action
    Inhibits vesicular transporter
  31. Catecholamine receptors
    • All receptors are metabotropic (G-protein coupled)
    • Affect ion channels directly or indirectly via second messenger pathways
    • Receptor activation is complex, can cause excitation in some neurons, inhibition in others
  32. Major mechanism for stopping synaptic action of monoamines
    • Reuptake into cell
    • Two types of pmCatecholamine transporters: dopamine and NE/E transporter
    • Importance: 1. Terminates synaptic action 2. Limits diffusion to other synapses 3. Recycles unmetabolized transmitter for packaging in vesicles and its reuse
  33. Catecholamine degradation
    • 2 enzymes: MAO and COMT found intra and extracellularly in neurons and other cells
    • MAO: monoamine oxidase
    • COMT: catechol-O-methyltransferase
  34. Dopamine synthesis
    • Synthesized from tyrosine
    • Tyrosine to Dopa: by tyrosine hydroxylase (usually saturated, which is why you give Dopa for Rx, not more tyrosine..)
    • Then Dopa to Dopamine: by L-aromatic amino acid decarboxylase
  35. Tyrosine hydroxilase
    • Rate-limiting enzyme in dopamine synthesis
    • It’s activity is saturated at normal levels of tyrosine in neuron
    • Tyrosine and pnehylalanine: cross the BBB via a single transporter – this transporter is also saturated at normal blood AA levels
    • Therefore, catecholamine synthesis cannot be increased by raising tyrosine levels!: !
  36. L-aromatic amino acid decarboxylase (AADC)
    • has broad specificity for amino acid substrates
    • also present in many cell types outside of the nervous system
  37. Carbidopa
    • doesn’t cross BBB
    • inhibits peripheral AADC to prevent conversion of Dopa to dopamine peripherally
    • peripheral dopamine affects gut and causes nausea/vomiting
  38. Dopamine source
    • Midbrain is the major source of dopaminergic neurons
    • Also some in hypothalamus
  39. Dopamine receptors
    • Metabotropic D1-5
    • Excitatory or inhibitory, depending on receptor type
  40. Dopamine inactivation
    • By reuptake
    • Via pmDopamine Transporter (DAT)
    • Pm Transporter specific for dopamine: inhibited by cocaine
    • Amphetamines interact with dopamine and NE transporters
    • Neurotoxin MPP is a substrate for MPDopamine transporter: selectively kills dopaminergic neurons when internalized
  41. Norepinephrine synthesis
    • Dopamine-Beta-Hydroxylase
    • Is unique: bound to inner surface of synaptic vesicle
    • NE is synthesized inside vesicle from dopamine: by Dopamine-beta-hydroxylase
    • Thus, uses vesicular monoamine transporter
  42. Norepinephrine source
    • Pons (locus ceruleus): is the major source of NE cell bodies for CNS. The locus ceruleus influences arousal.
    • Also in postganglionic sympathetic neurons
  43. Norepinephrine receptors
    Multiple types of alpha/beta adrenergic receptors
  44. Inactivation of Norepinephrine
    • Inactivation by reuptake via pmNE transporter (NET): Inhibited by several classes of antidepressants:
    • tricyclics – imipramine, amytriptyline and Selective NE reuptake Inhibitors (SNRIs) – venflaxine, reboxetine; and cocaine
    • These drugs inhibit NE, DA and SERT transporters to varying degrees
  45. Epinephrine synthesis
    • Pnehylalanine-N-methyltransferase converts NE to E
    • Requires NE to exit vesicle
    • Undergo conversion, and then transported back into the vesicle
  46. Epinephrine source
    • Few E-neurons in the CNS,
    • Epinephrine is primarily synthesized in adrenal medulla
  47. Epinephrine receptors
    Alpha/beta adrenergic receptors
  48. Degradation of monoamines
    by MAO and COMT
  49. MAO
    • Present in neurons and most mammalian cells
    • Intracellular and extracellular location
    • Intracellularly localized to outer mitochondrial membrane: degrades monoamines not protected inside vesicles by deamination to aldehyde
  50. Functions of MAO:
    • degrades monoamines in neurons/regulates general neurotransmitter level
    • Dietary monoamines act as “false neurotransmitters”
    • MAO also:
    • 1. decreases availability of dieatary monoamines in peripheral tissues (gut)
    • 2. prevents their entry across BBB
    • MAOa and MAOb forms: differ in CNS location, substrate specificity, pharmacology
  51. MAOa
    • Distribution:CNS and gut
    • Substrate specificity: all monoamines BUT preference serotonin > NE > Dopamine
    • Present in gut and liver to breakdown dietary monoamines
    • e.g. tyramine in cheese and transporter and concentrates in vesicles via the vesicular monoamine transporter where it displaces NE
    • Irreversibly inhibited by clorgyline: !
  52. MAOb
    • Distribution: CNS (astrocytes, serotonergic neurons, histaminergic neurons)
    • Specificity: all monoamines but preference for beta-phenylethylamine
    • Irreversibly inhibited by selegiline: !
  53. MAO inhibitors (MAOIs)
    • Nonspecific irreversible inhivitors: tranylcypromine, phenelzine, isocarboxazid
    • Newer MAOIs are more selective and reversible
    • Increase presynaptic concentration of neurotransmitters and prolong availability of released neurotransmitter
  54. Caution on dangerous interactions
    • When combined with foods containing tyramine (beer, red wine, cheese, salami, soy sauce, fava beans, liver), may result in release of large amounts of NE, inducing hypertensive crisis.
    • Why: MAO normally metabolizes tyramine in gut. Excess tyramine displaces NE in sympathetic vesicles and NE is released at synapses by reversal of the pmNE transporter
  55. COMT
    • Present in nervous system and peripheral tissues; present extracellularly in synaptic cleft and degrades neurotransmitter after release
    • Broad catechol substrate specificity
    • Methylates (SAM cofactor) one of the catechol hydroxyl groups
    • Inhibitors include: entacapone, tolcapone
  56. Indolamines: Serotonin synthesis
    • 5-hydroxytryptamine/5-HT
    • from tryptophan, an essential AA transported across BBB
    • synthesis similar to dopamine: tryptophan hydroxylase is rate-limiting enzyme
  57. Serotonin concentrated by
    Vesicular monoamine transporter
  58. Serotonin Source:
    • serotonergic cell bodies: are located in midline (raphe) nuclei of the pons and medulla
    • axons distribute widely to the cortex and spinal cord
  59. Serotonin Receptors
    • 14 different receptoras identified so far (5-HT1, etc): all metabotropic, except 5-HT3(inotropic) – different roles in brain function
    • All hallucinogenic drugs are 5HT2Apartial agonists
    • Many antipsychotics are 5-HT2A and D2 dopamine receptor antagonists
  60. Inactivation of serotonin
    • Synaptic action stopped primarily by reuptake: via specific pmSerotonin transporter (SERT)
    • Inhibitors: many antidepressants (SSRIs – selective serotonin reuptake inhibitors, tricyclics) bind with high affinity; also cocaine
    • These drugs inhibit NE, DA, and SERT transporters to varying degrees
    • Serotonin also metabolized: by MAO
  61. Histamine Synthesis
    From histidine (essential AA) via AADC
  62. Histamine Source:
    • Most histaminergic cell bodies are located in the
    • Hypothalamus: !
  63. Histamine vesicular and pm reuptake transporters
    Are presumed but have not been identified
  64. Histamine receptors
    2 sybtypes of metabotropic receptors
  65. Histamine metabolized by
    MAO and histamine methyltransferase
  66. Histamine Functions:
    • Involved in circuits in hypothalamus that control/maintain arousal
    • Common antihistamines cross BBB and are neuronal H1 receptor antagonists
    • They cause sedation. Newer, 2nd generation antihistamines (loratidine) don’t cross BBB and don’t sedate
  67. Acetylcholine (ACh)
    The first identified neurotransmitter by Otto Loewi working on vagus nerve
  68. ACh synthesis
    • Acetyl-CoA + Choline -> CoA + acetylcholine -> choline + acetate
    • One enzymatic step involving: choline acetyltransferase
  69. Choline:
    • derived primarily from the diet and is transported across the BBB
    • Alternatively, choline can also be synthesized from the membrane lipid phosphatidylcholine via phosphatidylethanolamine (requires folate and vitamin B12)
    • Synthesis of acetylcholine is limited by availability of choline
    • Choline enters neuron via pmCholine transporter
  70. Source of ACh
    • In CNS: cholinergic cell bodies are located primarily in nuclei in the pons and lower frontal lobe
    • Peripherally: all cranial nerve and spinal motoneurons, preganglionic sympathetic and parasympathetic neurons, and postganglionic parasympathetic neurons
  71. Vesicular ACh transporter
    • Concentrates ACh into vesicles
    • Is inhibited by vesamicol: causes depletion of ACh from vesicles
  72. ACh receptors
    • Ionotropic: nicotinic – composed of combinations of subunits, which could result in many potential types of inotropic ACh receptors
    • Mebatropic: muscarinic: 5 subtypes (G-protein coupled) identified M1-M5
  73. ACh Inactivation
    • Ionotropic ACh receptors desensitize rapidly
    • So ACh MUST be removed quickly fro synapses
    • Enzymatic degradation: major sroute for inactivation of ACh by acetylcholine esterase, which is located in the synaptic cleft (concentrated in postsynaptic membrane) and also intracellularly
    • The resulting choline (generated by degradation) undergoes reuptake via a pmCholine transporter
    • Reuptake is the major regulator for ACh synthesis
    • NO REUPTAKE TRANSPORTER FOR ACh, but have pmCholine transporter
  74. Anticholinesterases
    • Block enzymatic activity of acetylcholine esterase
    • Cause accumulation of ACh at synapses, aCh receptor desensitization and inactive receptors
  75. Reversible inhibitors of anticholinesterases:
    • Block activity for several hours or less
    • physostigmine and tacrine: Cross BBB!
    • Neostigmine and edrophonium: don not cross BBB
  76. Irreversible inhibitors of anticholinesterases
    • Completely inhibit ACh breakdown and require new synthesis of acetylcholine esterase to replenish normal enzymatic activity
    • These include: insecticies and nerve gases such as sarin, which can result in death within 5 mins due to respiratory failure
    • Antidote involves treatment with nicotinic and muscarinic antagonists (atropine)
  77. Other types of small molecule neurotransmitters
    • Purines: ATP, adenosine
    • Important neurotransmitter in pain system. Peripheral pain fibers have purinergic receptors damaged tissues release ATP, causing excitation!
    • Adenosine receptors: are metabotropic and caffeine is an antagonist
    • Membrane-soluble molecules: NO and arachidonic acid
  78. Neuroactive Peptides
    • Small polypeptides of 5-41 AAs act as NTs to adjacent neurons, they can enter the circulation to act as hormones on distant target organs in the body, and they act as neuromodulators of activity nad behavior by influencing release of other transmitters over long periods of time
    • Their synthesis, packaging into vesicles, processing in presynaptic terminals, and function are DIFFERENT from that of the small molecule NTs
  79. Neuropeptide synthesis
    • Requires DNA transctiprion and mRNA translation
    • To produce a protein
    • Generally neuropeptides are synthesized as large precursor polypeptides (prepropeptides): that are subsequently cleaved into smaller molecules in a multistep process.
    • Thus, each precursor may give rise to many different smaller peptides that each have bioactivity
    • Location of synthesis: in cell body on ribosomes, they subsequently are processed through the endoplasmic reticulum, and then are transferred to the Golgi Apparatus where they are packaged into vesicles.
    • Vesicles containing neuropeptides travel by axoplasmic transport down the axon to presynaptic terminals
  80. Presynaptic vesicles of neuropeptides
    Undergo caocium-dependent release
  81. Inactivation of neuropeptides
    • Is slow and depends on extracellular proteases, which results in long-lasting effects
    • There are NO reuptake transporters for neuropeptides: so they cannot re-enter the presynaptic terminal
    • Synaptic transmission with neuropeptides: therefore depends on a continuous supply from the cell body
  82. Neuropeptides additional info
    • Can be co-released with small molecule neurotransmitters from the same terminal
    • They utilize a large variety of receptors, which are metabotropic, G-protein coupled
  83. Mechanisms of action of some drugs at the synapse
    • 1.NT synthesis: levodopa is converted to dopamine and therefore increases the levels of dopamine; also MAO-I – selegiline
    • 2.Storage in Vesicles: reserpine – blocks the concentration of NT in vesicles – blocks vesiculat transport of NTs
    • 3.Ca entry: none really
    • 4.Neurotransmitter release: amphetamine, amantadine
    • 5.Binding to receptors: agonists – benzodiazepens, baclofen, opiods; antagonists – antipsychotics, pyridostigmine, edrophonium, naloxone
    • 6.Degradation in cleft, metabolism, or diffusion: MAO-I – selegiline; COMT-I – entacapone; AChE-I – neuromusc/CNS
    • 7.Reuptake transporter: cocaine, antidepressants – decrease the function of the reuptake transporter, therefore increasing the concentreation of NT in the synaptic cleft
    • 8.Recycling vesicles: no drugs really
    • 9.Modulation by presynaptic receptors
    • 10.Alpha-2 agonist/antagonist
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