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Overview / Objectives

  • Pathways involved in synthesis and metabolism of neurotransmitters
  • Describe their principle pathways in the CNS
  • Roles in CNS function (normal and abnormal)
  • A few examples of drugs affecting neurotransmitter systems in the CNS

Each stage of neurotransmission is a potential site of drug action

  1. Action potential in presynaptic nerve
  2. synthesis of transmitter
  3. Storage
  4. Metabolism
  5. Release
  6. Reuptake (nerve or glia)
  7. Degradation
  8. Receptor
  9. Receptor‐induced increase or decrease in ionic conductance
  10. Retrograde signaling

Revise all the different types of receptor!

Criteria for neurotransmitters

  1. Transmitter made/stored in vesicles
  2. Transmitter released upon nerve stimulation
  3. Action is terminated in some way
  4. Exogenous application mimics effects of nerve stimulation

Pathways in brain

  • Lots of neurological pathways
  • Learn about them in
  • Cholinergic cell bodies are very localised, specific
    • Although the axons themselves are widespread
  • Serotonin cell bodies in raphe nuclei, but axons extend very far off
  • Similar for dopamine with substantia nigra
  • Similar for noradrenaline with locus coerelus
  • Glutamate (excitatory) is not localised - very widespread cell bodies

Neurotransmitters ‐ a diverse range of chemicals

  • Amino acids
    • glutamate, GABA
  • “Classical” neurotransmitters
    • serotonin, dopamine, noradrenaline, adrenaline, acetylcholine
  • Neuropeptides
    • opioids (endorphin), tachykinins (substance P), neuropeptide Y (NPY)
  • Diffusible mediators
    • nitric oxide


Key markers

  • We use markers to identify whether neurons are cholinergic, dopaminergic, etc
  • See list in slide

Amino acid neurotransmitters

  • Essential for normal functioning of CNS
    • Excitation (glutamate) vs inhibition (GABA); need a good balance. These regulate fast neurotransmission.
  • Ionotropic receptors mediate rapid neurotransmission
    • Effects determined by ions, selective compounds
  • Fast excitatory = glutamate used to cause excitation and spread further AP down the next neuron
  • GABA, however, is coupled to a negative ion, lowering the membrane potential even more, making it more difficult for excitation of postsynaptic neuron to occur when another impulse comes in.

L‐Glutamate (Glu)

  • Major excitatory transmitter in mammalian CNS
  • Glu is vital for normal brain function
  • Involved in development, learning and memory, cognition, pain and nociception
  • Excessive activation of Glu receptors is neurotoxic
  • Widespread effects throughout the brain
  • Synthesised from glutamine using enzyme glutaminase; then glutamate is packaged into vesicles for released.
    • Glutamate is then transported via a transporter into astrocytes, then the astrocyte has glutamine synthase to break down the glutamate into glutamine. Glutamine then transported back into neuron for synthesis of glutamate (recycle).

Ionotropic Glu Receptors (iGluRs)

  • AMPA and Kainate are coupled to Na and K influx
  • NMDA mediates slightly slower responses (still millisecond), with Ca, Na and K
  • AMPA and kainate receptors mediate fast excitatory transmission
  • NMDA receptors mediate slower responses
    • voltage dependent,
    • effects mediated by Ca2+ entry
    • synaptic plasticity
    • Glycine is a co‐agonist
    • Voltage dependent Mg2+ channel block (Mg will block the NMDA receptor) - therefore we need the other receptors activated first to cause depolarisation to get rid of the Mg block

Requirements for these channels to go

  • Kainate and AMPA - just need glutamate
  • NMDA - needs glutamate + depolarisation (to get rid of Mg)

Big Problems with blockade of iGlu receptors

  • Normal excitatory function is vital; Glu is widespread - can't target certain part of brain; just blocks it everywhere
  • Competitive antagonists block Glu signalling at all synapses / regions
  • Competitive antagonists most effective at low Glu concentrations (after brain injury, Glu is very high, so drug is useless)
  • High affinity (slow off‐rate = lasting effects) --=> coma (MK‐801)
  • Moderate affinity --> hallucinations (PCP) or drowsiness (Ketamine)
  • (MK-801, PCP and ketamine are just drug names)

Metabotropic Glu Receptors

  • Table shows signal transduction pathways

Glutamatergic Neuron

  • No extracellular enzymes to metabolise Glu
    • Must be removed to prevent excitotoxicity
  • 5 subtypes of Glu transporter, with distinct pharmacology and localisation
    • EAAT1, 2 – glial (esp. astrocytes)
    • EAAT3‐5 – neuronal

Therapeutic relevance of Glu receptors and transporters?

  • iGluR blockade potentially beneficial in excitotoxicity BUT they have serious side‐effects
    • Seizures
    • PCP‐like effects
    • Memantine – used for Alzheimer’s disease (NMDA receptor antagonist)
  • mGluR modulation promising
    • Subtype selectivity difficult
  • EAAT modulation promising
    • Blockade ‐ subtype selectivity difficult
    • Modulation ‐ ?

γ‐Aminobutyric Acid (GABA)

  • Major inhibitory neurotransmitter in the mammalian CNS
  • Present in all areas of the CNS ~60‐80% of neurons may have GABA receptors
  • GABAergic neurons widespread in brain
  • Roles in regulation of respiratory, cardiovascular, visceral function, Learning and memory, seizures, schizophrenia, anxiety disorders
  • Hyperpolarizes neurons by triggering an increase in anion conductance

GABAergic Neuron

  • Glutamic acid decarboxylase (GAD) converts glutamate to GABA
  • GABAA – ionotropic
  • GABAB ‐ metabotropic
  • There's the similar recycling pathway through astrocyte with gaba-transaminase to convert gaba back to glutamate then glutamine

Common therapeutic drugs that affect GABA system in CNS

  • Benzodiazepines–positive allosteric modulators of GABAA receptor. Used as sedative, hypnotic and to prevent seizures (e.g. Diazepam)

Acetylcholine (ACh)

  • Interneurons and projection neurons
  • Basal forebrain nuclei project to forebrain structures
  • Septohippocampal projection
  • Short interneurons in the striatum
  • Synthesis:
    • Choline + acetyl CoA --ChAT--> Acetylcholine + CoA --AChE--> Choline + acetate

CNS roles for ACh

  • Memory
  • Attention
  • Movement
  • Addiction
  • Mood
  • Alzheimers patients – loss of memory related to degeneration of cholinergic neurons
  • Scopolamine (muscarinic antagonist) – induce memory deficit, reversed using donepezil (AChE inhibitor)
  • Muscarinic antagonist in poor memory prevents forgetting in low dose, gets back to normal at high dose
    • In good memory people, it causes memory loss

ACh receptors

  • Muscarinic: M1, M3, M5 are coupled to one set of signal transduction, while M2, M4 are coupled to a different set of signal transduction
  • Nicotinic receptors: are ligand-gated ion channels

CNS effects of nicotine

  • Generally via smoking tobacco
  • Involved in pleasure, reward and addiction
  • Stimulates release of dopamine in nucleus accumbens (pleasure centre in brain)
  • Regular nicotine exposure – changes in receptor numbers and sensitivity


Common therapeutic drugs that affect ACh system in CNS

  • Donepezil–AChEInhibitor–effectivein slowing down cognitive decline in Alzheimers Disease patients
  • Nicotine(gum,patches)–smokingcessation


Dopamine (DA)

  • Mesolimbic/Mesocortical pathways – ventral tegmental area – project to limbic (amygdala, nucleus accumbens) and cortical (frontal) structures
  • Nigrostriatal pathway – substantia nigra – project to striatum
  • Tuberoinfundibular pathway – arcuate nucleus (hypothalamus)
    • projections to pituitary
  • Others in retina, olfactory system
    • Important for ARx
  • Tyrosine --tyrosine hydroxylase (Rate limiting)--> Dihydroxyphenylalanine (DOPA) --Dopa decarboxylase -->dopamine


CNS roles for Dopamine

  • Movement
  • Memory
  • Mood
  • Reward
  • Addiction
  • Vomiting
  • All Dopamine Receptors are G‐protein coupled receptors
  • D1 family (D1, D5) – Gs coupled – increase AC
  • D2 family (D2, D3, D4) – Gi coupled – decrease AC

CNS Roles for Dopamine

  • MOVEMENT
    • Parkinson’s disease – degeneration of dopaminergic neurones in nigrostriatal pathway – dopamine deficiency
    • Neuroleptic medication – D2 antagonists
      • Motor dysfunction is major side effect
  • REWARD
    • Consumption of reward (nice food, wine, addictive drug) – causes pleasure – can be reduced with DA antagonist
      • Can reduce drug-seeking behaviour

Common therapeutic drugs that affect DA system in CNS

  • Schizophrenia – D2 antagonists – haloperidol
  • Parkinsons disease – L‐dopa (increases DA), carbidopa (AADC inhibitor), entacapone (COMT inhibitor), selegeline (MAOB inhibitor)
    • All these maintain dopamine effect

Noradrenaline (NA)

  • Select brainstem nuclei
  • Mainly locus coereleus (LC)
  • Terminals widespread (cortex, hippocampus)
  • Used by sympathetic neurons of autonomic nervous system
  • α, β adrenergic receptors
  • Tyrosine --tyrosine hydroxylase --> Dopa -- L-aromatic amino acid decarboxylase --> dopamine --dopamine b hydroxylase --> noradrenaline --PNMT--> adrenaline

CNS roles for NA

  • Sleep
  • Attention
  • Arousal (fear, stress)
  • Learning, memory
  • Mood (depression, anxiety)
  • Blood pressure regulation
  • NA is metabolised by Catechol‐O‐methyl transferase (COMT ) and Monoamine oxidase (MAO) enzymes
  • NA acts on α‐ and β‐ adrenergic receptors – G protein coupled receptors


NA transporter (NET)

  • Re‐uptake of NA from synaptic cleft
  • Similar structure to SERT, DAT
  • High levels of protein expression throughout brain (projections, nerve terminals)

Drugs which affect NET

  • Antidepressants – inhibit NET
    • venlafaxine
  • Cocaine – inhibits NET– maintain levels of NA
  • Amphetamines: are taken up by transporters (NET), and then VMAT, leading to leakage of transmitter out of vesicles

Common therapeutic drugs that affect NA in CNS

  • Antidepressants (TCAs, MAO Inhibitors, SNRIs)
  • Stimulants
    • Methylphenidate (ADHD)
    • Amphetamines (ADHD, narcolepsy)

Serotonin – 5 hydroxytryptamine (5HT)

  • tryptophan --tryptophan hydroxylase--> 5-hydroxytryptophan --L-aromatic amino acid decarboxylase --> 5HT --monoamine oxidase; aldehyde dehydrogenase --> 5HIAA

CNS roles for 5HT

  • Hallucinations
  • Behaviour
  • Sleep
  • Mood, emotion
  • Memory
  • Autonomic control
  • Migraine
  • Many receptor subtypes (14 so far). All except 5HT3 – GPCRs
  • All except 5HT-3 are GPCR (5HT3 is ion channel)

5HT and Sleep

  • Lesion of raphe nucleus in animals to deplete 5HT – reduce sleep
  • Injection of 5HT into animals can induce sleep
  • However – in humans
    • 5HT precursors (tryptophan, 5‐ hydroxytryptophan) – do not induce sleep in people with insomnia

5HT transporter (SERT)

  • Re‐uptake of 5HT from synaptic cleft
  • High levels of protein expression throughout brain (projections, nerve terminals)
  • Drugs which inhibit transport – can promote/prolong 5HT signalling

Drugs affecting SERT

  • MDMA (3,4‐methylenedioxy methamphetamine ‐ Ecstasy) ‐ Substrate for SERT, agonist at 5HT2 = Mood elevation, altered perception
  • Antidepressants – inhibitors of SERT
  • SSRIs (fluoxetine)
  • tricyclic antidepressants (desiprimine)
  • Cocaine – inhibits SERT

Common therapeutic drugs that affect 5HT system in CNS

  • Sumatriptan: 5‐HT1D agonist: migraine
  • Clozapine: 5HT2A/2C antagonist + dopamine:
    • antipsychotic
  • Fluoxetine:SSRI(more5‐HTinsynapse). Used to treat depression, OCD, relatively few side effects

Neuropeptides as neurotransmitters

  • More than 50 identified
  • Large molecules: chains of amino acids
  • Actions mediated by G‐protein coupled receptors

Eg

  • Opioids: Enkephalin, Dynorphin, β‐Endorphin
  • Tachykinins: Substance P, Substance K, Neuropeptide Y
  • Neuropeptides are made from gene - transcription and gene splicing. Prepeptide (large protein) is made, then cleaved. Cleaved thing is secreted then changed again. This is a relatively long process
    • Peptides and small‐molecule transmitters can coexist and be coreleased

Synthesis, release, and termination of action of neuropeptides

  • Precursors of neuropeptides, prohormones, are synthesized in the cell body and packaged in the Golgi apparatus into vesicles which are transported to presynaptic terminals by fast axonal transport along microtubules
  • Precursors are modified to become neuropeptides before reaching the terminal
  • Following exocytosis, the membranes of the large dense‐core vesicles are returned to the cell body for recycling. Inactivation also occurs via diffusion or enzymatic activity.
    • No recycling. They're broken down for peptidases in tissue.
    • Not many drugs use this - because peptides themselves get broken down in gut, and probably don't cross BBB (large molecules).

Nitric Oxide

  • Retrograde messengers as neurotransmitters
  • Non‐vesicular mechanisms
  • NO synthesis is induced at postsynaptic sites in neurons, after activation of the NMDA receptor, which causes calcium influx and activation of nNOS.
  • The gases diffuse through neuronal membranes of presynaptic terminals to influence subsequent transmitter release
  • Produced from L-arginine using nitric oxide synthase, of which there are several types (neural, endothelial. These are constitutively active, but increased on activation. iNOS is 100% inducible)
    • Arginine makes NO and citrulline
  • NO is a gas - diffuses. Doesn't act on receptors, but instead activates guanylyl cyclase enzyme, which causes effects via a signal transduction pathway


Summary

  • Glutamate, GABA – predomininantly fast synaptic transmission (some modulation – using metabotropic)
  • ACh – some fast (nicotinic), some modulation (muscarinic)
  • DA, NA, 5HT – modulation
  • Neuropeptides – modulation • Nitric oxide – modulation
  • Please Note:
    • There are many others – purines, histamine, endocannabinoids...