GABAergic and Glutamatergic neurotransmission

Neurotransmitters in the CNS

• GABA (gamma-amino butyric acid) = major inhibitory

• Glutamate = primary excitatory

GABA inhibitory NTs

• open K+ channels or Cl- to induce K+ efflux or Cl- influx

• loss of intracellular cations or gain of intracellular anions

• results in membrane hyperpolarization and decreased membrane resistance

• move membrane potential further below threshold value

• reduce ability of inward currents to depolarize membrane

Glutamate excitatory NTs

• may open cation-specific channels (Na+)

• net influx of Na+ ions

• depolarizes membrane

• excitatory (depolarizing) response

• can also close K+ leak channels to reduce outward flow of K+ and depolarize membrane

Inhibitory neurotransmitters do what to membranes?

hyperpolarize membranes

Direct effects of inhibitory neurotransmitters

induce net outward current by promoting influx of anions (ex. opening Cl- channel)

Indirect effects of inhibitory neurotransmitters

• induce net outward current by promoting efflux of cations (ex. opening K+ channel)

• G proteins, 2nd messengers

Glutamic acid decarboxylase

• GAD

• decarboxylates glutamate to produce GABA

• requires pyridoxal phosphate (vitamin B

VGAT

• vesicular GABA transporter

• transports GABA into vesicles

GATs

• GABA transporters

• remove GABA from extracellular space

• facilitates termination of GABA in synapse

GABA-T

• GABA-Transaminase

• converts GABA to SSA (succinic semiaaldehyde)

• SSA oxidized by SSA dehydrogenase to succinic acid to enter Krebs cycle (becomes alpha-ketoglutarate)

• regenerates glutamate from alpha-ketoglutarate

• irreversibly inhibited by vigabatrin

Types of GABA receptors

• Ionotropic

• Metabotropic

Ionotropic GABA receptors

• GABA_A and GABA_C

• bind GABA and open intrinsic Cl- channels

• pentameric transmembrane glycoprotein; ion pore surrounded by 5 subunits, each of which has four spanning membrane domains

• GABA binds to 2 sites in extracellular part of receptors

  • binding sites: interface of alpha and beta subunits

• inhibitory postsynaptic currents (IPSCs) activated by very brief (high-freq) bursts of GABA release at synapses

Metabotropic GABA receptors

• GABA_B

• heterodimeric G protein-coupled receptors

• activate neuronal potassium channels through second messengers

GABA_A

• numerous modulatory sites for other ligands/drugs binding

• activation requires binding of 2 GABA molecules, ne to each receptor site

GABA_C

• pentameric ligand-gated chloride channels

• distribution in CNS limited to retina

• receptors display distinct pharmacologic properties that differ from GABA_A

• no drugs in use target GABA_C

GABA binding site for GABA_A

• site near junction of alpha and beta subunits

• open chloride channel

Benzodiazepine GABA_A binding site

• allosteric site by clef between alpha and gamma subunits

• facilitates GABA binding and increases chloride channel opening

Barbiturate GABA_A binding site

• bind adjacent to alpha and beta subunits

• increase duration of chloride channel opening

Ethanol GABA_A binding site

• distinct site on ionophore

• enhances chloride influx

Ionophore binding sites for

• ethanol

• steroids

• inhalational anesthetics

Effects of GABA on GABA_A

Effect of drugs that inhibit GABA_A receptors

• produce seizures in animals

GABA_A mutations that impair activation are associated with

inherited human epilepsy

Where are GABA_A receptors located in PNS?

airway epithelium

Activation of GABA_A receptors may

• decrease neuronal excitation and impair CNS functions

• enhance smooth muscle relaxation (bronchodilation)

Metabotropic GABA receptors

• heterotrimeric G-protein coupled receptors

• affect neuronal ion currents through 2nd messengers

• expressed at lower levels

• GABA_B

• interacts with G proteins, leads to dissociation of Beta-Gamma subunit, which directly activates K+ channels and inhibits opening of voltage gated Ca2+ channels

• suppresses adenylyl cyclase, decreases cAMP

• modulate transmitter release by reducing Ca2+ influx

• obligate heterodimer of GABA_B1 and GABA_B2

  • each are 7 transmembrane spanning GPCR

Where are GABA_B receptors located in CNS?

postsynaptically and presynaptically (autoreceptors)

How do postsynaptic GABA_B receptors produce IPSPs?

through G protein activated inward rectifier K+ channels (GIRKS)

What does increased K+ efflux results in?

slow, long-lasting inhibitory postsynaptic potentials

What does reduced Ca2+ influx result in?

ability of GABA_B autoreceptors to inhibit presynaptic neurotransmitter release

Inhibitors of GABA metabolism

• Tiagabine

• Gamma-Vinyl GABA (vigabatrin)

Tiagabine

• competitive inhibitor of GABA transporter in neurons and glia

• selective for GAT-1

• increases synaptic and extrasynaptic GABA concentrations, non specific agonist both ionotropic and metabotropic GABA receptors

• 90% bioavailability

• highly protein bound

• metabolized by CYP3A4

• adverse: confusion, sedation, amnesia, ataxia, can potential other GABA_A receptor modulators (alcohol, benzodiazepines, barbiturates)

Gamma-Vinyl GABA

• vigabatrin

• suicide inhibitor of GABA-T

• blocks conversion of GABA to succinic semialdehyde

• increases GABA concentrations synaptic release

• used in treatment of epilepsy

• investigated for treatment of drug addiction, panic, OCD

• adverse: drowsiness, confusion, headache, bilateral visual field defects

GABA receptor agonists

• Muscimol

• bind directly to and activate GABA_A

• full agonist

GABA receptor Antagonist

• Biculline: competitive

• Picrotoxin: non-competitive

• all infuce epileptic convulsions

• exclusively used for research

Picrotoxin

• non- competitive inhibitor of GABA_A

• blocks ion pore

GABA inverse agonists

• Beta-carbolines

• elicit anxiogenic effects and convulsions

Benzodiazepine Agonists

• Alprazolam (Xanax)

• Clonazepam (Klonopin)

• Diazepam (Valium)

• Lorazepam (Ativan)

• end in “pam” or “lam” except Chlordiazepoxide

Benzodiazepine Antagonist

• Flumazenil (Romazocon)

Benzodiazepine Inverse agonist

• Beta-Carbolines

Nonbenzodiazepine hypnotic

• Zoldipem (ambien)

• imidazopyridine class potentiates GABA by binding GABA_A receptors at same locations as benzodiazepines

• short acting (15 min) used for treatment of insomnia and some brain disorders

Benzodiazepines

• modulators of the GABA_A receptor at allosteric binding sites to enhance GABAergic neurotransmission

• sedative, hypnotic, muscle relaxant, amnestic, and anxiolytic effects

• high affinity and selectivity, highly plasma bound

• act as positive allosteric modulators by enhancing GABA_A receptor channel gating in presence of GABA

• increase freq of channel opening when GABA conc low, slow receptor deactivation when GABA conc high

  • INCREASE net Cl- influx

• DO NOT activate native GABA_A receptors in absence of GABA

• shift dose response curve to left, increase potency

• lower margin of safety when co-administered with alcohols, other sedatives hypnotics

Benzodiazepine Clinical applications

• differ in onset of action, duration of effect

• tendency for rebounds when withdrawn

• used as sleep enhancers, anxiolytics (inhibit synapses), panic disorders, sedatives, anti-epileptics, muscle relaxants, alcohol withdraw symptoms

• intermittent use bc of potential development of tolerance, dependence, addiction

• used for brief uncomfortable procedures

• used prior to general anesthesia

• reduce skeletal muscle spasticity by enhancing inhibitory interneurons in spinal cord

  • treat muscle spasms in neuromuscular degenerative disorders

Points of discussion for Benzodiazepines

• induce comparable respiratory changes to that of natural sleep, no cardiovascular changes in healthy individuals

• for patients with pulmonary/cardiovascular disease, respiratory/cardiovascular depression may occur (medullar depression)

• patients with stroke, head trauma profoundly sedated

Diazepam clinical use

• anxiety

• epilepticus

• muscle relaxant

• IV general anesthetic

• alcohol withdraw

• long acting

Benzodiazepine adverse effects

• releated to therapeutic effects in undesirable settings

• amnesia, over-sedation, ataxia, sleep walking, sleep driving, sleep eating

• high doses rarely cause death unless administered with other drugs

  • ethanol: inhibits CYP3A4

  • CNS depressants, opioid analgesics, tricyclic anti-depressants

• arrythmias

• CNS depression

• drug dependence

• hypotension

• mild respiratory depression

How can benzodiazepine overdose be reversed?

Flumazenil

What is associated with chronic benzodiazepine use?

decreased efficacy of both benzodiazepines and barbiturates

possible decreased receptor density at synapse or uncoupling of receptor at GABA site

Up-regulation of receptors

• increase receptors

• sensitization

• by sustained reduction in NT release or long-term administration of receptor antagonist

Down-regulation of receptors

• desensitization

• decrease receptors

• by sustained blockade of NT reuptake or by long-term administration of receptor agonist

Barbiturate Agonists

• Phenobarbital (Luminal)

• Thiopental (Pentothal)

• Pentobarbital (Nembutal)

• end in tal

Barbiturates

• modulator of GABA_A receptor at allosteric binding sites to enhance GABAnergic neurotransmission

• large group of drugs used for control of epilepsy, general anesthetic induction, control of intracranial hypertension, causes sedation, loss of consciousness, amnesia

• GABAnergic transmission at motor neurons suppresses reflexes and relaxes muscles

• bind to specific site on receptors, NOT binding site

• enhance efficacy of GABA by increasing the time that the Cl- channel stays open, permitting more influx of Cl- ions

  • greater hyperpolarization, less excitability

• greater GABA-enhancing ability than benzodiazepines

• also affect excitatory neurotransmission by decreasing AMPA receptors by glutamate, reducing depolarization and excitability

Benzodiazepine overdoses are…

deeply sedating but rarely dangerous

Barbiturate overdose…

may induce profound hypnosis, coma, respiratory depression, death (more severe vs benzodiazepines)

Anesthetic Barbiturates

• thiopental, pentobarbital

• agonists at GABA_A and enhancers of GABA_A response

What determines a barbiturate’s duration of action?

rapidity with which it is redistributed from brain to other, less vascular compartments (muscle, fat)

Dose response curve of barbiturates

• linear dose-response effect

• progresses from sedation to respiratory depression, coma, death

Dose response curves of benzodiazepines

• ceiling effect

• precludes severe CNS depression following oral admin

• IV admin can produce anesthesia and mild respiratory depression

Baclofen

• only compound used clinically to target GABA_B receptors

• treat spasticity associated with motor neuron disease or spinal cord injury

• stimulates downstream 2nd messenger to act on Ca2+ and K+ channels

• may modulate pain, cognition (investigated for drug addiction)

Adverse effects of baclofen

• sedation, somnolence (drowsiness), ataxia

Non-prescription drugs that alter GABA physiology

• ethanol

• gamma-hydroxybutyric acid (GHB)

• Flunitrazepam (Rohypnol)

Binding of Glutamate to receptor induces:

• excitatory neuronal responses associated with motor neuron activation

• acute sensory responses

• development of elevated pain response (hyperalgesia)

• synaptic changes associated with memory formation

• cerebral neurotoxicity from brain ischemia and functional deficits from spinal cord

Glutamate synthesis

• 2 pathways

• Alpha-ketoglutarate (Krebs cycle) transaminated to glutamate in CNS terminals

• Glutamine produced and secreted by glia cells transported into nerve terminals and converted to glutamate by glutaminase

Ionotropic Glutamate receptors

• fast excitatory synaptic responses

• multi-subunit (possibly tetramer), cation-selective channels that permit flow of Na+, K+, Ca2+

• 3 main subtypes classified by selective agonists, arise from amino acid sequence of alternative splicing

  • AMPA

  • Kainate

  • NMDA

AMPA receptors

• located throughout CNS, particularly hippocampus (HPC) and cortex (CX)

• four subunits (GluR1-GluR4)

• results in Na+ influx, K+ efflux, Ca2+ inflex; excitatory postsynaptic depolarizations

• bind glutamate or AMPA

Kainate receptors

• expressed throughout CNS, particularly in hippocampus and cerebellum

• 5 subunits (GluR5, GluR6, GluR7, KA1, KA2)

• allow Na+ influx, K+ efflux (Ca2+ entry depends on subunits)

• bind Kainate and glutamate

NMDA receptors

• expressed in hippocampus, cortex, and spinal cord

• multi-subunit oligomeric complexes (NR1, NR2A, NR2B, NR2D)

• activation requires simultaneous binding of glutamate and glycine

• allows Na+ influx, Ca2+ influx, K+ efflux

• Mg2+ blocks channel pore in resting membrane

  • depolarization required to relieve block

  • can occur by post-syanptic action potentials or activation of adjacent AMPA/Kainate receptors

• bind glutamate + glycine OR NMDA + glycine

Metabotropic Glutamate receptors

• seven transmembrane spanning domain coupled to G proteins

• at least 8 subtypes

• 3 groups

Group I Metabotropic Glutamate Receptors

• cause neuronal excitation through phospholipase C and IP3 mediated release of Ca2+ or adenylyl cyclase activation and cAMP generation