GABAergic Transmission

how diazapine works

 - benzodiazepines enhance IPSPs, thereby making it even more difficult for the postsynaptic neuron to fire an action potential—this is clearly important for limiting the neuronal activity responsible for seizures.

• amplifies inhibition

balacing excitation and inhibition

• seizures are a good example of what happens when this balance is tipped in favor of excitation.

• sedation and comas are examples of what happens when this balance is tipped in favor of inhibition

Gaba in the synaptic cleft

can go through reuptake, ionotropic and metabatropic receptors,

Gaba synthesis

• γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the brain.

•  GABA is actually a derivative of glutamate, and is synthesized from the decarboxylation of glutamate by glutamic acid decarboxylase (GAD).

GAD isoforms

- there are two isoforms of GAD—GAD₆₅ and GAD₆₇, which are derived from separate genes.

Glutamic acid decarboxylase (GAD)

the enzymes for synthesis are made in soma and transferred to to presynaptic terminal through axonal transport

• localalized synthesis of GABA but not GAD

GAD67 v GAD65

 - GAD₆₇ is responsible for ~ 90% of the GABA production in the presynaptic terminal, whereas GAD₆₅ is responsible for boosting GABA production when there is a high demand.

 - palmitoylation of GAD₆₅ targets it to synaptic vesicles, where it synthesizes GABA next to the transporter that loads the vesicle—GAD₆₇ is thought to associate with synaptic vesicles  through a different mechanism.

GAD staining

 - because the GADs are only expressed in GABAegic synapses, they are a good marker of GABAergic (or inhibitory) neurons in the brain.

vesicular GABA transporter

- this is accomplished by the vesicular GABA transporter (vGAT), which uses the energy of the vesicle’s proton gradient to drive GABA into the vesicle against its concentration gradient.

 - chloride ions are thought to be exchanged in order to preserve the charge balance, although the mechanism is unclear.

GABA recycling and degradation

- aside from binding to GABA receptors, GABA in the synaptic cleft is either transported back into the presynaptic terminal or neighboring astrocytes via GABA transporters (GAT).

 - GABA can  be converted into glutamate by GABA transaminase (GABA-T) and returned to the presynaptic neuron as glutamine

inhibitory post synaptic current

the reversal potential is below threshold

what ionic gradients have the potential to be inhibitory

Potassium and Chloride

how can we determine which ion mediates this IPSC?

 - note that as there is no change in PSP when the membrane potential is held at the E_Cl (with a DC current injection)—this is consistent with an IPSC mediated by a chloride conductance.

chloride-based IPSC is mediated by what

ionotropic GABA receptor called the GABA_A receptor.

GABAa receptor

 - the GABA_AR is a pentameric channel, related to the nAChR, composed of a ligand binding domain and a transmembrane channel domain.

GABAaR subunit topology

 - GABA_A receptor subunits are four-pass transmembrane proteins (M1–4) with extracellular N- and C-termini.

 - the N-terminus, which contains a cys-loop, forms the GABA binding site.

 - the intracellular loop in between M3 and M4 is the site of intracellular regulation, mostly through phosphorylation.

GABA_AR subunits

 - although α, β, γ, δ, ρ, ε, θ, and π subunits have been described, most GABA_ARs are composed of two α subunits, two β subunits, and one of the other “modulatory” subunits (γ, δ, ε, and θ).

 - ρ subunits tend to form homopentamers, and are considered a separate class of GABA receptor—the GABA_C receptor.

most common species of GABAaR

 is the 2α₁,2β₂,γ₂ isoform.

alpha subunits of GABAaR

tend to form homopentamers, and are considered a separate class of GABA receptor—the GABA_C receptor.

formation of the GABA receptor

 - the GABA_AR is formed when five subunits coalesce around a central axis to form a pore—these subunits are arranged so that their M2 segments line the channel’s pore.

binding GABA

- like its cousin the nAChR, the GABA_A receptor binds its ligand (GABA) at the interfaces between the N-terminal ligand binding domains of its subunits.

 - specifically, GABA binds to residues located in the α and β subunits—this is why there are always at least two α and β subunits in each receptor.

 - this binding event is translated into the opening of the channel’s pore.

the GABA_A receptor  pore

 - the M2 segment of the GABA_A receptor lines the channel’s pore and contains residues responsible for chloride selectivity.

 - the M2 segments of anion channels (the GABA_AR and GlyR) are similar to their cation-passing cousins (the nAChR and 5-HT₃α₁) except that they have neutral residues at the -1 position—this region of the channel is thought to form a constriction that acts as a selectivity filter.

unitary GABA_AR-mediated currents

• in symmetrical chloride solutions, GABA_AR-mediated currents reverse at 0 mV—the slope of the I–V plot reveals a single channel conductance of 30 pS.

•  - as the chloride concentration is changed to alter E_Cl, the reversal potential changes accordingly—this indicates that the GABA_AR mediates a “pure” chloride conductance.

building the IPSC

• the macroscopic IPSC is simple the summation of many unitary GABA_AR-mediated currents.

• here, we can see several spontaneous IPSCs, with one blown up on a much shorter time scale—note the discrete current steps.

the benzodiazepine binding site

• however, benzodiazepines bind at the interface between the α and γ subunits.

• furthermore, not all α subunits   interact with benzodiazepines equally —only receptors containing α₁,α₂,α₃, and α₅ can be potentiated by benzodiazepines.

the effect of benzodiazepines

• although they interact directly with GABA_ARs, benzodiazepines do not increase the single channel conductance or the dwell time in the open state.

• instead, benzodiazepines increase the frequency that the channel opens in the presence of GABA—note that benzodiazepines don’t act as agonists, but instead potentiate the receptor’s response to GABA.

interneurons of the cortical microcircuit

inhibitory inputs, pyramidal cell, excitatory inputs

defining the interneurons

• interneurons are commonly defined by the calcium-binding  proteins that they express—calbindin (CB), calretinin (CR) and parvalbumin (PV).

• interneurons are also categorized by their shape and the region of the postsynaptic cell that they innervate

variety of GABAergic interneurons of the neocortex

• there is a tremendous variety of neurons in the brain—even a specific region such as the neocortex has many types of GABAergic interneurons.

inhibitory veto power

 - inhibitory synapses are often positioned so that they can override excitatory inputs—remember the synapses on the soma and AIS?

 - in the above recording, an inhibitory neuron was stimulated (black triangle) while the neuron fired a train of action potentials—note the interruption in the train associated with stimulating the interneuron.

the symmetrical synapse

Gray’s type II, they lack the large postsynaptic density seen in glutamatergic synapses/excitatory synapses

also have pleomorphic or ‘flat’ synaptic vesicles

molecular organization of the inhibitory synapse

Gephyrin is the primary scaffolding protein

gephyrin

- the central scaffold of the GABAergic synapse (and glycinergic synapses)

 - gephyrin forms a hexagonal, “honeycomb” lattice beneath the membrane that holds GABA_A receptors in place, along with many other proteins.  

types of inhibition

phasic and tonic inhibition

phasic inhibition

 - the GABA_A receptors bound to gephyrin across from the presynaptic terminal react to changes in the [GABA] due to synaptic release—this is called phasic inhibition.

tonic inhibition

there is a non-zero concnetration of ambient GABA present

 - however, a separate, extrasynaptic population of GABA_A receptors provides a steady inhibitory current due to ambient GABA in the extracellular space or spillover from the synapse—this is called tonic inhibition.

GAMMA subunit of GABA subunit

5th subunit (modulatory) of GABA subunit that allows benzodiazapine to bind

delta subunit of GAMMA receptor

associated with the uptake of ambient Gabba of tonic inhibition- ‘extra-synaptic GABA receptors’