A Novel Electrophysiological Measure for Fast GABA-Mediated Inhibition in Intact Circuits of Rat Hippocampal Brain Slices

Introduction:
The GABAA receptor (GABAAR) is an attractive, tractable drug discovery target. It remains unclear how native neural circuits of the hippocampus respond to drugs in this highly clinically relevant class. The CA1 region of the hippocampus is crucial for learning, memory and cognition, thus a key brain region to screen GABAergic compounds that may influence these processes. We developed a novel screen for GABAAR ligands, including general anesthetics, by measuring field inhibitory postsynaptic potentials (fIPSPs) in the CA1 area. While allowing many of the advantages of an in vitro preparation, field recordings mirror their in vivo counterparts, and unlike intracellular recordings, are minimally invasive to the neuron, typically remaining stable for many hours.
Methods:
24-28 day old Sprague Dawley rats were anesthetized and decapitated. Brains were dissected and submerged in chilled artificial cerebrospinal fluid (ACSF). 400 μm thick coronal slices were cut and maintained in ACSF bubbled with 95% O2 and 5% CO2. fIPSPs were evoked through a bipolar tungsten stimulating electrode placed in the stratum pyramidale (SP) of the CA1 region and recorded by microelectrode 300-400 μm away in the SP of CA1. GABAergic fIPSPs were isolated with NMDA and AMPA receptor antagonists (d-APV, NBQX, kynurenic acid). fIPSP dependence on GABAAR was confirmed by blockade in high dose GABAAR antagonist, picrotoxin (PTX). GABAergic ligands were applied to slices, and their effects on magnitude and decay kinetics of the fIPSP were measured. Ligands tested include: propofol, isoflurane, midazolam, diazepam, flumazenil and furosemide (FUR) and PTX.
Results:
Hippocampal GABAergic inhibition can be classified by its duration and sensitivity to allosteric modulators like benzodiazepines (BZPs). We characterized the CA1 fIPSP with compounds known to affect these parameters; a subset of our data is summarized here. FUR, a selective antagonist of GABAA-fast, dose-dependently reduces fIPSP amplitude and prolongs its decay, suggesting that the fIPSP is largely mediated by GABAA-fast synapses. In comparison, PTX, a non-selective GABAAR antagonist, depressed evoked fIPSP amplitude without modifying the fIPSP decay. fIPSPs are also sensitive to BZPs, including midazolam and diazepam, both of which enhanced fIPSP amplitude, and prolonged decay time. Flumazenil, a BZP antagonist, blocked these effects.
Conclusions: This method for studying synaptic inhibition has major advantages over conventional electrophysiological techniques: 1) it is extracellular, so key intracellular signaling molecules remain intact, 2) it detects changes in both tonic and phasic GABAAR mediated signaling, and finally 3) it is more stable and technically easier than whole-cell recording. Combining this fast, minimally cell invasive, neural population based approach affords a unique opportunity to assay multiple lead compounds for anesthetic efficacy in an intact, well characterized neural circuit with clear relevance to learning, memory and cognition.

Keywords:  Department of Biology
fIPSP
electrophysiology
field recording
inhibition
basket cell interneurons.
Thesis
Advisor: MacIver Bruce
Chen Xiaoke