Characterization of inhibitory GABA-A receptor activation during spreading depolarization in brain slice

Abstract

Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABAAR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABAAR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABAAR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl- decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABAAR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABAAR activation and suggest that intracellular Cl- loading is insufficient to generate excitatory GABAAR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABAAR antagonists, and the lack of inhibitory effect of GABAAR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABAA modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD.

Figure 1. Characterization of sIPSC during SD.

A. A representative recording of whole-cell current (I_m, top) and DC potential (DC, bottom) during SD. Whole-cell recordings were made at 0 mV to isolate GABA_AR mediated sIPSC. Right insets shows expanded spontaneous events during the periods indicated by a-d. The SD onset is defined by the onset of extracellular DC shift and is indicated by the arrow. There was a significant elevation of sIPSC frequency prior to SD arrival (b, Prodromal phase), followed by a transient suppression (c, late phase). B. Plots of mean sIPSC frequencies during SD are presented. Mean sIPSC frequency were calculated from 10 s bins during the initial 2 minutes, and later from 30 s bins. The arrow indicates the bin accompanying the onset of extracellular DC shift. Gabazine 10 µM (open squares) nearly completely eliminated sIPSC detection, and TTX 1 µM (open circle) decreased the initial sIPSC frequency increase as well as the degree of suppression. Note near complete elimination of sIPSC detection in the presence of gabazine (open squares). Statistical tests were performed between control and TTX. Control n = 9, Gabazine n = 6, TTX n = 7. *p<0.05, Control vs. TTX.

Figure 2. Comparison of sEPSC and sIPSC during SD.

A. Top: representative recordings of pairs of SDs recorded in whole-cell configurations from the same neuron, either at 0 mV or −45 mV. SD onsets were aligned based on DC potential shifts and the onset is indicated by the arrow. Bottom insets show expanded sIPSC and sEPSC recordings during baseline (a) and the SD late phase (b) from the same recordings. B. Quantitative analyses from 5 sets of paired sIPSC and sEPSC recordings, showing frequencies during baseline and the late SD phase. In two cases, recordings from the same neuron could be maintained through two rounds of SD, and in the other three cases a newly-patched CA1 neuron was used for the second recording in the pair. **p<0.01, ***p<0.005, paired t-test, n = 5 each.

Figure 3. Characterization of paired pulse ratio (PPR) of evoked IPSC during SD.

A-C. Characterization of PPR in control conditions. Pairs of IPSC (100 ms pulse interval, 0.1 Hz) were continuously evoked throughout whole-cell recordings of SD, and the PPR in different phases of SD were analysed. In the representative trace (A) evoked IPSCs are represented as sharp vertical lines. B. Single evoked IPSC pairs at corresponding time points are presented at an expanded time scale (a-c). Note the change in paired-pulse ratio as well as slow kinetics of IPSC during late phase (b). C. Quantitative analysis of PPR during control, SD late phase and following full recovery. ***p<0.005, n = 6. D&E The same experiments were conducted in the presence of the AMPA receptor antagonist DNQX (20 µM) to isolate monosynaptic components. D. Representative IPSCs are shown. Note that DNQX nearly completely abolished PPR changes during the late phase. E. Quantitative analysis of PPR changes. There was no significant change in PPR of monosynaptic IPSCs. n = 6.

Figure 4. Effects of propofol and gabazine on the large GABA_AR current during SD.

A&B Effects of propofol (200 µM) and gabazine (10 µM) on basal GABA transmission. Representative traces (A) and quantitative analysis (B) are shown. ****p<0.001, Control: n = 15, propofol n = 10 C&D Pharmacological characterization of the large GABA_AR current during SD. Gabazine nearly completely eliminated the early GABA_AR current, while propofol had little effect. Representative traces of early GABA_AR currents in control conditions as well as in the presences of gabazine and propofol, at the concentrations used in A&B. The SD onsets are defined by the onsets of extracellular DC shifts and are indicated by the arrows. D. Quantitative analysis of current amplitudes. Current amplitudes were determined from holding currents immediately before SD onset. Control: n = 9, gabazine: n = 8, propofol: n = 7, TTX: n = 7, **p<0.01 compared to others.

Figure 5. GABA_AR activation during SD contributes to cellular Cl⁻ influx.

A&B [Cl⁻]_o during SD were measured by using Cl⁻ selective electrodes. A. Representative [Cl⁻]_o change during SD, characterized as an Initial decrease and a subsequent slow overshoot response. Extracellular DC potential recorded from the reference barrel is also shown below. B. Pharmacology of [Cl⁻]_o was tested in repetitive SD. Gabazine (Gbz, 10 µM) significantly decreased [Cl⁻]_o decreases during SD, while propofol (Prop, 200 µM) was without effect. **p<0.01 C&D Extracellular volume changes were analysed by assessing bath applied TMA⁺ (0.5 mM) concentration changes. [TMA⁺]_o in slice were measured with TMA⁺ selective electrodes. Representative plots of [TMA⁺]_o and DC potential shift are shown in C and quantitative analysis of peak amplitudes are shown in D. Gabazine and propofol had no significant effect on [TMA⁺]_o responses.

Figure 6. Effects of GABA_AR potentiation and inhibition on the propagation rates and durations of SD.

A&B. Representative recording of SD generated in hippocampal CA1. A. Image sequence shows IOS signal during SD. Left top panel shows a raw image and others are ratio images of IOS. Scale bar: 400 µm. A KCl microinjection resulted in increased light transparency as indicated by increased IOS traveling across CA1 subregion (left panels, arrow indicate wavefront). SD arrival was indicated by a biphasic DC potential shift and enhanced IOS signal as indicated in B. C&D Effects of propofol (Prop, 200 µM) and gabazine (Gbz, 10 µM) on SD propagation rate and duration of DC shift (70% recovery) were evaluated by repetitively generated SD. In these experiments, 4 SDs were generated in single slices with different conditions (i.e. control, propofol, gabazine and wash, 10 minutes drug exposures or 20 minutes washout,>15 minutes recovery after SD). n = 5, ***p<0.005, * p<0.05. E&F Effects of THIP (10 µM) and picrotoxin (Ptx, 50 µM) on sIPSC and tonic current. n = 5, ** p<0.01. G&H Effects of THIP and Ptx on the SD propagation rate and DC duration. n = 5, **p<0.01.