Distinctions among GABAA and GABAB responses revealed by calcium channel antagonists, cannabinoids, opioids, and synaptic plasticity in rat hippocampus

Abstract

Rationale: Hippocampal interneurons release gamma-aminobutyric acid (GABA) and produce fast GABA(A)- and slow GABA(B)-inhibitory postsynaptic potentials (IPSPs). The regulation of GABA(B) eIPSPs or the interneurons that produce them are not well understood. In addition, while both micro-opioid receptors (microORs) and cannabinoid CB1R receptors (CB1Rs) are present on hippocampal interneurons, it is not clear how these two systems interact.

Objectives: This study tests the hypotheses that: (1) all interneurons can initiate both GABA(A) and GABA(B) inhibitory postsynaptic potentials; (2) GABA(B) responses are insensitive to mGluR-triggered, endocannabinoid (eCB)-mediated inhibitory long-term depression (iLTD); (3) GABA(B) responses are produced by interneurons that express microOR; and (4) CB1R-dependent and microOR-dependent response interact.

Materials and methods: Pharmacological and electrophysiological approaches were used in acute rat hippocampal slices. High resistance microelectrode recordings were made from pyramidal cells, while interneurons were stimulated extracellularly.

Results: GABA(B) responses were found to be produced by interneurons that release GABA via either presynaptic N-type or P/Q-type calcium channels but that they are insensitive to suppression by eCBs or eCB-mediated iLTD. GABA(B) IPSPs were sensitive to suppression by a microOR agonist, suggesting a major source of GABA(B) responses is the microOR-expressing interneuron population. A small eCB-iLTD (10% eIPSP reduction) persisted in conotoxin. eCB-iLTD was blocked by a microOR agonist in 6/13 slices.

Conclusions: GABA(B) responses cannot be produced by all interneurons. CB1R or microOR agonists will differentially alter the balance of activity in hippocampal circuits. CB1R- and microOR-mediated responses can interact.

Fig. 1

Conotoxin differentially suppresses GABA_A and GABA_B eIPSPs. a A 250-msec negative dc pulse is given at the beginning of the trace to assess passive cell properties. Arrows indicate points at which GABA_A and GABA_B amplitude were measured. The dashed line shows an exponential curve fitted to the initial falling phase of the GABA_A eIPSP and extrapolated through the total response. b GABA_B response obtained by subtracting the GABA_A eIPSP (calib.=1 mV). c Comparison of the GABA_A and GABA_B eIPSP reduction after addition of conotoxin to the bath perfusion (n=7). Traces: Gray trace eIPSP before conotoxin, black trace eIPSP after the application of conotoxin. In all figures, the traces shown are the average of ten traces. The conotoxin concentration was 250 nM in all figures. The calibrations for the traces are: x-axis, 2 mV; y-axis, 200 ms, unless otherwise stated

Fig. 2

Agatoxin suppression of GABA_A and GABA_B eIPSPs. a Comparison of the GABA_A and GABA_B eIPSP reduction after addition of 200 nM agatoxin to the bath perfusion (n=3). b Time course of isolated GABA_B response reduction by agatoxin (n=5). Traces: Gray trace eIPSP before agatoxin, black trace eIPSP after the application of agatoxin

Fig. 3

DAMGO depresses both responses. a Graph showing the time course of the suppression of GABA_A eIPSPs by DAMGO (100 or 500 nM; n=17). b Suppression of the isolated GABA_B response (with bicuculline 20 M, present as indicated. Gray trace eIPSP before DAMGO, black trace eIPSP after the application of DAMGO

Fig. 4

iLTD of GABA responses. a Example of a 1-s/100-Hz stimulus train; four were given, each 1 s after a 1-s depolarizing dc injection. b A 10-min DHPG application (black circles, n=27) reduces the amplitude of an eIPSP for more than 30 min (iLTD). Synaptic stimulation (white circles, two 100-Hz stimulus trains, 20 s apart, n=6) also induced robust iLTD. c iLTD is blocked by AM 251, 3 µM (black circles, n=4) and by LY367385 100 µM plus MPEP 5 µ;M (gray circles, n=4). d Synaptic stimulation after a 10-min exposure to DHPG caused a further decrease in the GABA_A response (n=5). Traces (Fig. 4a–c): light gray line control, black line 25 min after the application of DHPG or synaptic stimulation. Traces (Fig. 4d): light gray line control, dark gray line 25 min after the application of DHPG, black line 25 min after synaptic stimulation

Fig. 5

The GABA_B component of the eIPSP is unaltered by DHPG-mediated endocannabinoid mobilization. a Representative GABA_B response before (black trace) and 25 min after (gray trace) DHPG treatment (calib.=1 mV). b The isolated GABA_B is insensitive to a 10-min DHPG treatment (n=7); c percent reduction in GABA_A and GABA_B eIPSPs by DHPG application

Fig. 6

Differential effects of calcium channel antagonists on DAMGO-induced eIPSP suppression. DAMGO suppresses GABA_A eIPSPs to a greater extent in slices treated with conotoxin (white circles, n=4) than in slices preincubated in agatoxin (black circles, n=4)

Fig. 7

Differential effects of calcium channel antagonists on eCB-induced eIPSP suppression. a Pre-incubation with agatoxin does not reduce iLTD. White circles Control (not preincubated in agatoxin, n=27; same data as in Fig. 3b, white circles). Black circles Agatoxin, n=8). b Conotoxin does not entirely prevent iLTD induction measured 20 min after DHPG washout (n=5). c DHPG-induced iLTD is blocked byAM251 3 µM(n=3; calib. for inserts in b and c=1 mV). Traces before (gray) and after (black) DHPG application

Fig. 8

DAMGO treatment prevents DHPG-induced iLTD induction in a subset of slices. a Pooled data (n=6) of cells that did not show DHPG-induced iLTD in the presence of DAMGO. b Pooled data of cells (n=7) that did show DHPG-induced iLTD in the presence of DAMGO. c PPR comparison of cells that did (white bars) and those that did not (black bars) express iLTD in the presence of DAMGO. PPRs are shown for the groups in control solution (con), after adding DAMGO, after washing DHPG from the chamber (wash) and 10 min after adding naloxone (NAL, data not shown). PPR of eIPSPS in DAMGO differs from its value in control and naloxone conditions for the non-iLTD group, as indicated (*p<0.05; **p<0.001). Representative traces showing paired-pulse responses in control conditions, i.e., in DAMGO before DHPG treatment (gray) and 20 min after starting to wash DHPG (black)