Unitary synaptic currents between lacunosum-moleculare interneurones and pyramidal cells in rat hippocampus

Abstract

1. Unitary inhibitory postsynaptic currents (uIPSCs) were characterised between 23 synaptically coupled interneurones at the border of stratum radiatum and lacunosum-moleculare (LM) and CA1 pyramidal cells (PYR) using dual whole-cell recordings and morphological identification in rat hippocampal slices. 2. LM interneurones presented a morphology typical of stellate cells, with a fusiform soma as well as dendritic and axonal arborisations in stratum radiatum and lacunosum-moleculare. 3. Single spikes in interneurones triggered uIPSCs in pyramidal cells that were blocked by the GABA(A) antagonist bicuculline and mediated by a chloride conductance. The latency, rise time, duration and decay time constant of uIPSCs were a function of amplitude in all pairs, suggesting a homogeneity in the population sampled. 4. During paired pulse stimulation, individual LM-PYR connections exhibited facilitation or depression. The paired pulse ratio was inversely related to the amplitude of the first response. The transition from facilitation to depression occurred at 26 % of the maximal amplitude of the first uIPSC. Paired pulse depression was not modified by CGP 55845 and thus was GABA(B) receptor independent. 5. CGP 55845 failed to modify the amplitude of uIPSCs, suggesting an absence of tonic presynaptic GABA(B) inhibition at LM-PYR connections. 6. Increasing GABA release by repetitive activation of interneurones failed to induce GABA(B) IPSCs. With extracellular minimal stimulation, increasing stimulation intensity above threshold, or repetitive activation, evoked GABA(B) IPSCs, probably as a result of coactivation of several GABAergic fibres. 7. Thus, dendritic inhibition by LM interneurones involves GABA(A) uIPSCs with kinetics dependent on response amplitude and subject to GABA(B)-independent paired pulse plasticity.

Figure 1. Recordings and analysis of uIPSCs

Diagram of the experimental arrangement (A1). pyr, stratum pyramidale; rad, stratum radiatum; lm, stratum lacunosum-moleculare; dg, dentate gyrus. A pyramidal cell (A2) and a LM interneurone (A3) were recorded in the whole-cell patch-clamp configuration. B 1, definition of the parameters of uIPSC recorded in voltage-clamp mode from a pyramidal cell (Pyr) after an action potential induced by current injection in the LM interneurone (LM). B 2, measurement of the mean uIPSC. Each uIPSC was sampled into 100 ms events, taking as reference the peak of the presynaptic action potential, and averaged.

Figure 2. Camera lucida drawing of a synaptically connected LM interneurone and pyramidal cell pair labelled with biocytin

A, the soma and dendrites of the interneurone are shown in red, the axon in black. The pyramidal cell processes are shown in green. B, example of unitary synaptic currents recorded in this pyramidal cell following action potentials in the interneurone.

Figure 3. Properties of GABA_A uIPSCs between LM interneurones and pyramidal cells

A, in a connected LM-PYR pair, an action potential in the presynaptic interneurone (LM, bottom trace) triggers a hyperpolarisation of the pyramidal cell (Pyr) in current-clamp mode at resting membrane potential (V_m, top trace) or an outward uIPSC in voltage-clamp mode with the membrane held at -40 mV (V_h, second trace). The uIPSC was completely antagonised by bicuculline (25 μm). B, distribution of mean uIPSC amplitude and background noise (inset) for all recorded pairs n = 23. C-F, distribution of mean latency (C), duration and decay time constant (D), rise time (E) and failure rate (F) of uIPSCs for all pairs.

Figure 4. Analysis of uIPSC kinetics

A1, superposed unitary synaptic currents recorded from a pyramidal cell soma following single spikes in a LM interneurone (V_m -65 mV). Inset, onset of uIPSCs at higher time resolution (horizontal bar 1 ms, vertical bar 20 pA). A2 and A3, plots of uIPSC latency, rise time (A2), duration and decay time constant (A3) as a function of amplitude for the same pair as in A1. B 1 and B 2, plots of mean uIPSC latency, rise time (B 1), duration and decay time constant (B 2) versus normalised uIPSC amplitude for the entire population of LM-PYR pairs. The continuous line illustrates the linear fit (y = ax + b) for latency and dotted lines indicate monoexponential fits (y = a + b exp(-cx)) for the rise time, duration and decay time constant.

Figure 5. Voltage sensitivity of uIPSCs

A, superimposed mean uIPSCs recorded from a pyramidal cell at holding potentials between -40 and -90 mV (-10 mV increments). The reversal potential of the mean uIPSC in this cell was near -70 mV; (interneurone, V_m -75 mV). B, plot of peak amplitude of mean uIPSCs versus holding potential for all pairs tested (number above each point indicates number of pairs). C and D, graphs of mean duration and decay time constant (C), failure rate (inset in C), rise time and latency (D) versus membrane potential for all connections tested. Only failure rate was significantly increased at a holding potential of -70 mV (*P < 0.05).

Figure 6. Paired pulse modulation of uIPSCs

A, paired stimulations were delivered at 50 ms interval to LM interneurones at 0.2 Hz. Three different trials are illustrated for the same LM-PYR pair showing depression (A1), no change (A2) or facilitation (A3) of the second uIPSC amplitude compared to the first. B, graph of the paired pulse ratio (second uIPSC/mean first uIPSCs × 100) as a function of the amplitude of the first uIPSC (normalised to the largest first uIPSC in each pair), for all pairs tested in control conditions (•) and in presence of CGP 55845 (○) (B 1). Paired pulse depression (points under the dotted line) and paired pulse facilitation (points above the dotted line) can be clearly seen. In control conditions, the relationship was well fitted by an exponential equation y = a + b exp(-c x) where a= -76.5, b= 210.2 and c= -8.3 (P < 0.05; r= -0.7). Arrow on the x-axis indicates the transition point from facilitation to depression obtained from the exponential function. In control conditions this transition point was at 26 % of the maximal amplitude of the first uIPSC. The inset shows a similar analysis for paired pulse ratios at 5 s intervals and the continuous line represents the linear regression (r= 0.03, n= 4 pairs) obtained from the plot indicating an absence of paired pulse modulation at this interval. B 2, plot of second uIPSC as a function of first uIPSC for all pairs tested in control conditions (•) and in presence of CGP55845 (1 μm; ○). Amplitude was normalised to the mean first uIPSC in each pair. The continuous line represents the linear regression (P < 0.05; r= -0.23) in control conditions and the dashed line the linear regression obtained in presence of CGP55845 (P < 0.05; r= -0.24). C, histogram of the amplitude, latency, rise time, duration and decay time constant of the averaged second uIPSC expressed as a percentage of the averaged first uIPSC. Parameters were not significantly changed during paired pulse stimulation. D, superimposed traces from a different pair from that shown in A, showing that the averaged first uIPSC triggered by single presynaptic action potentials (bottom traces) was similar in control conditions (black trace) and in the presence of CGP 55845 (grey trace).

Figure 7. Recruitment of GABA_B slow IPSCs by extracellular stimulation

A, repetitive activation of individual LM interneurones failed to activate GABA_B currents. A burst of action potential potentials in a LM interneurone (-55 mV) evoked uIPSCs that summated in control conditions (A1) and were suppressed by addition of bicuculline. Increasing the number of presynaptic action potentials failed to recruit additional synaptic currents (A2). B-D, IPSCs evoked by extracellular stimulation. B, single pulse stimulations (0.5 ms, 0.2 Hz) at threshold intensity using a theta glass electrode in stratum radiatum evoked in pyramidal cells all-or-none fast IPSCs (T; B 1). Three traces with two IPSCs and a failure are superimposed. These minimally evoked IPSCs were completely blocked by bicuculline (B 2). However, increasing stimulation intensity to 2-4T recruited a slow IPSC (B 2 and D). Note different time scales in B 1 and B 2. C, a short 50 Hz train of stimulation at T intensity in bicuculline recruited slow IPSCs. Increases in stimulation intensity (1.5-2T) increased the slow IPSC amplitude. D, histogram of mean IPSC amplitude with single pulse and 50 Hz train of stimulation at different intensities in the presence of bicuculline. Number above each bar indicates the number of cells tested in each condition. Slow IPSCs were completely blocked by the GABA_B antagonist CGP 55845 (1 μm).