In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells

Abstract

In the adult hippocampus, two different forms of GABA(A) receptor-mediated inhibition have been identified: phasic and tonic. The first is due to the activation of GABA(A) receptors facing the presynaptic releasing sites, whereas the second is due to the activation of receptors localized away from the synapses. Because of their high affinity and low desensitization rate, extrasynaptic receptors are persistently able to sense low concentrations of GABA. Here we show that, early in postnatal life, between postnatal day (P) 2 and P6, CA1 and CA3 pyramidal cells but not stratum radiatum interneurons, express a tonic GABA(A)-mediated conductance. Block of the neuronal GABA transporter GAT-1 slightly enhanced the persistent GABA conductance in principal cells but not in GABAergic interneurons. However, in adulthood, a tonic GABA(A)-mediated conductance could be revealed in stratum radiatum interneurons, indicating that the ability of these cells to sense ambient GABA levels is developmentally regulated. Pharmacological analysis of the tonic conductance in principal cells demonstrated the involvement of beta2/beta 3, alpha 5 and gamma 2 GABA(A) receptor subunits. Removal of the tonic depolarizing action of GABA with picrotoxin, reduced the excitability and the glutamatergic drive of principal cells but did not modify the excitability of stratum radiatum interneurons. The increased cell excitability and synaptic activity following the activation of extrasynaptic GABA(A) receptors by ambient GABA would facilitate the induction of giant depolarizing potentials.

Figure 1. A tonic GABA_A-mediated current is present in CA1 and CA3 pyramidal cells

A, left, representative tracings of spontaneous GABA_A-mediated synaptic currents recorded from a CA1 principal cell in control conditions and in the presence of gabazine (GBZ, 0.5 μm) and gabazine plus picrotoxin (PTX, 100 μm). Holding potential, −60 mV. Right, all-point histograms of 500 ms traces from the cell recorded on the left. Control, light grey; gabazine, dark grey; gabazine plus picrotoxin, black. B, as in A, but from a CA3 pyramidal cell. C, summary data of tonic currents obtained from 14 CA1 and 14 CA3 pyramidal cells in the presence of different concentrations (0.2, 0.3 and 0.5 μm) of gabazine and 100 μm of picrotoxin. In this and in the following figures the error bars indicate s.e.m.

Figure 2. Contribution of the GABA transporter GAT-1 to tonic GABA_A-mediated conductance in CA1 and CA3 pyramidal cells

A, left, representative traces of spontaneous GABA_A-mediated currents recorded from a CA1 principal cell in control conditions, during application of the GAT-1 blocker NO-711 (10 μm) and NO-711 plus picrotoxin. Right, spontaneous events obtained in control conditions and in presence of NO-711, normalized and superimposed on the right (each trace is the average of 40–50 events). B, all-point histogram of 500 ms traces from the cell recorded on the left in control conditions (light grey), in the presence of NO-711 (dark grey) and NO-711 plus picrotoxin (black). C and D, as in A and B, but from a CA3 principal cell. E, summary data obtained from six CA1 and seven CA3 principal cells. F, decay kinetics (τ) of spontaneous events detected in control conditions (open columns), and in the presence of NO-711 (filled columns) from six CA1 and seven CA3 pyramidal cells. *P < 0.05.

Figure 3. The network activity contributes to GABA_A-mediated tonic conductance in pyramidal cells

A, left, representative traces of spontaneous GABA_A-mediated miniature synaptic currents recorded from a CA1 pyramidal cell in the presence of TTX (1 μm) alone (control), and during bath application of gabazine (GBZ) and gabazine plus picrotoxin. Right, all-point histograms of 500 ms traces recorded from the cell shown on the left. Control, white; gabazine, grey; gabazine plus picrotoxin, black. B, as in A, but from a CA3 principal cell. C, summary data of tonic currents obtained from five CA1 and eight CA3 pyramidal cells recorded in the presence of TTX.

Figure 4. Lack of a sustained tonic GABA_A-mediated conductance in stratum radiatum interneurons

A, left, representative traces of spontaneous GABA_A-mediated currents obtained from stratum radiatum interneurons in control conditions, during application of NO-711 (10 μm) and NO-711 plus picrotoxin. Right, spontaneous events obtained in control conditions and in the presence of NO-711, normalized and superimposed (each trace is an average of 50 responses). B, all-point histograms of 500 ms traces recorded on the left. C, summary data obtained from 19 interneurons. D, mean deactivation kinetic values (τ, of spontaneous events recorded in control conditions (n = 9) and in the presence of NO-711; n = 11; *P < 0.05). E, average values of tonic GABA_A-mediated conductance recorded from stratum radiatum interneurons in slices obtained from adult (n = 4) and newborn (n = 16) animals.

Figure 5. Different extrasynaptic GABA_A receptor subunits mediate tonic currents in CA1 principal cells

Left, inward and outward shifts in the holding current obtained in control conditions and in the presence of the α5 inverse agonist L-655,708 (5 μm, A), the β2/β3 enhancer ETMD (100 nm, B), the γ2 positive allosteric modulator flurazepam (FZP, 10 μm, C) and the δ antagonist THDOC (10 nm, D). Right, related all-point histograms for the traces shown on the left. The inset in C represent spontaneous events obtained in control conditions and in the presence of FZP, normalized and superimposed (each trace is the average of 60 responses).

Figure 6. Pharmacological characterization of GABA_A receptor subunits contributing to the tonic GABA_A-mediated conductance recorded in CA1 (filled columns) and CA3 (open columns) pyramidal cells

Data are presented as percentage control relative to the PTX-mediated shift in current amplitude (Wilcoxon matched-pair test, *P < 0.05; **P < 0.01). Etomidate (ETMD) (n = 8), L-655,708 (L-655, n = 6), flurazepam (FZP, n = 6) and allotetrahydrodeoxycorticosterone (THDOC) (n = 6).

Figure 7. Ambient GABA enhances spontaneous firing in CA1 principal cells but not in stratum radiatum interneurons

A, example trace recorded in cell-attached mode from a CA1 pyramidal cell (postnatal day 4) in control conditions and in the presence of picrotoxin. B, each column represents the mean firing frequency obtained from six CA1 principal cells in control conditions and during bath application of picrotoxin (*P < 0.05). C and D, as in A and B, but for stratum radiatum interneurons (n = 7).

Figure 8. Ambient GABA enhances pyramidal cell excitability

A, individual traces showing the firing patterns of a CA1 pyramidal cell in response to a steady depolarizing current pulse, before (upper) and during (lower) bath application of picrotoxin (PTX, 100 μm). In this cell, PTX induced a 6 mV membrane hyperpolarization. B, changes in firing frequency obtained by depolarizing CA1 principal cells in control conditions and during bath application of PTX. The firing rate was tested at the peak of PTX-induced membrane hyperpolarization (ranging in different cells from 2 to 10 mV, n = 12, ***P < 0.001). C and D, as in A and B but for stratum radiatum interneurons (n = 4, P > 0.5).

Figure 9. A tonic GABA_A-mediated conductance enhances the glutamatergic drive to principal cells

A, synaptic currents evoked at −50 mV in a CA1 pyramidal neuron by stimulation of the Schaffer collateral, before (thin line) and during (thick line) application of PTX. On the right the two currents are superimposed (each trace is the average of 15 responses). B, each column represents the mean amplitude of EPSCs evoked in CA1 principal cells by stimulation of the Schaffer collateral in control (open column) and during bath application of PTX (filled column, n = 6; *P < 0.05). C and D, as for A and C, but for EPSCs evoked in stratum radiatum interneurons (n = 7). Note that PTX failed to reduce the amplitude of EPSCs in interneurons. E, in the graph the amplitude of fEPSPs evoked by Schaffer collateral stimulation before and during (indicated by horizontal bar) application of PTX (100 μm) is plotted against time. Each point represents the mean of three responses. In the inset, fEPSPs recorded at the time indicated (each trace is the average of three responses). The reduction in amplitude of the fEPSP was associated with a small decrease in the amplitude of the afferent volley (12%). F, each column represents the mean amplitude of fEPSPs evoked by stimulation of the Schaffer collateral in control (open column) and during bath application of PTX (filled column, n = 9 slices from nine P3–P5 rats; **P < 0.01).