GABAergic inhibition suppresses paroxysmal network activity in the neonatal rodent hippocampus and neocortex

Abstract

In the adult cerebral cortex, the neurotransmitter GABA is strongly inhibitory, as it profoundly decreases neuronal excitability and suppresses the network propensity for synchronous activity. When fast, GABA(A) receptor (GABA(A)R)-mediated neurotransmission is blocked in the mature cortex, neuronal firing is synchronized via recurrent excitatory (glutamatergic) synaptic connections, generating population discharges manifested extracellularly as spontaneous paroxysmal field potentials (sPFPs). This epileptogenic effect of GABA(A)R antagonists has rarely been observed in the neonatal cortex, and indeed, GABA in the neonate has been proposed to have an excitatory, rather than inhibitory, action. In contrast, we show here that when fast GABAergic neurotransmission was blocked in slices of neonatal mouse and rat hippocampus and neocortex, sPFPs occurred in nearly half the slices from postnatal day 4 (P4) to P7 neocortex and in most slices from P2 to P7 hippocampus. In Mg(2+)-free solution, GABA(A)R antagonists elicited sPFPs in nearly all slices of P2 and older neocortex and P0 and older hippocampus. Mg(2+)-free solution alone induced spontaneous events in the majority of P2 and older slices from both regions; addition of GABA(A)R antagonists caused a dramatic increase in the mean amplitude, but not frequency, of these events in the hippocampus and in their mean frequency, but not amplitude, in the neocortex. In the hippocampus, GABA(A)R agonists suppressed amplitudes, but not frequency, of sPFPs, whereas glutamate antagonists suppressed frequency but not amplitudes. We conclude that neonatal rodent cerebral cortex possesses glutamatergic circuits capable of generating synchronous network activity and that, as in the adult, tonic GABA(A)R-mediated inhibition prevents this activity from becoming paroxysmal.

Fig. 1.

sPFPs elicited in neonatal hippocampus and neocortex by GABA_AR antagonists in normal ACSF.A–D, Left, A continuous 45 min record illustrating spontaneous activity before, during, and after superfusion of drug (drug presence indicated by horizontal bar abovethe trace). A–D, Right, A 4.5 sec record illustrating a single sPFP expanded from thetrace on the left. Postnatal age and region recorded [neocortex (Nctx); hippocampus, area CA3 (CA3)] are noted above eachtrace. Note the sharp spikes characterizing sPFPs in CA3 (A, B, right), and the similarity between sPFPs elicited by BMC and GBZ. BMC, 10 μmBMC; GBZ, 5 μm gabazine. Horizontal calibration: left, 10 min; right, 1 sec. Vertical calibration: A, B, 300 μV; C, 200 μV; D, 120 μV.

Fig. 2.

sPFPs elicited by GABA_AR antagonists in Mg²⁺-free ACSF. A–D, Left, A continuous 30 min record illustrating spontaneous activity before, during, and after superfusion of drug (drug presence indicated byhorizontal bar above the trace; concentrations as in Fig. 1). A–D, Right, A 4.5 sec record illustrating a single sPFP from the trace on theleft. Slices were bathed in Mg²⁺-free ACSF for at least 20 min by the beginning of each trace.Arrowheads point to some of the spontaneous events occurring in Mg²⁺-free ACSF alone, before addition of antagonist. Horizontal calibration: left, 400 sec;right, 1 sec. Vertical calibration: A, 400 μV; B, 200 μV; C, D, 150 μV.

Fig. 3.

Age-dependent changes in incidence, frequency, amplitude, and effective duration of sPFPs elicited by GABA_AR antagonists. See Materials and Methods and Results for definitions of these four parameters. Data are combined into four 2 d age groups. In normal ACSF, only 1 P0–P1 slice in each of the two regions generated sPFPs, and therefore analysis begins with the P2–P3 age group. A, The number of CA3 slices in the four age groups (P0–P1, P2–P3, P4–P5, and P6–P7) was 9, 24, 18, and 9, respectively, in normal ACSF and 19, 17, 14, and 8, respectively, in Mg²⁺-free ACSF. The number of neocortex slices tested was 5, 22, 17, and 11, respectively, in normal ACSF and 9, 17, 13, and 10, respectively, in Mg²⁺-free ACSF.B–D, The number of CA3 slices was 16, 17, and 9 (P2–P3, P4–P5, and P6–P7, respectively) in normal ACSF and 18, 17, 14, and 8 (P0–P1, P2–P3, P4–P5, and P6–P7, respectively) in Mg²⁺-free ACSF. The number of neocortex slices was 3, 7, and 7, respectively, in normal ACSF and 2, 14, 13, 10, respectively, in Mg²⁺-free ACSF. The statistical significance of age dependency is indicated by one asterisk for p < 0.05 (significant) or bytwo asterisks for p < 0.01 (highly significant); a marginally significant difference (p < 0.1) is indicated by anasterisk in parentheses. Significance values were calculated over the whole age range plotted, except inD, neocortex, where the decrease in effective duration was highly significant between P2–P3 and P6–P7 but only significant between P0–P1 and P6–P7. In general, the incidence and amplitude of sPFPs were higher in CA3 than in neocortex (note the different scales for CA3 and neocortex in C); in both regions, the incidence and frequency in Mg²⁺-free ACSF were higher than those in normal ACSF, but amplitudes were smaller.

Fig. 4.

GABA_AR antagonists added to Mg²⁺-free ACSF increased amplitudes (but not the frequency) of spontaneous events in the hippocampus and the frequency (but not amplitudes) in the neocortex. A, Representative cases from CA3 and neocortex. Each trace is a continuous 5 min record. In the CA3 slice illustrated, events before antagonists were added occurred in doublets (top). Note the different vertical calibration for the left andright panels. B, Summary of results from all slices tested. The y-axis indicates multiplicative change, compared with the control, on a logarithmic scale (0.2 log₁₀ units between grid lines). Data points and error bars denote geometric means and SEM, respectively (i.e., the means and SEM of the logarithms of the pairwise experimental/control ratios). For CA3, sample sizes for each of the three experimental conditions (drug, washout, and drug) were 17, 7, and 6, respectively. For the neocortex, sample sizes were 15 and 5, respectively. Significancesymbols are described in Figure 3. Drug concentrations were 10 μm BMC and 5 μm GBZ. .

Fig. 5.

The GABA_AR agonist muscimol depressed amplitudes (but not the frequency) of sPFPs occurring in 50 μm 4-AP (4AP). A, A representative experiment using 50 nm muscimol. Eachtrace is a continuous 2.5 min record. B, Summary of all cases tested with 100 nm muscimol. Graphic conventions are described in Figure 4. The number of slices tested was seven in each condition (with muscimol and after washout); with muscimol, in four of the seven slices the sPFPs were fully blocked and are therefore not included in the mean.

Fig. 6.

The NMDA receptor antagonist APV depressed the frequency, but not amplitudes, of sPFPs elicited by GABA_AR antagonists in Mg²⁺-free ACSF. A, A representative experiment using 10 μm APV. Eachtrace is a continuous 5 min record. Adding 10 μm CNQX in addition to APV blocked all events. Note the different vertical calibration for the left andright panels. Records in CA3 and neocortex were taken simultaneously from the same slice; neocortex-originating sPFPs propagated to CA3 and are visible as small-amplitude deflections on the CA3 records, but note that the two sets of events are not temporally correlated. B, Summary of all cases tested with APV and after washout. The number of slices tested was nine and five, respectively, in CA3 and four and four, respectively, in the neocortex; in one of the four neocortex slices sPFPs were fully blocked, and this slice is therefore not included in the mean. GBZ and BMC concentrations and graphic conventions are described in Figure 4.

Fig. 7.

The non-NMDA receptor antagonist CNQX also depressed the frequency of sPFPs elicited by GABA_AR antagonists in Mg²⁺-free ACSF. A, Representative cases tested with 10 μm CNQX. Eachtrace is a continuous 5 min (CA3) or 4 min (neocortex) record. Note that both vertical and horizontal calibrations are different for the right and left panels.B, Summary plot of all cases. The number of slices tested with CNQX and after washout was 10 and 8, respectively, in CA3 and 6 and 2, respectively, in the neocortex. Two of the 10 CA3 slices, in which the sPFPs were fully blocked, are not included in the mean. GBZ and BMC concentrations and graphic conventions are described in Figure 4.