Functional GABAergic synaptic connection in neonatal mouse barrel cortex

Abstract

Intracortical inhibition is crucial to proper functioning of the mature neocortex, yet, paradoxically, is reported to be rare or absent in the neonatal animal. We reexamined this issue by recording whole-cell postsynaptic currents (PSCs) of barrel cortex neurons in thalamocortical brain slices from neonatal mice. Monosynaptic, excitatory thalamocortical responses were elicited in layers V/VI neurons as early as postnatal day 0 (P0, the first 24 hr after birth) and in presumptive layer IV as early as P2. At very low stimulation frequencies, the monosynaptic response was invariably followed by a prolonged (up to 1 sec) synaptic barrage, which fatigued at stimulus repetition rates of 2/min or higher. This barrage consisted of postsynaptic responses to spiking activity in neighboring cortical cells, because (1) it could also be evoked by intracortical stimulation in coronal slices and (2) it was abolished by antagonists to NMDA receptors (NMDARs), even when NMDARs on the recorded cell were under a voltage-dependent block. Some of the larger polysynaptic events changed polarity at a negative reversal potential and were blocked by GABAA receptor (GABAAR) antagonists, with a concurrent enhancement of the extracellular field potential, indicating that they were GABAAR- mediated, CI-dependent inhibitory PSCs (IPSCs). We conclude that a network of functional intracortical GABAAR-mediated synaptic connections exists from the earliest postnatal ages, although it gives rise to responses that differ from mature IPSCs in reversal potential and latency.

Fig. 1.

Age-dependent changes in electrophysiological parameters. Resting potentials (top) and input resistance (bottom) of sampled cells, pooled into three age groups for layers V/VI (L. V/VI) cells (left), and into two age groups for layer IV (L. IV) cells (right). (The number of layer IV cells in the youngest age group was too small for analysis.) Thecenter line represents the median value; box andwhisker lines represent the 25–75th and 5–95th percentiles, respectively. Asterisks denote a statistical significance between one age group and the immediately preceding one, as determined by a two-tailed Mann–Whitney test, withp < 0.05 and p < 0.01 denoted byone asterisk and two asterisks, respectively. The number of cells included in each group is indicated inparentheses; the discrepancy between the numbers in the upper and lower panels reflects cells for which resting potentials could not be determined, as explained in Materials and Methods.

Fig. 2.

Monosynaptic and polysynaptic components of thalamocortical responses. A, Whole-cell postsynaptic current responses of a layer V/VI P2 neuron (P2, L. V/VI) to thalamocortical stimulation. Holding potential indicated to the left of the trace. The monosynaptic response (its peak marked by arrowhead) was followed by a barrage of long-latency events (selected events marked byasterisks), in this case lasting >600 msec. Boxed region is expanded in B. B, Superimposed responses to repeated thalamocortical stimulation, at an expanded time base. Compare the small variance in the latency of the monosynaptic component (onset marked by arrowhead) with the jitter in the onset of the polysynaptic events (double arrow).

Fig. 5.

Reversal potentials of unitary polysynaptic events. A, Whole-cell postsynaptic currents in a P5 neuron (P5, L. V/VI) that exhibited unitary polysynaptic events (largest events marked by asterisks) but no monosynaptic response. B, Records from a P6 neuron (P6, L. IV) that exhibited a monosynaptic response (onset marked by arrowheads) followed by a polysynaptic barrage (largest polysynaptic events marked by asterisks). The monosynaptic response was inward at all negative potentials and had a voltage dependency characteristic of NMDA receptor-mediated currents.C, The largest polysynaptic events in the P5 cell (asterisks in A) reversed at approximately −30 mV. D, The largest polysynaptic events in the P6 cell (asterisks in B) reversed at approximately −50 mV.

Fig. 3.

The long-latency PSCs depended on activation of presynaptic neurons through both NMDA and non-NMDA receptors.A, Whole-cell currents in response to thalamocortical stimulation of a P6 neuron (P6, L. IV), in control ACSF (light traces), after addition of 12.5 μm APV (marked by arrow) and after drug washout (heavy traces). Holding potentials indicated to the left of each set of traces. The NMDA antagonist reversibly blocked most of the polysynaptic responses, even at −130 mV, when the response could not have been mediated by NMDA receptors.B, Responses of a P7 neuron (P7, L. IV) before and after addition of 10 μm CNQX. The non-NMDA antagonist blocked much of the response at +70 mV, even though the late part of this response was probably mediated by NMDA receptors. Both drugs were clearly acting on cells presynaptic to the recorded neurons.

Fig. 7.

The larger polysynaptic events were GABAergic.A, Superimposed whole-cell current responses of a P3 neuron (P3, L. V/VI) to intracortical stimulation, in control ACSF (light trace) and after addition of 6 μm of the GABA_AR antagonist BMC (heavy trace). The polysynaptic events (asterisk) were blocked by the drug, revealing a barrage of small, presumably excitatory events. The monosynaptic response (arrowhead) was slightly enhanced, indicating no deterioration in the recording. B, Postsynaptic currents in a P7 neuron (P7, L. IV). Addition of 2 μm BMC (heavy trace) caused ade novo appearance of inward, presumably excitatory synaptic events (asterisk). C, D, Averaged field potentials recorded during the experiments shown in A andB, respectively. Heavy trace is the recording taken after drug washout, indicating that the BMC effect was reversible. In both experiments the drug caused a pronounced increase in field potential amplitude, indicating a disinhibitory effect of the drug and therefore an inhibitory effect of GABA.

Fig. 4.

Fatigue of the long-latency responses. Whole-cell postsynaptic responses of two layer IV neurons from different P6 animals (P6, L IV). A, Superimposed first, third, and fourth responses to thalamocortical stimulation at 33 mHz (2/min); note that the polysynaptic events (asterisks in first and third trials) are virtually gone by the fourth repetition of the stimulus. B, Representative responses to thalamocortical stimuli from three consecutive trains, delivered at 5.5 mHz and 33 mHz and again at 5.5 mHz. Note the dramatic but reversible fatigue of the polysynaptic responses (asterisks) at 33 mHz. Theboxed regions are expanded in C and D, showing that the monosynaptic responses (arrowheads) were unchanged or even enhanced when the polysynaptic events were maximally fatigued (heavy line represents fatigued response).

Fig. 6.

Reversal potentials plotted against synaptic conductance of 12 unitary PSCs. UPSCs in the P3–5 group reversed at or more positive to −30 mV, whereas in the P6–7 group they reversed negative to −40 mV. Synaptic conductances were on average higher (but not significantly so) in the older group as well.