NMDA receptors are the basis for persistent network activity in neocortex slices

Abstract

During behavioral quiescence the neocortex generates spontaneous slow oscillations that consist of Up and Down states. Up states are short epochs of persistent activity, but their underlying source is unclear. In neocortex slices of adult mice, we monitored several cellular and network variables during the transition between a traditional buffer, which does not cause Up states, and a lower-divalent cation buffer, which leads to the generation of Up states. We found that the resting membrane potential and input resistance of cortical cells did not change with the development of Up states. The synaptic efficacy of excitatory postsynaptic potentials mediated by non-NMDA receptors was slightly reduced, but this is unlikely to facilitate the generation of Up states. On the other hand, we identified two variables that are associated with the generation of Up states: an enhancement of the intrinsic firing excitability of cortical cells and an enhancement of NMDA-mediated responses evoked by electrical or optogenetic stimulation. The fact that blocking NMDA receptors abolishes Up states indicates that the enhancement in intrinsic firing excitability alone is insufficient to generate Up states. NMDA receptors have a crucial role in the generation of Up states in neocortex slices.

Keywords: Down state; Up state; arousal; cortex; slice; slow oscillation.

Fig. 1.

Low buffer generates spontaneous slow oscillations consisting of Up and Down states in neocortex slices. A: whole cell intracellular [membrane potential (V_m)] and field potential (FP) recordings from a cell in somatosensory cortex before and during low buffer. B: close-ups of Up states marked by asterisks in A. C: population data showing the abolishment of spontaneous Up states by application of d-AP5 (50 μM).

Fig. 2.

Effect of low buffer on input-output curves of electrically evoked synaptic responses. A: example showing averaged synaptic responses (V_m and FP) evoked at different intensities by a stimulating electrode placed in layers III and II during control buffer, low buffer, and subsequent addition of AP5. B: overlaid responses evoked by 1 intensity (20 μA) for the data shown in A. Arrows indicate short- and long-latency responses. C: population data showing the effects of low buffer on the amplitude of short-latency (2–15 ms) and long-latency (15–50 ms; reflecting the Up states) FP responses under normal conditions (no glutamate receptor antagonists). D: population data showing the effects of low buffer on AMPA FP responses (in the presence of AP5): peak amplitude (left) and time to peak (right). E: population data showing the effects of low buffer on NMDA FP responses (in presence of CNQX): peak amplitude (left) and area (right) of FP response (3–50 ms). Note that low buffer enhances NMDA responses. *P < 0.01. n.s., Not significant.

Fig. 3.

Examples depicting the effect of low buffer on AMPA responses. A: an experiment showing simultaneous FP and intracellular recordings of AMPA responses evoked by electrical stimulation at different intensities during control and low buffer. Right: overlay of responses evoked by 2 intensities for comparison. Bottom: arrows indicate a shift in the peak and a broadening of the FP response. B: another experiment showing intracellular responses as described in A. C: individual traces used to derive the average traces shown in B for the 50-μA stimulus intensity. Note the reduction in the slope of the excitatory postsynaptic potential (PSP) and the enhanced spiking during low buffer.

Fig. 4.

Examples depicting the effect of low buffer on NMDA responses. A: 1 FP experiment is shown. NMDA FP responses are evoked by electrical stimulation at different intensities during control and low buffer. Right: overlay of the responses evoked by 1 intensity for comparison and the normal response evoked before addition of CNQX to isolate NMDA responses. B, top and bottom: 2 additional experiments. Intracellular NMDA responses are evoked by electrical stimulation at different intensities during control and low buffer. Top right: overlay of responses evoked by 2 of the intensities for comparison. Bottom right: responses evoked after AP5.

Fig. 5.

Effect of low buffer on optogenetically evoked responses. A: an experiment showing simultaneous FP and intracellular AMPA responses evoked by blue light pulses at different intensities during control and low buffer in a Thy1 slice. Right: overlay of responses evoked by 2 intensities for comparison. B: another experiment showing simultaneous FP and intracellular NMDA responses evoked by blue light pulses at different intensities during control and low buffer in a Thy1 slice. Third panel from left: overlay of responses evoked by 2 intensities for comparison. Fourth panel: effect of AP5 on the responses evoked by the 5 intensities and 1 of the traces before AP5 (low buffer) for comparison. This cell responded directly to blue light (in the presence of glutamate receptor antagonists), indicating that it expressed Channelrhodopsin-2 (ChR2). C: a third experiment showing simultaneous FP and intracellular NMDA responses evoked by blue light pulses at different intensities during control and low buffer in a slice expressing ChR2 in thalamocortical fibers. Third panel from left: overlay of responses evoked by 2 intensities for comparison. Fourth panel: effect of AP5 and 1 of the traces before AP5 (low buffer) for comparison. AP5 abolished the intracellular responses and most of the FP response except for the short-latency fiber volley reflecting the direct activation of thalamocortical fibers.

Fig. 6.

Low buffer slightly suppresses the isolated inhibitory synaptic conductance (Gsyn) evoked by intracortical electrical stimulation. A: 2 examples showing the effect of low buffer on the inhibitory synaptic conductance derived from isolated inhibitory PSPs evoked at different V_m. B: population data showing the effects of low buffer on the peak amplitude of the isolated inhibitory synaptic conductance evoked by electrical stimulation. *P < 0.05.

Fig. 7.

Low buffer increases firing excitability. A: effect of low buffer on resting V_m. B: effect of low buffer on input resistance (R_in) estimated by measuring the voltage drop caused by negative current pulses (I_m) of different amplitudes. C: effect of low buffer on input resistance estimated by measuring the decay time constant (τ) after pulse offset. D: effect of low buffer on firing excitability estimated by measuring the number of spikes evoked by 500-ms positive current pulses of different amplitudes. *P < 0.05. E: same data shown in D but plotted as perievent time histograms with 100-ms bins.