Cortical inhibitory cell types differentially form intralaminar and interlaminar subnetworks with excitatory neurons

Abstract

The neocortical circuit is composed of excitatory principal neurons and inhibitory interneurons. Recent advances have established that multiple subnetworks of synaptically coupled excitatory neurons provide distinct pathways for information flow through the cortical circuit. Here we have investigated how inhibitory interneurons are incorporated into these excitatory subnetworks in the rat frontal cortex. In layer 5 (L5), the probability of reciprocal synaptic connections between pyramidal cells and fast-spiking (FS) interneurons was significantly higher than the probability of reciprocal connections between pyramidal cells and non-FS interneurons. Further, the amplitude of synaptic currents in reciprocally connected FS/pyramidal cell pairs was larger than that in pairs connected only in one direction. To examine interlaminar connection specificity, we stimulated layer 2/3 (L2/3) pyramidal cells, using focal glutamate puff stimulation, and recorded evoked EPSCs in L5 cells. Stimulation of L2/3 cells evoked EPSCs in L5 non-FS cells more frequently than in L5 FS cells. Dual recordings from L5 interneurons and neighboring pyramidal cells revealed that connected non-FS/pyramidal cell pairs were more likely to share excitatory inputs from L2/3 cells than were unconnected cell pairs. On the other hand, the connectivity between L5 FS and pyramidal cell pairs did not affect the common input probability from L2/3. Our results suggest that L5 inhibitory interneurons form distinct intralaminar and interlaminar subnetworks with pyramidal cells, depending on inhibitory cell types.

Figure 1.

Firing patterns and morphologies of L5 inhibitory interneurons. A, Voltage responses to positive and negative current pulse injections in FS and non-FS interneurons (pulse duration is 1 s, amplitudes are ±200 pA). The asterisk indicates rebound burst spikes generated by negative current pulse injection. B, Morphological reconstructions of FS (left and middle cell) and non-FS interneurons (right cell). Black and gray indicate dendrites and axons, respectively.

Figure 2.

Intralaminar connections between pyramidal cells and inhibitory interneurons in layer 5. A, Detection of EPSCs and IPSCs in a cell pair of pyramidal cell and interneuron. Left, Unitary EPSCs (lower trace) recorded in an interneuron held at −80 mV and induced by a single action potential in a presynaptic pyramidal cell. Right, Unitary IPSCs (upper trace) in a pyramidal cell held at 0 mV evoked by a single action potential in an interneuron. PC, Pyramidal cell; IN, interneuron. B, Percentage of cell pairs (consisting of a pyramidal cell and either an FS or non-FS cell) connected one way from pyramidal cell to interneuron, connected one way from interneuron to pyramidal cell, or reciprocally connected.

Figure 3.

Synaptic strength depends on the connectivity between pyramidal and FS cells. Distribution of IPSCs (left) and EPSCs (right) amplitudes from FS to pyramidal cell and pyramidal to FS cell obtained in cell pairs connected one way or reciprocally. Bars in the graph indicates means.

Figure 4.

Synaptic connection properties from L2/3 pyramidal cells to L5 interneurons. A, Detections of EPSCs in L5 interneurons by glutamate puff stimulation to L2/3 pyramidal cells. The triangles and circle show pyramidal cells and interneuron. ** indicates EPSCs evoked at relatively constant timings following glutamate stimulation of the L2/3 pyramidal cell (left traces). An example of a case where glutamate stimulation generated no EPSCs is shown in the traces at right. Arrowheads indicate the timing of puff stimulation onset. B, Interlaminar connection maps to L5 FS and non-FS interneurons. Cell positions were measured as vertical distance from the L1 and L2/3 border. Gray, Regression lines. C, Cumulative probabilities of the depth location of presynaptic L2/3 pyramidal cells. The data for the L5 pyramidal subtypes were obtained from our previous study.

Figure 5.

Divergent connection selectivity from L2/3 pyramidal cells to L5 pyramidal cells and interneurons. A, Dual whole-cell recordings from pyramidal cell and interneuron in the layer 5, while focal glutamate stimulations were applied to L2/3 pyramidal cells. The triangles and circle indicate pyramidal cells and interneurons, respectively. B, EPSCs inductions in the cell pair of pyramidal cell and interneuron. Left, Common inputs induced by L2/3 cell stimulation (indicated by double asterisks). Middle and right, EPSCs observed only in one cell (single asterisks). Arrowheads indicate the timing of stimulation onset. C, Upper box, Observed connection probabilities from L2/3 to L5 cells in each cell types. Lower box, Calculation of common input probabilities assuming nonselective cases. Data for connection probabilities from L2/3 to L5 pyramidal subtypes were obtained from previous our study. D, The common input probabilities were compared between L5 cell pairs of pyramidal cell and interneuron. Data are means ± SD. **p < 0.01.