Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex

Abstract

GABAergic interneurons in different cortical areas play important roles in diverse higher-order cognitive functions. The heterogeneity of interneurons is well characterized in different sensory cortices, in particular in primary somatosensory and visual cortex. However, the structural and functional properties of the medial prefrontal cortex (mPFC) interneurons have received less attention. In this study, a cluster analysis based on axonal projection patterns revealed four distinct clusters of L6 interneurons in rat mPFC: Cluster 1 interneurons showed axonal projections similar to Martinotti-like cells extending to layer 1, cluster 2 displayed translaminar projections mostly to layer 5, and cluster 3 interneuron axons were confined to the layer 6, whereas those of cluster 4 interneurons extend also into the white matter. Correlations were found between neuron location and axonal distribution in all clusters. Moreover, all cluster 1 L6 interneurons showed a monotonically adapting firing pattern with an initial high-frequency burst. All cluster 2 interneurons were fast-spiking, while neurons in cluster 3 and 4 showed heterogeneous firing patterns. Our data suggest that L6 interneurons that have distinct morphological and physiological characteristics are likely to innervate different targets in mPFC and thus play differential roles in the L6 microcircuitry and in mPFC-associated functions.

Keywords: axonal projection; interneurons; layer 6; medial prefrontal cortex.

Figure 1

Morphological analysis of L6 inhibitory neurons using unsupervised cluster analysis. (A) Dendrogram obtained from a cluster analysis based on morphological parameters reveals four clusters of L6 interneurons (n = 48). The color map below the dendrogram indicates the standardized values of the corresponding parameters (listed on the right) of individual neurons, in which red represents values above the mean, white represents the mean, and blue represents values below the mean. Pie chart shows the percentage contribution of each cluster. (B) Representative axodendritic morphologies of the four clusters. Dendrites are shown in red and axons in blue. A L5: axonal distribution in L5; A Pia-Soma: axonal distribution from Pia-Soma; A L1/2/3: axonal distribution in L1-L3; A WM: axonal distribution in WM; A Soma-WM: axonal distribution from Soma-WM; Soma position: relative soma position in L6; A L6: axonal distribution in L6; A H/V: axon horizontal/vertical.

Figure 2

mPFC L6 interneurons display a strong correlation between soma position and axonal projection pattern. (A) Relative soma position in L6 of interneurons of the four morphological clusters. (B) Correlation between soma position of individual L6 interneurons and their axonal distribution from pia to soma. (C) Relative axonal distributions of the neurons from different clusters are shown. Top: from pia to soma; Bottom: from soma to WM.

Figure 3

Reconstruction overlays and density maps of inhibitory mPFC L6 interneuron axons based on 4 morphological clusters. Left: All interneurons are aligned with their soma position represented as yellow dots; axons are shown in blue. Middle: 2D density maps of different clusters, the outer border of gray isosurfaces show the 80 percentile of the axonal density. Right: vertical axonal distribution of L6 interneuron clusters. The individual curves show the average axon density distribution along the vertical axis; bin size in the x axis: 50 μm in vertical direction. Dashed lines indicate layer borders. Results from 5 L1/2/3-projecting interneurons, 15 L5-projecting interneurons, 18 locally projecting L6 interneurons, and 10 L6/WM-projecting interneurons are shown in panel A, B, C, and D, respectively.

Figure 4

Comparison of electrophysiological parameters in morphological clusters of L6 interneurons in rat mPFC. (A) Representative examples of L6 interneurons of the four morphological clusters. Axons are given in blue, the somatodendritic domain in red. (B) Corresponding firing pattern of the neurons that are shown in A (10-spike train and the first AP at rheobase current injection). (C) Statistical analysis of the active electrophysiological properties of mPFC L6 interneurons in the four morphological clusters. A Wilcoxon–Mann–Whitney test was performed for the significant difference among clusters. P values are shown above these two clusters (examples in A and B).

Figure 5

Comparison of morphological and electrophysiological parameters of L6 interneurons in rat mPFC. (A) A combined unsupervised hierarchical cluster analysis based on both morphological and electrophysiological parameters revealed four clusters. The cut-off for significant clusters is 70% of the maximum linkage distance. Colored lines in each dendrogram represent the color code from the morphological clusters in (B). Colored boxes on each line show the morphology clusters the neurons belong to. The color map below the dendrogram indicates the standardized values of the corresponding parameters (listed on the right) of individual neurons, in which red represents values above the mean, white represents the mean, and blue represents values below the mean (see Fig. 1). (B) Representative examples of L6 inhibitory neurons based on combined morphological/electrophysiological parameters. Axons are given in blue, the somatodendritic domains in red. Corresponding firing pattern are shown below each example morphology.

Figure 6

Neurochemical marker expression of individual L6 interneurons in rat mPFC. Whole-cell patch-clamp recordings were made with simultaneous biocytin and Alexa 594 filling (red) to identify the location of patched neurons. Immunostaining was performed after a brief fixation period in PFA to check the expression of PV (green), Prox1 (purple), and SOM (blue) of the patched neurons. Representative examples of interneurons expressing PV (A), Prox1 (D), and somatostatin (G) with morphological reconstructions (B, E, H) and firing pattern (C, F, I, left: the first 10 spikes traces; right: the firing traces at the current 100 pA above the threshold) are shown. Axon is labeled in blue, soma and dendrites in red.

Figure 7

Schematic summary of the potential targets of the four types of interneurons in L6 of mPFC. P: pyramidal cells; upr. P: upright pyramidal cells; inv. P: inverted pyramidal cells. L1/2/3 projecting interneurons (red) are, in our study, Martinotti-like interneurons projecting mainly to the superficial layers and potentially targeting distal dendrites of excitatory neurons from different layers. L5-projecting interneurons (purple) are mostly FS interneurons and they potentially inhibit L5 basal dendrites of excitatory neurons and L6 upright pyramidal cells. Locally projecting interneurons (blue) are more like basket cells, potentially targeting basal dendrites of different L6 excitatory neurons. The last type, L6/WM-projecting interneurons (green), is located in the deep L6 and preferentially targets WM. Potential synaptic connections are labeled as small puncta (yellow filling) in different layers.