Perineuronal Nets Regulate the Inhibitory Perisomatic Input onto Parvalbumin Interneurons and γ Activity in the Prefrontal Cortex

Abstract

Parvalbumin-expressing (PV+) interneurons play a key role in the maturation and synchronization of cortical circuitry and alterations in these inhibitory neurons, especially in the medial prefrontal cortex (mPFC), have been found in different psychiatric disorders. The formation of perineuronal nets (PNNs) around many of these interneurons at the end of the critical periods reduces their plasticity and sets their connectivity. Consequently, the presence of PNNs must have an important impact on the synaptic input and the physiology of PV+ cells. In the present study, we have found that in adult male mice, prefrontocortical PV+ cells surrounded by PNNs show higher density of perisomatic excitatory and inhibitory puncta, longer axonal initial segments (AISs), and higher PV expression when compared with PV+ cells lacking PNNs. In order to better understand the impact of PNNs on the connectivity and physiology of PV+ interneurons in the mPFC, we have digested enzymatically these structures and have found a decrease in the density of inhibitory puncta on their perisomatic region but not on the PV+ perisomatic puncta on pyramidal neurons. Moreover, extracellular recordings show that the digestion of PNNs induces a decrease in γ activity, an oscillation dependent on PV+ cells, in the mPFC of anesthetized mice. Our results suggest that the presence of PNNs enwrapping PV+ cells regulates their inhibitory input and has a potent influence on their activity. These results may be relevant for psychiatric research, given the alterations in PNNs, PV+ interneurons and their physiology described in different mental disorders.SIGNIFICANCE STATEMENT Parvalbumin-expressing (PV+) interneurons are surrounded by specializations of the extracellular matrix, the perineuronal nets (PNNs). PNNs regulate the development and plasticity of PV+ cells and, consequently, their presence must influence their synaptic input and physiology. We have found, in the adult prefrontal cortex (PFC), substantial differences in the structure and connectivity of PV+ interneurons depending on the presence of PNNs. The depletion of PNNs from the PFC has also a potent effect on the connectivity of PV+ cells and on neural oscillations that depend on these cells. These findings are relevant to understand the role of PNNs in the adult brain and in certain psychiatric disorders in which alterations in PNNs and PV+ interneurons have been described.

Keywords: basket cell; interneuron; perineuronal net; prefrontal cortex; γ oscillation.

Figure 1.

The presence or absence of PNNs surrounding PV+ interneurons influences PV expression in their somata and their perisomatic innervation. A, Schematic drawing of a coronal slide, highlighting the area analyzed and a representative immunostaining of the expression of PV and WFA in PrL. The white arrowhead points to a PV+ cell lacking PNN. Scale bar, 10 μm. B, Graph showing the expression of PV in PV+ cells surrounded versus not surrounded by PNNs. C, Distribution graph showing the positive correlation of PV fluorescence intensity and WFA fluorescence intensity. D, High-magnification single confocal planes comparing the inhibitory perisomatic innervation on PV+ cells depending on the presence or absence of PNNs. White arrowheads point to representative puncta. Scale bar, 4 μm. E, Graphs showing the lower density of perisomatic puncta expressing inhibitory markers on PNN– PV cells. F, Distribution graph showing the positive correlation of PV fluorescence intensity and density of inhibitory perisomatic puncta. G, Single confocal planes of VGLUT1-expressing perisomatic puncta on PV+ cells. Scale bar, 4 μm. H, Graph showing the density of VGLUT1+ puncta on the perisomatic region of PV+ cells. I, Graph comparing the ratio of excitatory/inhibitory perisomatic puncta on PV+ cells. J, Representative images of the AIS from PV+ cells. White arrowheads point to the AIS. Scale bar, 6 μm. K, Graph comparing the length of the AIS in PV+ cells surrounded versus not surrounded by PNNs. *p < 0.05, **p < 0.01.

Figure 2.

The presence of PNNs does not influence the density of perisomatic puncta coming from CCK⁺ basket cells or extracortical origin on PV+ interneurons. A, Representative confocal planes showing CB1r-immunoreactive perisomatic puncta on PV+ cells. Scale bar, 4 μm. B, Graph comparing the density of CB1r-expressing puncta on PV+ enwrapped by versus lacking PNNs. C, Single confocal planes showing the density of VGLUT2-expressing puncta on the perisomatic region of PV+ cells. Scale bar, 4 μm. D, Graph showing the density of VGLUT2+ perisomatic puncta on PV+ cells, comparing those surrounded by PNNs versus those lacking these extracellular matrix structures.

Figure 3.

Timeline of PNNs digestion after the intracranial injection of ChABC revealed by WFA labeling. A, Microphotographs showing labeled PNNs in the mPFC of control and ChABC-injected mice. Dashed lines indicate the limits of ChABC effects. Scale bar, 300 μm. B, Insets from the squares in PrL of panel A showing the digestion and the subsequent partial recovery of PNNs. Scale bar, 80 μm.

Figure 4.

Effects of ChABC on PV expression and perisomatic puncta in the PrL. A, Scheme of experimental timeline and drawing of a coronal slide, highlighting the area of study. B, Representative confocal stacks of PV immunostaining in the PrL. C, Graph showing the PV fluorescence intensity in PrL. D, Single confocal planes showing SYN-expressing puncta (white arrowheads) surrounding the soma of a PV+ cell. Scale bar, 5 μm. E, Graphs showing that ChABC treatment induces a decrease in the density of SYN-expressing puncta on the perisomatic region of PV+ cells. F, Confocal reconstruction (three planes) of PV+ cells and their AIS. Arrowheads point to AIS stained by AnKG. Scale bar, 8 μm. G, Graph comparing the length of the AIS in PV+ cells after penicillinase and ChABC injection. H, Single confocal planes showing the density of excitatory (VGLUT1+) and inhibitory (VGAT+) perisomatic puncta (white arrowheads) on PV+ cells. Scale bar, 5 μm. I, Graphs showing the ChABC induced decrease in the density of inhibitory but not excitatory puncta on the perisomatic region of PV+ cells. J, Histograms showing that decrease in inhibitory puncta occurs both in low PV and high PV cells. K, Graph showing the significant decrease induced by ChABC on the density of puncta coexpresssing VGAT and PV in the perisomatic region of PV+ cells. L, Graphs showing the ratio between the densities of VGLUT1/VGAT-expressing puncta. M, Schematic drawing showing the area of analysis highlighted by a gray square. N, Single confocal planes showing PV-expressing puncta (white arrowheads) on the perisomatic region of pyramidal neurons. Scale bar, 5 μm. O, Graph showing the effects of ChABC treatment on the density of PV+ perisomatic puncta on pyramidal neurons. P, Graph showing intensity of CamkII fluorescence in the cell body of pyramidal neurons. *p < 0.05, **p < 0.01.

Figure 5.

ChABC effects on the neuropil of the PrL. A, Schematic drawing of the area imaged. B, Representative confocal planes showing SYN-expressing puncta in the neuropil. Scale bar, 6 μm. C, Graph showing the effects of ChABC on the density of SYN+ puncta in the neuropil. D, Representative confocal planes of VGLUT1+ and VGAT+ puncta. Scale bar, 6 μm. E, Graphs showing the effects of ChABC on the density of VGLUT1+ and VGAT+ puncta.

Figure 6.

PNNs digestion decreases the fluorescence intensity of FosB in PrL. A, Schematic drawing of the area imaged. B, Confocal stack (three confocal planes) of PrL of the mPFC showing the expression of PV, CamkII, and FosB. White arrowheads point to higher magnification microphotographs right of each stack showing the FosB expression in PV and CamkII cells. Scale bar, 25 and 3 μm. C, Graph showing the decrease of the FosB expression in PV cells and CamkII cells in the ChABC animals. ***p < 0.001.

Figure 7.

PNNs digestion alters γ oscillations in PrL during sensory induced cortical activity. A, Scheme of experimental design. B, Example of LFP recording in PrL, tail-pinch onset time point is highlighted with a dotted red line. C, Power spectrum of frequencies from 0.5 to 250 Hz in PrL in a representative case where an average of 10 tail-pinch effects is shown (line, power spectra; shaded area, 95% confidence interval). D, Fitting of linear models over the grand average of the γ power spectra for all experiments. E, Box plots of high γ and fast γ oscillations normalized energy in the PrL. F, MI of high γ and fast γ oscillations in the PrL. G, Confocal stack (three planes) on the PrL showing c-Fos expression of PV interneurons and pyramidal neurons after tail-pinch stimulation. White arrowheads point to representative cells. Scale bar, 25 μm. H, Plots showing the fluorescence intensity of c-Fos in PV interneurons and pyramidal neurons. **p < 0.01.