Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex

Abstract

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Keywords: Circadian; Diurnal; Medial prefrontal cortex; OTX2; Parvalbumin; Perineuronal nets.

Figure 1.. PNN and PV intensity vary across the diurnal cycle.

(a) Image showing WFA+ and PV+ immunolabeling. White arrows are double-labeled cells (63X). (b) Total WFA intensity in all WFA+ cells was elevated at ZT12 and ZT18 compared with ZT0, and there was a light/dark difference. (c) Similarly, WFA intensity around PV+ cells was elevated at ZT12 and ZT18 compared with ZT0, and there was a light/dark difference. (d) Total PV intensity was decreased in neurons at ZT6 and ZT12 compared with ZT0, and there was a trend for a light/dark difference, (e) PV cell intensity in PV+ cells surrounded by WFA+ was decreased at ZT6 compared with ZT0, and there was a light/dark difference. Data are mean ± SEM; N-size: ZT0, N = 10; ZT6, N = 8; ZT12, N = 7; ZT18, N = 8. *p < 0.05 for individual ZTs compared with ZT0 (individual bars) or for light vs. dark comparison (at base of graph); #p < 0.10.

Figure 2.. The oxidative stress marker 8-oxo-dG varies in PV cells with or without PNNs across the diurnal cycle.

(a) Image showing 8-oxo-dG+, WFA+, and PV+ immunolabeling. White arrows are triple-labeled cells; yellow arrow is single-labeled 8-oxo-dG cell (63X). (b) Total 8-oxo-dG was decreased at all ZTs compared with ZT0, and there was a light/dark difference. (c) Triple-labeled 8-oxo-dG+/WFA+/PV+ cells demonstrated a decrease at all ZTs compared with ZT0, and there was a light/dark difference. (d) Double-labeled 8-oxo-dG+/WFA+ cells demonstrated a decrease at all ZTs compared with ZT0, and there was a light/dark difference. (e) Double-labeled 8-oxo-dG+/PV+ cells demonstrated a decrease at all ZTs compared with ZT0, and there was a light/dark difference, (f) 8-oxo-dG+/PV+ cells showed higher oxidative stress levels than 8-oxo-dG+/WFA+ cells at ZT6 and ZT12. Each group of double-labeled cells was normalized to its own ZT0 value; dotted line is normalized ZT0 value. Data are mean ± SEM; N-size: ZT0, N = 4; ZT6, N = 4; ZT12, N = 3; ZT18, N = 4. For B-E, *p < 0.05 compared with ZT0 for individual ZTs compared with ZT0 (individual bars) or for light vs. dark comparison (at base of graph); for (F), *p < 0.05 compared with 8-oxo-dG/WFA+ cells within the same ZT.

Figure 3.. The number of glutamatergic puncta apposing PV neurons with PNNs increases at ZT18, and PV cell volume increases at ZT18

(a) An immunolabeled PV neuron (red) is surrounded by a WFA-labeled PNN (blue) in a representative confocal micrograph from the mPFC. The PV neuron is receiving appositions from both vGLUT1-labeled glutamatergic puncta (magenta) and GAD65/67-labeled GABAergic puncta (green), (b) The Imaris Surfaces segmentation tool was used to render the PV neuron (gray) and WFA-labeled PNN (blue). The Imaris Spots segmentation tool was used to segment GAD65/67 (green arrows) and vGLUT1-labeled (magenta arrows) puncta that met size and location criteria. Scale bar = 5 µm. In WFA+/PV+ cells: (c) Number of inhibitory puncta (GAD65/67) were similar at ZT6 and ZT18. (d) Number of excitatory puncta (vGLUT1) apposing PV neurons was higher at ZT18 compared with ZT6. (e) The ratio of GAD65/67:vGLUT1 puncta trended toward a decrease at ZT18 compared with ZT6. (f) The volumes of the three-dimensional surfaces rendered with Imaris software through the middle of the PV neurons were larger at ZT18 compared with those at ZT6. In WFA−/PV+ cells: (g) Number of inhibitory puncta (GAD65/67) was similar in the ZT6 and ZT18 groups, (h) Number of excitatory puncta (vGLUT1) was similar at ZT6 and ZT18. (i) The ratio of GAD65/67:vGLUT1 puncta was similar at ZT6 and ZT18. (j) The volumes of the three-dimensional surfaces rendered with Imaris software were similar at ZT6 and ZT18. Data are mean ± SEM; N = 4/group. *p < 0.05 compared with ZT6; #p < 0.10 compared with ZT6.

Figure 4.. The AMPA:NMDA ratio increases at ZT18.

(a) Representative traces of the AMPA (black) and NMDA (green) components at ZT6 and ZT18. Scale bar represents 10 pA, 10ms. (b) AMPA:NMDA ratio of mEPSCs evoked from PNN-surrounded fast-spiking cells in the prelimbic mPFC increases during the dark phase at ZT18. (c) Average time constants of decay of the NMDA component of mEPSCs evoked from PNN surrounded PV FSIs in the prelimbic mPFC. Data are mean ± SEM; Number of rats: ZT6 N = 7; ZT18 N = 8. One cell per rat was used for each experiment so that reported N-sizes represent the number of animals. *p < 0.05; #p < 0.10 compared with ZT6.

Figure 5.. OTX2 staining in PV+ cells and WFA+/PV+ cells trend toward increase at ZT18.

(a) Image showing OTX2+, WFA+, and PV+ immunolabeling. White arrows are triple-labeled cells and yellow arrowhead is double-labeled OTX2+/PV+ cell (63X). (b) Total number of OTX2+ cells was similar at ZT6 and ZT18. (c) Number of OTX2+/WFA+ cells was similar at ZT6 and ZT18. (d) Number of OTX2+/PV+ cells showed a strong trend toward increase at ZT18 compared with ZT6. (e) Number of OTX2+/WFA+/PV+ cells showed a trend toward increase at ZT18 compared with ZT6. Data are mean ± SEM; N = 6-7/group. #p < 0.10 compared with ZT6.