Circadian Rhythms of Perineuronal Net Composition

Abstract

Perineuronal nets (PNNs) are extracellular matrix (ECM) structures that envelop neurons and regulate synaptic functions. Long thought to be stable structures, PNNs have been recently shown to respond dynamically during learning, potentially regulating the formation of new synapses. We postulated that PNNs vary during sleep, a period of active synaptic modification. Notably, PNN components are cleaved by matrix proteases such as the protease cathepsin-S. This protease is diurnally expressed in the mouse cortex, coinciding with dendritic spine density rhythms. Thus, cathepsin-S may contribute to PNN remodeling during sleep, mediating synaptic reorganization. These studies were designed to test the hypothesis that PNN numbers vary in a diurnal manner in the rodent and human brain, as well as in a circadian manner in the rodent brain, and that these rhythms are disrupted by sleep deprivation. In mice, we observed diurnal and circadian rhythms of PNNs labeled with the lectin Wisteria floribunda agglutinin (WFA+ PNNs) in several brain regions involved in emotional memory processing. Sleep deprivation prevented the daytime decrease of WFA+ PNNs and enhances fear memory extinction. Diurnal rhythms of cathepsin-S expression in microglia were observed in the same brain regions, opposite to PNN rhythms. Finally, incubation of mouse sections with cathepsin-S eliminated PNN labeling. In humans, WFA+ PNNs showed a diurnal rhythm in the amygdala and thalamic reticular nucleus (TRN). Our results demonstrate that PNNs vary in a circadian manner and this is disrupted by sleep deprivation. We suggest that rhythmic modification of PNNs may contribute to memory consolidation during sleep.

Keywords: circadian rhythms; extracellular matrix; memory consolidation; perineuronal nets; psychiatric disorders; sleep.

Figure 1.

Diurnal rhythms of PNNs in the mouse hippocampus. Analysis of WFA+ PNNs across the 24-h cycle in male mice housed in a 12/12 h LD cycle revealed a diurnal rhythm of WFA+ PNNs in hippocampal sectors CA1 (A) CA2/3 (B), CA4 (C), and the DG (D) with peaks at ∼ZT20 and troughs at ∼ZT8. Error bars represent SD. Representative low-magnification images of WFA labeling in the mouse hippocampus at ZT8 (E) and ZT20 (F).

Figure 2.

Diurnal rhythms of PNNs in the mouse amygdala. Diurnal rhythms of WFA+ PNNs were observed in the lateral amygdala (A) basal amygdala (B), and central amygdala (C) with peaks at ∼ZT20 and troughs at ∼ZT8. Error bars represent SD. Representative low-magnification images of WFA labeling in the mouse amygdala at ZT8 (D) and ZT20 (E).

Figure 3.

Diurnal rhythms of PNNs in the mouse prefrontal cortex. Diurnal rhythms of WFA+ PNNs were observed in the infralimbic superficial (A) prelimbic superficial (B), infralimbic deep (C), and prelimbic deep (D) layers of the mouse, with peaks at ∼ZT0 and troughs at ∼ZT8. Error bars represent SD. Representative low-magnification images of WFA labeling in the mouse prefrontal cortex at ZT8 (E) and ZT20 (F).

Figure 4.

Diurnal rhythms of PNNs in the mouse habenula. Analysis of WFA+ PNNs across the 24-h cycle in male mice housed in a 12/12 h LD cycle revealed a diurnal rhythm of WFA+ PNNs in the lateral habenula (A) and medial habenula (B), with a peak at ∼ZT0 and trough at ∼ZT8 for the lateral habenula, and a peak at ∼ZT16 and trough at ∼ZT8 for the medial habenula. Error bars represent SD. Representative low-magnification images of WFA labeling in the mouse habenula at ZT8 (C) and ZT20 (D).

Figure 5.

Diurnal rhythms of PNNs in the mouse TRN. Analysis of WFA+ PNNs across the 24-h cycle in male mice housed in a 12/12 h LD cycle revealed a diurnal rhythm of WFA+ PNNs in the TRN (A) with a peak at ∼ZT20 and a trough at ∼ZT8. Error bars represent SD. Representative low-magnification images of WFA labeling in the mouse TRN at ZT8 (B) and ZT20 (C).

Figure 6.

Circadian rhythms of PNNs in the mouse brain. Circadian rhythms in the density of WFA+ PNNs were observed in mice housed in constant darkness. In the hippocampus, these rhythms were similar to the diurnal rhythms observed in the CA regions and the DG (A–D); circadian rhythms in the density of WFA+ PNNs in the mouse prefrontal cortex also paralleled the observed diurnal rhythms in these regions, with peaks at ∼CT0 and troughs at ∼CT8 (E–H), with the exception of the deep layers of the IL cortex, which showed a peak at ∼CT20 and trough at CT8 (F). Circadian rhythms of WFA+ PNN densities were also observed in the lateral, basal, and central amygdala nuclei in constant darkness, with a peak at ∼CT16 and a trough at ∼CT6 (I–K). Circadian rhythms of WFA+ PNN densities in the lateral and medial habenula and TRN paralleled diurnal PNN rhythms in these regions (L–N). Error bars represent SDs.

Figure 7.

Sleep deprivation prevents PNN decreases. Five hours of sleep deprivation, from lights on (7 A.M.) to 12 P.M. following auditory fear conditioning, resulted in rapid extinction of fear memory (A), along with significantly higher numerical density of WFA+ PNNs in the hippocampus (B). Representative photomicrographs of the hippocampus labeled with WFA from a control mouse (C) and a sleep-deprived mouse (D). Scale bar = 1000 μm. Similar increases in densities of WFA+ PNNs in SD mice were also observed in the amygdala (E), habenula (F), and prefrontal cortex (G). Error bars represent 95% confidence intervals.

Figure 8.

Cathepsin-S diurnal rhythms in the mouse hippocampus. Diurnal rhythms in densities of cathepsin-S-IR cells were observed in CA1 (A), CA2/3 (B), CA4 (C), and the DG (D) in mice, with expression peaking during the middle of the light cycle, when WFA+ PNN numbers are low in these regions, and decreasing during the dark cycle, when WFA+ PNN densities are high. Error bars represent SD. Representative photomicrographs of the hippocampus labeled with cathepsin-S at ZT8 (E) and ZT20 (F).

Figure 9.

Cathepsin-S diurnal rhythms in the mouse amygdala and prefrontal cortex. Diurnal rhythms in densities of cathepsin-S-IR cells were observed in the lateral amygdala (A), basal amygdala (B), and central amygdala (C), with expression peaking during the middle of the light cycle, when WFA+ PNN numbers are low in these regions, and decreasing during the dark cycle, when WFA+ PNN densities are high. Similar diurnal rhythms were also observed in the infralimbic cortex superficial layers (D), prelimbic cortex superficial layers (E), infralimbic cortex deep layers (F), and prelimbic cortex deep layers (G). Error bars represent SD.

Figure 10.

Cathepsin-S is expressed in microglia and eliminates PNN labeling. Significant reduction in WFA+ PNNs is observed after 3 h of cathepsin-S incubation (A–C) and a complete absence of PNN labeling after 24 h (D–F). Error bars represent 95% confidence interval. Scale bars = 1000 μm. Dual fluorescence immunohistochemistry demonstrated that the vast majority of cathepsin-S-IR cells in the mouse hippocampus co-express the microglial marker IBA1 (G–N). Scale bar = 50 μm.

Figure 11.

Diurnal rhythms of PNNs in the human brain. WFA+ PNN numbers vary with TOD in the human brain. Photomicrograph depicting PNN labeling by WFA lectin in the human amygdala during the day (A) and at night (B). WFA+ PNNs displayed a significant day/night difference in the human amygdala (C), with peaks PNN numbers at noon and midnight, and troughs at 4 A.M. and 8 P.M. D, Photomicrograph depicting PNN labeling in the human TRN during the day (E) and at night (F). Significant day/night differences were observed in total numbers of WFA+ PNNs in the TRN (G). Quartic regression plots revealed a dual peak rhythm in the TRN that is antiphase to the rhythm observed in the amygdala (H). Error bars represent 95% confidence intervals.

Figure 12.

Microglial expression of cathepsin-S may modify PNNs to allow for memory consolidation during sleep. In the mouse hippocampal sector CA1, diurnal rhythms in the numerical density of WFA+ PNNs decreases during the day as mice sleep, reaching the lowest density in WFA+ PNN numbers between ZT4–ZT10 (green curved line). This coincides with the peak expression of cathepsin-S (red curved line) and the reported daytime decrease in LTP (blue circles; from Chaudhury et al., 2005). In comparison, the numerical density of WFA+ PNNs peaks during the dark at ∼ZT20 during the active period for nocturnal mice, coinciding with the low point of cathepsin-S immunoreactivity in this region as well as the reported increase in LTP at night in mice (pink circles; from Chaudhury et al., 2005). These results suggest that cathepsin-S modifies PNN composition, coinciding with decreased TLP during sleep, to allow for memory consolidation, and PNN composition is restored during the active wake periods to allow for optimal encoding of novel information.