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 (WFA1 PNNs) in several brain regions involved in emotional memory processing. Sleep deprivation prevented the daytime decrease of WFA1 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, WFA1 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.

Background There are many contradictory findings on the role of the hormone ghrelin in aversive processing, with studies suggesting that ghrelin signaling can both inhibit and enhance aversion. Here, we characterize and reconcile the paradoxical role of ghrelin in the acquisition of fearful memories. Methods We used enzyme-linked immunosorbent assay (ELISA) to measure endogenous acyl-ghrelin and corticosterone at time points surrounding auditory fear learning. We used pharmacological (systemic and intra-amygdala) manipulations of ghrelin signaling and examined several aversive and appetitive behaviors. We also used biotin-labeled ghrelin to visualize ghrelin binding sites in coronal brain sections of amygdala. All work was performed in rats. Results In unstressed rodents, endogenous peripheral acyl-ghrelin robustly inhibits fear memory consolidation through actions in the amygdala and accounts for virtually all inter-individual variability in long-term fear memory strength. Higher levels of endogenous ghrelin after fear learning were associated with weaker long-term fear memories, and pharmacological agonism of the ghrelin receptor during the memory consolidation period reduced fear memory strength. These fear-inhibitory effects cannot be explained by changes in appetitive behavior. In contrast, we show that chronic stress, which increases both circulating endogenous acyl-ghrelin and fear memory formation, promotes both a profound loss of ghrelin binding sites in the amygdala and behavioral insensitivity to ghrelin receptor agonism. Conclusions These studies provide a new link between stress, a novel type of metabolic resistance, and vulnerability to excessive fear memory formation and reveal that ghrelin can regulate negative emotionality in unstressed animals without altering appetite.

Accumulating evidence supports the role of sleep in synaptic plasticity and memory consolidation. One line of investigation, the synaptic homeostasis hypothesis, has emphasized the increase in synaptic strength during waking, and compensatory downsizing of (presumably less frequently used) synapses during sleep. Conversely, other