Perineuronal Nets in the Adult Sensory Cortex Are Necessary for Fear Learning

Abstract

Lattice-like structures known as perineuronal nets (PNNs) are key components of the extracellular matrix (ECM). Once fully crystallized by adulthood, they are largely stable throughout life. Contrary to previous reports that PNNs inhibit processes involving plasticity, here we report that the dynamic regulation of PNN expression in the adult auditory cortex is vital for fear learning and consolidation in response to pure tones. Specifically, after first confirming the necessity of auditory cortical activity for fear learning and consolidation, we observed that mRNA levels of key proteoglycan components of PNNs were enhanced 4 hr after fear conditioning but were no longer different from the control groups 24 hr later. A similar pattern of regulation was observed in numbers of cells surrounded by PNNs and area occupied by them in the auditory cortex. Finally, the removal of auditory cortex PNNs resulted in a deficit in fear learning and consolidation.

Keywords: Chondroitinase ABC; PTSD; auditory cortex; conditioning; consolidation; extracellular matrix; hearing; memory; perceptual learning; plasticity.

Figure 1. Muscimol mediated inactivation of auditory cortex before auditory fear conditioning decreased fear expression observed 24 and 48 hours later

Muscimol or saline was injected into the auditory cortex of mice 15–30 minutes prior to Pavlovian auditory fear conditioning. Mice were fear-conditioned and no differences were observed in fear acquisition (A) in saline and muscimol groups. B depicts fluorescent bodipy-muscimol in the auditory cortex. Decreased fear expression was observed in muscimol injected mice 24 hours (C) after fear conditioning with significant differences between groups during CS 1–5 and CS11–15 (D) and 48 hours after fear conditioning (E) during CS1–5(F). *P < 0.05 vs. vehicle. All values are means ±SEM.

Figure 2. Muscimol mediated inactivation of auditory cortex after auditory fear conditioning decreased fear expression observed 24 and 48 hours later

Muscimol or saline was injected into the auditory cortex of mice within 30 minutes after Pavlovian auditory fear conditioning. No differences were observed in fear acquisition (A) in saline and muscimol groups. Decreased fear expression was observed in muscimol injected mice 24 hours (B) after fear conditioning with significant differences between groups during CS1–5, CS 6–10 and CS 11–15 (C) and 48 hours after fear conditioning (D) during CS1–5, CS 6–10 and CS 11–15 (E). *P < 0.05 vs. vehicle. All values are means ±SEM.

Figure 3. mRNA levels of genes encoding lecticans were enhanced 4 hours after fear conditioning but returned to baseline levels 24 hours later

mRNA levels of aggrecan were significantly higher at 4 hours after fear conditioning compared to 2 hours after fear conditioning (A). Brevican mRNA levels were highest at 4 hours after fear conditioning and significantly higher than the home cage group and unpaired group (B). mRNA levels of neurocan were significantly higher at 4 hours after fear conditioning as compared to home cage group, tone only group and 24 hours after fear conditioning (C). *P < 0.05 vs. vehicle. All values are means ±SEM.

Figure 4. PNN expression was specifically enhanced 4 hours after tone-shock paired fear conditioning but returned to baseline levels 24 hours later

Area of expression of PNNs across the auditory cortex was highest at 4 hours after fear conditioning, but not different from controls by 24 hours after fear conditioning (A). In a separate group of animals sacrificed at 4 hours after tone experience (B), area of expression of PNNs was highest for the paired group as compared to home cage controls, and groups receiving only tone presentation or unpaired stimulation. Numbers of cells surrounded by PNNs are higher at 4 hours after fear conditioning than home cage group and the group sacrificed 24 hours after fear conditioning (C). In the same group of animals as in (B), the number of cells surrounded by PNNs was highest in the paired group 4 hours after auditory fear conditioning as compared to the home cage controls, tone alone and unpaired group 4 hours after tone experience (D). *P < 0.05 vs. vehicle. All values are means ±SEM.

Figure 5. Removal of PNNs in the auditory cortex using ChABC before auditory fear conditioning decreased fear expression observed 24 and 48 hours later, but did not decrease fear expression 30 minutes later

ChABC or saline was injected into the auditory cortex of mice 72 hours prior to Pavlovian auditory fear conditioning. Mice were fear-conditioned and no differences were observed in fear acquisition (A) or fear expression 30 minutes after fear conditioning (B) between saline and ChABC groups (C). Decreased fear expression was observed in ChABC injected mice in comparison to controls at 24 hours after fear conditioning (D) wherein significant differences between groups were observed during CS1–5 and CS6–10 (E) and at 48 hours after fear conditioning during CS1–5 and CS11–15(F). *P < 0.05 vs. vehicle. All values are means ±SEM

Figure 6. Removal of PNNs in the auditory cortex after fear conditioning, results in decreased fear expression observed after 24 and 48 hours but not 30 minutes after fear conditioning

ChABC or saline was injected into the auditory cortex of mice within 30 minutes after auditory fear conditioning. No differences were observed in fear acquisition (A) or fear expression 30 minutes after fear conditioning (B) between saline and ChABC groups (C). Decreased fear expression was observed in ChABC injected mice in comparison to controls at 24 hours after fear conditioning (D) wherein significant differences between groups were observed during CS1–5, CS6–10 and CS11–15 (E) and at 48 hours after fear conditioning (F). No significant differences between groups were observed after binning according to CS number (G). *P < 0.05 vs. vehicle. All values are means ±SEM