Selective disruption of perineuronal nets in mice lacking Crtl1 is sufficient to make fear memories susceptible to erasure

Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase ABC reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hyaluronan and proteoglycan link protein 1, Hapln1), which is upregulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. Therefore, the organization of chondroitin sulphate proteoglycan into perineuronal nets is the key event in the control of central nervous system plasticity by the extracellular matrix. In addition, using chondroitinase ABC treatment targeting chondroitin sulfate proteoglycans, previous studies showed that the maturation of the extracellular matrix makes fear memories resistant to deletion. Since mice lacking the cartilage link protein Crtl1 display normal chondroitin sulfate proteoglycans levels but impaired chondroitin sulfate proteoglycans condensation in perineuronal nets, we asked whether the presence of perineuronal nets in the adult brain is responsible for the appearance of persistent fear memories by investigating fear extinction in Crtl1-KO mice. We found that mutant mice displayed fear memory erasure after an extinction protocol as revealed by analysis of freezing and pupil dynamics. Fear memory erasure did not depend on passive loss of retention. Moreover, we demonstrated that, after extinction training, conditioned Crtl1-KO mice display no neural activation in the amygdala (Zif268 staining) in comparison to control animals. Taken together, our findings suggest that the aggregation of chondroitin sulfate proteoglycans into perineuronal nets regulates the boundaries of the critical period for fear extinction.