Very long-term memories may be stored in the pattern of holes in the perineuronal net

Abstract

A hypothesis and the experiments to test it propose that very long-term memories, such as fear conditioning, are stored as the pattern of holes in the perineuronal net (PNN), a specialized ECM that envelops mature neurons and restricts synapse formation. The 3D intertwining of PNN and synapses would be imaged by serial-section EM. Lifetimes of PNN vs. intrasynaptic components would be compared with pulse-chase (15)N labeling in mice and (14)C content in human cadaver brains. Genetically encoded indicators and antineoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. Further techniques suggested include genetic KOs, better pharmacological inhibitors, and a genetically encoded snapshot reporter, which will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, drive expression of effector genes to mark those cells, and allow selective excitation, inhibition, or ablation to test their functional importance. The snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Finally, biosynthesis of PNN components and proteases would be imaged.

Keywords: extracellular matrix; genetically encoded reporters; memory; plasticity; protein turnover.

Fig. 1.

Perineuronal nets. (A) Cat cerebral cells stained with reduced silver nitrate (75). (B) Dog cortical cells stained by the Bethes method (76). (C) WFA- and synapsin-stained cultured cortical neurons (77). (D) Confocal image of a rat somatosensory cortex interneuron labeled with fluorescent VVA (78). (E) WFA-stained somata and proximal dendrites of rat neurons (79).

Fig. 2.

The structure of the PNN. Hyaluronan, secreted by membrane-bound HA synthase (HAS), binds to members of the lectican family (aggrecan, brevican, versican, and neurocan) and is cross-linked by link proteins and tenascin-R to form supramolecular aggregates on the surface of neurons. Reproduced from ref. with permission of John Wiley & Sons, Inc. CS-GAG, chondroitin sulfate – glycosaminoglycans.

Fig. 3.

Cartoon depicting one of the many ways to engineer a light- and Ca²⁺-triggered transcriptional readout to serve as a snapshot reporter of neuronal activity. Many different permutations of the three fusions are possible, provided that no single fusion contains (Gal4 + VP16), (CaM + M13), or (PhyB + PIF6). PhyB, PIF6, and red light may be replaced by CRY2, CIBN, and blue light, respectively. Nuclear localization signals may have to be provided. For simplicity, binding of the Gal4 chimera to its cognate DNA is shown only after final assembly of the three-hybrid. IRES, internal ribosomal entry site.