Developing a genetic reporter to longitudinally study perineuronal nets in vivo

Perineuronal nets (PNNs) are highly organized structures of extracellular matrix associated with different neuronal populations throughout the CNS. In the cerebral cortex, they are mostly associated with parvalbumin (PV) interneurons. However, not all PV interneurons are enwrapped by these extracellular matrix structures. This heterogeneity is further accentuated by the biochemical composition of PNNs, which can vary according to the region of the CNS, during development, and in pathological conditions. PNNs mature along with postnatal circuit refinements led by experience and act as brakes of plasticity during adulthood. Indeed, PNNs stabilize adult neuronal circuits, which is fundamental to sustaining PNN-associated neuron computations or stabilizing long-term memories. Moreover, alterations in PNNs have been found in several neurodevelopmental and neurodegenerative disorders, underlining the potential fundamental role of PNNs in the proper functioning of brain circuits. Much of the current knowledge of PNNs and their key functions in the CNS derives from applying methodologies for their manipulation. Via their enzymatic degradation, genetic deletion, or overexpression, many PNN fundamental functions have been described. However, the underlying mechanisms have not been extensively elucidated. This gap in the literature is mainly due to the inability to study PNNs and PNN-associated neurons in vivo specifically. In this thesis, a genetic methodology to visualize PNNs in vivo has been developed. By fusing one of the fundamental PNN components, the link protein Hapln1 to the fluorophore mRuby2, a fluorescent PNN reporter suitable for calcium imaging applications, has been delivered through an adeno-associated virus (AAV). Through in vitro and in vivo validation steps, it has been demonstrated that the fusion protein Hapln1-mRuby2 retained the function of the native Hapln1. Indeed, Hapln1-mRuby2 inserted in PNNs has enabled the labeling of these extracellular matrix structures. To provide enough contrast between PNNs and the diffuse extracellular matrix in in vivo PNN imaging sessions, a conditional version of the PNN reporter has been developed. The injection of the conditional AAV in the visual cortex of adult mice provided the first in vivo visualization of genetically labeled PNN, demonstrating the feasibility of simultaneously performing PNN-associated neuron activity recordings. However, the expression of the conditional AAV induced a slight increase in PNN density, likely due to the overexpression of Hapln1-mRuby2. To further optimize the PNN fluorescent reporter, changing regulating elements of the expression cassette of Hapln1-mRuby2 could be a promising strategy. Moreover, the retention of the function of the native Hapln1 by the fluorescent PNN reporter Hapln1-mRuby2 might pave the way for generating a transgenic mouse line expressing fluorescent PNNs.