Background

The extracellular spaces of the central nervous system are filled by an amorphous interstitial matrix that is composed of dense network of proteoglycans, hyaluronic acid (HA), tenascins and link proteins, and is contiguous with well-organized lattice-like structures called PNNs that enclose the somata, axons and dendrites of PV expressing interneurons in the cerebral cortex (Härtig et al., 1999). The lattice of PNNs is composed of chondroitin sulfate proteoglycans (CSPGs), HA, tenascins, various link proteins (Deepa et al., 2006), etc. Owing to the high density of sulfated proteoglycans, PNNs are highly negatively charged (Morawski et al., 2015). The development of PNN coincides with the closure of ocular dominance critical period and studies have shown reinstatement of ocular dominance plasticity upon removal of the PNNs thereby suggesting inhibitory role of PNNs on neuronal plasticity (Pizzorusso et al., 2002). PNNs are also suggested to modulate the biophysical properties PV interneurons (Balmer, 2016, Favuzzi et al., 2017, Tewari et al., 2018), and are prone to degradation by ECM remodeling enzymes. Most notably, under pathological conditions including epilepsy (McRae et al., 2012, Rankin-Gee et al., 2015, Dubey et al., 2017, Tewari et al., 2018, Patel et al., 2019), schizophrenia and Alzheimer disease (Testa et al., 2018) ECM remodeling is common.

One of the major challenges in reporting on the status of PNNs is the quantification of the structural integrity of their lattice-like assembly. Essentially all published studies use the CSPG binding lectin, WFA, as non-specific PNN marker to visualize PNNs, and use WFA intensity to quantify the overall CSPG levels without detailed analysis of individual PNN’s architecture (Slaker et al., 2015 and 2016, Lensjø et al., 2017, Ueno et al., 2018). We developed a method to quantify the structural integrity of individual PNNs using high-magnification fluorescence imaging. This method also minimizes the errors due to procedure-related variations in the WFA intensity. After acquiring multichannel high magnification images (40x objective lens with 5 digital zoom) of individual PNNs, a polyline is drawn around the periphery of the cell. The fluorescence intensity of WFA along this line shows a discrete pattern of peaks separated by low-intensity valleys. Each peak represents the continuous CSPG and the gap between the two consecutive peaks represent a hole in the PNN (Tewari et al., 2018). The average number of peaks and average width of holes can be derived from the WFA intensity data points and are reflective of the PNN’s structural integrity. The peaks in the line profile can be counted manually or by using clampfit program (Molecular Devices).

A vast majority of studies, which explore the function of PNNs and their influence on the neuronal physiology, use ChABC, which is a bacterial enzyme that cleaves the chondroitin sulfate side chains thereby dismantling the PNN assembly (Dityatev et al., 2007, Balmer, 2016, Testa et al., 2018). The most common approach in such studies involves degradation of PNN in-vivo/in-situ brain slices/primary cultures and studying the function afterward by comparing two different populations of samples. However, it would be advantageous to record from PNN bearing neurons during the real-time degradation of PNNs to evaluate the physiological role of PNN on neuronal function. Such studies are largely absent in the literature. Here, we report two variants of experimental PNN degradation in the brain slices, which reliably degrade PNNs to allow studying their function. The first variant includes degradation of PNNs by incubating brain slices with ChABC in an incubation chamber followed by performing experiments to compare their properties with non-treated controls. The other variant is to study the baseline physiology in the presence of intact PNNs followed by superfusing ChABC solution and recording the functional activity during and after PNN depletion in the experimental set-up. Both the variants involve controlled degradation of PNNs and identifiable traces of degraded PNNs around the cells can be observed to confirm the presence/absence of PNNs on the experimental cell/tissue after post-experimental WFA staining of the samples. These methods provide unparalleled advantages to study the physiological functions associate with the PNNs.