Expression of Hyaluronan and Perineuronal Nets in Hippocampal Neurons in vivo and in vitro

Abstract

Hyaluronan (HA) is a polysaccharide which is ubiquitously expressed in the extracellular matrix (ECM) of all tissues in the body. In the central nervous system (CNS), HA forms the backbone of specialised ECM structures termed perineuronal nets (PNNs). These reticulated structures envelop the soma and proximal dendrites of a specific subtype of GABAergic interneuron, named parvalbumin-positive (PV+) interneurons. The onset of PNNs in the developing CNS signifies the start of a ‘critical period’ in postnatal synaptic development, and the mature expression of PNNs have been shown to reduce chemical and anatomical neuroplasticity. The hippocampus is a neural region where neuroplasticity is important for facilitating learning and memory. Thus, we aimed to investigate the change in PNN and PV+ interneuron expression in the developing rat hippocampus. Further, while it was initially believed that astrocytes are integral for the synthesis of HA in the CNS, including within PNNs, more recent findings suggest that neurons also have the capacity for the synthesis of HA and PNNs independent of astrocytes. Thus, our second aim was to demonstrate the expression of HA, PNNs and hyaluronan synthases (HASs, enzymes required to synthesise HA), in dissociated hippocampal neuron cultures devoid of astrocytes. Our experiments used immunohistochemistry to label the expression of PNNs and PV+ interneurons in the principal cell layer of several hippocampal subregions (CA1-CA4, and dentate gyrus), in coronal sections collected from Sprague Dawley rat pups aged postnatal day (P) 3, 7, 14 and 21. Similarly, we used immunocytochemistry to label the expression of HA, PNNs, PV+ interneurons and HASs in dissociated hippocampal cultures generated from embryonic rats. These cultures were collected at days in vitro (DIV) 1, 3, 7, 14 and 21. The in situ studies demonstrated the earliest expression of PV+ interneurons at P3, and PNNs from P14 onwards. By P21, the vast majority of PV+ interneurons expressed PNNs, but the majority of PNN-bearing neurons across the different hippocampal subregions did not express PV immunoreactivity. In vitro, HA and the HASs, HAS2 and HAS3, were observed on hippocampal neurons as early as DIV1, thus suggesting hippocampal neurons can synthesise HA independent of astrocytes. In conclusion, our study demonstrates the time course of PNN development in the developing rat hippocampus, and the capacity for hippocampal neurons to synthesise HA independent of astrocytes.