Releasing the peri-neuronal net to patch-clamp neurons in adult CNS

Abstract

The extracellular matrix of adult neural tissue contains chondroitin sulphated proteogylcans that form a dense peri-neuronal net surrounding the cell body and proximal dendrites of many neuronal classes. Development of the peri-neuronal net beyond approximately postnatal day 17 obscures visualization and often access by patch electrodes to neuronal membranes with the result that patch clamp recordings are most readily obtained from early postnatal animals. We describe a technique in which the surface tension of a sucrose-based medium promotes partial dissociation of thin tissue slices from adult tissue. Surface tension spreads the tissue and loosens the peri-neuronal net from neuronal membranes within minutes and in the absence of proteolytic enzymes. Furthermore, the extent of dissociation can be controlled so as to maintain the overall slice structure and allow identification of specific cell classes. Excellent structural preservation of neurons and dendrites can be obtained and full access by patch electrodes made possible for current- or voltage-clamp recordings in tissue well beyond the development of peri-neuronal nets. We demonstrate the feasibility of using this approach through patch recordings from neurons in the brainstem and cerebellum of adult gymnotiform fish and in deep cerebellar nuclei of rats as old as 6 months.