Entorhinal cortex lesion studied with the novel dye Fluoro-Jade

Abstract

We used the fluorescent dye Fluoro-Jade, capable of selectively staining degenerating neurons and their processes, in order to analyze degenerative effects of transecting the hippocampus from its main input, the entorhinal cortex in vivo and in organotypical hippocampal slice culture. Degenerating fibers stained with Fluoro-Jade were present as early as 1 day postlesion in the outer molecular layer of the dentate gyrus and could be detected up to 30 days postlesion. However, the intensity of the Fluoro-Jade staining in the outer molecular layer faded from postlesional day 20 onward. Punctate staining, various cells and neural processes became visible in this area suggesting that degenerating processes were phagocytosed by microglial cells or astrocytes. We conclude that Fluoro-Jade is an early and sensitive marker for studying degenerating neurites in the hippocampal system.

Introduction

The afferent connections of the hippocampus and in particular its entorhinal input, the perforant pathway, have been widely used for studies of postlesional reorganization in the adult central nervous system [7], [33]. Following lesion of the entorhinal cortex or transection of the perforant pathway, degenerative and regenerative changes have been reported to occur in the former termination zone of these neurons [8], [10], [14], [22], [23], [24], [33], [34].

Ipsilateral entorhinal lesion leads to an almost complete denervation of the middle and outer molecular layers of the fascia dentata. Degenerating axon terminals have been observed to be present in the denervated region during the first 2–4 days, followed by morphological and functional changes in astrocytes and microglia [1], [17], [18], [19].

In addition, to the anterograde axonal changes in the deafferented outer molecular layers, it has been shown that the reorganization taking place in the hippocampal formation following entorhinal lesion includes transneuronal changes [5], [6], [28]. These changes occur mainly on dendrites of receiving dentate gyrus and hippocampal neurons [4], [15], but also are found within intrahippocampal connections outside the denervated termination zone of the perforant pathway [3], [4], [26]. Retraction of granule cell dendrites and parvalbumin-containing GABAergic inhibitory neurons dendrites from the perforant path termination zone and reduction of CA1 pyramidal cell spines have been reported to occur parallel to the process of axonal degeneration within the first 10 days after lesion [3], [28]. Both mechanisms play an important role in the remodeling of the hippocampus following entorhinal lesions and its subsequent functional consequences [4], [21], [27]. The aim of this study was to analyze in vivo and in vitro the degenerating effects of perforant path transection with the novel fluorescent dye Fluoro-Jade. This fluorescent marker has been shown to label degenerating neurons after a variety of neurotoxic treatments [2], [11], [12], [14], [20], [29], [32]. We also used organotypic hippocampal slice cultures (OHSC) from young postnatal rats (days 10–12) to examine the neurodegenerative effects of perforant path transection in this in vitro model. We chose this model because it maintains the organization and synaptic circuitry of the intact hippocampus and closely resembles in vivo conditions [34].