Differentiation of granule cells in relation to GABAergic neurons in the rat fascia dentata

Summary Golgi impregnation was used to study the dendritic differentiation of granule cells in the rat fascia dentata. The impregnated granule cells were gold-toned allowing for a fine structural study of the same identified neurons and of the input synapses onto their cell bodies and dendrites. Due to the long postnatal formation of these cells it was possible to describe a sequence of maturational stages coexisting on the same postnatal day (P5). Characteristic features of the dendritic development of granule cells were i) occurrence of varicose swellings along the dendrites, ii) growth cones on dendritic tips, iii) transient formation of basal dendrites, and iv) progressive development of dendritic spines. Incoming synapses on the differentiating granule cells were mainly found on dendritic shafts. Their membrane specializations were symmetric. At least some of these symmetric synapses were GABAergic because immunostaining of Vibratome sections from the same postnatal stage (P5) demonstrated a well-developed GABAergic axon plexus in the fascia dentata (antibodies against glutamate decarboxylase (GAD), the GABA synthesizing enzyme). Electron microscopy of the immunostained axon plexus revealed numerous GABAergic terminals that formed symmetric synaptic contacts, mainly on shafts of differentiating dendrites but also on cell bodies of granule cells. Our results thus indicate that the plexus of inhibitory GABAergic axons is already well developed at a stage when the target neurons, the granule cells, are still being formed.     

Synaptic extensions from the mossy fibers of the fascia dentata

Summary In the hilar region of the rat hippocampus, filamentous extensions have been observed to originate from the en passant synaptic expansions on the so-called mossy fibers, which are the axons of the dentate granule cells. These extensions range in length from about 1 m to 30 m, are often branched, and appear to contact the processes of various cell types in the hilar region. In 28-day-old rats, there are between 4 and 9 such extensions from most mossy fiber expansions, and the total length of the extensions from any one expansion is on the order of 75 m. Analysis of serial electron micrographs through normal and Golgi-impregnated mossy fibers has confirmed that these extensions are, indeed, presynaptic processes. Each contains one or more vesicle-rich foci along its length, and is associated with asymmetric membrane specializations. At these sites, the extensions are in synaptic contact with dendrites and dendritic spines of, as yet, unknown origin. A quantitative analysis of these extensions in Golgi material from rats at different ages indicates that they reach their greatest length around 14 days and then decline to adult values by 28 days.This work was supported in part by grant NS-10943 and was carried out while the author was in receipt of a U.S. Public Health Service post-doctoral fellowship (F32 NSO 5765-02)     

Desensitization-like changes in GABA receptor binding of rat fascia dentata after entorhinal lesion

Destruction of the hippocampal perforant path fibers reduced the binding of [3H]GABA to membranes prepared from the rat fascia dentata. This result could be detected 1--4 months after surgery, but not in 10 days or less. Such a delayed response appears most compatible with a transsynaptic effect on GABA receptors. Values of low affinity KD and Bmax decreased by about 65%, but no changes were detected in high affinity binding, Hill slope or pharmacological specificity. These findings are consistent with a desensitization of postsynaptic low affinity GABA receptors, possible caused by excessive release of GABA.     

Differential regulation of chondroitin sulfate proteoglycan mRNAs in the denervated rat fascia dentata after unilateral entorhinal cortex lesion

Following brain trauma, chondroitin sulphate proteoglycans (CSPGs) are enriched at injury sites and in denervated areas. At injury sites, CSPGs are regarded as inhibitors of axonal regeneration because of their growth inhibitory properties. In areas of denervation their role is less clear, since they are enriched in zones of sprouting, i.e. zones of axonal growth. To identify CSPGs expressed in a denervated brain area and to quantify changes in their mRNA expression, neurocan, brevican, NG2, phosphacan and aggrecan mRNA were analyzed in the rat fascia dentata following entorhinal denervation. Laser microdissection was combined with quantitative RT-PCR to measure mRNA changes specifically within the denervated portion of the molecular layer (1 h, 6 h, 10 h, 12 h, 1 d, 2 d, 3 d, 4 d, 7 d and 14 d post-lesion). Changes in glial fibrillary protein mRNA were measured at the same time points and used as lesion control. This approach revealed a differential regulation of CSPG mRNAs in the denervated zone: neurocan, brevican and NG2 mRNA were upregulated with a maximum around 2 days post-lesion. In contrast, aggrecan mRNA levels reached a maximum 7 days post-lesion and phosphacan mRNA levels were not significantly altered. Taken together, our data reveal a temporal pattern in CSPG mRNA expression in the denervated fascia dentata. This suggests specific biological functions for CSPGs during the denervation-induced reorganization process: whereas the early increase in CSPGs in the denervated zone could influence the pattern of sprouting, the late increase of aggrecan mRNA suggests a different role during the late phase of reorganization.     

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetyl cholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.     

Localization of CRMP5 mRNA by in situ hybridisation during development of the mouse forebrain

The expression of the collapse response mediator protein CRMP5 in the prenatal mouse is largely unknown. Evidence suggests that CRMP family members play important roles in neurite outgrowth, and CRMP5 is known to modulate outgrowth of processes in oligodendrocytes through signalling via neuropilin-1 and SemaA. Furthermore, CRMP family members function in axon regeneration after injury and are implicated in the early stages of Alzheimer's disease. Despite these findings relatively little is known about the specific roles these proteins play. The aim of the present study was to evaluate CRMP5 expression in the developing mouse forebrain using in situ hybridisation. Serial coronal sections of brain from E12.5 to E18.5 were analysed. We found highly specific patterns of expression which were restricted to the post-mitotic layers of both the ganglionic eminence and neocortex, and an additional domain of strong expression in the pyramidal layers of the hippocampus in all prenatal ages. Our results are therefore consistent with a role for CRMP5 in process extension. Interestingly, our results also revealed a temporal switch in high-expression levels from the ganglionic eminence to the cortex at a critical time during tangential cell migration. However, the pattern of expression appeared more representative of a general permissiveness for neurite outgrowth rather than one which is restricted to a particular cell subset or cell class. Additionally, expression was also found during periods predominated by neurogenesis and not neurite extension. We conclude that expression of CRMP5 is consistent with a dynamic implicit role in forebrain development.     

Modification of the distribution of receptive field orientation in cats by selective visual exposure during development

Summary A novel procedure was used to rear kittens under conditions of controlled visual experience: one eye was exposed only to vertical lines while the other eye, simultaneously, was exposed only to horizontal lines. Stimuli were presented in a mask which the animals wore whenever they were in a lighted environment. At the conclusion of the rearing period elongated receptive fields of units recorded from the visual cortex of these animals were oriented either horizontally or vertically. This is in contrast to the full complement of receptive field orientations found in normal cats. Furthermore, units with vertically oriented fields were predominantly or exclusively activated by the eye which had been exposed to vertical lines, while neurons with horizontally oriented fields were predominantly or exclusively activated by the eye which had been exposed to horizontal lines. Normally, 80–90% of the neurons in the visual cortex of the cat are activated by both eyes. The consequences of this controlled visual experience provide evidence for highly selective modification of neuronal connectivity by environmental conditions prevailing during development. Sufficient control of the visual environment was achieved to allow direct comparison between single unit response characteristics and the specific stimuli presented during rearing.Note added in press: This aspect of the work is being pursued further. (Spinelli, D.N., Hirsch, H.V.B.: Genesis of receptive field shapes in single units of cat's visual cortex. Federation Proceedings 30, 615 Abs (1971)).