Institute of Anatomy, University of Freiburg, PO Box 111, D-79001 Freiburg, Germany
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Abstract |
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Introduction |
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One way to analyze structural changes in synaptic structures is to monitor their formation during ontogenetic development. Synapses form when axonal terminals arrive at their target cells, and it has been of major interest to find out to what extent axonal terminals and their neuronal activity are involved in the differentiation of postsynaptic structures such as dendrites and spines. Early Golgi studies have in fact provided evidence for an inductive role of afferent axons in the formation of post-synaptic spines (Valverde, 1967, 1968
; Hámori, 1973
). However, it remains an open question whether or not neuronal activity is required. Studies in monkeys revealed that the lack of visual experience does not alter the rate of synaptogenesis in the visual cortex of these animals (Bourgeois et al., 1989
, 1999
; Bourgeois and Rakic, 1996
).
To this end, we have studied the development of the dendritic arbor and dendritic spines of granule cells in slice cultures of rat hippocampus. Previous studies had shown that hippocampal neurons develop in an organotypic manner under these culture conditions (Gähwiler, 1981, 1984
; Frotscher and Gähwiler, 1988
; Caeser and Aertsen, 1991
; Frotscher et al., 1990
, 1995
; Heimrich and Frotscher, 1991
; Zafirov et al., 1994
) and that entorhinal afferents, when supplied by a co-culture of entorhinal cortex, terminate as normal on the distal granule cell dendrites in the outer molecular layer of the dentate gyrus (Frotscher and Heimrich, 1993
, 1995
; Heimrich and Frotscher, 1993
; Li et al., 1993
). This allowed us to study the role of the entorhinal input, a major source of granule cell afferent innervation, in the development of granule cell dendrites and spines by comparing granule cells developed in single slice cultures of hippocampus and in entorhino-hippocampal co-cultures (Drakew et al., 1999
). The role of neuronal activity could be tested by applying the sodium channel blocker tetrodotoxin (TTX, 1 µM) to the culture medium.
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The Layer-specific Termination of the Entorhino-hippocampal Projection Develops In Vitro and Does Not Require Neuronal Activity |
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We were struck by the high precision with which the entorhinal fibers were found to terminate in their appropriate layers, the outer molecular layer of the fascia dentata and the stratum lacunosum-moleculare of the hippocampus proper (Frotscher and Heimrich, 1993; Li et al., 1993
) (Figure 1
). Biocytin-labeled entorhinal axons formed a sharp border towards the inner molecular layer known to contain the axons of hippocampal neurons, largely mossy cells. Electron microscopic studies revealed that the entorhinal terminals as normal established asymmetric synapses with dendritic spines and shafts. Blockade of neuronal activity by application of TTX did not alter the layer-specific termination of entorhinal fibers. These findings are in line with our recent studies which have provided evidence that specific cellcell interactions and interactions with the extracellular matrix are important for the layer-specific termination and the branching pattern of entorhinal afferents. Thus, by applying a double-labeling approach, we were able to show that early generated pioneer neurons, CajalRetzius cells in the outer molecular layer and in stratum lacunosum-moleculare, are transient targets of ingrowing entorhinal axons, keeping them in their correct termination zones before they establish their definitive synapses with the distal dendrites of granule cells (Del Rio et al., 1997
; Ceranik et al., 1999
). Reelin, an extracellular matrix glycoprotein synthesized and secreted by CajalRetzius cells (D'Arcangelo et al., 1997
), was found to have an effect on the collateralization pattern of entorhinal fibers (Del Rio et al., 1997
) and thus on the number of synaptic contacts formed (Borell et al., 1999
).
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Entorhinal Fibers Shape the Granule Cell Dendritic Arbor |
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Golgi-impregnated granule cells were easily identified and differentiated from other dentate neurons by their characteristic small cell body located in the granular layer and by their cone-shaped dendritic arbor extending into the molecular layer (Lübbers and Frotscher, 1987; Heimrich and Frotscher, 1991
; Zafirov et al., 1994
) (Figure 2
). All dendrites were densely covered with spines. The axon, the mossy fiber, originated from the basal pole of the cell body and invaded the hilar region (Figure 2
).
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What is the role of neuronal activity in dendritic differentiation? When we compared granule cell dendritic length in TTX-treated and untreated co-cultures of entorhinal cortex and hippocampus, we did not observe a difference (Figure 3A). Thus, some as yet unknown trophic factor, but not neuronal activity mediated by entorhinal axons, seems to be essential for postsynaptic dendritic differentiation. In our deafferentation experiments in adult animals we found that N-methyl-D-aspartate (NMDA) receptor blockade prevented the retraction of parvalbumin-positive basket cell dendrites following entorhinal lesion (Nitsch and Frotscher, 1992
). Although these two processes, dendritic differentiation in development and dendritic retraction following deafferentation, can hardly be compared, both of them indicate that glutamate release and subsequent glutamate receptor activation, resulting from synaptic activity or neuronal damage, are unlikely to promote dendritic elongation of dentate neurons. In line with this conclusion, Mattson et al. found that glutamate receptor blockade significantly increased dendritic growth of cultured hippocampal neurons (Mattson et al., 1988
).
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Role of Neuronal Activity in the Differentiation of Granule Cell Dendritic Spines |
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In the TTX-treated cultures, we observed a significant increase in long spines or dendritic filopodia (Figures 2B and 3C,D). Such filopodia are generally regarded as a feature of immature neurons. Fiala et al. have recently shown that the number of synapses on filopodia decreases during postnatal development (Fiala et al., 1998
). There was also a decrease in the percentage of shaft synapses with increasing age and an increase in the percentage of spine synapses. The authors concluded that filopodia recruit shaft synapses that later give rise to synapses on spines. Our data indicate that neuronal activity plays a role in this maturation process. McKinney et al. recently noticed a similar increase in filopodia of CA1 pyramidal cells following NMDA receptor blockade (McKinney et al., 1999
).
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Conclusions |
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Notes |
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Address correspondence to: M. Frotscher, Institute of Anatomy, University of Freiburg, Albertstraße 17, D-79104 Freiburg, Germany. Email: frotsch{at}uni-freiburg.de.
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References |
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