Cerebral Cortex

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Cerebral Cortex, Vol. 10, No. 10, 946-951, October 2000
© 2000 Oxford University Press

Role of Afferent Innervation and Neuronal Activity in Dendritic Development and Spine Maturation of Fascia Dentata Granule Cells

M. Frotscher, A. Drakew and B. Heimrich

Institute of Anatomy, University of Freiburg, PO Box 111, D-79001 Freiburg, Germany

	Abstract

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
By using slice cultures of hippocampus as a model, we have studied the development of dendritic spines in fascia dentata granule cells. We raised the question as to what extent spine development is dependent on a major afferent input to these neurons, the fibers from the entorhinal cortex and neuronal activity mediated by these axons. Our results can be summarized as follows: (i) the entorhino-hippocampal projection develops in an organotypic manner in co-cultures of entorhinal cortex and hippocampus. Like in vivo, entorhinal fibers, labeled by anterograde tracing with biocytin, terminate in the outer molecular layer of the fascia dentata. (ii) The layer-specific termination of entorhinal fibers is not altered by the blockade of neuronal activity with tetrodotoxin. Likewise, the differentiation of the dendritic arbor of postsynaptic granule cells does not require neuronal activity. Blockade of neuronal activity did not affect the mean spine number of granule cell dendrites in entorhino-hippocampal co-cultures, but led to a relative increase in thin, long filiform spines that are characteristic of immature neurons. (iii) The maturation of the granule cell dendritic arbor is, however, controlled by the afferent fibers from the entorhinal cortex in an activity-independent manner. In single slice cultures of hippocampus lacking entorhinal input, Golgi-impregnated granule cells have much shorter, less branched dendrites when compared with granule cells in entorhino-hippocampal co-cultures. This reduction in dendritic length in granule cells lacking entorhinal input results in a lower mean total number of spines per neuron, but the mean number of spines per µm is not reduced in the absence of entorhinal innervation. These results indicate that innervation by fibers from the entorhinal cortex, but not neuronal activity mediated via these axons, is essential for the normal development of the granule cell dendritic arbor. Neuronal activity is required, however, for the maturation of dendritic spines.

	Introduction

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
It has been assumed for a long time that neuronal plasticity is accompanied by changes in synaptic structures. Many studies have focused on dendritic spines, which are known to be major postsynaptic elements of many neurons in the central nervous system. It could, in fact, be shown that a de novo formation of spines and changes in the structure of spines and synapses take place in long-term potentiation (LTP), a well-established paradigm of neuronal plasticity which has often been related to learning and memory (Lee et al., 1980; Desmond and Levy, 1990; Schuster et al., 1990; Geinisman et al., 1991, 1993, 1996; Hosokawa et al., 1995; Buchs and Muller, 1996; Trommald et al., 1996; Andersen and Soleng, 1998; Engert and Bonhoeffer, 1999).

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.

	The Layer-specific Termination of the Entorhino-hippocampal Projection Develops In Vitro and Does Not Require Neuronal Activity

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
The preparation and maintenance of hippocampal slice cultures and of co-cultures of hippocampus with entorhinal cortex have been described in previous studies (Frotscher and Heimrich, 1993, 1995; Heimrich and Frotscher, 1994; Frotscher et al., 1995; Drakew et al., 1999), and the reader is referred to these articles for further details. For the experiments reported here, slice cultures were prepared from neonate (postnatal days 0–1) rat brains. In order to trace the termination of entorhinal axons in co-cultured slices of hippocampus, small crystals of biocytin were placed onto the superficial layers of the entorhinal culture. Following incubation for a further 2 days, the cultures were fixed, sectioned horizontally at 50 µm and further processed following a protocol of a heavy metal-intensified diaminobenzidine reaction.

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 cell–cell 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, Cajal–Retzius 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 Cajal–Retzius 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).

View larger version (160K): [in this window] [in a new window]   Figure 1.  Development of the entorhino-hippocampal projection in vitro. Biocytin-labeled entorhinal fibers (black) from an entorhinal co-culture terminate as in vivo in the outer molecular layer (OML) of the fascia dentata. The inner molecular layer (IML), known to contain commissural and associational fibers, is devoid of labeled axons. The arrow points to a small cell with a prominent horizontal dendrite, probably a Cajal-Retzius cell that was retrogradely labeled with biocytin following the injection of the tracer into the entorhinal culture. This entorhino-hippocampal co-culture was incubated in the presence of TTX, indicating that the lack of neuronal activity does not interfere with the pathfinding of entorhinal axons. For better visualization of the granule cell layer (GCL), cell bodies are Nissl-counterstained. Scale bar: 25 µm.

 
We conclude from these studies that the activity of neither the presynaptic nor of the postsynaptic neurons plays a major part in the formation of the layer-specific entorhino-hippocampal projection—at least not during development. As far as the path-finding of entorhinal axons is concerned, available evidence suggests a guidance role of hippocampal Cajal–Retzius cell axons which were found to give rise to an early pioneer projection to the entorhinal cortex (Frotscher, 1998; Ceranik et al., 1999).

	Entorhinal Fibers Shape the Granule Cell Dendritic Arbor

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
Next, we raised the question of the role of entorhinal fibers in the differentiation of their postsynaptic partners, the granule cell dendrites. For this purpose, either entorhino-hippocampal co-cultures or single cultures of hippocampus lacking entorhinal input were Golgi-impregnated to stain individual granule cells with their dendrites and spines. We applied a section Golgi technique (Frotscher, 1992) and a gold-toning procedure (Fairén et al., 1977) in these experiments. Total dendritic length, branching index and total number of spines were analyzed in camera lucida drawings of the impregnated granule cells (Drakew et al., 1999). In order to document potential differences between different dendritic segments, the Sholl method was applied (Sholl, 1955; Drakew et al., 1999).

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).

View larger version (101K): [in this window] [in a new window]   Figure 2.  (A) Photomontage of a Golgi-impregnated granule cell in a slice culture of hippocampus. Note the cell type-specific bipolar differentiation of this neuron, with the dendrites extending into the molecular layer and the axon (arrow) invading the hilar region. This culture was incubated in the presence of TTX. Scale bar: 20 µm. (B) Higher magnification of granule cell dendrites from a hippocampal slice culture incubated in the presence of TTX. Note the abundance of thin, filiform spines lacking a spine head (arrows). Scale bar: 10 µm.

 
Granule cells in single slice cultures of hippocampus incubated in vitro for 20 days had significantly shorter and less branched dendrites than their counterparts in entorhino-hippocampal co-cultures (Drakew et al., 1999). However, we noticed that granule cells in single cultures gave rise to significantly more short primary dendrites originating directly from the cell body. An exuberant number of short primary dendrites, including basal dendrites invading the hilus, is a feature of immature granule cells (Seress and Pokorny, 1981; Lübbers and Frotscher, 1988). It appears that the entorhinal input, and probably other inputs as well, are needed for the full differentiation of the granule cell dendritic arbor. A similar growth-promoting effect of entorhinal axons on granule cell dendrites was recently observed in dissociated cultures (Kossel et al., 1997). Interestingly enough, we noticed a retraction of parvalbumin-immunopositive dendrites of dentate basket cells following removal of the entorhinal input in adult animals (Nitsch and Frotscher, 1991, 1992). The entorhinal afferents in the outer molecular layer thus seem to be important for full maturation and maintenance of granule cell and basket cell distal dendritic portions extending into this layer.

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).

View larger version (18K): [in this window] [in a new window]   Figure 3.  Total dendritic length per granule cell (A), density of spines (B), length of spines (C) and percentage of filiform spines (D) in control cultures and cultures incubated in the presence of TTX. Data represented as mean + SEM. *Statistically significant (P < 0.05).

 

	Role of Neuronal Activity in the Differentiation of Granule Cell Dendritic Spines

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
We regard it as a major finding of the present series of experiments that blockade of neuronal activity with TTX did not alter the mean total number of spines on dentate granule cells. Spine density was not affected as both mean dendritic length and mean total number of spines per neuron were similar in the TTX-treated and control group (Figure 3B). We conclude that the impulse flow mediated via the perforant path, at least as far as it can be blocked by the application of TTX to the slice cultures, is not essential for the formation of spines on postsynaptic granule cells. However, for full synaptic development, the presence of these afferents seems to be required, as the total number of spines on granule cells is reduced in single slice cultures of hippocampus due to a reduction in total dendritic length (see above). In adult animals, there is a significant loss of dendritic spines on granule cell dendrites following removal of entorhinal fibers by a lesion of the entorhinal cortex (Parnavelas et al., 1974).

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).

	Conclusions

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
The formation of contacts between nerve cells essentially requires axonal pathfinding, target recognition and synapse formation. Our findings on the entorhino-dentate synaptic connection suggest that these very complex processes are governed by the sequential action of a variety of factors (Figure 4).

View larger version (14K): [in this window] [in a new window]   Figure 4.  Schematic diagram summarizing the factors governing the pathfinding of entorhinal axons, the dendritic differentiation of granule cells, and the maturation of spines on granule cell dendrites in organotypic co-cultures of entorhinal cortex and hippocampus from neonate rat pups. (A) Axons originating from the entorhinal cortex (EC) are guided to the outer molecular layer (OML) of the fascia dentata by pioneer axons of Cajal-Retzius (CR) cells. CR cells synthesize and secrete the glycoprotein reelin (stippled zone) that induces branching of entorhinal terminals (Del Rio et al., 1997). The entorhinal fibers establish transient synaptic contacts with CR cells until the distal dendrites of late-generated granule cells are available for synaptic contact. IML, inner molecular layer; GCL, granule cell layer. (B) Entorhinal axons are essential for the full dendritic differentiation of postsynaptic granule cells. In the absence of entorhinal input (in single slice cultures of hippocampus), granule cells display much shorter dendrites extending into all directions (left cell in black). In entorhino-hippocampal co-cultures, granule cells differentiate their characteristic cone-shaped dendritic arbor; basal dendrites extending into the hilus underneath the granule cell layer are rare or absent. Dendrites extend as far as to the outer molecular layer (right cell in black). This trophic influence of the entorhinal fibers does not require neuronal activity. (C) Neuronal activity is required, however, for the full maturation of dendritic spines. In the presence of TTX, a significantly larger number of long, thin filiform spines lacking a characteristic spine head are found (upper spine originating from the dendritic shaft, D).

 

1. Axonal pathfinding, target recognition and terminal collateralization are controlled by interaction of the growing axonal tip with membrane-bound or soluble molecules (Tessier-Lavigne and Goodman, 1996) in its environment. We previously provided evidence that entorhinal axons are guided to the hippocampus by pioneer axons of early generated Cajal–Retzius cells projecting to the entorhinal cortex (Ceranik et al., 1999). Dendrites and cell bodies of hippocampal Cajal–Retzius cells located in the termination zones of entorhino-hippocampal fibers were found to be essential for target recognition (Del Rio et al., 1997). Reelin, a glycoprotein synthesized by Cajal–Retzius cells (D'Arcangelo et al., 1995, 1997), controls terminal collateralization and synapse formation (Del Rio et al., 1997; Borell et al., 1999). As detailed in the present report, neuronal activity does not seem to be involved in these processes. Our results are in line with a recent report by Verhage et al., who found that a complete loss of transmitter secretion in Munc 18-1 deleted mice does not prevent normal formation of fiber pathways and morphologically defined synapses (Verhage et al., 2000).
   
2. Not neuronal activity, but as yet unknown trophic interactions between entorhinal axons and their target dendrites are required for the full development of the granule cell dendritic arbor. Glutamate, released from entorhinal terminals, seems to restrict dendritic growth (Mattson et al., 1988).
   
3. The actual number of dendritic spines formed on granule cell dendrites is determined not by neuronal activity, but by the presence of entorhinal axons which control dendritic growth (see factor 2 above). However, the differentiation of spines, i.e. the transformation of long, thin spines or filopodia into mature spines exhibiting a spine head, is influenced by neuronal activity. These latter findings, together with data from the literature, indicate that the impulse flow at a spine synapse may lead to a number of structural changes, including an increased turnover of spines (Engert and Bonhoeffer, 1999), changes in spine shape (Fischer et al., 1998), recruitment of additional transmitter receptors (Liao et al., 1999; Petralia et al., 1999; Shi et al., 1999), outgrowth of spinules Schuster et al., 1990) and the formation of ‘perforated’ synapses (Geinisman, 2000). Finally, it needs to be shown to what extent the present findings in slice culture are valid under the more complex in vivo conditions, where a variety of additional factors may modify dendritic development.
   

	Notes

Top Abstract Introduction The Layer-specific Termination... Entorhinal Fibers Shape the... Role of Neuronal Activity... Conclusions Notes References

 
The authors wish to thank M. Winter for her help with the figures. The present study was supported by grants from the Deutsche Forschungs-gemeinschaft (SFB 505, TP A3 and A8).

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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