Institute of Anatomy, Humboldt University Berlin (Charité), 10098 Berlin and , 1 Department of Anatomy and Cellular Neurobiology, University Ulm, 89081 Ulm, Germany
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Abstract |
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Introduction |
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During the first month after lesion, there is a profound initial loss of synapses, followed by the sprouting of unlesioned afferent fibers and the formation of new synapses within the deafferented zone (Matthews et al., 1976a,b
; Steward and Vinsant, 1983
). In addition to the well-known sprouting of excitatory cholinergic axons originating in the septum (Lynch et al., 1972
; Nadler et al., 1977
), there is now morphological evidence for the sprouting of a commissural projection which terminates in the dentate outer molecular layer (Deller et al., 1995a
,b
). This projection has been characterized, at least in part, as GABAergic (Deller et al., 1995b
), suggesting that the lesioned excitatory axons are partially replaced by sprouting inhibitory fibers. This finding supports earlier data on increased glutamic acid decarboxylase immunoreactivity in the dentate gyrus following entorhinal cortex lesion (Goldowitz et al., 1982
). Physiological studies revealed strong synaptic inhibition of dentate granule cells following entorhinal cortex lesion. Stimulation of these cells in the molecular layer resulted in a minimized field potential (Clusmann et al., 1994
). Immunocytochemical studies have shown lesion-induced alterations of various presynaptic proteins related to synaptogenesis like synapsin, growth-associated protein 43 (Masliah et al., 1991
; Melloni et al., 1994
) and synaptosomal-associated protein 25 (Geddes et al., 1990
). Postsynaptic proteins such as postsynaptic density protein 95 (Sampedro et al., 1982
), the excitatory glutamate receptor protein NMDAR1 (Gazzaley et al., 1997
) or GABAA receptor ß-subunits (Mizukami et al., 1997
) are also affected by the lesion.
In order to further elucidate specific molecular changes that might be induced by or result in these morphological and functional alterations in the dentate gyrus following deafferentiation, we analyzed the distribution of the peripheral membrane protein gephyrin, which is also localized at postsynaptic sites. This polypeptide has been shown to link both glycine receptors (Kirsch et al., 1993; Meyer et al., 1995
) and GABAA receptors (Craig et al., 1996
; Sassoè-Pognetto and Wässle, 1997
; Essrich et al., 1998
; Giustetto et al., 1998
;) to the subsynaptic cytoskeleton (Kirsch et al., 1991
; Kirsch and Betz, 1995
). Moreover, pharmacological studies on differentiating spinal cord neurons revealed that gephyrin clusters form in response to glycine receptor activation (Kirsch and Betz, 1998
). Therefore we investigated whether alterations of the excitatory input to the dentate gyrus could also induce changes in the distribution of gephyrin at inhibitory synapses in the molecular layer. Our study produces evidence that the cell type and layer-specific distribution of different gephyrin clusters detected in the normal dentate gyrus is altered upon deafferentiation. These findings suggest a reorganization of specific GABAergic synapses in the dentate gyrus in response to entorhinal cortex lesion.
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Materials and Methods |
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Seventy adult male Wistar rats (250350 g body wt), housed under standard laboratory conditions, were used in this study. The experimental animals were divided into three groups: animals with a unilateral electrolytic lesion of the entorhinal cortex (n = 33), sham-operated animals (n = 31) and naive controls (n = 6). For stereotaxic surgery, the rats were deeply anesthetized with a mixture of 25% Ketavet, 6% Rompun and 2.5% Vetranquil in 0.9% sterile NaCl (2.5 ml/kg body wt i.p.). A standard electrocoagulator was used to make an unilateral cut in the frontal plane between the entorhinal cortex and hippocampus. We used the following coordinates measured from lambda: AP +1.2; L +3.1 to +6.1; V down to the base of the skull (Paxinos and Watson, 1986). Four single pulses (2.5 µA), 3 s each, were given. Sham operations were performed in the same manner, but the electrocoagulator was inserted only 1.5 mm into the cortex. The lesions were assessed by visual control, Nissl staining and staining for acetylcholinesterase according to the method of Mesulam et al. (Mesulam et al., 1987
).
Fixation Protocols
Cryo-fixation
One set of experimental animals was killed at days 2, 5, 8, 14, 28 (four lesioned and four sham-operated animals at each time point) and 21 after lesion (six lesioned andsix sham-operated animals) plus three naive control animals. The animals were deeply anesthetized with an overdose of the anesthetic mixture described above and then decapitated. The brains were removed, immediately frozen in liquid nitrogen and stored at 80°C.
Perfusion fixation
Another set of experimental animals was killed at days 14 (three lesioned and three sham-operated animals), 28 and 84 after lesion (two lesioned and one sham-operated animal at each time point) plus three naive control animals. These rats were deeply anesthetized with an overdose of the anesthetic mixture and were perfused through the ascending aorta with 30 ml 0.9% NaCl followed by 200 ml 2% paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4). The brains were removed and post-fixed for 1 h in the same fixative. After incubation in 30% sucrose in PB for cryoprotection, the tissue was frozen in liquid nitrogen and stored at 80°C.
The cytosolic calcium-binding proteins, which are excellent markers for the different cell populations in the hippocampus, cannot be detected by immunohistochemistry in cryo-fixed tissue (Nitsch and Klauer, 1989). In order to combine the immunocytochemical detection of gephyrin with different cell markers we had to perfuse the tissue. Immunolabeling of gephyrin using monoclonal antibody (mAb) 7a is rather sensitive to prolonged aldehyde fixation, which results in a complete loss of antigenicity after 24 h of fixation (Kirsch and Betz, 1993
). This phenomenon might also account for the differences in immunostaining of gephyrin in perfusion-fixed sections (Araki et al., 1988
; van den Pol and Gorcs, 1988
). Using extensive aldehyde fixation, Araki et al. found only a weak signal in the brain, whereas the lower brain stem and spinal cord showed a clear immunosignal using mAb 7a (Araki et al., 1988
). It is well known that gephyrin shows a high expression in the brain stem and spinal cord (van den Pol and Gorcs, 1988
; Colin et al., 1998
). Therefore it is reasonable to assume that sites of high gephyrin expression are less sensitive to aldehyde fixation. This might be due to the presence of a higher number of antigens within prominent gephyrin clusters, which are, as noted, abundant in the brain stem and spinal cord. The staining of large clusters in the dentate gyrus (see below) might also be less affected by aldehyde fixation. To avoid extensive loss of anitgenicity we used only 2% paraformaldehyde and brief post-fixation. This reduced the antigenicity of smaller, less intensely stained clusters, but did not effect the overall distribution pattern of gephyrin.
Antibodies
The mAb 7a (Pfeiffer et al., 1984) was used for gephyrin detection. Rabbit antisera against parvalbumin (PV 28) and calbindin D28k (CB 38) were purchased from SWANT (Bellinzona, Switzerland). Parvalbumin is a specific marker for a subpopulation of GABAergic interneurons and the calbindin antiserum stains granule cells in the dentate gyrus (Celio, 1990
). Secondary antibodies were purchased from Biotrend (Cologne, Germany) (Cy3 goat anti-mouse IgG) and Molecular Probes (Eugene, OR) (ALEXA 488 goat anti-mouse IgG, ALEXA 568 goat anti-rabbit IgG). Horseradish-peroxidase goat anti-mouse IgG was purchased from American Qualex (La Mirada, CA).
Membrane Preparation
Crude membranes from adult rat brainstem and hippocampus were prepared according to Nadler et al. (Nadler et al., 1994), with minor modifications. Briefly, the tissues were homogenized in 10 volumes of homogenization buffer containing 50 mM TrisHCl (pH 7.4) and a protease-inhibitor cocktail (1 tablet/25 ml TrisHCl; Boehringer Mannheim, Germany), and centrifuged at 1000 g for 10 min at 4°C. The resulting supernatants were centrifuged at 12 000 g for 20 min at 4°C, pellets were resuspended in 1 ml homogenization buffer and recentrifuged. The final pellets were resuspended in 0.4 ml homogenization buffer, homogenized once more and stored at 80°C.
Western Blotting
Membrane preparations were diluted in sample buffer [50 mM TrisHCl, pH 7.0, 83 mM dithiothreitol, 1% sodium dodecyl sulphate (SDS), 10% glycerol, 0.001% bromphenol blue], boiled for 5 min and subjected to SDSpolyacrylamide gel electrophoresis on 10% gels. Following electrophoresis, proteins were electrotransferred onto polyvinyldifluoride membranes (Millipore, Eschborn, Germany) and blocked with 1% Blocking Reagent (Boehringer Mannheim, Germany) in Tween buffer (PB, 0.1% Tween 20). Incubation with primary antibody mAb 7a, diluted 1:1000 in the blocking solution, lasted 16 h and membranes were subsequently washed for 2 x 5 min in Tween buffer, then for 10 min under high-stringency conditions (2 M urea, 0.1 M glycine, 1% Triton X-100, pH 7.5) and finally washed for a further 5 min in Tween buffer. Blots were incubated for 1 h with secondary antibody (horseradish-peroxidase goat anti-mouse IgG) diluted 1:20 000 in Tween buffer containing 2% normal rat serum. The addition of rat serum abolished cross-reactivity of the anti-mouse antibodies with other antigens both on Western blots and on brain tissue. After three final washes in Tween buffer, the bound antibody was visualized by enhanced chemiluminescence (Amersham, Braunschweig, Germany).
Immunohistochemistry
Cryo-fixed brains were processed as follows: 20 µm cryostat sections were thaw-mounted onto 3-aminopropyltriethoxysilane-coated slides and fixed for 5 min in 4% paraformaldehyde in PB. After three washes in PB, sections were pre-incubated in blocking solution (5% normal goat serum, 0.5% Triton X-100, PB) for 30 min. mAb 7a was diluted 1:100 in blocking solution and sections were incubated for 16 h at 4°C. Sections were rinsed three times with PB and incubated for 1 h at room temperature with Cy3 goat anti-mouse IgG diluted 1:500 in PB. After final washes, sections were mounted in Vectashild (Vector Labs, CA). The perfusion-fixed tissue was processed using the following protocol. The tissue was sectioned on a cryostat at 40 µm thickness and processed free-floating, beginning with three washes in PB, followed by preincubation with the above-mentioned blocking solution for 1 h. mAb 7a (1:100) was applied separately or together with either parvalbumin antiserum or calbindin antiserum (both diluted 1:1000) simultaneously. ALEXA 488 goat anti-mouse IgG and ALEXA 568 goat anti-rabbit IgG were diluted 1:200 in 2% normal rat serum in PB. Incubation time was prolonged to 3 h at room temperature. All other steps were performed as described above. Controls included omitting primary antibodies (for parvalbumin and calbindin antisera) or replacing it with unrelated isotype antibody (for mAb 7a).
Confocal Laser Scanning Microscopy and Quantitative Immunofluorescence Analysis
Confocal microscopy was performed using a Leica DM IRBE microscope (Leica Camera AG, Solms, Germany) equipped with an argon/krypton laser. Images were acquired using ScanWare 5.10. Cy3 and ALEXA 568 fluorochromes were visualized using an excitation wavelength of 568 nm. ALEXA 488 fluorochrome was excited using a wavelength of 488 nm. Double-labeled sections were scanned by the simultaneous application of both excitation wavelengths. Controls included dual-channel recordings of single-labeled sections as well as dual-channel recordings of a double-labeled single antigen to detect bleed-through phenomena and possible shifts between the detected signals.
Cryo-fixed sections from rat brains were used for the quantitative analysis of gephyrin immunolabeling. Images consisting of extended focus projections of three optical sections separated by 4.5 µm (magnification 100x) were made from the dentate gyrus of each section. These images were used to determine relative fluorescence intensities within the molecular layers. An image analysis program (Image Pro Plus 1.0, Media Cybernetics, MD) was used to analyze adjacent areas of the inner (IML) and middle (MML) molecular layer of each image. The relative pixel intensity of the IML was taken as a reference for the values obtained for the MML. Four separate regions were chosen within one section of the dentate gyrus of each animal. Four lesioned and four sham-operated animals were analyzed at each survival time (n = 4). All measurements were calculated as relative values and a mean relative intensity was determined for each animal. Across-group comparisons were made using the MannWhitney U-test at a significance level of P < 0.05. Brain sections from animals killed 21 days after lesion and from corresponding sham-operated control animals (n = 6) were used for the detailed quantitative analysis of gephyrin-positive clusters. Three sections of each brain were analyzed. High-magnification images (magnification 1000x) were acquired from the IML and the adjacent MML from inside three randomly chosen areas of the dentate gyrus of each section. All confocal parameters were kept constant. Image analysis was performed to determine the concentration of gephyrin-positive clusters. A visually established pixel intensity threshold was applied to remove the contribution of the unlabeled portion of the image. This allowed for the analysis of single objects corresponding to individual gephyrin-positive clusters. Relative values for the MML were calculated using the values from the IML as a reference. Across-group comparisons were analyzed using the Mann Whitney U-test at a significance level of P < 0.05.
Double-immunolabeled brain sections of perfusion-fixed animals at 14 days after lesion and corresponding sham-operated controls (n = 3) were used for the analysis of gephyrin-positive clusters and their co-localization with calbindin- and parvalbumin-positive structures. Five sets of two simultaneously scanned confocal images (magnification 400x) were acquired from each section at the same z-position with all confocal parameters kept constant. Three sections were prepared from each brain. A visually established pixel intensity threshold was set and the logical operation AND was used to combine the regions of the MML of corresponding binary images. This resulted in images displaying only gephyrin-positive structures co-localized with calbindin- or parvalbumin-positive structures. The value for co-localization was calculated as the total number of pixels representing co-localized gephyrin-positive clusters per total number of pixels representing calbindin- or parvalbumin-positive structures within the region of the MML. Across-group comparisons were analyzed using the MannWhitney U-test at a significance level of P < 0.05. Two series of 45 pairs of images depicting gephyrin-positive clusters and calbindin- or parvalbumin-positive dendrites (magnification 400x) were used to analyze the differential distribution of the various sizes of gephyrin clusters in relation to granule cells and parvalbumin-positive interneurons (n = 45). The threshold was optimized for each pair of images. Objects representing gephyrin-positive clusters were divided into four groups according to their size and the binary images, which selectively displayed one group of objects, were combined with the corresponding binary images displaying all dendritic structures. The resulting objects were regarded as co-localized when at least 80% of the area overlapped. The images depicting the dendritic structures were rotated by 90° and also combined with the images displaying gephyrin objects. The resulting number of objects was considered to represent the level of random co-localization. Statistical analysis was performed using the MannWhitney U-test at a significance level of P < 0.05.
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Results |
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Western blots of crude membrane fractions from rat brain stem and hippocampus were incubated with mAb 7a. The antibody stained a single band of ~92 kDa in both preparations (Fig. 1). This corresponds to the molecular weight described for gephyrin (Pfeiffer et al., 1984
), showing that mAb 7a selectively detects gephyrin in the hippocampus as well.
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Partial denervations of the molecular layer were achieved by knife cut transections of the medial part of the perforant pathway. These transections lead to specific denervations of the middle molecular layer of the dentate gyrus (Fig. 2). The extent of the lesion in all animals was determined by evaluating horizontal sections of the hippocampal formation stained for AchE (Fig. 2
) or Nissl. All lesioned animals showed a distinct altered staining pattern in the MML of the dentate gyrus. The patterns of AchE staining and Nissl staining in the dentate gyrus of sham-operated control animals were indistinguishable from those of unoperated control animals.
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Single labeling for gephyrin in sections prepared from cryo-fixed and perfusion-fixed brains showed a difference in staining intensity (Figs 3, 5) which was reported for the mAb 7a antibody in other systems (Kirsch and Betz, 1993
). Although it appeared that it was mostly the labeling in the granule cell and molecular layers that was affected by the fixation protocol (cf. Fig. 3A and 3B
), there was no difference in the general distribution of the immunofluorescence signal (Figs 3A,B, 5A,C,E,G,I,K,M
). High-magnification images demonstrated that the majority of clusters in the molecular layer revealed less staining intensity in perfusion-fixed tissue compared to cryo-fixed tissue (cf. Fig. 3C,D
). However, larger clusters which were distributed along distinct dendrites still showed a very intense immunofluorescence signal.
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Distribution of Gephyrin in Normal Adult Rat Dentate Gyrus: Cell-specific Localization of Gephyrin
In order to specify the neuron population that gave rise to gephyrin IR in the dentate gyrus double-immunofluorescence labeling was performed using mAb 7a in combination with antibodies against parvalbumin, to identify parvalbumin-containing, GABAergic neurons or against calbindin, to identify dentate granule cells. These experiments revealed that the dendrites of parvalbumin-positive neurons co-localized with large, intensely stained, gephyrin-positive clusters in the molecular layer (Fig. 4AC) and in the polymorph layer (Fig. 4DF
). The somata of parvalbumin-positive neurons were frequently associated with smaller gephyrin-positive clusters (Fig. 4DF
). The localization of gephyrin on granule cells was analyzed on sections stained for gephyrin (Fig. 4G
) and calbindin (Fig. 4H
). Calbindin-positive dendrites were mostly associated with small, gephyrin-positive clusters of minor staining intensity than the prominent gephyrin-clusters along the dendrites of parvalbumin-positive neurons. The somata of calbindin-positive granule cells displayed also a small dot-like gephyrin IR (Fig. 4I
). The quantitative analysis supported this finding. The majority of large gephyrin-positive clusters is specifically localized on parvalbumin-positive dendrites. Calbindin-positive dendrites co-localize predominantly with smaller clusters (Fig. 6D
).
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Horizontal sections of the temporal hippocampal formation obtained from lesioned, sham-operated and naive control animals that were immunostained for gephyrin were analyzed to investigate whether the layer- and cell-specific distribution of gephyrin-positive postsynaptic membrane specializations in the rat dentate gyrus is altered in the deafferented dentate gyrus. Two days after unilateral perforant path transection, no obvious change in immunolabeling was observed in the molecular layer of the deafferented dentate gyrus (Fig. 5B) when compared to sham-operated or naive controls, which all displayed the typical trilaminar pattern of gephyrin immunolabeling described above (Fig. 5A,C,E,G,I,K,M
). An increase in the intensity of immunolabeling could be observed from 5 days after lesion onwards in the deafferented MML (Fig. 5D,F
). At 14 days after lesion, the transition from the IML to the MML was clearly visible as a sudden change in the level of immunofluorescence intensity (Fig. 5H
). At 21 and 28 days after lesion (Fig. 5J,L,N
) the transition between the MML and OML also became evident. In cryo-fixed sections we frequently detected a band of rather intense immunofluorescence labeling between these two layers (Fig. 5J,L
). A similar pattern of immunostaining could be observed on sections prepared from perfusion-fixed brains of animals killed 28 and 84 days after lesion, the longest survival time investigated in our study (Fig. 5N,O
). The sections of cryo-fixed brains were used to determine the relative intensity levels. The mean intensity in the MML (in relation to the IML) was compared between the lesioned and control groups at different time points. Statistical analysis (MannWhitney U-test) demonstrated that the relative immunofluorescence intensity in the MML was altered in each of the lesioned animal groups including the group that survived for 2 days, in comparison to the MML of the corresponding sham-operated group or the naive control group. This was so even in the case of random comparison. At 2 days after lesion a slight but significant decrease in gephyrin immunolabeling intensity was evident, whereas a significant increase in immunofluorescence intensity could be demonstrated from 8 days after lesion onwards (Fig. 6A
). High-magnification images from brain sections of animals that survived until 21 days after lesion and corresponding sham-operated controls were used for a more detailed analysis. The individual gephyrin-positive clusters were divided into four different groups according to their size. The analysis showed that only the very small clusters showed a significant increase in number in the deafferented MML compared to the MML of the sham-operated controls (Fig. 6B
). To quantify the changes of gephyrin in relation to the different cell groups we analyzed brain sections of perfusion-fixed animals that were simultaneously stained for gephyrin and calbindin or parvalbumin. This revealed that the relation between gephyrin-positive structures and parvalbumin-positive structures was not altered in the MML following deafferentiation. In contrast, calbindin-positive dendrites of granule cells displayed a significant increase of co-localized gephyrin-positive clusters (Fig. 6C
). This increase most likely relates to the specific increase of the very small gephyrin-positive clusters as found in the analysis of cryo-fixed sections (Fig. 6B
).
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Discussion |
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Three major findings were made: (i) anti-gephyrin IR is distributed in a layer-specific manner in the molecular layer of the dentate gyrus; (ii) dendrites of granule cells and parvalbumin-positive interneurons are associated with different sized types of gephyrin-positive clusters; (iii) deafferentation of the dentate gyrus by perforant path transection leads to a specific increase in density of granule cell dendrite-associated gephyrin-positive clusters within the deafferented zone of the molecular layer.
Layer-specific Distribution of Gephyrin in the Dentate Gyrus
Based on qualitative and quantitative immunofluorescence methods we found that gephyrin IR was distributed in a layer-specific manner in the dentate gyrus. Gephyrin immunolabeling intensity was highest in the OML. In the MML the intensity was significantly lower and the layer closest to the granule cell somata, the IML, showed the lowest level of gephyrin IR. Since gephyrin is known to cluster inhibitory receptors such as glycine and GABAA receptors (Craig et al., 1996; Sassoè-Pognetto and Wässle, 1997
; Giustetto et al., 1998
; Essrich et al., 1998
), the differential distribution of gephyrin might indicate that the inhibitory input onto granule cell dendrites mediated by the associated receptor increases with the distance to the cell somata. This is consistent with the varying density of GABAergic innervation onto granule cells in the dentate gyrus (Halasy and Somogyi, 1993
). Several GABAergic afferent systems terminate within specific layers of the dentate gyrus. The proximal dendritic segments of granule cells are innervated by inhibitory axo-axonic and basket cells. Various interneurons which participate in the formation of the commissural/associational system have been characterized based on their location in the dentate gyrus and also based on the part of the molecular layer they innervate. Thus, they either innervate the IML or the MML and OML exclusively (Freund and Buzsáki, 1996
).
The presence of three sublayers within the molecular layer, distinguishable by the various levels of gephyrin IR, correspond to the different termination zones of the major afferent systems: the commissural/associational projection and the entorhino-dentate projection. Whereas the IML receives its major input via the former projection, the MML and OML are predominantly innervated by axons originating from cells in the entorhinal cortex. This excitatory input can be further divided into medial and lateral parts terminating in the MML and OML respectively (Ruth et al., 1982, 1988
). A GABAergic commissural projection is also part of this layer-specific innervation of the MML and OML (Deller et al., 1995a
). One might speculate that the layer-specific input to the dentate molecular layer is reflected by the differential distribution of gephyrin.
Cell-specific Distribution of Gephyrin in the Dentate Gyrus
The qualitative and quantitative data demonstrated that gephyrin clusters vary in size and that the majority of the larger clusters specifically co-localizes with parvalbumin-positive interneurons. As we also found large clusters distributed along unidentified dendrites it is likely that these clusters are associated with other interneurons as well. Granule cell somata and dendrites were mainly associated with smaller, gephyrin-positive clusters. The difference in size of gephyrin-positive clusters might indicate differences in the structural organization of the synaptic contact. Parvalbumin-positive neurons would thus receive a much stronger and/or more consistent inhibitory input along their dendritic extensions, mediated by the gephyrin-associated receptors, than granule cells. Granule cells, on the other hand, seem to receive a layer-specific input which increases towards their distal dendritic segment and is most prominent in the outer part of the molecular layer. A correlation between synaptic strength and the size of the postsynaptic receptor region has just recently been demonstrated (Lim et al., 1999). The existence of different-sized gephyrin clusters has also been demonstrated (Craig et al., 1997); these authors described two different sizes of gephyrin clusters in cultured hippocampal neurons. This variability of gephyrin clusters in size and distribution can be also found in other neuronal cell populations. Alvarez et al. have shown that gephyrin clusters do appear in distinct sizes and shapes and even show a differential distribution within single cells of the spinal cord (Alvarez et al., 1997
).
Gephyrin has been described as a molecule that anchors inhibitory receptors at synaptic sites. Immunocytochemical studies have shown a close correlation of gephyrin with different GABAA receptor subunits (Sassoè-Pognetto and Wässle, 1997; Giustetto et al., 1998
). Essrich et al. have provided evidence that the majority of GABAA receptors requires gephyrin as an anchoring molecule in combination with the
2 subunit (Essrich et al., 1998
). GABAA receptor subunits show a distinct regional distribution and also a cell-specific expression in the rat brain (Gao and Fritschy, 1994
; Fritschy and Möhler, 1995
; Sperk et al., 1997
). As the subunit composition defines the class of GABAA receptor, subpopulations of hippocampal neurons differ in the class of receptor they predominantly express. Therefore, one is tempted to speculate that although gephyrin anchors a variety of different GABAA receptors, the size and/or structural composition of gephyrin clusters depends on the type of receptor anchored at the synapse.
Increased Concentration of Small Gephyrin Clusters after Perforant Path Transection
Deafferentation of the MML of the dentate gyrus led to an increase of small gephyrin-positive clusters in the deafferented dendritic field of granule cells. Following a slight initial reduction of gephyrin IR, a continuous increase in gephyrin-positive clusters was observed from 5 days after lesion onwards. After 2 weeks, a rather constant, elevated level of gephyrin immunolabeling was reached which varied only slightly until 4 weeks after lesion, a time point when the process of synaptic reorganization is almost complete (Matthews et al., 1976a,b
). The process of degeneration, reactive sprouting and synaptogenesis is accompanied by a gradual shrinkage of the molecular layer until 14 days after lesion when this effect has reached its maximum level (Steward and Vinsant, 1983
). Any change in the density of a component within this layer might therefore be only due to tighter packing. This should affect all components in the shrinking layer equally and proportional to their normal pattern of density. The observation that only the group of the very small gephyrin-positive clusters displayed significant changes following lesion contradicts the possibility that we are only dealing with a shrinkage effect. Another important point relates to our finding that there is an increase of gephyrin-positive structures restricted to calbindin-positive dendrites. Within the same animals the number of gephyrin-positive structures on parvalbumin-positive dendrites is not changed. The reduced staining intensity in perfusion-fixed material resulted from the reduced immunofluorescence intensity of the small clusters. The differences in staining due to the different fixation protocols still allowed for the detection of a postlesional increase of small clusters on calbindin-positive dendrites. Thus we have reason to assume that our results do not conflict with tissue shrinkage or differences in the fixation protocols.
The altered pattern of gephyrin immunolabeling which is still detectable 3 months following lesion demonstrates that the increase of gephyrin-positive clusters in the deafferented zone reflects a permanent effect. It is known that a very small part of the entorhino-dentate projection that is destroyed upon lesion of the entorhinal cortex is comprised of GABAergic afferents (Germroth et al., 1989, 1991
). Deller et al. provided evidence for the sprouting of a GABAergic projection in the MML and OML of the dentate gyrus in response to lesion of the perforant path (Deller et al., 1995a
,b
). Therefore the initial reduction of gephyrin IR in the deafferented zone of the dentate gyrus might result from the loss of the inhibitory GABAergic input from the entorhino-dentate projection. In consequence, the increase in number of gephyrin-positive clusters might then be triggered by the sprouting of the GABAergic commissural projection. The observation of a cell-type specific localization of different sized gephyrin clusters and GABAA receptor subtypes would imply that only a certain type of receptor is formed in response to the lesion, which then resulted in a differential effect of the newly formed inhibitory connections onto the subpopulations of hippocampal neurons. This phenomenon might account for the diminished field potential detected upon stimulation of fibers terminating on the distal part of granule cell dendrites (Clusmann et al., 1994
). The increase of inhibitory synapses in the deafferented zone, triggered by the sprouting of a GABAergic projection, might be the morphological correlate to this physiological function. The specific increase of the small gephyrin-positive clusters which are predominantly associated with calbindin-positive granule cell dendrites is in line with this idea and would explain the physiological imbalance between excitation and inhibition. This would then be due to changes of the inhibitory input on granule cells following reorganization of the dentate molecular layer. In fact, sprouting inhibitory fibers have been shown to establish GABAergic synaptic contacts with the spines of granule cell dendrites (Deller et al., 1995b
). The formation of large, gephyrin-positive clusters localized on parvalbumin-positive dendrites is not triggered by the increase of inhibitory terminals. As there is no overall significant change of gephyrin-positive structures along these dendrites following lesion, this cell type does not receive new afferent input from sprouting inhibitory fibers at all. Thus the formation of new synaptic contacts between sprouting inhibitory projections and the deafferented dendrites is not just a filling of the holes but a highly layer- and cell-specific process which might be responsible for the physiological alterations within the denervated dentate gyrus. Considering this, the sprouting response of afferent fibers cannot be addressed simply as a trial for functional recovery.
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Notes |
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Address correspondence to Robert Nitsch, Institute of Anatomy, Department of Cell- and Neurobiology, Humboldt University Hospital Charité, 10098 Berlin, Germany. Email: robert.nitsch{at}charite.de.
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