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Heterogeneity of the commissural projection to the rat dentate gyrus: a Phaseolus vulgaris leucoagglutinin tracing study
Pergamon
PII:
Neuroscience Vol. 75, No. 1, pp. 111–121, 1996 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/96 $15.00+0.00 S0306-4522(96)00255-2
HETEROGENEITY OF THE COMMISSURAL PROJECTION TO THE RAT DENTATE GYRUS: A PHASEOLUS VULGARIS LEUCOAGGLUTININ TRACING STUDY T. DELLER,*† R. NITSCH‡ and M. FROTSCHER* *Institute of Anatomy, University of Freiburg, Freiburg, P.O. Box 111, D-79001, Freiburg, Germany ‡Institute of Anatomy, Humboldt University Berlin (Charite´), D-10098, Berlin, Germany Abstract––The commissural and associational projections to the rat dentate gyrus are believed to be anatomically homologous fiber systems. They are often referred to as the so-called commissural/ associational system of the dentate gyrus. However, whereas characteristic laminar termination patterns within the molecular layer of the dentate gyrus have been described for the different cells of origin of the associational projection, the axons of the different cell types of commissural neurons have long been believed to terminate exclusively within the inner molecular layer. Only recently, a previously unknown commissural projection to the outer molecular layer of the dentate gyrus was described and the question was raised whether the commissural fibers could exhibit a heterogeneity similar to that of the associational projections. Using the anterograde tracer Phaseolus vulgaris leucoagglutinin, which labels individual axons and their collaterals, we have studied the termination pattern of commissural axons in the dentate gyrus of the septal hippocampus. At least four different commissural fiber types could be revealed on the basis of their laminar termination pattern: fibers to the inner molecular layer (type 1), fibers to the outer molecular layer (type 2), fibers terminating throughout the molecular layer (type 3), and fibers terminating in both the granule cell layer and the molecular layer (type 4). These observations demonstrate a previously underestimated heterogeneity of the commissural projection. In addition, there is a great deal of parallelism between the different commissural and associational fibers, pointing to a coordinated action of the two systems in the two hippocampi. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: hippocampal commissure, anterograde tracing, limbic connections, fascia dentata, entorhinal cortex lesion.
The commissural and associational projections to the rat dentate gyrus are among the best studied fiber systems of the hippocampus7,9,12,13,21,26,30,31,32,48,49,50,52 (see Ref. 4 for review). The two projections have been considered homologous, since it appears to be a general principle of hippocampal organization6 that neurons participating in the associational projection also contribute to the commissural projection. The cells of origin of the two projections are located in the polymorph layer of the hilus,6,24,31,51 and several groups of neurons have been identified that contribute to both: glutamate-immunoreactive mossy cells,2,17,38,40,46 somatostatin-54 and neuropeptide Y (NPY)-containing9 neurons that appear to be subpopulations of GABAergic neurons,28 and several GABAergic neurons that were not known to contain neuropeptides.32,39,41,42 Characteristic laminar termination patterns throughout the molecular layer of the dentate gyrus have been described for the different cells of origin †To whom correspondence should be addressed. Abbreviations: NPY, neuropeptide Y; PB, phosphate buffer; PHAL, Phaseolus vulgaris leucoagglutinin.
of the associational projection,18,22,23,45 whereas the inner molecular layer has long been considered the only termination zone of the different commissural cell types.7,15,21,25,2930 We recently demonstrated that commissural fibers are not restricted to the inner molecular layer13 and that the hilar/ perforant pathway (HIPP) cell23 appears to have a commissural collateral that terminates in the same hippocampal layer in the contralateral hippocampus as the associational collateral on the ipsilateral side.13 This raised the question whether other hilar neurons with associational and commissural axon collaterals also have corresponding ipsilateral and commissural termination zones. In the present study, we used the Phaseolus vulgaris leucoagglutinin (PHAL) anterograde tracing technique, which demonstrates the entire course of anterogradely labeled axons on the single fiber level19 and makes it possible to describe the exact trajectory and termination pattern of commissural axons. With this method we have analysed the commissural projection to the septal part of the dentate gyrus and have compared the termination pattern of commissural fibers there with that of their associational counterparts.
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CA1, CA3 hippocampal subfields CA1, CA3 DG dentate gyrus Fi fimbria GCL granule cell layer H hilus
HICAP HIPP IML OML
hilar/commissural-associational-pathway hilar/perforant-pathway inner molecular layer outer molecular layer
Fig. 1. PHAL injection sites in the dentate gyrus and commissural projections to the dentate molecular layer. (a) Typical PHAL injection site located in the crest of the dentate gyrus. Granule cells and hilar neurons have taken up the tracer. The associational projection to the inner molecular layer (arrow) and the associational projection to the outer molecular layer (arrowheads) are labeled. (b) Schematic drawing of the various PHAL injection sites in and close to the dentate gyrus. (c) Dentate gyrus contralateral to the injection site illustrated in a. The commissural projection to the inner molecular layer (arrows) and the commissural projection to the outer molecular layer (arrowheads) are labeled. Scale bars = 400 µm (a, c); 800 µm (b).
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Fig. 2. Camera lucida drawings of commissural fibers to the inner molecular layer (type 1) and outer molecular layer (type 2) of the dentate gyrus. (a) Typical commissural fiber to the inner molecular layer (IML): commissural fiber type 1. The axon reaches the inner molecular layer via the granule cell layer and all axon collaterals are restricted to the inner molecular layer. (b–d) Commissural fibers to the outer molecular layer (OML): commissural fiber type 2. These axons pass through the inner molecular layer without branching and terminate exclusively within the outer molecular layer. Three fiber types may be differentiated on the basis of the morphology of their axonal arbor: axons with collaterals arranged in a bundle (b; fiber type 2a), axons with collaterals that branch off collaterals in the middle one-third of the molecular layer or send recurrent axons to this zone (c; fiber type 2b), and axons with very long collaterals that run in parallel with the granule cell layer (d; fiber type 2c). Scale bars = 50 µm (a–d).
EXPERIMENTAL PROCEDURES
Fourteen male and female Sprague–Dawley rats (250– 350 g; Harlan Winkelmann, Borchen, Germany) housed under standard laboratory conditions were used in this
study. Surgical procedures were performed under deep anesthesia, in agreement with the German legislation on the use of laboratory animals (Nembutal, 50 mg/kg body weight). PHAL (2.5% in 10 mM phosphate buffer (PB), pH 7.8; Vector Laboratories, Burlingame, CA, U.S.A.) was
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iontophoretically delivered19 into the hilar area of the dentate gyrus using a glass micropipette (tip diameter 15–30 µm; 5 µA positive current applied every other 5 s for 20–30 min). All animals received PHAL injections into the septal part of the dentate gyrus (coordinates from bregma: AP "3.8, L 1.6, V "3.8).37 The animals were allowed to survive for 10 days following the injection of the anterograde tracer. After this time period the rats were deeply anesthetized with Nembutal and were transcardially perfused with a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 15% picric acid in 0.1 M PB (pH 7.4). Brains were removed and postfixed for 2 h in glutaraldehyde-free fixative. One-hundred-micrometer-thick sections were cut with a Vibratome and washed in PB. Immunocytochemistry was used to visualize the PHALcontaining axons. Free-floating sections were incubated at 4)C in biotinylated goat anti-PHAL (1:400), 1% normal horse serum, 0.1% NaN3, 0.5% Triton X-100 in 0.1 M PB for two days. After rinsing the sections in PB, they were incubated in the avidin–biotin–peroxidase complex (ABC Elite, Vector Labs) for 3 h. Following subsequent washes, the sections were immersed for 5–10 min in a nickel/ diaminobenzidine solution (0.05% 3,3*-diaminobenzidine, 0.02% nickel ammonium chloride, 0.024% cobalt chloride, 0.001% H2O2), which resulted in a dark blue reaction product. Sections were then placed on gelatin-coated slides, dehydrated in ethanol, mounted in Eukitt and investigated under the light microscope. In order to analyse individual commissural fibers in the dentate molecular layer, the axonal arbor as visualized in single sections was drawn with the aid of a camera lucida (final magnification #400). A total of 65 axons and axon fragments of commissural fibers (types 2–4) were drawn from the experimental material and selected axons were chosen for the illustration of the different commissural fiber types (Figs 2–4).
frontal sections of the hippocampus. Injection sites varied between 500 and 800 µm in their longitudinal extent. The ipsilateral associational projections to the inner one-third of the molecular layer and to the outer molecular layer were labeled3,4,12 on the side of the injection (Fig. 1a).
RESULTS
The commissural fibers terminating in the inner molecular layer have been described in detail earlier.7,9,12,13,2126,30,31,32,48,49,50,52 The overwhelming majority of commissural fibers belongs to this axon type. PHAL tracing revealed that these axons frequently branch in the granule cell layer, thereby forming several collaterals that often run in parallel until they arborize throughout the inner molecular layer (Fig. 2a).13
Injection sites
PHAL injections were placed into the hilus of the dentate gyrus in the septal part of the hippocampus (see Experimental Procedures). The injection site usually covered major portions of the hilar area and occasionally included CA3 pyramidal neurons and frequently some granule cells (Fig. 1a, b). Injection sites varied in diameter between 100 and 300 µm. An increased immunocytochemical background staining could be observed for an additional 100–400 µm beyond the central injection site. In this peripheral zone of the injection site, no PHAL-labeled cells could be observed. The septotemporal extent of the injection sites was reconstructed using consecutive
Laminar termination of commissural fibers In the dentate gyrus contralateral to the injection, commissural fibers could be found in all layers (Fig. 1c). The ‘‘classical’’ commissural fiber band in the inner molecular layer contained the largest number of PHAL-labeled fibers and appeared to be similar in density to the associational fiber plexus on the injection side (Fig. 1a, c). In contrast, only few commissural fibers were present in the outer molecular layer, considerably less than on the injection side (Fig. 1a, c). The axonal arborization of selected PHAL-labeled commissural axons was drawn with a camera lucida and analysed for its laminar distribution throughout the granule cell layer and molecular layer. The fiber courses of PHAL-labeled commissural fibers through the fimbria–fornix1,10 and through the hilar area13 have been described earlier and will not be described in detail in the present paper. Fibers terminating in the inner molecular layer (fiber type 1)
Fibers terminating in the outer molecular layer (fiber type 2) We recently described another group of commissural fibers that pass through the granule cell layer and inner molecular layer without branching. This
Fig. 3. Commissural fibers to the molecular layer (type 3). (a) Commissural fiber that terminates throughout the molecular layer (type 3). A camera lucida drawing of this fiber is shown in b; higher magnifications of this fiber are shown in c and d. The arrow points to the ‘‘classical’’ commissural projection in the inner molecular layer. (b) Camera lucida drawing of the commissural fiber illustrated in a, demonstrating the arborization pattern of this commissural axon within the subzones of the dentate molecular layer. (c) Higher magnification of the commissural fiber illustrated in a and b. Note that the recurrent axon collateral (framed area) reaches the inner fiber plexus and continues within this zone. Framed area is shown at higher magnification in d. (d) Higher magnification of the rectangle in c. The long arrows indicate varicosities formed by this recurrent collateral. The short arrows indicate commissural fibers of the **classical’’ commissural fiber plexus in the inner molecular layer. Scale bars = 70 µm (a, b); 25 µm (c); 15 µm (d).
Heterogeneity of commissural fibers to the rat dentate gyrus
Fig. 3.
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group of fibers reaches the hippocampal fissure and arborizes throughout the outer molecular layer.13 Most of the commissural fibers that did not belong to the commissural fiber type 1 were of this type. Based on the large sample of fibers obtained in the present study, this fiber type is not homogeneous. Three fiber subtypes could be differentiated on the basis of the morphology of their axonal arbor: axons with collaterals arranged in a bundle (Fig. 2b, fiber type 2a), axons that branch off collaterals in the middle onethird of the molecular layer or send recurrent axons to this zone (Fig. 2c, fiber type 2b), and axons with very long collaterals that run in parallel with the granule cell layer (Fig. 2d, fiber type 2c). Taken together as a group (fiber type 2), the number of axons and boutons appeared to be highest throughout the outer one-third of the molecular layer and somewhat lower in the middle one-third (Fig. 5). Fibers terminating throughout the molecular layer (type 3) In the present study, we observed a further commissural fiber type that terminates throughout the entire molecular layer. An example of this fiber type is illustrated in Fig. 3. This commissural fiber passes through the dense commissural fiber plexus in the inner molecular layer (Fig. 3a–c), but gives off a recurrent collateral to the inner molecular layer that leaves a major axon branch in the middle one-third of the molecular layer (Fig. 3c, d). This collateral enters the main commissural fiber plexus in the inner molecular layer, where it is indistinguishable from other PHAL-labeled commissural fibers (Fig. 3d). Only few commissural fibers belonged to this commissural fiber type. The number of axons and boutons appeared to be evenly distributed throughout the molecular layer (Fig. 5). Fibers terminating in the granule cell layer and molecular layer (type 4) A fourth commissural fiber type was observed that displayed a large number of collaterals in the granular layer (Fig. 4): the main axon of these fibers entered the granule cell layer from the hilus, ramified primarily within the granule cell layer, but extended a few long axon collaterals up to the hippocampal fissure (Fig. 4a, b). At higher magnification, numerous boutons are visible mainly in the granule cell layer (Figs 4c, d, 5). DISCUSSION
Methodological considerations Earlier studies on the commissural projection used anterograde degeneration or transport (with autoradiography), which do not depict individual commissural fibers. The anterograde tracer PHAL19 allows for the identification of projections at the level
of single fibers. Using this technique, we recently found a previously unknown commissural projection to the outer molecular layer of the dentate gyrus,13 demonstrating that anterograde tracing with PHAL is a useful tool in order to analyse the terminal arborization pattern of individual fibers. However, in comparison with slice preparations for analysis of the associational projection employing intracellular staining of individual neurons,23 the PHAL technique has certain shortcomings that should be kept in mind here. First, with the PHAL technique, numerous fibers and their collaterals are labeled. Therefore, a direct correlation between the different commissural fiber types described in the present study and their cells of origin cannot be made. Nevertheless, based on the well known anatomical homology of the commissural and associational projections,6,13 putative cells of origin for the different commissural fiber types may be deduced. Second, only portions of the axons are present in the 100-µm-thick sections that we analysed, whereas the axonal arbor is much more complete in the 400-µm-thick slices that were used for intracellular labeling of local collaterals. Nevertheless, using PHAL, we frequently found long axon segments that could be followed through the entire extent of the hippocampus.11 Where necessary, we investigated consecutive sections in order to ensure that we did not miss larger portions of the distal axonal arbor. Taken together, we think that the fairly large number of single axons (n = 65; see Experimental Procedures) analysed in the present study allows us to characterize the different commissural fiber types, which in view of their long trajectories cannot be studied by intracellular staining in acute slices. Third, the possiblity has to be considered that some of the tracer was retrogradely transported into contralateral hilar neurons and that some of these neurons could send axons into the outer molecular layer. The commissural PHAL-labeled fibers we observed in the outer molecular layer would then be part of the retrogradely labeled associational projection to the outer molecular layer. It is clear, however, that in such a case intensely PHAL-labeled neuronal cell bodies would have to be present in the contralateral hilus. Since none were observed in our material, we conclude that the PHAL-labeled fibers in the outer molecular layer did not arise from local neurons. Laminar termination of commissural fibers and their putative cells of origin Since the commissural fiber types proposed in the present study show preference for specific laminae in the dentate (Fig. 5) and similar laminar termination patterns characterize associational axons,23 we will discuss our results in view of this knowledge. The ‘‘classical’’ commissural termination zone in the inner molecular layer is the target of the majority of commissural fibers (commissural fiber type 1; Fig. 5) and, previously, all commissural axons
Heterogeneity of commissural fibers to the rat dentate gyrus
Fig. 4. Commissural fibers to the granule cell layer and molecular layer (type 4). (a) Commissural fiber that terminates primarily within the granule cell layer and extends axon collaterals to the molecular layer (type 4). The arrow points to the ‘‘classical’’ commissural projection in the inner molecular layer. A camera lucida drawing of this fiber is shown in b; higher magnifications of the same fiber are shown in c and d. (b) Camera lucida drawing of the fiber illustrated in a, showing its arborization pattern within the layers of the dentate. (c) Higher magnification of the fiber illustrated in a and b. Note that the main axon comes from the hilar area and arborizes within the granule cell layer. Long axon collaterals continue through the inner molecular layer and enter the outer molecular layer. The arrow points to the commissural fiber plexus in the inner molecular layer. The middle of the illustration is shown at higher magnification in d. (d) Higher magnification of part of c. The arrows indicate varicosities formed by the collaterals within the granule cell layer. Scale bars = 100 µm (a, b); 25 µm (c); 10 µm (d).
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Fig. 5. Laminar preference of commissural fibers to the rat fascia dentata. Relative densities of fibers and boutons formed by the different fiber types within the layers of the dentate are illustrated with increasing grades of grey.
have been believed to terminate in this layer.4,7,9,12,13,21,26,30,31,32,48,49,52 On the associational side, at least three different cells are believed to project to this zone (Fig. 6): glutamateimmunoreactive hilar mossy cells,8,17,38,46 glutamatecontaining CA3c pyramidal cells16,34 and the GABAimmunoreactive hilar/commissural–associational pathway (HICAP) cell.23 Commissural fibers of this type terminate on the spines of granule cell dendrites7,21,26,30,3150,52 and on the shafts of inhibitory basket cell dendrites.12,43 Commissural fibers of type 2 terminate in the outer molecular layer13 (Fig. 5). The termination pattern of this fiber type corresponds to the axonal arborization pattern of the GABA-immunoreactive HIPP cell23 (Fig. 6). The hilar/perforant pathway cell is strikingly similar to the members of the somatostatin and/or NPY-containing population of hilar neurons,5,9,23,27,33 and these neurons do indeed participate in the commissural projection. At least 4–5% of somatostatin-containing cells54 and 2% of NPY neurons9 have a commissural collateral. The small number of somatostatin and NPY neurons participating in the commissural projection is compatible with the relatively small number of commissural axons observed to reach the outer molecular layer as compared to the large number of associational fibers reaching the corresponding layer on the injection side. Commissural fibers of type 3 terminate throughout the molecular layer (Fig. 5). This termination pattern corresponds to the local axonal arborization of a GABA-immunoreactive neuron45 that these authors called the molecular layer axon (MOLAX) cell18
(Fig. 6). In fact, the axonal arbor of this cell is very similar to the fiber illustrated in Fig. 3. Thus, collaterals of the molecular layer axon cell enter the inner molecular layer and participate in the formation of the fiber plexus there, while other collaterals extend up to the hippocampal fissure, where they arborize. Commissural fibers of type 4 terminate in the granule cell layer and extend some axonal collaterals into the molecular layer (Fig. 5). No associational cell type is known that has an axon with a similar termination pattern (Fig. 6). Dentate basket cells23 and hilar and supragranular chandelier cells23,44 are known to terminate in the granule cell layer. However, both cell types have a very characteristic axon morphology, differing from commissural fiber type 4. Basket cell axons surround the somata of granule cells, whereas the axons of axo-axonic cells run parallel to granule cell axons, and thus perpendicular to the granule cell layer. Moreover, neither cell type sends long axon collaterals into the outer molecular layer. Therefore, a corresponding associational cell type may exist that has so far escaped detection. This comparison of the commissural and associational fiber types suggests that the parallelism of the two projections may have been underestimated. As demonstrated earlier,6 neurons participating in associational connections may have a very similar commissural projection. The difference between the two fiber systems appears to be quantitative rather than qualitative. Whereas almost as many cells appear to project to the inner molecular layer ipsilaterally and contralaterally (fiber type 1), the number of cells that project outside this zone (fiber types 2–4) appears to be much larger on the ipsilateral side.
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Fig. 6. Commissural fibers in the rat fascia dentata and their associational correlates.
Implications for the sprouting of commissural fibers after entorhinal lesion Commissural fibers to the inner molecular layer (type 1 fibers) are said to sprout into the denervated outer molecular layer after entorhinal cortex lesion20,35,36,53 (see Ref. 47 for review). In other words, they are supposed to leave their appropriate lamina and form contacts on dendritic segments formerly occupied by the entorhinal fibers. However, by tracing the sprouted fibers with PHAL we were unable to find a massive translaminar expansion of the main commissural fiber plexus into the former entorhinal termination zone.11 In contrast, we
noticed that the few commissural fibers normally terminating in the outer molecular layer (type 2 fibers) show lamina-specific sprouting within their appropriate layer in response to the deafferentation.11 This finding raised the question to what extent the ‘‘classical’’ commissural fibers in the inner molecular layer participate in the reinnervation of the denervated outer molecular layer. Further studies of the commissural fiber plexus in the inner molecular layer revealed that it expands by approximately 40 µm after entorhinal cortex lesion, only a very small number of fibers growing further out.14 The latter fibers suggested the existence of an as yet unknown commissural fiber type capable of
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sprouting into the denervated outer molecular zone.14 In the present paper, we have demonstrated a commissural fiber type that terminates throughout the molecular layer in normal animals (type 3, Fig. 3) and at the same time participates in the formation of the ‘‘classical’’ commissural fiber plexus in the inner molecular layer. Since this fiber type does not respect laminar boundaries in the dentate molecular layer, collaterals of these axons might be the ones sprouting into the outer molecular layer in response to an entorhinal lesion. The small number of type 3 fibers
found in our material would also explain why only few fibers leaving the inner plexus after an entorhinal lesion could be observed.14 The numerous type 1 fibers, however, remain in the inner molecular layer and respect its laminar boundary. Acknowledgements—The authors thank A. Schneider, S. Nestel, M. Winter and R. Kovacs for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Fr 620/4-2, Leibniz Program, Ni 344/1-1, Ni 344/5-1).
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(1981) Associational and commissural collaterals of neurons in the hippocampal formation (hilus fasciae dentatae and subfield CA3). Brain Res. 212, 287–300. Leranth C. and Frotscher M. (1987) Cholinergic innervation of hippocampal GAD- and somatostatinimmunoreactive commissural neurons. J. comp. Neurol. 261, 33–47. Leranth C., Malcolm A. J. and Frotscher M. (1990) Afferent and efferent synaptic connections of somatostatinimmunoreactive neurons in the rat fascia dentata. J. comp. Neurol. 295, 111–122. Li X.-G., Somogyi P., Ylinen A. and Buzsa´ki G. (1994) The hippocampal CA3 network: an in vivo intracellular labeling study. J. comp. Neurol. 339, 181–208. Lynch G. S., Gall C., Rose G. and Cotman C. W. (1976) Changes in the distribution of the dentate gyrus associational system following unilateral or bilateral entrohinal lesion in the adult rat. Brain Res. 110, 57–71. Lynch G. S., Stanfield B. and Cotman C. W. (1973) Developmental differences in postlesion axonal growth in the hippocampus. Brain Res. 59, 155–168. Paxinos G. and Watson C. (1986) Atlas of the Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego. Ribak C. E., Seress L. and Amaral D. G. (1985) The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus. J. Neurocytol. 14, 835–857. Ribak C. E., Seress L., Peterson G. M., Seroogy K. B., Fallon J. H. and Schmued L. C. (1986) A GABAergic inhibitory component within the hippocampal commissural pathway. J. Neurosci. 6, 3492–3498. Scharfman H. E. and Schwartzkroin P. A. (1988) Electrophysiology of morphologically identified mossy cells of the dentate hilus recorded in guinea pig hippocampal slices. J. Neurosci. 810, 3812–3821. Schwerdtfeger W. K. and Buhl E. H. (1986) Various types of non-pyramidal hippocampal neurons project to the septum and contralateral hippocampus. Brain Res. 386, 146–154. Seress L. and Ribak C. E. (1983) GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons. Expl Brain Res. 50, 173–182. Seress L. and Ribak C. E. (1984) Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition. J. Neurocytol. 13, 215–225. Soriano E. and Frotscher M. (1989) A GABAergic axo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway. Brain Res. 503, 170–174. Soriano E. and Frotscher M. (1993) GABAergic innervation of the rat fascia dentata: a novel type of interneuron in the granule cell layer with extensive axonal arborization in the molecular layer. J. comp. Neurol. 334, 385–396. Soriano E. and Frotscher M. (1994) Mossy cells of the rat fascia dentata are glutamate-immunoreactive. Hippocampus 4, 65–70. Steward O. (1991) Synapse replacement on cortical neurons following denervation. Cerebral Cortex 9, 81–132. Swanson L. W. and Cowan W. M. (1977) An autoradiographic study of the organization of the efferent connections of the hippocampal formation of the rat. J. comp. Neurol. 172, 49–84. Swanson L. W., Sawchenko P. E. and Cowan W. M. (1981) Evidence for collateral projections by neurons in Ammon’s horn, the dentate gyrus, and the subiculum—a multiple retrograde labeling study in the rat. J. Neurosci. 1, 548–559. Swanson L. W., Wyss J. M. and Cowan W. M. (1978) An autoradiographic study of the organization of intrahippocampal association pathways in the rat. J. comp. Neurol. 181, 681–716. Voneida T. J., Vardaris R. M., Fish S. E. and Reiheld C. T. (1981) The origin of the hippocampal commissure in the rat. Anat. Rec. 201, 91–103. Zimmer J. (1971) Ipsilateral afferents to the commissural zone of the fascia dentata, demonstrated in decommissurated rats by silver impregnation. J. comp. Neurol. 142, 393–416. Zimmer J. (1973) Extended commissural and ipsilateral projections in the postnatally de-entorhinated hippocampus and fascia dentata demonstrated in rats by silver impregnation. Brain Res. 64, 293–311. Zimmer J. and Laurberg S. and Sunde N. (1983) Neuroanatomical aspects of normal and transplanted hippocampal tissue. In Neurobiology of the Hippocampus (ed. Seifert W.), pp. 39–64. Academic Press, San Diego.
. (Accepted 23 April 1996)
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Neuroscience Vol. 75, No. 1, pp. 111–121, 1996 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/96 $15.00+0.00 S0306-4522(96)00255-2
HETEROGENEITY OF THE COMMISSURAL PROJECTION TO THE RAT DENTATE GYRUS: A PHASEOLUS VULGARIS LEUCOAGGLUTININ TRACING STUDY T. DELLER,*† R. NITSCH‡ and M. FROTSCHER* *Institute of Anatomy, University of Freiburg, Freiburg, P.O. Box 111, D-79001, Freiburg, Germany ‡Institute of Anatomy, Humboldt University Berlin (Charite´), D-10098, Berlin, Germany Abstract––The commissural and associational projections to the rat dentate gyrus are believed to be anatomically homologous fiber systems. They are often referred to as the so-called commissural/ associational system of the dentate gyrus. However, whereas characteristic laminar termination patterns within the molecular layer of the dentate gyrus have been described for the different cells of origin of the associational projection, the axons of the different cell types of commissural neurons have long been believed to terminate exclusively within the inner molecular layer. Only recently, a previously unknown commissural projection to the outer molecular layer of the dentate gyrus was described and the question was raised whether the commissural fibers could exhibit a heterogeneity similar to that of the associational projections. Using the anterograde tracer Phaseolus vulgaris leucoagglutinin, which labels individual axons and their collaterals, we have studied the termination pattern of commissural axons in the dentate gyrus of the septal hippocampus. At least four different commissural fiber types could be revealed on the basis of their laminar termination pattern: fibers to the inner molecular layer (type 1), fibers to the outer molecular layer (type 2), fibers terminating throughout the molecular layer (type 3), and fibers terminating in both the granule cell layer and the molecular layer (type 4). These observations demonstrate a previously underestimated heterogeneity of the commissural projection. In addition, there is a great deal of parallelism between the different commissural and associational fibers, pointing to a coordinated action of the two systems in the two hippocampi. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: hippocampal commissure, anterograde tracing, limbic connections, fascia dentata, entorhinal cortex lesion.
The commissural and associational projections to the rat dentate gyrus are among the best studied fiber systems of the hippocampus7,9,12,13,21,26,30,31,32,48,49,50,52 (see Ref. 4 for review). The two projections have been considered homologous, since it appears to be a general principle of hippocampal organization6 that neurons participating in the associational projection also contribute to the commissural projection. The cells of origin of the two projections are located in the polymorph layer of the hilus,6,24,31,51 and several groups of neurons have been identified that contribute to both: glutamate-immunoreactive mossy cells,2,17,38,40,46 somatostatin-54 and neuropeptide Y (NPY)-containing9 neurons that appear to be subpopulations of GABAergic neurons,28 and several GABAergic neurons that were not known to contain neuropeptides.32,39,41,42 Characteristic laminar termination patterns throughout the molecular layer of the dentate gyrus have been described for the different cells of origin †To whom correspondence should be addressed. Abbreviations: NPY, neuropeptide Y; PB, phosphate buffer; PHAL, Phaseolus vulgaris leucoagglutinin.
of the associational projection,18,22,23,45 whereas the inner molecular layer has long been considered the only termination zone of the different commissural cell types.7,15,21,25,2930 We recently demonstrated that commissural fibers are not restricted to the inner molecular layer13 and that the hilar/ perforant pathway (HIPP) cell23 appears to have a commissural collateral that terminates in the same hippocampal layer in the contralateral hippocampus as the associational collateral on the ipsilateral side.13 This raised the question whether other hilar neurons with associational and commissural axon collaterals also have corresponding ipsilateral and commissural termination zones. In the present study, we used the Phaseolus vulgaris leucoagglutinin (PHAL) anterograde tracing technique, which demonstrates the entire course of anterogradely labeled axons on the single fiber level19 and makes it possible to describe the exact trajectory and termination pattern of commissural axons. With this method we have analysed the commissural projection to the septal part of the dentate gyrus and have compared the termination pattern of commissural fibers there with that of their associational counterparts.
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CA1, CA3 hippocampal subfields CA1, CA3 DG dentate gyrus Fi fimbria GCL granule cell layer H hilus
HICAP HIPP IML OML
hilar/commissural-associational-pathway hilar/perforant-pathway inner molecular layer outer molecular layer
Fig. 1. PHAL injection sites in the dentate gyrus and commissural projections to the dentate molecular layer. (a) Typical PHAL injection site located in the crest of the dentate gyrus. Granule cells and hilar neurons have taken up the tracer. The associational projection to the inner molecular layer (arrow) and the associational projection to the outer molecular layer (arrowheads) are labeled. (b) Schematic drawing of the various PHAL injection sites in and close to the dentate gyrus. (c) Dentate gyrus contralateral to the injection site illustrated in a. The commissural projection to the inner molecular layer (arrows) and the commissural projection to the outer molecular layer (arrowheads) are labeled. Scale bars = 400 µm (a, c); 800 µm (b).
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Fig. 2. Camera lucida drawings of commissural fibers to the inner molecular layer (type 1) and outer molecular layer (type 2) of the dentate gyrus. (a) Typical commissural fiber to the inner molecular layer (IML): commissural fiber type 1. The axon reaches the inner molecular layer via the granule cell layer and all axon collaterals are restricted to the inner molecular layer. (b–d) Commissural fibers to the outer molecular layer (OML): commissural fiber type 2. These axons pass through the inner molecular layer without branching and terminate exclusively within the outer molecular layer. Three fiber types may be differentiated on the basis of the morphology of their axonal arbor: axons with collaterals arranged in a bundle (b; fiber type 2a), axons with collaterals that branch off collaterals in the middle one-third of the molecular layer or send recurrent axons to this zone (c; fiber type 2b), and axons with very long collaterals that run in parallel with the granule cell layer (d; fiber type 2c). Scale bars = 50 µm (a–d).
EXPERIMENTAL PROCEDURES
Fourteen male and female Sprague–Dawley rats (250– 350 g; Harlan Winkelmann, Borchen, Germany) housed under standard laboratory conditions were used in this
study. Surgical procedures were performed under deep anesthesia, in agreement with the German legislation on the use of laboratory animals (Nembutal, 50 mg/kg body weight). PHAL (2.5% in 10 mM phosphate buffer (PB), pH 7.8; Vector Laboratories, Burlingame, CA, U.S.A.) was
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iontophoretically delivered19 into the hilar area of the dentate gyrus using a glass micropipette (tip diameter 15–30 µm; 5 µA positive current applied every other 5 s for 20–30 min). All animals received PHAL injections into the septal part of the dentate gyrus (coordinates from bregma: AP "3.8, L 1.6, V "3.8).37 The animals were allowed to survive for 10 days following the injection of the anterograde tracer. After this time period the rats were deeply anesthetized with Nembutal and were transcardially perfused with a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 15% picric acid in 0.1 M PB (pH 7.4). Brains were removed and postfixed for 2 h in glutaraldehyde-free fixative. One-hundred-micrometer-thick sections were cut with a Vibratome and washed in PB. Immunocytochemistry was used to visualize the PHALcontaining axons. Free-floating sections were incubated at 4)C in biotinylated goat anti-PHAL (1:400), 1% normal horse serum, 0.1% NaN3, 0.5% Triton X-100 in 0.1 M PB for two days. After rinsing the sections in PB, they were incubated in the avidin–biotin–peroxidase complex (ABC Elite, Vector Labs) for 3 h. Following subsequent washes, the sections were immersed for 5–10 min in a nickel/ diaminobenzidine solution (0.05% 3,3*-diaminobenzidine, 0.02% nickel ammonium chloride, 0.024% cobalt chloride, 0.001% H2O2), which resulted in a dark blue reaction product. Sections were then placed on gelatin-coated slides, dehydrated in ethanol, mounted in Eukitt and investigated under the light microscope. In order to analyse individual commissural fibers in the dentate molecular layer, the axonal arbor as visualized in single sections was drawn with the aid of a camera lucida (final magnification #400). A total of 65 axons and axon fragments of commissural fibers (types 2–4) were drawn from the experimental material and selected axons were chosen for the illustration of the different commissural fiber types (Figs 2–4).
frontal sections of the hippocampus. Injection sites varied between 500 and 800 µm in their longitudinal extent. The ipsilateral associational projections to the inner one-third of the molecular layer and to the outer molecular layer were labeled3,4,12 on the side of the injection (Fig. 1a).
RESULTS
The commissural fibers terminating in the inner molecular layer have been described in detail earlier.7,9,12,13,2126,30,31,32,48,49,50,52 The overwhelming majority of commissural fibers belongs to this axon type. PHAL tracing revealed that these axons frequently branch in the granule cell layer, thereby forming several collaterals that often run in parallel until they arborize throughout the inner molecular layer (Fig. 2a).13
Injection sites
PHAL injections were placed into the hilus of the dentate gyrus in the septal part of the hippocampus (see Experimental Procedures). The injection site usually covered major portions of the hilar area and occasionally included CA3 pyramidal neurons and frequently some granule cells (Fig. 1a, b). Injection sites varied in diameter between 100 and 300 µm. An increased immunocytochemical background staining could be observed for an additional 100–400 µm beyond the central injection site. In this peripheral zone of the injection site, no PHAL-labeled cells could be observed. The septotemporal extent of the injection sites was reconstructed using consecutive
Laminar termination of commissural fibers In the dentate gyrus contralateral to the injection, commissural fibers could be found in all layers (Fig. 1c). The ‘‘classical’’ commissural fiber band in the inner molecular layer contained the largest number of PHAL-labeled fibers and appeared to be similar in density to the associational fiber plexus on the injection side (Fig. 1a, c). In contrast, only few commissural fibers were present in the outer molecular layer, considerably less than on the injection side (Fig. 1a, c). The axonal arborization of selected PHAL-labeled commissural axons was drawn with a camera lucida and analysed for its laminar distribution throughout the granule cell layer and molecular layer. The fiber courses of PHAL-labeled commissural fibers through the fimbria–fornix1,10 and through the hilar area13 have been described earlier and will not be described in detail in the present paper. Fibers terminating in the inner molecular layer (fiber type 1)
Fibers terminating in the outer molecular layer (fiber type 2) We recently described another group of commissural fibers that pass through the granule cell layer and inner molecular layer without branching. This
Fig. 3. Commissural fibers to the molecular layer (type 3). (a) Commissural fiber that terminates throughout the molecular layer (type 3). A camera lucida drawing of this fiber is shown in b; higher magnifications of this fiber are shown in c and d. The arrow points to the ‘‘classical’’ commissural projection in the inner molecular layer. (b) Camera lucida drawing of the commissural fiber illustrated in a, demonstrating the arborization pattern of this commissural axon within the subzones of the dentate molecular layer. (c) Higher magnification of the commissural fiber illustrated in a and b. Note that the recurrent axon collateral (framed area) reaches the inner fiber plexus and continues within this zone. Framed area is shown at higher magnification in d. (d) Higher magnification of the rectangle in c. The long arrows indicate varicosities formed by this recurrent collateral. The short arrows indicate commissural fibers of the **classical’’ commissural fiber plexus in the inner molecular layer. Scale bars = 70 µm (a, b); 25 µm (c); 15 µm (d).
Heterogeneity of commissural fibers to the rat dentate gyrus
Fig. 3.
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group of fibers reaches the hippocampal fissure and arborizes throughout the outer molecular layer.13 Most of the commissural fibers that did not belong to the commissural fiber type 1 were of this type. Based on the large sample of fibers obtained in the present study, this fiber type is not homogeneous. Three fiber subtypes could be differentiated on the basis of the morphology of their axonal arbor: axons with collaterals arranged in a bundle (Fig. 2b, fiber type 2a), axons that branch off collaterals in the middle onethird of the molecular layer or send recurrent axons to this zone (Fig. 2c, fiber type 2b), and axons with very long collaterals that run in parallel with the granule cell layer (Fig. 2d, fiber type 2c). Taken together as a group (fiber type 2), the number of axons and boutons appeared to be highest throughout the outer one-third of the molecular layer and somewhat lower in the middle one-third (Fig. 5). Fibers terminating throughout the molecular layer (type 3) In the present study, we observed a further commissural fiber type that terminates throughout the entire molecular layer. An example of this fiber type is illustrated in Fig. 3. This commissural fiber passes through the dense commissural fiber plexus in the inner molecular layer (Fig. 3a–c), but gives off a recurrent collateral to the inner molecular layer that leaves a major axon branch in the middle one-third of the molecular layer (Fig. 3c, d). This collateral enters the main commissural fiber plexus in the inner molecular layer, where it is indistinguishable from other PHAL-labeled commissural fibers (Fig. 3d). Only few commissural fibers belonged to this commissural fiber type. The number of axons and boutons appeared to be evenly distributed throughout the molecular layer (Fig. 5). Fibers terminating in the granule cell layer and molecular layer (type 4) A fourth commissural fiber type was observed that displayed a large number of collaterals in the granular layer (Fig. 4): the main axon of these fibers entered the granule cell layer from the hilus, ramified primarily within the granule cell layer, but extended a few long axon collaterals up to the hippocampal fissure (Fig. 4a, b). At higher magnification, numerous boutons are visible mainly in the granule cell layer (Figs 4c, d, 5). DISCUSSION
Methodological considerations Earlier studies on the commissural projection used anterograde degeneration or transport (with autoradiography), which do not depict individual commissural fibers. The anterograde tracer PHAL19 allows for the identification of projections at the level
of single fibers. Using this technique, we recently found a previously unknown commissural projection to the outer molecular layer of the dentate gyrus,13 demonstrating that anterograde tracing with PHAL is a useful tool in order to analyse the terminal arborization pattern of individual fibers. However, in comparison with slice preparations for analysis of the associational projection employing intracellular staining of individual neurons,23 the PHAL technique has certain shortcomings that should be kept in mind here. First, with the PHAL technique, numerous fibers and their collaterals are labeled. Therefore, a direct correlation between the different commissural fiber types described in the present study and their cells of origin cannot be made. Nevertheless, based on the well known anatomical homology of the commissural and associational projections,6,13 putative cells of origin for the different commissural fiber types may be deduced. Second, only portions of the axons are present in the 100-µm-thick sections that we analysed, whereas the axonal arbor is much more complete in the 400-µm-thick slices that were used for intracellular labeling of local collaterals. Nevertheless, using PHAL, we frequently found long axon segments that could be followed through the entire extent of the hippocampus.11 Where necessary, we investigated consecutive sections in order to ensure that we did not miss larger portions of the distal axonal arbor. Taken together, we think that the fairly large number of single axons (n = 65; see Experimental Procedures) analysed in the present study allows us to characterize the different commissural fiber types, which in view of their long trajectories cannot be studied by intracellular staining in acute slices. Third, the possiblity has to be considered that some of the tracer was retrogradely transported into contralateral hilar neurons and that some of these neurons could send axons into the outer molecular layer. The commissural PHAL-labeled fibers we observed in the outer molecular layer would then be part of the retrogradely labeled associational projection to the outer molecular layer. It is clear, however, that in such a case intensely PHAL-labeled neuronal cell bodies would have to be present in the contralateral hilus. Since none were observed in our material, we conclude that the PHAL-labeled fibers in the outer molecular layer did not arise from local neurons. Laminar termination of commissural fibers and their putative cells of origin Since the commissural fiber types proposed in the present study show preference for specific laminae in the dentate (Fig. 5) and similar laminar termination patterns characterize associational axons,23 we will discuss our results in view of this knowledge. The ‘‘classical’’ commissural termination zone in the inner molecular layer is the target of the majority of commissural fibers (commissural fiber type 1; Fig. 5) and, previously, all commissural axons
Heterogeneity of commissural fibers to the rat dentate gyrus
Fig. 4. Commissural fibers to the granule cell layer and molecular layer (type 4). (a) Commissural fiber that terminates primarily within the granule cell layer and extends axon collaterals to the molecular layer (type 4). The arrow points to the ‘‘classical’’ commissural projection in the inner molecular layer. A camera lucida drawing of this fiber is shown in b; higher magnifications of the same fiber are shown in c and d. (b) Camera lucida drawing of the fiber illustrated in a, showing its arborization pattern within the layers of the dentate. (c) Higher magnification of the fiber illustrated in a and b. Note that the main axon comes from the hilar area and arborizes within the granule cell layer. Long axon collaterals continue through the inner molecular layer and enter the outer molecular layer. The arrow points to the commissural fiber plexus in the inner molecular layer. The middle of the illustration is shown at higher magnification in d. (d) Higher magnification of part of c. The arrows indicate varicosities formed by the collaterals within the granule cell layer. Scale bars = 100 µm (a, b); 25 µm (c); 10 µm (d).
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Fig. 5. Laminar preference of commissural fibers to the rat fascia dentata. Relative densities of fibers and boutons formed by the different fiber types within the layers of the dentate are illustrated with increasing grades of grey.
have been believed to terminate in this layer.4,7,9,12,13,21,26,30,31,32,48,49,52 On the associational side, at least three different cells are believed to project to this zone (Fig. 6): glutamateimmunoreactive hilar mossy cells,8,17,38,46 glutamatecontaining CA3c pyramidal cells16,34 and the GABAimmunoreactive hilar/commissural–associational pathway (HICAP) cell.23 Commissural fibers of this type terminate on the spines of granule cell dendrites7,21,26,30,3150,52 and on the shafts of inhibitory basket cell dendrites.12,43 Commissural fibers of type 2 terminate in the outer molecular layer13 (Fig. 5). The termination pattern of this fiber type corresponds to the axonal arborization pattern of the GABA-immunoreactive HIPP cell23 (Fig. 6). The hilar/perforant pathway cell is strikingly similar to the members of the somatostatin and/or NPY-containing population of hilar neurons,5,9,23,27,33 and these neurons do indeed participate in the commissural projection. At least 4–5% of somatostatin-containing cells54 and 2% of NPY neurons9 have a commissural collateral. The small number of somatostatin and NPY neurons participating in the commissural projection is compatible with the relatively small number of commissural axons observed to reach the outer molecular layer as compared to the large number of associational fibers reaching the corresponding layer on the injection side. Commissural fibers of type 3 terminate throughout the molecular layer (Fig. 5). This termination pattern corresponds to the local axonal arborization of a GABA-immunoreactive neuron45 that these authors called the molecular layer axon (MOLAX) cell18
(Fig. 6). In fact, the axonal arbor of this cell is very similar to the fiber illustrated in Fig. 3. Thus, collaterals of the molecular layer axon cell enter the inner molecular layer and participate in the formation of the fiber plexus there, while other collaterals extend up to the hippocampal fissure, where they arborize. Commissural fibers of type 4 terminate in the granule cell layer and extend some axonal collaterals into the molecular layer (Fig. 5). No associational cell type is known that has an axon with a similar termination pattern (Fig. 6). Dentate basket cells23 and hilar and supragranular chandelier cells23,44 are known to terminate in the granule cell layer. However, both cell types have a very characteristic axon morphology, differing from commissural fiber type 4. Basket cell axons surround the somata of granule cells, whereas the axons of axo-axonic cells run parallel to granule cell axons, and thus perpendicular to the granule cell layer. Moreover, neither cell type sends long axon collaterals into the outer molecular layer. Therefore, a corresponding associational cell type may exist that has so far escaped detection. This comparison of the commissural and associational fiber types suggests that the parallelism of the two projections may have been underestimated. As demonstrated earlier,6 neurons participating in associational connections may have a very similar commissural projection. The difference between the two fiber systems appears to be quantitative rather than qualitative. Whereas almost as many cells appear to project to the inner molecular layer ipsilaterally and contralaterally (fiber type 1), the number of cells that project outside this zone (fiber types 2–4) appears to be much larger on the ipsilateral side.
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Fig. 6. Commissural fibers in the rat fascia dentata and their associational correlates.
Implications for the sprouting of commissural fibers after entorhinal lesion Commissural fibers to the inner molecular layer (type 1 fibers) are said to sprout into the denervated outer molecular layer after entorhinal cortex lesion20,35,36,53 (see Ref. 47 for review). In other words, they are supposed to leave their appropriate lamina and form contacts on dendritic segments formerly occupied by the entorhinal fibers. However, by tracing the sprouted fibers with PHAL we were unable to find a massive translaminar expansion of the main commissural fiber plexus into the former entorhinal termination zone.11 In contrast, we
noticed that the few commissural fibers normally terminating in the outer molecular layer (type 2 fibers) show lamina-specific sprouting within their appropriate layer in response to the deafferentation.11 This finding raised the question to what extent the ‘‘classical’’ commissural fibers in the inner molecular layer participate in the reinnervation of the denervated outer molecular layer. Further studies of the commissural fiber plexus in the inner molecular layer revealed that it expands by approximately 40 µm after entorhinal cortex lesion, only a very small number of fibers growing further out.14 The latter fibers suggested the existence of an as yet unknown commissural fiber type capable of
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sprouting into the denervated outer molecular zone.14 In the present paper, we have demonstrated a commissural fiber type that terminates throughout the molecular layer in normal animals (type 3, Fig. 3) and at the same time participates in the formation of the ‘‘classical’’ commissural fiber plexus in the inner molecular layer. Since this fiber type does not respect laminar boundaries in the dentate molecular layer, collaterals of these axons might be the ones sprouting into the outer molecular layer in response to an entorhinal lesion. The small number of type 3 fibers
found in our material would also explain why only few fibers leaving the inner plexus after an entorhinal lesion could be observed.14 The numerous type 1 fibers, however, remain in the inner molecular layer and respect its laminar boundary. Acknowledgements—The authors thank A. Schneider, S. Nestel, M. Winter and R. Kovacs for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Fr 620/4-2, Leibniz Program, Ni 344/1-1, Ni 344/5-1).
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. (Accepted 23 April 1996)