HIPPOCAMPUS, VOL. 2, NO. 2, PAGES 99-106, APRIL 1992

Tracing of Axonal Connections by Rhodamine-Dextran-Amine in the Rat Hippocampal-Entorhinal Cortex Slice Preparation Carolyn L. Boulton, Dorothea v. Haebler, and Uwe Heinemann Institut fur Neurophysiologie, Zentrum fur Physiologie und Pathophysiologie d e r Universitat zu Koln Robert-Koch StraBe 39, 5000 Koln 41, Germany

ABSTRACT In order to demonstrate axonal connections preserved in rat temporal cortex slices the authors used rhodamine-dextran-amine as a tracer. The slices contained the neocortical areas Te2 and Te3, the medial and lateral entorhinal cortices (MEC and LEC), the subicular regions, and the dentate gyrus and hippocampus proper. Rhodamine-dextran-amine crystals were placed by microinjection into a given area. Following this local lesioning the dye was permitted to diffuse and migrate intraaxonally in antero- and retrograde directions for about 8 hours. The slices were then formaldehyde-fixed and analyzed by fluorescence microscopy. Most of the known connections within and between the entorhinal cortex and the hippocampus and dentate gyrus were preserved in the slice preparation, provided that the slices were cut with a near horizontal orientation corresponding to plates 99-108 in Paxinos and Watson (1986). Only the lateral perforant path between the LEC and the hippocampus could not be followed to its full extent. The authors conclude that most aspects of the intrinsic synaptic organization of the temporal lobe can be reliably studied in hippocampal-entorhinal cortex slice preparations. Key words: fluorescent tracer, neuronal circuitry, perforant path, anatomical organization

In recent years complex slice preparations have become popular for studies of synaptic connectivity, circuit properties, and pathological alterations. The combined hippocampal-entorhinal cortex slice was introduced by this laboratory (Walther et al., 1986), but as applies for other complex slice preparations, the results obtained were criticized for the obvious risk that not only extrinsic afferent and efferent pathways but also intrinsic connections of the involved structures were cut (Amaral and Witter, 1989). Electrophysiological studies have demonstrated the existence of functional pathways between the entorhinal cortex and the hippocampus and dentate gyrus (Walther et al., 1986; Jones and Heinemann, 1988; Wilson et al., 1988; Dreier and Heinemann, 1990). Electrical stimulations delivered to the subiculum result in the generation of field potentials in the MEC (Jones, 1987). Epileptiform activity appears to propagate from the MEC to the hippocampal formation via the dentate gyrus (Jones and Heinemann, 1988), and epileptiform activity recorded in the CA3 region of the hippocampus is intricately coupled to seizure-like activity occurring in the entorhinal cortex (Wilson et al., 1988). There is, however, a complete absence of anatomical data to support the electroCorrespondence and reprint requests to: D. v. Haebler or U. Heinemann, Institut fur Neurophysiologie, Zentrum fur Physiologie und Pathophysiologie der Universitat zu Koln, Robert-Koch StraRe 39, 5000 Koln 41, Germany.

physiological findings obtained in this particular slice preparation, and for this reason we have used the fluorescent dye rhodamine-dextran-amine to demonstrate that the connectivity present in vivo is preserved in the in vitro slice preparation. Fluorescent axonal tracers have been used to demonstrate connectivity in the central nervous system but suffer from problems of low fluorescence yield, fast signal fading, and uptake by axons of passage. Fluorochromes such as fluorescein or rhodamine have been conjugated with dextran and lysine to yield the strongly fluorescent dextran-amines (Gimlich and Braun, 1985). These compounds are efficiently taken up by damaged neuronal structures, hence allowing selective tracing of pathways after lesioning (Glover et al., 1986). Limited uptake into nerve terminals has also been observed (Glover et al., 1986). The fluorescent dextran-amine is rapidly transported in the cytoplasm away from the injury site in both anterograde and retrograde axonal directions to give a complete picture of the neuronal structures (Glover et al., 1986; Fritzsch et al., 1989; Schmued et al., 1990).

METHODS Preparation of combined entorhinal cortexhippocampal slices

Female Wistar rats of approximately 130-180 g body weight were used in all experiments (n = 26). Combined entorhinal cortex-hippocampal slices were prepared with a

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100 HIPPOCAMPUS VOL. 2, NO. 2, APRIL 1992 McIlwain tissue chopper or a Campden Vibroslice (Loughborough, UK) using the technique described by Dreier and Heinemann (1990). The animals were decapitated under deep ether anesthesia and the brains rapidly removed to ice-cold (4°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl 124, KCl 3, NaHC03 26, Na2HP04 1.2.5, CaClz 1.6, MgS04 1.8, glucose 10. The pH was maintained at 7.4 by oxygenating the solution with 95% 0 2 / 5 % COz. For experiments where slices were prepared using a McIlwain tissue chopper, the cerebellum was removed and a midsagittal cut made to divide the two cerebral hemispheres. The rostra1 two thirds of the hemispheres were separated from the caudal thirds, which were subsequently placed on moistened filter paper, where all diencephalic and midbrain structures were carefully removed to leave tissue blocks containing the hippocampal regions and neocortex. The caudal aspect of the cortex was placed facing the blade of the tissue chopper. The dorsal third of the tissue block was cut away before five to seven slices 400 pM thick were prepared. With slices prepared using a Camden vibroslice, the cerebellum was removed and a coronal cut made at the level of the anterior pituitary. A further horizontal cut was then made across the dorsal surface of the brain to give a flat surface, which was subsequently fixed to the perspex carrier of the vibroslice using cyanoacrylate adhesive (Loctite, UK). Slices of 400 p M thickness comprising the ventral hippocampal region, neocortex, and amygdala of both hemispheres were then prepared and divided at the midline by hand. The slices were rapidly removed to an interface chamber and perfused with oxygenated ACSF at 36 5 1°C at a rate of 1.7-1.9 mL/min. A period of approximately 1 hour was allowed to elapse before dye application to allow equilibration of the slices and to allow axons damaged as a result of the preparation to seal up (Glover et al., 1986). Application of rhodamine-dextran-amine

Injections of the rhodamine-dextran-amine were performed in vitro. We used the compound tetramethylrhodamine-dex-

tran-amine (MW 10,000; Molecular Probes Inc., Eugene, OR), termed Fluoro-Ruby by Schmued et al. (1990), in all experiments. The application was made with a crystal of the rhodamine-dextran-amine gently inserted by hand into the tissue on the tip of a microinjection needle. After dye application, some diffusion of the dye into the perfusate occurs, rendering a quantitative measure of the dye application virtually impossible. The applications were made in the middle and outer thirds of the molecular layer of the dentate gyrus and in the dentate hilus; in the pyramidal cell layer of CAI and CA3; in the subiculum; in the angular bundle; in the superficial layers (IIII), in the deep layers (V-VI), or in all layers of the medial entorhinal cortex (MEC); and in the superficial and deep layers of the lateral entorhinal cortex (LEC). Following the injections, slices were superfused for a further 8-16 hours before being removed for fixation. Preparation for microscopy

The combined slices were fixed overnight at 6 4 ° C with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). They were transferred to a solution of 30% sucrose in 0.1 M

phosphate buffer until fully infiltrated (2-3 hours) and then sectioned to SO pM thickness using a Leitz Rotary freezing microtome. Sections were mounted on gelatin-coated slides, air-dried, and coverslipped using the non-fluorescent mounting medium UV-Inert (Serva, Heidelberg, Germany). Subsequently the slides were stored in the dark at 643°C. Sections were examined using an Orthoplan Leitz microscope fitted with Leitz fluorescence equipment. Rhodamine-dextranamine fluorescence was visualized using a filter system with excitation frequency of 530-560 nm, and a barrier filter frequency of 580 nm. Neuronal structures appeared luminous orange-red under these filters. Photographs were taken using a Leitz Orthomat-W microscope-mounted camera onto Ilford HP5 400 ASA film.

RESULTS Application of rhodamine-dextran-amine provided intense fluorescent labeling, which could be clearly visualized for up to 6 weeks after the experiment. The injection site provided a center of dense fluorescence from which labeled axons emerged. These could be traced over distances of up to 4 mm from the injection site. The dextran-amine appeared to travel much more readily in the anterograde, rather than the retrograde, axonal direction. Fine cellular details were apparent, including dendritic trees, spines, and dendritic varicosities. Figure 1 illustrates examples of dentate granule cells (Fig. 1A) and entorhinal pyramidal cells (Fig. 1B). The maximal distance over which the tracer traveled had usually been reached after 8-10 hours postinjection, which may correlate to the reduced integrity of the slice preparation after this period. Perfusion periods greater than 10 hours frequently resulted in a loss of the dye from the neuronal elements, so that shorter perfusion periods of around 8 hours were generally used. Application of a crystal of rhodamine-dextran-amine into the CA3 pyramidal cell layer and adjacent mossy fiber bundle (Fig. 2) retrogradely labeled cells in the hilar region of the dentate gyrus, the mossy fiber plexus, and dentate granule cells (n = 20 slices). Occasionally, dye injection at this site resulted in the labeling of ectopic granule cells, which are the only projection neurons in the dentate molecular layer. In addition, fibers that might be commissural fibers were observed passing between the hilus and the fimbria, and fibers that might be Schaffer collaterals were noted from the hilus passing in the stratum radiatum of CA1. Application of rhodamine-dextran-amine into the molecular layer of the dentate gyrus fluorescently labeled the dendrites and somas of granule cells and their axonal plexus (mossy fibers) in the hilus (n = 25 slices). The fluorescence observed in the mossy fiber bundle could be followed as far as the CA2 region. Infrequently, other cell types could be identified in the dentate molecular layer. Polymorphic cells in the hilar region of the dentate gyms were always labeled. In addition, dye injection into the dentate molecular layer labeled fibers that crossed the hippocampal fissure and could be traced as far as the subiculum. Polymorphic cells were labeled following injection of the fluorescent marker into the dentate hilus, as were pyramidal cells in CA3 and parts of the mossy fiber bundle (n = 15 slices). Retrograde labeling of granule cells was routinely ob-

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Fig. 1 . Photomicrographs of rhodamine-dextran-amine-labeledneurons in an entorhinal cortex-hippocampal slice preparation. Note the extensive and apparently complete labeling of the different neuronal structures, including dendritic spines and varicosities. (A) Retrogradely labeled dentate granule cells following injection into the CA3 pyramidal cell and mossy fiber layer. (B) Pyramidal cells in layer I11 of the MEC after dye injection into layer I1 of this region. Calibration bar, 25 P.M.

served. Commissural fibers and Schaffer collaterals were also stained by dye injection into this area. Introducing the fluorescent label into the subiculum revealed the presence of a large number of axons running between this area and the dentate gyrus, the presubiculum, the parasubiculum, and MEC (n = 24 slices). The latter pathway ran from layer I1 of the MEC, through the angular bundle, across the subiculum, and terminated in the middle third of the molecular layer of the dentate gyrus, a projection that clearly corresponds to the perforant path as described by Cajal (1911) and Lorente de NO (1933). Depositing the dye crystals at the border of the subiculum and the presubiculum labeled fibers of the perforant path running either directly across the hippocampal fissure and into the dentate gyrus, or along the transverse axis in the stratum moleculare to innervate CAI and to enter the dentate gyrus. The middle or outer thirds of the molecular layer of the dentate gyrus were fluorescently labeled, indicating that fibers from the MEC and the LEC were present (Fig. 3). In addition, injection of the fluorescent marker into the subiculum labeled the angular bundle and the alvear path, seen as a dense bundle of axons, which could be traced for up to 4 mm. Introduction of crystals of rhodamine-dextran-amine into

the presubiculum or the parasubiculum labeled mainly local axons but also fibers running through this region to the middle third of the dentate molecular layer (n = 11 slices). A large number of fibers running toward the alvear path were also evident after dye applications to this region. Injection of the fluorescent marker into either the LEC or the MEC gave a clear picture of the cell layers present in each region (shown in Figs. 4 and 5). A widespread dendritic network was observed in layer I, in which no cells were present. Stellate cells could be seen in layer 11, and horizontal bipolar and tripolar cells were also infrequently observed in this layer, particularly in the LEC. The definition of the cortical layers by the fluorescent complex was such that layers IIa and IIb in the LEC could easily be identified. Pyramidal cells were present in layer 111 and smaller pyramids in layer IV. The cell sparse lamina dissecans, lying between layers 111 and IV, was also distinguishable (Fig. 4). Layers V and VI showed pyramidal cells and the occasional horizontally oriented cell placed in the bundle of fibers lying alongside the external capsule toward the angular bundle. Placement of a dye crystal into lamina I or I1 of the MEC and LEC labeled the apical dendrites of cells with somas situated in the deeper layers. As a result, labeled cell bodies

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Fig. 2 . Dye injection into the CA3 pyramidal cell and adjacent mossy fiber layer resulting in retrograde labeling of the dentate granule cells, their axonal plexus in the hilar region (h), and the mossy fiber bundle (mf). Anterograde labeling of the mossy fiber bundle is evident as far as the transition from regio inferior to regio superior. Additionally, labeling of Schaffer collateral fibers (sc) and commissural fibers (cf) can be observed. ml, molecular layer. Calibration bar, 100 km.

were present in each cell layer. Fluorescence was observed in a widespread dendritic network in layer I and in vertically oriented stellate and pyramidal cells in layers I1 and 111. The axonal plexus of these cells was labeled, such that fibers were observed running to the deeper layers (IV, V, and V1) of these regions. Horizontally oriented fibers passing through the lamina dissecans were also fluorescently labeled. When rhodamine-dextran-amine was placed into layers I1 and I11 of the MEC (n = 33), a dense network of fibers was observed running through the subiculum and into the middle third of the dentate gyrus (Fig. 4). The fibers appeared to arise from stellate cells in layer TI and pyramidal cells in layer 111 of the MEC. In the cell-sparse layer I, fibers were observed to run along the pial surface from the MEC through the parasubiculum and the presubiculum toward the dentate gyrus (Fig. 4). Placement of rhodamine-dextran-amine crystals into the superficial layers of the LEC again showed the widespread dendritic fields in layer I, but failed to show the bundle of fibers observed in the MEC that corresponded to the perforant path (n = 40 slices). Individual fibers in layer 1/11 could be observed traveling to the MEC. Fibers traveling in layer I1 beyond the rhinal fissure and into the perirhinal cortex were more strongly labeled. Only neurons with somas situated where dye crystals were placed in the deeper cell layers of the LEC were fluorescently labeled. Injection of the rhodamine-dextran-amine into layers

V or VI of either the MEC or the LEC labeled a large number of horizontally oriented fibers running within the layers toward the angular bundle from the MEC, or to the MEC from the LEC (n = 22 slices; Fig. 5).

DISCUSSION Rhodamine-dextran-amine is a fluorescent complex with a relatively stable, high fluorescence yield. It can be applied by iontophoresis, injected intracellularly, or directly applied to the preparation as dry crystals or paste and gives a complete labeling of neuronal cell components, including dendritic spines, axon collaterals, and dendritic varicosities. The fluorescent complex is rapidly transported in both retro- and anterograde directions (Glover et al., 1986). However, in our hands and in those of Schmued et al. (1990), anterograde axonal labeling was more successful than retrograde. Reduced integrity of the cells in v i m with increasing superfusion times may cause a release of the fluorescent marker from the cells, limiting the time over which transport or diffusion can be allowed to occur. This may be useful for determining loss of cell integrity induced by, for example, status epilepticus-like activity (Dreier and Heinemann, 1990). The entorhinal cortex is a major relay area for the input and output of information to and from the hippocampal formation and subicular complex. In view of the importance of this site in relation to hippocampal and temporal lobe pathophysiology, the entorhinal-hippocampal slice preparation

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Fig. 3 . Fibers emerging from the subiculum (s) cross the hippocampal fissure into the outer third and, to a lesser extent, to the middle third of the dentate molecular layer (ml). This demonstrates that there are indeed lateral perforant path fibers in the dentate gyrus, although these cannot be traced further retrogradely than the subiculum. Calibration bar: 100 pm.

may prove extremely useful in the study of temporal lobe epilepsies, as the epileptogenic characteristics shown in the in vitro slice preparation appear to mimic those observed in vivo in a manner not seen with other in vitro preparations (Dreier and Heinemann, 1990). In particular, following longterm seizure activity, a state of pharmacoresistant recurrent discharges occurs in the combined slice preparation, which appears to be the correlate of status epilepticus in vivo (Dreier and Heinemann, 1990). Both the entorhinal cortex and the hippocampus show Hebbian and non-Hebbian mechanisms of long-term potentiation (LTP; Bliss and Lomo, 1973; Alonso et al., 1990), such that the combined slice preparation may prove useful for the study of LTP. The rapid transitions between different seizure states in this preparation may be a reflection of the plasticity of the hippocampal and temporal lobe regions. In addition, the high intrinsic rhythmicity of cells in layer I1 of the entorhinal cortex may contribute to its extreme susceptibility to epileptogenesis and may underly the plastic properties of the entorhinal cortex (Alonso and Llinas, 1989). The potential applications of the combined slice prepa-

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ration hence make it of great interest to demonstrate the preserved connectivity in vitro. In our experiments, injections of the rhodamine-dextranamine into the entorhinal cortex-hippocampal slices successfully labeled projections from the MEC running through the angular bundle and subiculum to the middle third of the dentate gyrus, a projection corresponding to the medial perforant path (Cajal, 1911; Lorente de N6, 1933). This pathway was observed only after the fluorescent label was injected into layers I1 and I11 of the MEC, where stellate cells (layer 11) and pyramidal cells (layer 111) are found. This site of origin of the perforant path has been widely described (Hjorth-Simonsen and Jeune, 1972; Schwartz and Coleman, 1981; Witter et al., 1989). Stellate cells of layer I1 appear to project mainly toward the dentate gyrus, whereas cells in layer I11 project toward the CAI region (Steward and Scoville, 1976). In accordance with previous studies, we observed that the fibers of the perforant path ran along the transverse axis of the dentate gyrus in the stratum moleculare of CAI, where they either crossed the hippocampal fissure into the dentate gyms or continued within the CA1 region. This latter pathway, innervating regio superior, has been termed the temporo-ammonic tract (Hjorth-Simonsen and Jeune, 1972; Witter et al., 1989). In addition to the perforant path fibers originating in the MEC, we clearly identified a projection from the superficial MEC that ran along the pial surface through the parasubiculum, presubiculum, and subiculum before crossing the hippocampal fissure into the dentate gyrus. This projection was recognized by Kohler (1988) and appears to represent an alternative route of innervation of the parahippocampal regions and the dentate gyms by the perforant path. Injections of the rhodamine-dextran-amine into layers I1 or 111 of the LEC, or into deeper cell layers, never resulted in the labeling of fibers to the outer third of the dentate molecular layer, which has been identified as the site of termination of lateral perforant path fibers from the LEC (Steward, 1976). This suggests that the axons of the lateral perforant path have been interrupted by the plane of the slices. It is possible that our slices, prepared from the ventral hippocampus, contained only a limited amount of LEC, which would explain the absence of the lateral perforant path. The innervation of the outer third of the dentate molecular layer by fibers from the subiculum is entirely consistent with this view, as the perforant path fibers enter the hippocampus through the subiculum preferentially at the level of their major terminal distribution (Amaral and Witter, 1989). The fact that the lateral perforant path is not preserved in this preparation is of relevance with respect to glutamatergic transmission in the dentate gyrus. The contribution of NMDA receptors to synaptic transmission is stronger in the medial than in the lateral perforant path (Mody et al., 1988; Stanton et al., 1989; Dahl et al., 1990; Heinemann et al., 1990; Lambert and Jones, 1990; Stabel et al., 1990). The fact that MEC activation leads to EPSPs with considerable NMDA receptor contribution is in line with these findings (Lambert and Jones, 1990). Additional sites of origin of the perforant path in layers IV and VI of the MEC and LEC have been proposed by Kohler (1985) following anterograde and retrograde labeling studies.

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Fig. 4. Widespread axonal labeling after injection of the rhodamine-dextran-amine into layer I11 of the MEC (mec). The different cortical cell layers, including the cell sparse lamina dissecans, are easily visible. Note that layer 111 injections result in labeling of the cell somas in all cell layers, excluding layer I , where only dendritic branches are seen. Perforant path fibers are clearly observed running from the MEC through the angular bundle (ab), crossing the subiculum (s) and the hippocampal fissure (hf) into the middle third of the molecular layer (ml) of the dentate gyrus. Another projection can be traced from the MEC running in layer I through the parasubiculum (pas), presubiculum (prs), and subiculum toward the dentate gyrus. Much more prominent is the projection from the MEC to the alvear path (ap), which appears as a smooth bundle of axons and can be traced as far as the CAI region. Calibration bar, 100 pm.

Axons run from these layers in the external capsule to the angular bundle, crossing the subiculum to pass along the transverse axis of the dentate gyrus before entering the molecular layer, where they innervate the outer two thirds of this layer in a manner similar to the traditionally recognized perforant path connections (Kohler, 1985; Amaral and Witter, 1989). We have observed a fiber projection arising from the deep layers of the LEC and MEC and running alongside the external capsule toward the angular bundle. However, again probably due to the angle of the slice, this pathway could not be labeled in its entirety, and we conclude that it is not a functional pathway in this preparation. In summary, the neuronal connectivity that was originally described by Cajal (191 1) and Lorente de NO (1933) appears to be mainly intact in the slice preparation, although input from the lateral entorhinal cortex has not been demonstrated. The medial perforant path projects from the entorhinal cortex to the dentate gyrus. From here, granule cells project to the regio inferior (CA2 and CA3), which connects to the regio superior (CAI) via the Schaffer collateral fibers. The presence of these largely excitatory pathways has clearly been demonstrated. The electrophysiological data derived from studies of the entorhinal cortex-hippocampal slice preparation is in complete agreement with our findings, and would

suggest that the fiber projections that we have observed are indeed functional in this preparation. The entorhinal cortex appears to possess strong reciprocal, recurrent excitatory connections and rather weak inhibitory ones (Schwartz and Coleman, 1981; Misgeld and Frotscher, 1986; Germroth et al., 1989; Jones and Lambert, 1990), rendering it a region highly vulnerable to increased excitability and possibly to epileptogenesis (Jones and Lambert, 1990). The neuronal circuitry that exists between the hippocampdl regions and the entorhinal cortex is such that there are reciprocal connections linking all areas, and the feedforward and feedback loops form the basis of hippocampal regulation (Steward, 1976; Witter et al., 1989). The neuronal connectivity present in the combined entorhinal cortex-hippocampal slice preparation renders it a useful preparation to study input from the medial entorhinal cortex, especially in relation to studies of limbic and temporal lobe seizures. However, as detailed in the excellent review by Amaral and Witter (1989), the transverse hippocampal entorhinal cortex slice, although appearing to preserve lamellar connectivity, does not take account of the large number of connections that are present along the septotemporal axis of the hippocampal and parahippocampal regions. Evidently, results obtained using the transverse entorhinal cortex-hippocampal slice must be eval-

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Fig. 5. Cell layers in the MEC demonstrated by dye injection into layer 11. Note the distinct columnar projections running into a horizontally projecting fiber bundle. ps, pial surface; ec, external capsule. Calibration bar, 100 pm.

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Tracing of axonal connections by rhodamine-dextran-amine in the rat hippocampal-entorhinal cortex slice preparation.

In order to demonstrate axonal connections preserved in rat temporal cortex slices the authors used rhodamine-dextran-amine as a tracer. The slices co...
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