THE JOURNAL OF COMPARATIVE NEUROLOGY 2 9 5 : l l l - 1 2 2 (1990)
Merent and Efferent Synaptic Connections of Somatostatin-ImmunoreactiveNeurons in the Rat Fascia Dentata CSABA LERANTH, ANDREW J. MALCOLM, AND MICHAEL FROTSCHER Yale University, School of Medicine, Section of Neuroanatomy, and Department of Obstetrics and Gynecology, New Haven, Connecticut 06510 (C.L.); The University of British Columbia, MRC Regulatory Peptide Group, Department of Physiology, Vancouver, B.C., Canada (A.J.M.); Institute of Anatomy, University of Frankfurt, Frankfurt a.M., Federal Republic of Germany (M.F.)
ABSTRACT The aim of this study was to determine whether somatostatin (SS)-immunoreactive neurons of the rat fascia dentata are involved in specific excitatory circuitries that may result in their selective damage in models of epilepsy. Synaptic connections of SS-immunoreactive neurons were determined at the electron microscopic level by using normal and colchicine pretreated rats. Vibratome sections prepared from both fascia dentata of control animals and from rats that had received an ipsilateral lesion of the entorhinal cortex 30-36 hours before sacrifice were immunostained for SS by using a monoclonal antibody (SS,). Correlated light and electron microscopic analysis demonstrated that many SS-immunoreactive neurons in the hilus send dendritic processes into the outer molecular layer of the fascia dentata, and dendrites of the same neurons occupy broad areas in the dentate hilar area. The majority of SS-immunoreactive axon terminals form symmetric synapses with the granule cell dendrites in the outer molecular layer and also innervate deep hilar neurons. Via their dendrites in the outer molecular layer, the SS-immunoreactive neurons receive synaptic inputs from perforant pathway axons which were identified by their anterograde degeneration following entorhinal lesions. The axons from the entorhinal cortex are the first segment of the main hippocampal excitatory loop. The hilar dendrites of the same SS-immunoreactive cells establish synapses with the mossy axon collaterals which represent the second member in this excitatory neuronal chain. These observations suggest that SS-immunoreactive neurons in the dentate hilar area may be driven directly by their perforant path synapses and via the granule cells which are known to receive a dense innervation from the entorhinal cortex. These observations demonstrate that SSimmunoreactive neurons in the hilar region are integrated in the main excitatory impulse flow of the hippocampal formation. K e y words: perforant pathway, mossy fiber collaterals, hilar neurons, feed-forward inhibition, feed-back inhibition, epilepsy
Intracerebroventricular injection of the neurotoxin kainic acid (Coyle et al., '78; Nadler et al., '78) as well as sustained electric stimulation of the perforant pathway (Sloviter and Damiano, '81) result in epileptiform convulsions similar to those of patients suffering from epilepsy. Electrophysiological observations suggest that the convulsions are the result of decreased recurrent inhibition (Sloviter and Damiano, '81). The primary victims of both experimental manipulations are those hippocampal neurons that receive massive excitatory inputs from mossy fibers of the fascia dentata granule cells (Sloviter and Damiano, '81). These cells are the o 1990 WILEY-LISS, INC.
CA3 pyramids and a population of hilar neurons (Ram6n y Cajal, 1893; Lorente de N6, '34; Amaral, '78; Amaral and Dent, '81). On the basis of their afferent and efferent synaptic connections, several types of hilar neurons could be involved in a recurrent inhibitory pathway. These include somatostatin (SS)-immunoreactive cells (Kohler and ChanPalay, '82; Morrison et al., '82; Bakst et al., '85, '86; Sloviter, '87; Sloviter and Nilaver, '87; Rapp and Amaral, '88), GABAergic (Seress and Ribak, '83; Frotscher et al., '841, Accepted December 21,1989.
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112 vasoactive intestinal polypeptide-containing (Leranth et al., '84), and cholecystokinin-containing (Leranth and Frotscher '86; Leranth et al., '88) neurons. However, according to the observation of Sloviter ('87), only the SS-immunoreactive neurons in the hilar region disappear after sustained electric stimulation of the perforant pathway. That a loss in the hilar SS-containing cells is associated with pathological seizure activity is further supported by the report of Robbins et al. ('87) of a selective loss of SS-immunoreactive neurons in the hilar area of patients with temporal lobe epilepsy. The question is whether the hilar SS-immunoreactive neurons are more sensitive for hyperstimulation than other nonpyramidal neurons of this area, or as an alternative, the SS-immunoreactive neurons are more than other hilar neurons involved in excitatory circuitries that results in their selective damage by nonphysiological hyperstimulation. In the present study, we examined SS-immunoreactive neurons in the hilar region for their involvement in himocampal excitatory pathways. We have focussed on synaptic connections with entorhinal fibers and mossy fibers, which both belong to the main (trisynaptic) excitatory pathway of the hippo cam pa^ formation (Andersen et al., 371). In tion, we will describe the synapses formed by SS-immunoreactive terminals with neurons in the fascia dentata. Y
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minutes in 1% sodium borohydride (Kosaka et al., '86) and rinsed three times for 15 minutes in PB.
Immunostaining for SS Free-floating (40 pm thick) vibratome sections were incubated for 48 hours at 4°C with a monoclonal antibody (SS,) directed against somatostatin 14. The antibody was diluted (1:1,000) in P B containing 0.17 sodium azide and 1% normal horse serum. The production and the detailed specification of this antibody has been described elsewhere (Buchan et al., '85). After four 15 minute rinses in PB, the tissue-bound primary antibody was visualized according to the ABC technique of Hsu et al. ('81) by using the Vectastain ABC Kit (Vector Laboratories Burlingame, CA) and a final diaminobenzidine (DAB) processing (14.5 mg DAB, 165 yl 0.3$0 H,O, in 25 ml P B for 5-10 minutes). The incubations with secondary biotinylated IgG and ABC were performed a t room temperature for 1.5-2 hours.
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MATERIALS AND METHODS Animals Eight adult (250-300 g) male and female Sprague Dawley rats were used for the present study. The animals were housed in the Yale Animal Care Facility under standard laboratory conditions: 2OoC ambient temperature; artificial 12 hour light/dark cycle; standard rat chow and tapwater ad libitum. All surgical procedures were performed under methohexital anesthesia (Brevital, 50 mg/kg B.W., i.p.). On four rats, the perforant pathway was transected unilaterally by using a glass knife from a dorsal approach 30-36 hours before sacrifice. To enhance the SS immunostaining in peripheral dendrites (Petrusz et al., '77), these rats were re-anesthetized, and given a colchicine injection (80 pg in 20 yl saline) into the lateral ventricle 20 hours before sacrifice. The remaining animals, which were used to describe the efferent connections of the dentate hilar SS neurons. were untreated.
Fixation and tissue preparation Rats were sacrificed under ether anesthesia by transcardial perfusion of 50 ml isotonic saline followed by 250 ml fixative containing 4 5 paraformaldehyde, 0.1 % ' glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate buffer (PB), pH 7.35. After 10 minutes of perfusion, the brains were removed and the middle portions of the hippocampi were dissected by making two horizontal cuts perpendicular to the hippocampal longitudinal axis. These blocks were then postfixed for 2 hours in glutaraldehyde-free fixative, and 40 wm vibratome sections were cut perpendicular to the hippocampal longitudinal axis. Following a 2 hour rinse in PB, sections were transferred to 0.5 ml of 10% sucrose in P B and allowed to equilibrate. To enhance antibody penetration, the sections were freeze-thawed by immersing the vials in liquid nitrogen until the sections were frozen. The sections were then thawed at 4OC. T o remove residual unbound aldehydes, sections were incubated for 20
Electron microscopic tissue processing After the immunostaining procedure, the vibratome sections were rinsed in PB, osmicated for 30 minutes in 1% Oso4 made in PB, (during which were stained with 1% uranyl acetate in 70% ethanol for 30 minutes), and flat embedded between aluminium foil and a glass coverslip (Leranth and FehBr, '83). Following light microscopic examination and photography, the flat-embedded sections were re-embedded for ultramicrotomy. Silver sections containing the selected areas of the fascia dentata were collected on single-slot grids coated with Formvar film, which were then examined in a Philips CM-10 electron microscope.
RESULTS Light microscopy of SS neurons and terminals The results of the present study confirm previous reports on the distribution of SS-immunoreactive neurons and axon terminals in the fascia dentata (see beginning of paper). Immunoreactivity for SS was found in neurons of the nonpyramidal type. The majority of SS-immunoreactive neurons (35-40 neurons per one 40 pm vibratome section of the fascia dentata) were located below the granule cell layer, i.e., in the polymorphic layer, and in the deep hilar region (zone 4 of Amaral, '78; Fig. la,b). In rare instances (one or two cells in one vibratome section), pyramidal-shaped SSimmunoreactive neurons could also be observed. These cells were embedded in the lower third of the granule cell layer and may correspond to the pyramidal basket cell (Fig. lc; cf. Ribak and Seress, '83). The dendrites of SS-immunoreactive neurons, particularly those from colchicine-pretreated animals, could be followed for a long distance. Some of the dendritic trees, mostly of those SS-immunoreactive neurons that occupy the deep hilar area, extended throughout the entire hilus. The most important feature of the majority of the SS-immunoreactive neurons was that they possessed another process, a dendrite that passed through the granule cell layer and branched in the upper third of the molecular layer (Fig. 1).This held true for both multipolar neurons in the hilus and pyramidal-shaped cells located just underneath the granular layer. In addition to the SS-immunoreactive somata and dendrites, numerous SS-immunoreactive axons and terminals were also observed in the fascia dentata. These axons were
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Fig. 1. Light micrographs taken of 40 pm thick vibratome sections from the upper (a and b) and lower (c) blades of the rat fascia dentata immunostained for SS. a and b show SS-immunoreactive hilar multipolar neurons and dendrites that arborize in both the molecular layer (labeled with small arrows) and hiliis. Panel c shows a pyramidal-shaped SS-immunoreactiveneuron (arrow) in a trimmed block. The molecular layer dendrite of this neuron is still in the block while its hilar area
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dendrites have already been sectioned for ultrastructural analysis. Note in panel a the abundant network of SS-immunoreactive axons in the outer third of the molecular layer (M) and a less dense network of SS-immunopositive axons in the hilus (Hi). Gr-granule cell layer. Ca in panel b labels a capillary in the lower horder of the hipporampal fissure. Magnification in aand b = x40; c = x10.
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most densely packed in the outer third of the molecular layer with a lower density of SS-immunoreactive boutons in the middle third of the molecular layer. The least abundant density of the SS-immunoreactive axons was detected in the lower third of the molecular layer and among granule cells. A moderate number of SS-immunoreactive boutons were found in the hilar region (Fig. la).
Electron microscopy of SS-positive terminals The hilar area contains a moderate number of SSimmunoreactive axons and terminals. Electron microscopic analysis of these terminals demonstrated that they establish exclusively symmetric synapses. Both axo-somatic (Fig. 2) and axo-dendritic synapses could be distinguished. Somatostatin-immunoreactive axons did not form synapses with large spines or thorny excrescences as was previously observed for cholecystokinin-immunoreactive axons in this area (Leranth and Frotscher, '86). However, SS-immunoreactive boutons were observed in a few cases to form synapses with hilar neurons which were also postsynaptic to mossy fiber collaterals (Fig. Za). These latter synapses were found mostly on primary dendrites. In the granule cell layer, SS-immunoreactive axons formed synapses with the principal cells. Without exception, these synaptic contacts were symmetric (Gray type 11). The SS-immunoreactive axons formed synapses only occasionally with the granule cell somata (Fig. 3). The overwhelming majority of the unmyelinated SS-immunoreactive axons pass through the granule cell layer and establish synapses with the shafts of the granule cell dendrites in the middle third of the molecular layer (Fig. 4).Corresponding to the light microscopic observation, the upper molecular layer contains the most dense population of SS-immunoreactive boutons. In this area the majority of SS-immunoreactive axon terminals establish symmetric synapses with the head or neck of spines (Fig. 5 ) . In most cases these same spines are synaptic targets of other, unlabeled axon terminals that form asymmetric synapses (Fig. 5a-c). They most likely arise from neurons in the entorhinal cortex (e.g., Matthews et al., '76). Parts of this correlated light and electron microscopic study were performed on colchicine pretreated rats which had received an entorhinal lesion. The main goal of this series of experiments was to elucidate the afferent connections of the dentate SS-immunoreactive neurons. Specifically, two well-characterized sets of axon terminals which most likely are excitatory (Andersen et al., '71) were examined: the mossy fiber collaterals of the granule cells and the perforant pathway terminals. The giant terminal boutons of the mossy fiber collaterals are easily recognized in the electron microscope because they possess characteristic ultrastructural features (Blackstad and Kjaerheim, '61; Hamlyn, '62). The degenerating boutons located in the outer molecular layer of the dentate gyrus most likely represented axon terminals of transected perforant pathway fibers originating in the entorhinal cortex (Steward and Scoville '76). Seven SS-immunoreactive neurons that had dendrites in the hilar area as well as a dendrite reaching the outer molecular layer (cf. Fig. 1) were examined by electron microscopy to determine their afferent connections with the mossy axon collaterals and degenerating perforant pathway axon terminals. In our previous study of the cholinergic innervation of hilar neurons we found that the SS-immunoreactive neurons in this region are heavily innervated by mossy fiber collaterals, in addition to their cholinergic afferents (Fig. 3
C. LERANTH ET AL. in Leranth and Frotscher, '87). In the present study, using a different SS antibody, the same synaptic arrangement (Fig. 6) has again been observed on the hilar dendrites of those SS-immunoreactive neurons which also send a dendritic process to the outer molecular layer. Further ultrastructural analysis of the afferent, connections of these SS-immunoreactive dendrites in the outer molecular layer demonstrated that virtually all of the terminals that are presynaptic are degenerating (Fig. 7). The synaptic contacts formed by the degenerating perforant pathway terminals on the aspinous SS-immunoreactive dendrites were always asymmetric. However, the majority of degenerating terminals established asymmetric synapses with immunonegative spines, most likely arising from granule cell dendrites (cf. Matthews et al., '76).
DISCUSSION The purpose of this study was to obtain new information on the efferent- and particularly on the afferent connections of SS-immunoreactive neurons in the hilar region. A schematic drawing in Figure 8 shows the suggested connections of these cells. Most nongranule cells in the dentate gyrus are immunoreactive for glutamate decarboxylase (Ribak et al., '78, '81; Somogyi et al., '83, '84; Frotscher et al., '84; Lubbers et al., '85; Lubbers ann Frotscher, '87) or GABA (Storm-Mathisen et al., '83; Somogyi et al., '84; Gamrani et al., '86),and there is evidence that GABA is colocalized with neuropeptides, including SS, in many of these cells (Somogyi et al., '84; Kosaka et al., '88). Most likely these neuropeptidecontaining neurons are inhibitory because GABA has been found to exert a powerful inhibitory action in the fascia dentata (e.g., Storm-Mathisen and Ottersen, '84). Therefore, the possible functional role of the dentate gyrus SS-immunoreactive neurons can be considered as that of a special subtype of inhibitory GABA cells. This view is further supported by the observation of Pitman and Siggins ('81),who found that SS hyperpolarizes hippocampal pyramidal cells in vitro. However, there are conflicting results in the literature which will be discussed below. Confirming earlier hypotheses based on light microscopic observations of the distribution pattern of the fascia dentata SS-immunoreactive boutons (Kohler and Chan-Palay '82; Morrison et al., '82; Bakst et al., '85, '86; Sloviter and Nilaver '87), this ultrastructural study demonstrated that while a portion of SS-immunoreactive axon terminals do in fact establish symmetric synaptic contacts on hilar neurons (not shown in Fig. 8), the majority of SS-immunoreactive axon terminals connect with the distal dendrites and spines of granule cells, again forming symmetric synaptic membrane specializations (see also Milner and Bacon, '89). This is the area which is occupied by perforant pathway axons originating in the entorhinal cortex (Ram& y Cajal, 1893). However, the entorhinal fibers mainly establish asymmetric contacts on granule cell dendritic spines (Matthews et al., '76). The present study, of course, does not provide direct evidence that these SS-immunoreactive axons in the outer molecular layer are derived from the SS-positive cells located in the hilus. However, the majority of the SSimmunoreactive axons seem to arise from ipsilateral SSimmunopositive cells because kainic acid injection into the hilus (Bakst et al., '86) and sustained electric stimulation of the perforant pathway (Sloviter, '87), destroy hilar SSimmunoreactive neurons on the side of the stimulation and
DENTATE SOMATOSTATIN NEURONS
Fig. 2. Electron micrographs demonstrate symmetric synapses (arrowheads) formed by SS-immunopositive axon terminals with somata (S) of deep hilar neurons. A mossy axon collateral (M) forms an asymmetric synapse with the initial part of the dendrite of one of the hilar neurons (a).Bars = 1 fim.
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Fig. 3. Electron micrographs of the granule cell layer of the rat fascia dentata immunostained for SS. One of the SS-immunoreactive s o n s traversing this layer synapses with the soma of a granule cell (G). b is an enlargement of the synapse indicated on a. D is an SS-immunopositive dendrite. Bars = 1pm.
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Fig. 4. Electron micrographs (a-c) demonstrate axo-dendritic symmetric synapses (arrowheads) between SS-immunopositive axon terminals and granule cell dendritic shafts (D)in the middle third of the molecular layer. Bars = 1pm.
result in a loss of SS-immunopositive fibers in the outer molecular layer. Furthermore, it is possible that a minor population of these boutons represent axon terminals of commissural SS-immunoreactive neurons located in the contralateral hilar region because some hilar SS-immunoreactive cells are retrogradely labelled following tracer injection into the contralateral hilar region (Zimmer et al., '83; Leranth and Frotscher, '87). Somatostatin-immunoreactiveneurons in the hippocampus have been considered as a subtype of inhibitory GABAergic cell because as many as 90% of the somatostatin hilar cells were found to colocalize GABA (Somogyi et al., '84; Kosaka et al., '88). Sloviter ('87), on the other hand, found that GABA-immunoreactive cells, in contrast to SS-positive cells, were not reduced in number in experimental epilepsy. In either case, our data suggest that the SS-positive neurons
act on the granule cells by establishing symmetric synapses with dendritic segments in the outer molecular layer that are preferentially contacted by entorhinal afferents (cf. Frotscher, '88). This laminar topography suggests that the SS netirons modulate the entorhinal input. Furthermore, SS-positive boutons frequently form synapses with the neck or base of the same spines that have established asymmetric synapses with immunonegative boutons, most likely of entorhinal origin (Matthews et al., '76). Such a synaptic arrangement may allow the first fine tuning of the hippocampal excitatory signal loop, by setting the threshold for the incoming entorhinal excitatory signals. If we assume that the present SS-containing cells are also GABAergic (Somogyi et al., '84; Kosaka et al., '88)' we may conclude that they serve both feed-forward and feed-back inhibitory processes in the fascia dentata (cf. Andersen, "75; Buzsiiki, '84). The
Fig. 5. Electron micrographs depicting SS-immunoreactive axons in the outer third of the molecular layer. T h e reaction product-containing axon terminals establish symmetric synapses (arrowheads) with the heads (a-d,f) or necks ( e )of dendritic spines. In many cases the same
structure postsynaptic to a SS-immunopositive houton is also a target of
another axon terminal (A in a-c) which forms a n asymmetric synaptic contact. Panel d shows a small SS-immunoreactive dendrite (D) in this superficial molecular layer. Bars = 1 pm.
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Fig. 6. Electron micrographs of the dentate hilus of a colchicine treated rat showing SS-immunopositive dendritic processes (a and b) and the soma of a SS-immunoreactive neuron ( c ) . T h e two SSimmunoreactive dendrites establish asymmetric synapses (arrowheads) with the boutons of mossy axon (M) collaterals. Note the postsynaptic complex spine of the SS immunoreactive dendrite in panel b. There are
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several spines (S) invaginated into the mossy axon terminal (M) shown in panel c, including a somatic spine (Sl) of a SS-immunoreactive hilar area neuron. The same mossy axon terminal forms multiple asymmetric synapses (arrowheads) with a small SS-immunopositive dendrite separated by unit membranes from the SS-immunopositive soma. Bars = l hm.
Fig. 7. Electron micrographs (a-c) demonstrate numerous degenerating axon terminals (black arrows) in the outer third of the dentate molecular layer in a colchicine pretreated rat following perforant pathway transection. The majority of the degenerating boutons form asymmetric synaptic contacts with the shaft of granule cell- (Gd) or
SS-immunoreartive dendrites (D), while the others form asymmetric synapses with granule cell dendritic spines (black and white arrows). Note that virtually all of the axons that establish synaptic connections with the SS-immunoreactive dendrites are degenerated. Bars = 1 pm.
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121 parvalbumin-immunoreactive neurons which also establish synaptic contacts with entorhinal afferents (Zipp et al., '89) and mossy fiber collaterals (Nitsch, Soriano, and Frotscher, in preparation). However, these cells may be better protected against overexcitation because they contain a calciumbinding protein (Sloviter, '89; Scharfman and Schwartzkroin, '89). It is of interest in this context that only few SS-immunoreactive neurons in the hippocampus contain the calcium-binding protein parvalbumin (Kosaka et al., '88). In conclusion, this study has shown that hilar SS-positive neurons are integrated in the main excitatory pathway of the hippocampal formation. At present we can only speculate that they exert a modulatory effect on the impulse flow from the entorhinal cortex to the hippocampus.
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ACKNOWLEDGMENTS
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The authors thank Marya Shanabrough for expert technical assistance and Dr. C.E. Ribak for the very useful detailed criticism of the manuscript. This research was supported by NIH grant NS 26068 (C.L.).
Mac
Ma
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Fig. 8. Schematic illustration of the suggested synaptic interconnections between entorhinal afferents (Ea), granule cells (Gc), and SSimmunoreactive neurons (SS)in the rat fascia dentata. Entorhinal afferents can exert excitatory actions (+ j on both granule cell- and SS-immunoreactive dendrites located in the upper third of the molecular layer. T h e SS-immunoreactive cells can also be stimulated (+) by collaterals (Mac) of t h e granule cell's mossy axons (Ma). Axon terminals (SSaj of t h e SS-immunoreactive hilar neurons in t urn may inhibit (-1 granule cells by both a feed-forward and a feed-back mechanism through axo-dendritic connections in t he external molecular layer.
feed-forward circuitry would be mediated by SS-immunoreactive neurons that are activated directly by entorhinal afferents terminating on their dendrites in the molecular layer. On the other hand, the feed-back loop would involve granule cells that give off collaterals which contact SSimmunoreactive neurons in the hilus (Fig. 6) and the SS axons forming synapses with the granule cell dendrites. Finally, we have demonstrated that the same SSimmunoreactive neurons are postsynaptic to entorhinal afferents and mossy axon collaterals (Fig. 8). Both of these axons are known to exert a powerful excitatory action (Andersen et al., '66, '711, probably releasing glutamate (Storm-Mathisen et al., '83) as their neurotransmitter. This involvement of the SS-immunoreactive hilar neurons into the main excitatory circuitry of the hippocampal formation (Andersen et al., '71) offers one possible explanation for their specific excitotoxicity related loss in temporal lobe epilepsy (Robbins et al., '87) and following sustained electric stimulation of the perforant pathway (Olney, '84; Sloviter, '86, '87). A single perforant pathway stimulus can doubly excite the same SS-immunoreactive neuron, first, directly by synapses on their dendrites in the outer molecular layer (Fig. 7); and secondly, the SS-immunoreactive neurons can again be activated indirectly by the same perforant pathway stimulus, within the time of one synaptic delay. This second excitatory impulse will arrive at the SS-immunoreactive cells via the mossy fiber collaterals (Fig. 8). A similar synaptic arrangement has recently been observed in dentate
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C. LERANTH ET AL. Rapp, P.R., and D.G. Amaral (1988) The time of origin of somatostatinimmunoreactive neurons in the rat hippocampal formation. Dev. Brain. Res. 41:231-239. Rihak, C.E., and L. Seress (1983) Five types of basket cell in the hippocampal dentate gyrus: A combined Golgi electron microscopic study. J. Neurocytol. 12577-597. Ribak, C.E., J.E. Vaughn, and K. Saito (1978) Immunocytochemical localization of glutamic acid decarhoxylase in neuronal somata following colchicine inhibition of axonal transport. Brain Res. 140:315-332. Ribak, C.E., J.E. Vaughn, and R.P. Barber (1981) Immunocytochemical localization of GABAergic neurons at the electron microscopic level. J. Histochem. 13:555-582. Robbins, R.J., T. Adrian, N. de Lanerolle, J. Kim, S. Halovnik, and D. Spencer (1987) Hippocampal somatostatin, CCK, and V1P levels in patients with temporal lobe epilepsy. SOC.Neurosci. Abstr. 13:366. Scharfman, H.I., and P.A. Schwartzkroin (1989) Protection of hilar cells from prolonged stimulation by intracellular calcium chelation. Science 246:257260. Seress, L., and C.E. Ribak (1983) GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons. Exp. Brain Res. 50:173-182. Sloviter, R.S. (1986) On the role of seizure activity and endogenous excitatory amino acids in mediating seizure-associated hippocampal damage. In R. Schwarcz and Y. Ben Ari (eds): Excitatory Amino Acids and Epilepsy. New York Plenum Press, pp. 669-671. Sloviter, R.S. (1987) Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy. Science 235:73-76. Sloviter, R.S. (1989) Calcium-binding protein (Calbindin-D28k) and parvalhumin immunocytochemistry: Localization in the rat hippocampus with specific reference to selective vulnerability of hippocampal neurons to seizure activity. J. Comp. Neurol. 280.183-196. Sloviter, R.S., and R.P. Damiano (1981) Sustained electrical stimulation of the perforant path duplicates kainate-induced electrophysiological effects and hippocampal damage in rats. Neurosci. Lett. 24:279-284. Sloviter, R.S., and G. Nilaver (1987) Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide-, and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat. J. Comp. Neurol. 256:42-60. Somogyi, P., A.J. Hodgson, A.D. Smith, M.G. Nunzi, A. Gorio, and J.-Y. Wu (1984) Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin or cholecystokininimmunoreactive material. J. h-eurosci. 4:2590-2603. Somogyi, P., A.D. Smith, M.G. Nunzi, A. Gorio, H. Takagi, and J.-Y. Wu (1983) Glutamate decarboxylase immunoreactivity in the hippocampus of the cat: Distribution of immunoreactive synaptic terminals with special reference to the axon initial segments of pyramidal neurons. J. Neurosci. 3:1450-1468. Steward, O., and S.A. Scoville (1976) Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat. J. Comp. Neurol. I69:347-370. Storm-Mathisen, J., and O.P. Ottersen (1984) Neurotransmitters in the hippocampal formation. In F. Reinoso-Subrez, and C. Ajmone-Marsan (eds): Cortical Integration. New York Raven Press, pp. 105-130. Storm-Mathisen, J., A.K. Leknes, A.T. Bore, J.L. Vaaland, P. Edminson, F.-M.S. Haug, and O.P. Ottersen (1983) First visualization of glutamate and GABA in neurones hy immunocytochemistry. Nature (London) 301:517-520. Zimmer, J., S. Laurberg, and N. Sunde (1983) Neuroanatomical aspects of normal and transplanted hippocampal tissue. In W. Seifert (ed):Neurobiology of the Hippocampus. New York Academic Press, pp. 39-64. Zipp, F., R. Nitsch, E. Soriano, and M. Frotscher (1989) Entorhinal fibers form synaptic contacts on parvalhumin-immunoreactive neurons in the rat fascia dentata. Brain Res. 495:161-166.
Merent and Efferent Synaptic Connections of Somatostatin-ImmunoreactiveNeurons in the Rat Fascia Dentata CSABA LERANTH, ANDREW J. MALCOLM, AND MICHAEL FROTSCHER Yale University, School of Medicine, Section of Neuroanatomy, and Department of Obstetrics and Gynecology, New Haven, Connecticut 06510 (C.L.); The University of British Columbia, MRC Regulatory Peptide Group, Department of Physiology, Vancouver, B.C., Canada (A.J.M.); Institute of Anatomy, University of Frankfurt, Frankfurt a.M., Federal Republic of Germany (M.F.)
ABSTRACT The aim of this study was to determine whether somatostatin (SS)-immunoreactive neurons of the rat fascia dentata are involved in specific excitatory circuitries that may result in their selective damage in models of epilepsy. Synaptic connections of SS-immunoreactive neurons were determined at the electron microscopic level by using normal and colchicine pretreated rats. Vibratome sections prepared from both fascia dentata of control animals and from rats that had received an ipsilateral lesion of the entorhinal cortex 30-36 hours before sacrifice were immunostained for SS by using a monoclonal antibody (SS,). Correlated light and electron microscopic analysis demonstrated that many SS-immunoreactive neurons in the hilus send dendritic processes into the outer molecular layer of the fascia dentata, and dendrites of the same neurons occupy broad areas in the dentate hilar area. The majority of SS-immunoreactive axon terminals form symmetric synapses with the granule cell dendrites in the outer molecular layer and also innervate deep hilar neurons. Via their dendrites in the outer molecular layer, the SS-immunoreactive neurons receive synaptic inputs from perforant pathway axons which were identified by their anterograde degeneration following entorhinal lesions. The axons from the entorhinal cortex are the first segment of the main hippocampal excitatory loop. The hilar dendrites of the same SS-immunoreactive cells establish synapses with the mossy axon collaterals which represent the second member in this excitatory neuronal chain. These observations suggest that SS-immunoreactive neurons in the dentate hilar area may be driven directly by their perforant path synapses and via the granule cells which are known to receive a dense innervation from the entorhinal cortex. These observations demonstrate that SSimmunoreactive neurons in the hilar region are integrated in the main excitatory impulse flow of the hippocampal formation. K e y words: perforant pathway, mossy fiber collaterals, hilar neurons, feed-forward inhibition, feed-back inhibition, epilepsy
Intracerebroventricular injection of the neurotoxin kainic acid (Coyle et al., '78; Nadler et al., '78) as well as sustained electric stimulation of the perforant pathway (Sloviter and Damiano, '81) result in epileptiform convulsions similar to those of patients suffering from epilepsy. Electrophysiological observations suggest that the convulsions are the result of decreased recurrent inhibition (Sloviter and Damiano, '81). The primary victims of both experimental manipulations are those hippocampal neurons that receive massive excitatory inputs from mossy fibers of the fascia dentata granule cells (Sloviter and Damiano, '81). These cells are the o 1990 WILEY-LISS, INC.
CA3 pyramids and a population of hilar neurons (Ram6n y Cajal, 1893; Lorente de N6, '34; Amaral, '78; Amaral and Dent, '81). On the basis of their afferent and efferent synaptic connections, several types of hilar neurons could be involved in a recurrent inhibitory pathway. These include somatostatin (SS)-immunoreactive cells (Kohler and ChanPalay, '82; Morrison et al., '82; Bakst et al., '85, '86; Sloviter, '87; Sloviter and Nilaver, '87; Rapp and Amaral, '88), GABAergic (Seress and Ribak, '83; Frotscher et al., '841, Accepted December 21,1989.
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112 vasoactive intestinal polypeptide-containing (Leranth et al., '84), and cholecystokinin-containing (Leranth and Frotscher '86; Leranth et al., '88) neurons. However, according to the observation of Sloviter ('87), only the SS-immunoreactive neurons in the hilar region disappear after sustained electric stimulation of the perforant pathway. That a loss in the hilar SS-containing cells is associated with pathological seizure activity is further supported by the report of Robbins et al. ('87) of a selective loss of SS-immunoreactive neurons in the hilar area of patients with temporal lobe epilepsy. The question is whether the hilar SS-immunoreactive neurons are more sensitive for hyperstimulation than other nonpyramidal neurons of this area, or as an alternative, the SS-immunoreactive neurons are more than other hilar neurons involved in excitatory circuitries that results in their selective damage by nonphysiological hyperstimulation. In the present study, we examined SS-immunoreactive neurons in the hilar region for their involvement in himocampal excitatory pathways. We have focussed on synaptic connections with entorhinal fibers and mossy fibers, which both belong to the main (trisynaptic) excitatory pathway of the hippo cam pa^ formation (Andersen et al., 371). In tion, we will describe the synapses formed by SS-immunoreactive terminals with neurons in the fascia dentata. Y
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minutes in 1% sodium borohydride (Kosaka et al., '86) and rinsed three times for 15 minutes in PB.
Immunostaining for SS Free-floating (40 pm thick) vibratome sections were incubated for 48 hours at 4°C with a monoclonal antibody (SS,) directed against somatostatin 14. The antibody was diluted (1:1,000) in P B containing 0.17 sodium azide and 1% normal horse serum. The production and the detailed specification of this antibody has been described elsewhere (Buchan et al., '85). After four 15 minute rinses in PB, the tissue-bound primary antibody was visualized according to the ABC technique of Hsu et al. ('81) by using the Vectastain ABC Kit (Vector Laboratories Burlingame, CA) and a final diaminobenzidine (DAB) processing (14.5 mg DAB, 165 yl 0.3$0 H,O, in 25 ml P B for 5-10 minutes). The incubations with secondary biotinylated IgG and ABC were performed a t room temperature for 1.5-2 hours.
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MATERIALS AND METHODS Animals Eight adult (250-300 g) male and female Sprague Dawley rats were used for the present study. The animals were housed in the Yale Animal Care Facility under standard laboratory conditions: 2OoC ambient temperature; artificial 12 hour light/dark cycle; standard rat chow and tapwater ad libitum. All surgical procedures were performed under methohexital anesthesia (Brevital, 50 mg/kg B.W., i.p.). On four rats, the perforant pathway was transected unilaterally by using a glass knife from a dorsal approach 30-36 hours before sacrifice. To enhance the SS immunostaining in peripheral dendrites (Petrusz et al., '77), these rats were re-anesthetized, and given a colchicine injection (80 pg in 20 yl saline) into the lateral ventricle 20 hours before sacrifice. The remaining animals, which were used to describe the efferent connections of the dentate hilar SS neurons. were untreated.
Fixation and tissue preparation Rats were sacrificed under ether anesthesia by transcardial perfusion of 50 ml isotonic saline followed by 250 ml fixative containing 4 5 paraformaldehyde, 0.1 % ' glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate buffer (PB), pH 7.35. After 10 minutes of perfusion, the brains were removed and the middle portions of the hippocampi were dissected by making two horizontal cuts perpendicular to the hippocampal longitudinal axis. These blocks were then postfixed for 2 hours in glutaraldehyde-free fixative, and 40 wm vibratome sections were cut perpendicular to the hippocampal longitudinal axis. Following a 2 hour rinse in PB, sections were transferred to 0.5 ml of 10% sucrose in P B and allowed to equilibrate. To enhance antibody penetration, the sections were freeze-thawed by immersing the vials in liquid nitrogen until the sections were frozen. The sections were then thawed at 4OC. T o remove residual unbound aldehydes, sections were incubated for 20
Electron microscopic tissue processing After the immunostaining procedure, the vibratome sections were rinsed in PB, osmicated for 30 minutes in 1% Oso4 made in PB, (during which were stained with 1% uranyl acetate in 70% ethanol for 30 minutes), and flat embedded between aluminium foil and a glass coverslip (Leranth and FehBr, '83). Following light microscopic examination and photography, the flat-embedded sections were re-embedded for ultramicrotomy. Silver sections containing the selected areas of the fascia dentata were collected on single-slot grids coated with Formvar film, which were then examined in a Philips CM-10 electron microscope.
RESULTS Light microscopy of SS neurons and terminals The results of the present study confirm previous reports on the distribution of SS-immunoreactive neurons and axon terminals in the fascia dentata (see beginning of paper). Immunoreactivity for SS was found in neurons of the nonpyramidal type. The majority of SS-immunoreactive neurons (35-40 neurons per one 40 pm vibratome section of the fascia dentata) were located below the granule cell layer, i.e., in the polymorphic layer, and in the deep hilar region (zone 4 of Amaral, '78; Fig. la,b). In rare instances (one or two cells in one vibratome section), pyramidal-shaped SSimmunoreactive neurons could also be observed. These cells were embedded in the lower third of the granule cell layer and may correspond to the pyramidal basket cell (Fig. lc; cf. Ribak and Seress, '83). The dendrites of SS-immunoreactive neurons, particularly those from colchicine-pretreated animals, could be followed for a long distance. Some of the dendritic trees, mostly of those SS-immunoreactive neurons that occupy the deep hilar area, extended throughout the entire hilus. The most important feature of the majority of the SS-immunoreactive neurons was that they possessed another process, a dendrite that passed through the granule cell layer and branched in the upper third of the molecular layer (Fig. 1).This held true for both multipolar neurons in the hilus and pyramidal-shaped cells located just underneath the granular layer. In addition to the SS-immunoreactive somata and dendrites, numerous SS-immunoreactive axons and terminals were also observed in the fascia dentata. These axons were
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Fig. 1. Light micrographs taken of 40 pm thick vibratome sections from the upper (a and b) and lower (c) blades of the rat fascia dentata immunostained for SS. a and b show SS-immunoreactive hilar multipolar neurons and dendrites that arborize in both the molecular layer (labeled with small arrows) and hiliis. Panel c shows a pyramidal-shaped SS-immunoreactiveneuron (arrow) in a trimmed block. The molecular layer dendrite of this neuron is still in the block while its hilar area
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dendrites have already been sectioned for ultrastructural analysis. Note in panel a the abundant network of SS-immunoreactive axons in the outer third of the molecular layer (M) and a less dense network of SS-immunopositive axons in the hilus (Hi). Gr-granule cell layer. Ca in panel b labels a capillary in the lower horder of the hipporampal fissure. Magnification in aand b = x40; c = x10.
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most densely packed in the outer third of the molecular layer with a lower density of SS-immunoreactive boutons in the middle third of the molecular layer. The least abundant density of the SS-immunoreactive axons was detected in the lower third of the molecular layer and among granule cells. A moderate number of SS-immunoreactive boutons were found in the hilar region (Fig. la).
Electron microscopy of SS-positive terminals The hilar area contains a moderate number of SSimmunoreactive axons and terminals. Electron microscopic analysis of these terminals demonstrated that they establish exclusively symmetric synapses. Both axo-somatic (Fig. 2) and axo-dendritic synapses could be distinguished. Somatostatin-immunoreactive axons did not form synapses with large spines or thorny excrescences as was previously observed for cholecystokinin-immunoreactive axons in this area (Leranth and Frotscher, '86). However, SS-immunoreactive boutons were observed in a few cases to form synapses with hilar neurons which were also postsynaptic to mossy fiber collaterals (Fig. Za). These latter synapses were found mostly on primary dendrites. In the granule cell layer, SS-immunoreactive axons formed synapses with the principal cells. Without exception, these synaptic contacts were symmetric (Gray type 11). The SS-immunoreactive axons formed synapses only occasionally with the granule cell somata (Fig. 3). The overwhelming majority of the unmyelinated SS-immunoreactive axons pass through the granule cell layer and establish synapses with the shafts of the granule cell dendrites in the middle third of the molecular layer (Fig. 4).Corresponding to the light microscopic observation, the upper molecular layer contains the most dense population of SS-immunoreactive boutons. In this area the majority of SS-immunoreactive axon terminals establish symmetric synapses with the head or neck of spines (Fig. 5 ) . In most cases these same spines are synaptic targets of other, unlabeled axon terminals that form asymmetric synapses (Fig. 5a-c). They most likely arise from neurons in the entorhinal cortex (e.g., Matthews et al., '76). Parts of this correlated light and electron microscopic study were performed on colchicine pretreated rats which had received an entorhinal lesion. The main goal of this series of experiments was to elucidate the afferent connections of the dentate SS-immunoreactive neurons. Specifically, two well-characterized sets of axon terminals which most likely are excitatory (Andersen et al., '71) were examined: the mossy fiber collaterals of the granule cells and the perforant pathway terminals. The giant terminal boutons of the mossy fiber collaterals are easily recognized in the electron microscope because they possess characteristic ultrastructural features (Blackstad and Kjaerheim, '61; Hamlyn, '62). The degenerating boutons located in the outer molecular layer of the dentate gyrus most likely represented axon terminals of transected perforant pathway fibers originating in the entorhinal cortex (Steward and Scoville '76). Seven SS-immunoreactive neurons that had dendrites in the hilar area as well as a dendrite reaching the outer molecular layer (cf. Fig. 1) were examined by electron microscopy to determine their afferent connections with the mossy axon collaterals and degenerating perforant pathway axon terminals. In our previous study of the cholinergic innervation of hilar neurons we found that the SS-immunoreactive neurons in this region are heavily innervated by mossy fiber collaterals, in addition to their cholinergic afferents (Fig. 3
C. LERANTH ET AL. in Leranth and Frotscher, '87). In the present study, using a different SS antibody, the same synaptic arrangement (Fig. 6) has again been observed on the hilar dendrites of those SS-immunoreactive neurons which also send a dendritic process to the outer molecular layer. Further ultrastructural analysis of the afferent, connections of these SS-immunoreactive dendrites in the outer molecular layer demonstrated that virtually all of the terminals that are presynaptic are degenerating (Fig. 7). The synaptic contacts formed by the degenerating perforant pathway terminals on the aspinous SS-immunoreactive dendrites were always asymmetric. However, the majority of degenerating terminals established asymmetric synapses with immunonegative spines, most likely arising from granule cell dendrites (cf. Matthews et al., '76).
DISCUSSION The purpose of this study was to obtain new information on the efferent- and particularly on the afferent connections of SS-immunoreactive neurons in the hilar region. A schematic drawing in Figure 8 shows the suggested connections of these cells. Most nongranule cells in the dentate gyrus are immunoreactive for glutamate decarboxylase (Ribak et al., '78, '81; Somogyi et al., '83, '84; Frotscher et al., '84; Lubbers et al., '85; Lubbers ann Frotscher, '87) or GABA (Storm-Mathisen et al., '83; Somogyi et al., '84; Gamrani et al., '86),and there is evidence that GABA is colocalized with neuropeptides, including SS, in many of these cells (Somogyi et al., '84; Kosaka et al., '88). Most likely these neuropeptidecontaining neurons are inhibitory because GABA has been found to exert a powerful inhibitory action in the fascia dentata (e.g., Storm-Mathisen and Ottersen, '84). Therefore, the possible functional role of the dentate gyrus SS-immunoreactive neurons can be considered as that of a special subtype of inhibitory GABA cells. This view is further supported by the observation of Pitman and Siggins ('81),who found that SS hyperpolarizes hippocampal pyramidal cells in vitro. However, there are conflicting results in the literature which will be discussed below. Confirming earlier hypotheses based on light microscopic observations of the distribution pattern of the fascia dentata SS-immunoreactive boutons (Kohler and Chan-Palay '82; Morrison et al., '82; Bakst et al., '85, '86; Sloviter and Nilaver '87), this ultrastructural study demonstrated that while a portion of SS-immunoreactive axon terminals do in fact establish symmetric synaptic contacts on hilar neurons (not shown in Fig. 8), the majority of SS-immunoreactive axon terminals connect with the distal dendrites and spines of granule cells, again forming symmetric synaptic membrane specializations (see also Milner and Bacon, '89). This is the area which is occupied by perforant pathway axons originating in the entorhinal cortex (Ram& y Cajal, 1893). However, the entorhinal fibers mainly establish asymmetric contacts on granule cell dendritic spines (Matthews et al., '76). The present study, of course, does not provide direct evidence that these SS-immunoreactive axons in the outer molecular layer are derived from the SS-positive cells located in the hilus. However, the majority of the SSimmunoreactive axons seem to arise from ipsilateral SSimmunopositive cells because kainic acid injection into the hilus (Bakst et al., '86) and sustained electric stimulation of the perforant pathway (Sloviter, '87), destroy hilar SSimmunoreactive neurons on the side of the stimulation and
DENTATE SOMATOSTATIN NEURONS
Fig. 2. Electron micrographs demonstrate symmetric synapses (arrowheads) formed by SS-immunopositive axon terminals with somata (S) of deep hilar neurons. A mossy axon collateral (M) forms an asymmetric synapse with the initial part of the dendrite of one of the hilar neurons (a).Bars = 1 fim.
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Fig. 3. Electron micrographs of the granule cell layer of the rat fascia dentata immunostained for SS. One of the SS-immunoreactive s o n s traversing this layer synapses with the soma of a granule cell (G). b is an enlargement of the synapse indicated on a. D is an SS-immunopositive dendrite. Bars = 1pm.
DENTATE SOMATOSTATIN NEURONS
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Fig. 4. Electron micrographs (a-c) demonstrate axo-dendritic symmetric synapses (arrowheads) between SS-immunopositive axon terminals and granule cell dendritic shafts (D)in the middle third of the molecular layer. Bars = 1pm.
result in a loss of SS-immunopositive fibers in the outer molecular layer. Furthermore, it is possible that a minor population of these boutons represent axon terminals of commissural SS-immunoreactive neurons located in the contralateral hilar region because some hilar SS-immunoreactive cells are retrogradely labelled following tracer injection into the contralateral hilar region (Zimmer et al., '83; Leranth and Frotscher, '87). Somatostatin-immunoreactiveneurons in the hippocampus have been considered as a subtype of inhibitory GABAergic cell because as many as 90% of the somatostatin hilar cells were found to colocalize GABA (Somogyi et al., '84; Kosaka et al., '88). Sloviter ('87), on the other hand, found that GABA-immunoreactive cells, in contrast to SS-positive cells, were not reduced in number in experimental epilepsy. In either case, our data suggest that the SS-positive neurons
act on the granule cells by establishing symmetric synapses with dendritic segments in the outer molecular layer that are preferentially contacted by entorhinal afferents (cf. Frotscher, '88). This laminar topography suggests that the SS netirons modulate the entorhinal input. Furthermore, SS-positive boutons frequently form synapses with the neck or base of the same spines that have established asymmetric synapses with immunonegative boutons, most likely of entorhinal origin (Matthews et al., '76). Such a synaptic arrangement may allow the first fine tuning of the hippocampal excitatory signal loop, by setting the threshold for the incoming entorhinal excitatory signals. If we assume that the present SS-containing cells are also GABAergic (Somogyi et al., '84; Kosaka et al., '88)' we may conclude that they serve both feed-forward and feed-back inhibitory processes in the fascia dentata (cf. Andersen, "75; Buzsiiki, '84). The
Fig. 5. Electron micrographs depicting SS-immunoreactive axons in the outer third of the molecular layer. T h e reaction product-containing axon terminals establish symmetric synapses (arrowheads) with the heads (a-d,f) or necks ( e )of dendritic spines. In many cases the same
structure postsynaptic to a SS-immunopositive houton is also a target of
another axon terminal (A in a-c) which forms a n asymmetric synaptic contact. Panel d shows a small SS-immunoreactive dendrite (D) in this superficial molecular layer. Bars = 1 pm.
DENTATE SOMATOSTATIN NEURONS
Fig. 6. Electron micrographs of the dentate hilus of a colchicine treated rat showing SS-immunopositive dendritic processes (a and b) and the soma of a SS-immunoreactive neuron ( c ) . T h e two SSimmunoreactive dendrites establish asymmetric synapses (arrowheads) with the boutons of mossy axon (M) collaterals. Note the postsynaptic complex spine of the SS immunoreactive dendrite in panel b. There are
119
several spines (S) invaginated into the mossy axon terminal (M) shown in panel c, including a somatic spine (Sl) of a SS-immunoreactive hilar area neuron. The same mossy axon terminal forms multiple asymmetric synapses (arrowheads) with a small SS-immunopositive dendrite separated by unit membranes from the SS-immunopositive soma. Bars = l hm.
Fig. 7. Electron micrographs (a-c) demonstrate numerous degenerating axon terminals (black arrows) in the outer third of the dentate molecular layer in a colchicine pretreated rat following perforant pathway transection. The majority of the degenerating boutons form asymmetric synaptic contacts with the shaft of granule cell- (Gd) or
SS-immunoreartive dendrites (D), while the others form asymmetric synapses with granule cell dendritic spines (black and white arrows). Note that virtually all of the axons that establish synaptic connections with the SS-immunoreactive dendrites are degenerated. Bars = 1 pm.
DENTATE SOMATOSTATIN NEURONS
121 parvalbumin-immunoreactive neurons which also establish synaptic contacts with entorhinal afferents (Zipp et al., '89) and mossy fiber collaterals (Nitsch, Soriano, and Frotscher, in preparation). However, these cells may be better protected against overexcitation because they contain a calciumbinding protein (Sloviter, '89; Scharfman and Schwartzkroin, '89). It is of interest in this context that only few SS-immunoreactive neurons in the hippocampus contain the calcium-binding protein parvalbumin (Kosaka et al., '88). In conclusion, this study has shown that hilar SS-positive neurons are integrated in the main excitatory pathway of the hippocampal formation. At present we can only speculate that they exert a modulatory effect on the impulse flow from the entorhinal cortex to the hippocampus.
--I-I-
SSa
ACKNOWLEDGMENTS
I
The authors thank Marya Shanabrough for expert technical assistance and Dr. C.E. Ribak for the very useful detailed criticism of the manuscript. This research was supported by NIH grant NS 26068 (C.L.).
Mac
Ma
*
Fig. 8. Schematic illustration of the suggested synaptic interconnections between entorhinal afferents (Ea), granule cells (Gc), and SSimmunoreactive neurons (SS)in the rat fascia dentata. Entorhinal afferents can exert excitatory actions (+ j on both granule cell- and SS-immunoreactive dendrites located in the upper third of the molecular layer. T h e SS-immunoreactive cells can also be stimulated (+) by collaterals (Mac) of t h e granule cell's mossy axons (Ma). Axon terminals (SSaj of t h e SS-immunoreactive hilar neurons in t urn may inhibit (-1 granule cells by both a feed-forward and a feed-back mechanism through axo-dendritic connections in t he external molecular layer.
feed-forward circuitry would be mediated by SS-immunoreactive neurons that are activated directly by entorhinal afferents terminating on their dendrites in the molecular layer. On the other hand, the feed-back loop would involve granule cells that give off collaterals which contact SSimmunoreactive neurons in the hilus (Fig. 6) and the SS axons forming synapses with the granule cell dendrites. Finally, we have demonstrated that the same SSimmunoreactive neurons are postsynaptic to entorhinal afferents and mossy axon collaterals (Fig. 8). Both of these axons are known to exert a powerful excitatory action (Andersen et al., '66, '711, probably releasing glutamate (Storm-Mathisen et al., '83) as their neurotransmitter. This involvement of the SS-immunoreactive hilar neurons into the main excitatory circuitry of the hippocampal formation (Andersen et al., '71) offers one possible explanation for their specific excitotoxicity related loss in temporal lobe epilepsy (Robbins et al., '87) and following sustained electric stimulation of the perforant pathway (Olney, '84; Sloviter, '86, '87). A single perforant pathway stimulus can doubly excite the same SS-immunoreactive neuron, first, directly by synapses on their dendrites in the outer molecular layer (Fig. 7); and secondly, the SS-immunoreactive neurons can again be activated indirectly by the same perforant pathway stimulus, within the time of one synaptic delay. This second excitatory impulse will arrive at the SS-immunoreactive cells via the mossy fiber collaterals (Fig. 8). A similar synaptic arrangement has recently been observed in dentate
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