Neuroscience Letters, 118 (1990) 33-36
33
Elsevier Scientific Publishers Ireland Ltd. NSL 07175
Increased somatostatin and enkephalin-like immunoreactivity in the rat hippocampus following hippocampal kindling B e n e d i k t e W a n s c h e r 1, J o r n K r a g h 1, D a v i d I. B a r r y 1, T o m Bolwig 1 a n d J e n s Z i m m e r 2 INeurobiology Research Group. Department of Psychiatry, Rigsho~pitalet, Copenhagen (Denmark) and 2pharmaBiotec, Institute of Neurobiology, University of Aarhus, Aarhus (Denmark)
(Received 15 December 1989; Revised version received6 June 1990; Accepted 8 June 1990) Key words: Fasciadentata; Mossy fibers; Perforant path; Cholecystokinin
As neuropeptides may play a role in the electrical kindling model of epileptogenesis, hippocampal somatostatin, Met-enkephalin and cholecystokinin were studied by immunocytochemistryin rats 24 h following full hippocampal kindling (three stage 5 seizures). As control animals we used sham-kindled rats, unoperated rats and rats subjected to a single electroshock-induced seizure. In addition, the distribution of septohippocampal, cholinergic fibers and hippocampal mossy fibers were studied by histochemistry. The important finding was that after kindling there was, as compared to unoperated control, (1) a marked increase of somatostatin immunoreactivityin cell bodies in the dentate hilus and their presumed projections area in the outer parts of the dentate molecular layer, and (2) a marked increase of Met-enkephalin immunoreactivity in hippocampal mossy fiber terminals. We found no evidenceof aberrant sprouting of mossy fiber collaterals in the fascia dentata.
Kindling is an animal model of epileptogenesis in which initially subconvulsive electrical or chemical stimulation results in progressively more intense seizure activity until the same stimulation eventually produces generalized seizures [18]. Although the mechanisms underlying kindling are still unclear, G A B A systems seem important [9, 11] and a number of neuropeptides may be involved in the process [7, 8, 12, 14, 16, 24]. Attention has focused on the role of opioid peptides and somatostatin in the induction and maintenance of kindling. Changes in hippocampal enkephalin/dynorphin and somatostatin have been reported after amygdala kindling [8, 9, 14], lidocaine-kindling [7], and after druginduced convulsions in rats [24], in seizure-sensitive gerbils [19] and after lesion- and kainic acid-induced seizures in mice [4]. Peptides derived from prodynorphin are synthesised in the granule cells of the hippocampal dentate gyrus and located in the mossy fiber pathway which arises from the granule cells [13, 15]. Mossy fiber sprouting in the dentate gyrus has also been reported after amygdala kindling [22] and in the hippocampus of epileptic humans [20]. In the present study of fully hippocampal kindled rats we looked for evidence of mossy fiber collateral sprouting using the histochemical T i m m stain, changes in cholinergic fibers using acetylcholinesterase (ACHE) histoCorrespondence: J. Zimmer, PharmaBiotec, Institute of Neurobiology, University of Aarhus, DK-8000 Aarhus C, Denmark.
0304-3940/90/$ 03.50 (~ 1990 Elsevier Scientific Publishers Ireland Ltd.
chemistry, and for changes in cholecystokinin (CCK), Met-enkephalin (m-Enk) and somatostatin (SS) using immunocytochemistry. The study was performed on male Sprague-Dawley rats (ALAB, Stockholm). Under equithesin anesthesia and using coordinates according to the stereotaxic atlas of Paxinos and Watson [17], bipolar stainless steel electrodes (o.d. 0.25 mm; SNEX-100, Rhodes Medical Instruments Inc., CA, U.S.A.) were implanted bilaterally in the caudal hippocampus (CA3: coordinates from bregma AP - 2 . 8 mm, M L 5.0 mm, DV - 6 . 4 mm) and amygdala (AP - 2 . 8 mm, M L 5.0 mm, DV - 8 . 5 mm). Unipolar silver electrodes were positioned over the hindlimb area of the cortex (AP - 1 . 0 mm, ML - 2 . 0 mm). Eight stainless steel anchor screws were screwed into the skull, the 9 electrode leads and an earth lead were connected to a 10-pin Teflon socket, and the whole embedded in dental cement so as to form a head plug. The inner surface of the scalp was rinsed with chlorhexidine and the cut surface glued to the head plug with tissue glue (Histoacryl Blue, Braun Melsungen, A.G., F.R.G.). A two week recovery period was then allowed prior to the initiation of stimulations. Kindling was induced by stimulating the rats each day for 36 days by 2 s trains of 1 ms pulses, 60 Hz, 4 ~ 1 2 0 /zA, in the left hippocampus using a constant current stimulator. The stimulation current for each rat was set at 10% above the threshold for inducing afterdischarge. The response to each stimulus was recorded by means
34 of a color video system with electroencephalographic recordings from the different brain regions superimposed on the image. The assessment of seizure grade made from the recordings using the severity scale 0-5 of Racine [18], modified to include falling on the back without prior rearing as grade 5 [3]. The rats were considered fully kindled, having had 3 grade 5 seizures (generalised clonic motor seizure). Twenty-four hours after the final stimulation, the kindled rats (n = 5) were deeply anesthetized by pentobarbital and killed by transcardial perfusion with a fixative consisting of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.3). Sham kindled rats (n=6), unoperated rats (n= 5) and rats subjected to a single electroshockinduced convulsion (alternating current for 0.3 s, 50 Hz, 50 mA, through electrodes in the earholes; n = 3) served as controls. After fixation the brains were removed and stored for 2 h in the fixative. The brains were then blocked and sectioned frontally on an Oxford vibratome at 50/~m. Sections including the hippocampus and fascia dentata were immunohistochemically stained using antibodies against CCK, SS and m-Enk as previously reported [27]. To obtain identical conditions during the immunohistological reaction, sections from normal control animals were processed together with sections from the kindled rats.
In addition, two kindled rats, one normal rat and one rat subjected to a single electroshock were perfused with a sulphide solution (5.86 g Na2S and 5.95 g Nail2_ POa'H20 in 1 litre distilled water). The brains were halved along the midline and horizontal and frontal, 30 am thick cryostat sections cut from the two hemispheres, respectively. Of the 4 or 5 parallel series of sections obtained from each hemisphere one series was stained with Thionine for ordinary cell staining, and one or two series with the histochemical Timm sulphide silver method [6, 23] in order to visualize hippocampal mossy fiber terminals. The last series was stained histochemically for acetylcholinesterase (ACHE) [5] in order to demonstrate the cholinergic, AChE-positive septohippocampal projection. There were no differences in staining between the 3 control groups, i.e. the unoperated, the sham kindled and the electroshock treated rats. In contrast the kindled rats displayed a hitherto undescribed marked and bilaterally located increase in somatostatin immunoreactivity of cell bodies in the dentate hilus (CA4) and fibers and terminal-like structures in the outer parts of the dentate molecular layer (Fig. la, b). The cellular and terminallike SS-immunoreactivity in the hippocampus proper (CA3 and CAI) was not different between the kindled and the various control rats. The kindled rats also dis-
Fig. 1. a, b: increase of somatostatin immunoreactivity in cell bodies in the dentate hilus (h) and in the perforant path zones in the outer parts of the dentate molecular layer (m) in fully kindled rat (b), compared to normal, adult rat (a). c, d: increase of met-enkephalin immunoreactivity of the hippocampal mossy fiber terminals after kindling (d) compared to normal, adult rat (c). Abbreviations: CAI, hippocampal field CAI;: CA3, hippocampal field CA3; g, granule cells of fascia dentata; h, dentate hilus (CA4); m, molecular layer of fascia dentata; mr, mossy fiber layer, × 16~6.
35 played a marked increase of m-Enk immunoreactivity in hippocampal mossy fiber terminals in the dentate hilus and the mossy fiber layer of CA3 (Fig. lc, d). Except for a slight decrease corresponding to the mossy fiber terminal fields in the dentate hilus and CA3 no differences were observed in the CCK immunoreactivity between the kindled and the control rats. The density and distribution of AChE staining appeared unaffected by kindling. The same was true for the histochemical Timm staining. There were thus no apparent changes between the kindled and the different control animals in neither the density nor the distribution of the Timm staining in the lateral perforant path zones in the molecular layer of fascia dentata and CA3, the commissural-associational zones in the inner part of the dentate molecular layer and in str. radiatum and oriens of CA3 and CA1, or the mossy fiber terminal zones in the dentate hilus and the CA3 mossy fiber layer. We in particular looked for, but did not find, any increase in Timm stained, aberrant supragranular mossy fiber terminals, which has been reported by Sutula et al. after kindling, or even nonkindling low-frequency stimulation of the perforant path [22]. Even in areas, where supragranular, mossy fibers normally do appear in small numbers, as for example near the tips of the lateral and medial blades of the dentate granule cell layer or in the so-called crest region where the two blades meet, the kindled and control rats were indistinguishable. As the increase in m-Enk and SS immunoreactivity was absent in the rats subjected to a single electroshockinduced convulsion, we assume that the increase reflects changes associated with kindling rather than an effect of the seizure per se. This is in line with previous findings after amygdala kindling [8, 12, 14]. Regarding the increase in SS-immunoreactivity observed specifically for the SS-reactive neurons in the dentate hilus and what is assumed to be their fibers and terminals in the outer parts of the dentate molecular layer [1, 2], this is to our knowledge the first immunocytochemical demonstration of such an increase at the cellular and terminal field level in relation to kindling. The SS-reactive hilar neurons are known for their particular susceptibility to cerebral ischemia [10], and seizures induced by perforant path stimulation [21]. Already within the first 24 h after transient cerebral ischemia the SS-reactive cells in the dorsal fascia dentata show signs of degeneration, including eosinophilia, positive staining with acid fuchsin and intensification and homogenization of the SS-staining in their cell bodies, and most of the cells are gone after 3~4 days [10]. As opposed to these degenerative changes, which as mentioned have also been observed after electric and kainic acid-induced seizures, there was no apparent loss of SS-reactive hilar
cells in the fully kindled rats. In contrast, the SS-immunoreactivity of both the cell bodies and their presumed fibers and terminals in the outer parts of the dentate molecular layer was greatly increased. The functional significance of the SS increase is not known at present. Neither is it known why this increase exclusively affected the dentate gyrus and not the hippocampus proper, but it is noted of course that the increase in enkephalin/dynorphin immunoreactivity of the lateral perforant path and the mossy fibers is also closely related to or involve the dentate granule cells. These cells are the primary receivers of the entorhinal perforant pathways, the major extrinsic, afferent input to the hippocampus. The granule cells convey the impulses received via this projection onto the dentate hilar neurons and the CA3 pyramidal cells via the mossy fibers. Due to their location the SS-reactive hilar neurons are likely to be activated by the mossy fibers, and by means of their own axons they can influence the activity in the dentate perforant path zones. The SS-reactive hilar neurons may accordingly play a central regulatory role both in the normal and the kindled rats, in relation to dentate seizure mechanisms. While the increased immunoreactivity for enkephalin in the rat mossy fibers observed after kainic acid or lesion-induced seizures has been shown to be associated with increased levels of m R N A for proenkephalin A [25, 26], prodynorphin m R N A have been reported to decrease in the rat dentate gyrus after kindling [15]. This shift in the expression of different peptides, including a decrease of CCK immunoreactivity corresponding to the mossy fiber zone, is discussed in detail by Gall in an experimental study on the mouse [4]. The increased cellular and axonal SS-immunoreactivity described here could be the result of an increased production of somatostatin by increased synthesis of its pro-forms and/or an increased transformation of these to the immunocytochemically detected form(s) of somatostatin. Alternatively there could be a decreased degradation of somatostatin in the kindled rats. Proper answering of these questions must await studies of the expression of somatostatin m R N A after kindling, similar to the ones performed by Gall and co-workers [4, 26] on enkephalin following seizures. This study was supported by the Danish MRC, the Lundbeck Foundation, and the Danish State Biotechnology Program. 1 Bakst, I., Avendano, C., Morrison, J.H. and Amaral, D.G.. An experimental analysis of the origins of the somatostatin immunoreactive fibers in the dentate gyrus of the rat, J. Neurosci., 6 (1986) 1452 1462.
36 2 Bakst, I., Morrison, J.H. and Amaral, D.G., The distribution of somatostatin-like immunoreactivity in the monkey hippocampal formation, J. Comp. Neurol., 236 (1985) 423-442. 3 Barry, D.I., Kikvadze, I., Brundin, P., Bolwig, T.G., Bj6rklund, A. and Lindvall, O., Grafted noradrenergic neurons suppress seizure development in kindling-induced epilepsym, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8712-8715. 4 Gall, C., Seizures induce dramatic and distinctly different changes in enkephalin, dynorphin, and CCK immunoreactivities in mouse hippocampal mossy fibers, J. Neurosci., 8 (1988) 1852-1862. 5 Geneser-Jensen, F.A., and Blackstad, T.W., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig, I. Entorhinal area, parasubiculum, and presubiculum, Z. Zellforsch., 114 (1971) 460-481. 6 Haug, F.-M.S., Heavy metals in the brain. A light microscope study of the rat with Timm's sulphide silver method. Methodological considerations and cytological and regional staining patterns, Adv. Anat. Embryol. Cell Biol., 47 (1973) I--71. 7 Higuchi, T., Shah, K.R., Sikand, G.S., West, M. and Friesen, H.G., Changes in immunoreactive somatostatin in brain following lidocaine-induced kindling in rat, Neuropharmacology, 23 (1984) 1311-1314. 8 Higuchi, T., Yamazaki, O., Takazawa, A. et al., Effects of carbamazepine and valproic acid on brain immunoreactive somatostatin and gamma-aminobutyric acid in amygdaloid-kindled rats, Eur. J. Pharmacol., 125 (1986) 169-175. 9 Itagaki, S., Uemura, S. and Kimura, H., GABAergic system and amygdaloid kindling studied by immunohistochemistry using antibody against GABA, Jpn. J. Psychiatry Neurol., 40 (1986) 341344. 10 Johansen, F.F., Zimmer, J. and Diemer, N.H., Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-I pyramidal cell loss. Acta Neuropathol., 73 (1987) 110-114. 11 Kamphuis, W., Wadman, W.J., Buijs, R.M. and Silva, F.H., Decrease in number of hippocampal gamma-aminobutyric acid (GABA) immunoreactive cells in the rat kindling model of epilepsy, Exp. Brain Res., 64 (1986) 491-495. 12 Kato, N., Higuchi, T., Friesen, H.G. and Wada, J.A., Changes of immunoreactive somatostatin and beta-endorphin content in rat brain after amygdaloid kindling, Life Sci., 32 (1983) 2415-2422. 13 McGinty, J.F., Henriksen, S.J., Goldstein, A., Terenius, L. and Bloom, F.E., Dynorphin is contained within hippocampal mossy fibers: immunochemical alterations after kainic acid administration and colchicine-induced neurotoxicity, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 589-593.
14 McGinty, J.F., Kanamatsu, T., Obie, J., Dyer, R.S., Mitchell, C.L~ and Hong, J.S., Amygdaloid kindling increases enkephalin-like immunoreactivity but decreases dynorphin-A-like immunoreactivity in rat hippocampus, Neurosci. Lett., 71 (1986) 31-36. 15 Morris, B.J., Haarmann, I., Kempter, B., Hollt, V. and Herz, A., Localization of prodynorphin messenger RNA in rat brain by in situ hybridization using a synthetic oligonucleotide probe, Neurosci. Lett., 69 (1986) 104-108. 16 Morris, B.J., Moneta, M.E., Bruggencate, G.T. and Hollt, V., Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling, Neurosci. Lett., 80 (1987) 298-302. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, London, 1986. 18 Racine, R.J., Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 19 Randall, J.L., Hong, J.S., McGinty, J.F., Lomax, P., Increased enkephalin and dynorphin immunoreactivity in hippocampus of seizure sensitive Mongolian gerbils, Brain Research, 401 (1987) 353-358. 20 Represa, A., Tremblay, E., and Ben-Ari, Y., Kainate binding sites in the hippocampal mossy fibers: localization and plasticity, Neuroscience, 20 (1987) 739-748. 21 Sloviter, R.S., Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy, Science, 235 (1987) 73--76. 22 Sutula, T., He, X.X., Cavazos, J. and Scott, G., Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science, 239 (1988) 1147-1150. 23 Timm, F., Zur Histochemie der Schwermetalle. Das Sulfid-SilberVerfahren, Dtseh. Z. ges. gerichtl. Med., 47 (1958) 428-481. 24 Vindrola, O., Asai, M., Zubieta, M. and Linares, G,, Brain content of immunoreactive [LeuS]enkephalin and [MetS]enkephalin after pentylenetetrazol-induced convulsions, Eur. J. Pharmacol., 90 (1983) 85-89. 25 White, J.D. and Gall, C.M., Differential regulation ofneuropeptide and proto-oncogene mRNA content in the hippocampus following recurrent seizures, Mol. Brain Res., 3 (1987) 21-29. 26 White, J.D., Gall, C.M. and McKelvy, J.F., Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus, J. Neurosci., 7 (1987) 753-759. 27 Zimmer, J. and Sunde, N., Neuropeptides and astroglia in intracerebral hippocampal transplants: an immunohistochemicat study in the rat, J. Comp. Neurol., 227 (1984) 331-347.
33
Elsevier Scientific Publishers Ireland Ltd. NSL 07175
Increased somatostatin and enkephalin-like immunoreactivity in the rat hippocampus following hippocampal kindling B e n e d i k t e W a n s c h e r 1, J o r n K r a g h 1, D a v i d I. B a r r y 1, T o m Bolwig 1 a n d J e n s Z i m m e r 2 INeurobiology Research Group. Department of Psychiatry, Rigsho~pitalet, Copenhagen (Denmark) and 2pharmaBiotec, Institute of Neurobiology, University of Aarhus, Aarhus (Denmark)
(Received 15 December 1989; Revised version received6 June 1990; Accepted 8 June 1990) Key words: Fasciadentata; Mossy fibers; Perforant path; Cholecystokinin
As neuropeptides may play a role in the electrical kindling model of epileptogenesis, hippocampal somatostatin, Met-enkephalin and cholecystokinin were studied by immunocytochemistryin rats 24 h following full hippocampal kindling (three stage 5 seizures). As control animals we used sham-kindled rats, unoperated rats and rats subjected to a single electroshock-induced seizure. In addition, the distribution of septohippocampal, cholinergic fibers and hippocampal mossy fibers were studied by histochemistry. The important finding was that after kindling there was, as compared to unoperated control, (1) a marked increase of somatostatin immunoreactivityin cell bodies in the dentate hilus and their presumed projections area in the outer parts of the dentate molecular layer, and (2) a marked increase of Met-enkephalin immunoreactivity in hippocampal mossy fiber terminals. We found no evidenceof aberrant sprouting of mossy fiber collaterals in the fascia dentata.
Kindling is an animal model of epileptogenesis in which initially subconvulsive electrical or chemical stimulation results in progressively more intense seizure activity until the same stimulation eventually produces generalized seizures [18]. Although the mechanisms underlying kindling are still unclear, G A B A systems seem important [9, 11] and a number of neuropeptides may be involved in the process [7, 8, 12, 14, 16, 24]. Attention has focused on the role of opioid peptides and somatostatin in the induction and maintenance of kindling. Changes in hippocampal enkephalin/dynorphin and somatostatin have been reported after amygdala kindling [8, 9, 14], lidocaine-kindling [7], and after druginduced convulsions in rats [24], in seizure-sensitive gerbils [19] and after lesion- and kainic acid-induced seizures in mice [4]. Peptides derived from prodynorphin are synthesised in the granule cells of the hippocampal dentate gyrus and located in the mossy fiber pathway which arises from the granule cells [13, 15]. Mossy fiber sprouting in the dentate gyrus has also been reported after amygdala kindling [22] and in the hippocampus of epileptic humans [20]. In the present study of fully hippocampal kindled rats we looked for evidence of mossy fiber collateral sprouting using the histochemical T i m m stain, changes in cholinergic fibers using acetylcholinesterase (ACHE) histoCorrespondence: J. Zimmer, PharmaBiotec, Institute of Neurobiology, University of Aarhus, DK-8000 Aarhus C, Denmark.
0304-3940/90/$ 03.50 (~ 1990 Elsevier Scientific Publishers Ireland Ltd.
chemistry, and for changes in cholecystokinin (CCK), Met-enkephalin (m-Enk) and somatostatin (SS) using immunocytochemistry. The study was performed on male Sprague-Dawley rats (ALAB, Stockholm). Under equithesin anesthesia and using coordinates according to the stereotaxic atlas of Paxinos and Watson [17], bipolar stainless steel electrodes (o.d. 0.25 mm; SNEX-100, Rhodes Medical Instruments Inc., CA, U.S.A.) were implanted bilaterally in the caudal hippocampus (CA3: coordinates from bregma AP - 2 . 8 mm, M L 5.0 mm, DV - 6 . 4 mm) and amygdala (AP - 2 . 8 mm, M L 5.0 mm, DV - 8 . 5 mm). Unipolar silver electrodes were positioned over the hindlimb area of the cortex (AP - 1 . 0 mm, ML - 2 . 0 mm). Eight stainless steel anchor screws were screwed into the skull, the 9 electrode leads and an earth lead were connected to a 10-pin Teflon socket, and the whole embedded in dental cement so as to form a head plug. The inner surface of the scalp was rinsed with chlorhexidine and the cut surface glued to the head plug with tissue glue (Histoacryl Blue, Braun Melsungen, A.G., F.R.G.). A two week recovery period was then allowed prior to the initiation of stimulations. Kindling was induced by stimulating the rats each day for 36 days by 2 s trains of 1 ms pulses, 60 Hz, 4 ~ 1 2 0 /zA, in the left hippocampus using a constant current stimulator. The stimulation current for each rat was set at 10% above the threshold for inducing afterdischarge. The response to each stimulus was recorded by means
34 of a color video system with electroencephalographic recordings from the different brain regions superimposed on the image. The assessment of seizure grade made from the recordings using the severity scale 0-5 of Racine [18], modified to include falling on the back without prior rearing as grade 5 [3]. The rats were considered fully kindled, having had 3 grade 5 seizures (generalised clonic motor seizure). Twenty-four hours after the final stimulation, the kindled rats (n = 5) were deeply anesthetized by pentobarbital and killed by transcardial perfusion with a fixative consisting of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.3). Sham kindled rats (n=6), unoperated rats (n= 5) and rats subjected to a single electroshockinduced convulsion (alternating current for 0.3 s, 50 Hz, 50 mA, through electrodes in the earholes; n = 3) served as controls. After fixation the brains were removed and stored for 2 h in the fixative. The brains were then blocked and sectioned frontally on an Oxford vibratome at 50/~m. Sections including the hippocampus and fascia dentata were immunohistochemically stained using antibodies against CCK, SS and m-Enk as previously reported [27]. To obtain identical conditions during the immunohistological reaction, sections from normal control animals were processed together with sections from the kindled rats.
In addition, two kindled rats, one normal rat and one rat subjected to a single electroshock were perfused with a sulphide solution (5.86 g Na2S and 5.95 g Nail2_ POa'H20 in 1 litre distilled water). The brains were halved along the midline and horizontal and frontal, 30 am thick cryostat sections cut from the two hemispheres, respectively. Of the 4 or 5 parallel series of sections obtained from each hemisphere one series was stained with Thionine for ordinary cell staining, and one or two series with the histochemical Timm sulphide silver method [6, 23] in order to visualize hippocampal mossy fiber terminals. The last series was stained histochemically for acetylcholinesterase (ACHE) [5] in order to demonstrate the cholinergic, AChE-positive septohippocampal projection. There were no differences in staining between the 3 control groups, i.e. the unoperated, the sham kindled and the electroshock treated rats. In contrast the kindled rats displayed a hitherto undescribed marked and bilaterally located increase in somatostatin immunoreactivity of cell bodies in the dentate hilus (CA4) and fibers and terminal-like structures in the outer parts of the dentate molecular layer (Fig. la, b). The cellular and terminallike SS-immunoreactivity in the hippocampus proper (CA3 and CAI) was not different between the kindled and the various control rats. The kindled rats also dis-
Fig. 1. a, b: increase of somatostatin immunoreactivity in cell bodies in the dentate hilus (h) and in the perforant path zones in the outer parts of the dentate molecular layer (m) in fully kindled rat (b), compared to normal, adult rat (a). c, d: increase of met-enkephalin immunoreactivity of the hippocampal mossy fiber terminals after kindling (d) compared to normal, adult rat (c). Abbreviations: CAI, hippocampal field CAI;: CA3, hippocampal field CA3; g, granule cells of fascia dentata; h, dentate hilus (CA4); m, molecular layer of fascia dentata; mr, mossy fiber layer, × 16~6.
35 played a marked increase of m-Enk immunoreactivity in hippocampal mossy fiber terminals in the dentate hilus and the mossy fiber layer of CA3 (Fig. lc, d). Except for a slight decrease corresponding to the mossy fiber terminal fields in the dentate hilus and CA3 no differences were observed in the CCK immunoreactivity between the kindled and the control rats. The density and distribution of AChE staining appeared unaffected by kindling. The same was true for the histochemical Timm staining. There were thus no apparent changes between the kindled and the different control animals in neither the density nor the distribution of the Timm staining in the lateral perforant path zones in the molecular layer of fascia dentata and CA3, the commissural-associational zones in the inner part of the dentate molecular layer and in str. radiatum and oriens of CA3 and CA1, or the mossy fiber terminal zones in the dentate hilus and the CA3 mossy fiber layer. We in particular looked for, but did not find, any increase in Timm stained, aberrant supragranular mossy fiber terminals, which has been reported by Sutula et al. after kindling, or even nonkindling low-frequency stimulation of the perforant path [22]. Even in areas, where supragranular, mossy fibers normally do appear in small numbers, as for example near the tips of the lateral and medial blades of the dentate granule cell layer or in the so-called crest region where the two blades meet, the kindled and control rats were indistinguishable. As the increase in m-Enk and SS immunoreactivity was absent in the rats subjected to a single electroshockinduced convulsion, we assume that the increase reflects changes associated with kindling rather than an effect of the seizure per se. This is in line with previous findings after amygdala kindling [8, 12, 14]. Regarding the increase in SS-immunoreactivity observed specifically for the SS-reactive neurons in the dentate hilus and what is assumed to be their fibers and terminals in the outer parts of the dentate molecular layer [1, 2], this is to our knowledge the first immunocytochemical demonstration of such an increase at the cellular and terminal field level in relation to kindling. The SS-reactive hilar neurons are known for their particular susceptibility to cerebral ischemia [10], and seizures induced by perforant path stimulation [21]. Already within the first 24 h after transient cerebral ischemia the SS-reactive cells in the dorsal fascia dentata show signs of degeneration, including eosinophilia, positive staining with acid fuchsin and intensification and homogenization of the SS-staining in their cell bodies, and most of the cells are gone after 3~4 days [10]. As opposed to these degenerative changes, which as mentioned have also been observed after electric and kainic acid-induced seizures, there was no apparent loss of SS-reactive hilar
cells in the fully kindled rats. In contrast, the SS-immunoreactivity of both the cell bodies and their presumed fibers and terminals in the outer parts of the dentate molecular layer was greatly increased. The functional significance of the SS increase is not known at present. Neither is it known why this increase exclusively affected the dentate gyrus and not the hippocampus proper, but it is noted of course that the increase in enkephalin/dynorphin immunoreactivity of the lateral perforant path and the mossy fibers is also closely related to or involve the dentate granule cells. These cells are the primary receivers of the entorhinal perforant pathways, the major extrinsic, afferent input to the hippocampus. The granule cells convey the impulses received via this projection onto the dentate hilar neurons and the CA3 pyramidal cells via the mossy fibers. Due to their location the SS-reactive hilar neurons are likely to be activated by the mossy fibers, and by means of their own axons they can influence the activity in the dentate perforant path zones. The SS-reactive hilar neurons may accordingly play a central regulatory role both in the normal and the kindled rats, in relation to dentate seizure mechanisms. While the increased immunoreactivity for enkephalin in the rat mossy fibers observed after kainic acid or lesion-induced seizures has been shown to be associated with increased levels of m R N A for proenkephalin A [25, 26], prodynorphin m R N A have been reported to decrease in the rat dentate gyrus after kindling [15]. This shift in the expression of different peptides, including a decrease of CCK immunoreactivity corresponding to the mossy fiber zone, is discussed in detail by Gall in an experimental study on the mouse [4]. The increased cellular and axonal SS-immunoreactivity described here could be the result of an increased production of somatostatin by increased synthesis of its pro-forms and/or an increased transformation of these to the immunocytochemically detected form(s) of somatostatin. Alternatively there could be a decreased degradation of somatostatin in the kindled rats. Proper answering of these questions must await studies of the expression of somatostatin m R N A after kindling, similar to the ones performed by Gall and co-workers [4, 26] on enkephalin following seizures. This study was supported by the Danish MRC, the Lundbeck Foundation, and the Danish State Biotechnology Program. 1 Bakst, I., Avendano, C., Morrison, J.H. and Amaral, D.G.. An experimental analysis of the origins of the somatostatin immunoreactive fibers in the dentate gyrus of the rat, J. Neurosci., 6 (1986) 1452 1462.
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