- Email: [email protected]
Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor (CRF) in the brain of a novel epileptic mutant rat, Ihara's genetically epileptic rat (IGER)
Brain Research 776 Ž1997. 255–260
Short communication
Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor Ž CRF. in the brain of a novel epileptic mutant rat, Ihara’s genetically epileptic rat Ž IGER. Yoshinari Takahashi a
a, )
, Miyuki Sadamatsu a , Hirohiko Kanai a , Akira Masui a , Shigeru Amano b , Nobuo Ihara c , Nobumasa Kato a
Department of Psychiatry, Shiga UniÕersity of Medical Science, Seta Tsukinowacho, Otsu 520-21, Japan b Department of Pathology, Shiga UniÕersity of Medical Science, Otsu 520-21, Japan c Institute of ICR Research, Tanabe 610-03, Japan Accepted 2 September 1997
Abstract Ihara’s genetically epileptic rat ŽIGER. is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures. In the present study, the possible changes of three neuropeptides, neuropeptide Y ŽNPY., somatostatin ŽSRIF. and corticotropinreleasing factor ŽCRF., in the brains of IGER were investigated. Increased contents of immunoreactive ŽIR. NPY were found only in the hippocampus of 2-month IGERs before developing convulsive seizures, while similar increases of IR-NPY were discovered in the striatum and pyriform and entorhinal cortex as well as hippocampus in 8-month IGERs with repetitive seizures. There were no significant differences in the brain contents of IR-SRIF and IR-CRF between IGERs and the controls at both ages. These findings indicate an enhanced rate of NPY synthesis in this experimental model of epilepsy which may play a critical role in the development of epileptogenesis. q 1997 Elsevier Science B.V. Keywords: Ihara’s genetically epileptic rat; Neuropeptide Y; Somatostatin; Corticotropin-releasing factor; Epilepsy
Ihara’s genetically epileptic rat ŽIGER. is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures ŽFig. 1. w1x. Spontaneous generalized convulsions develop in almost all male rats starting from 4 to 5 months of age without any external stimuli, and the frequency of the seizures increased with aging. The seizures usually begin with face and head myoclonus, followed by rearing and generalized tonic and clonic seizures ŽGTCS.. Microdysgenesis, such as abnormal neuronal clustering, neuronal disarrangement or interruption of pyramidal neurons in the hippocampal formation, was found in the young rats without GTCS, indicating this microdysgenesis to be genetically determined. The mossy fiber sprouting in the dentate gyrus ŽDG. was obvious in the hippocampus of aged IGER after repetitive GTCS. Electrographic recording during GTCS demonstrated that sustained spike discharges emerged at the hippocampus and then propagated to the neocortex w1x.
)
Corresponding author. Fax: q81 Ž775. 439698; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 1 1 9 - 0
Recently, changes in several neuropeptides have been found in the brains of rats with seizure susceptibility. A possible causative role of neuropeptide Y ŽNPY. in animal epilepsy has especially received a considerable interest in numerous recent publications w12–14,19–21,23x. The kainate-induced limbic seizures w14x or electrically induced status epilepticus w20x have been reported to cause a longlasting elevation of immunoreactive ŽIR. NPY, especially noted in the hippocampus. In the chronic stage after kainate-induced seizures and pentylenetetrazol ŽPTZ. kindling, pronounced increases of NPY immunoreactivity and prepro-NPY mRNA have been found in the DG including hippocampal mossy fibers and their sprouting w12x. It is thus suggested that enhanced biosynthesis of NPY in the hippocampus may contribute to the elevated seizure susceptibility in these animal models of epilepsy. The finding that amygdaloid kindling resulted in the elevation of IR-somatostatin ŽSRIF. contents in several brain regions w9x was further confirmed with audiogenic seizure-susceptible rats w10x and PTZ kindled rats w18x. Corticotropin-releasing factor ŽCRF. was originally identified as a hypothalamic stimulator of corticotropin secretion
256
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
and subsequently shown to produce a potent behavioral activation and excitation of hippocampal pyramidal cells w24x. In addition, it was reported that the intraventricular injection of CRF induced spontaneous seizure activity in rats similar to amygdaloid kindling w4x. In the present study, the possible changes of these three neuropeptides, NPY, SRIF and CRF, were investigated in the brains of IGERs at two different ages: 2 months of age without spontaneous seizures and 8 months of age with repetitive seizures. IGERs were bred in the Department of Pathology,
Shiga University of Medical Science, as reported previously w1x. Seven male IGERs at 2 months of age Žbefore seizure development. and 4 male IGERs at 8 months of age Žafter seizures developed. were used in the present study. Male IGER and female Wistar rats were mixbred ŽF-1., and seven male offspring at 2 months and four male offspring at 8 months served as controls. There was no difference in body weight between young IGERs Ž245 " 15 g; mean " SD. and young controls Ž227 " 19 g., and between old IGERs Ž475 " 43 g. and old controls Ž463 " 29 g..
Fig. 1. A: male IGER at 8 months. B1: forelimb clonus with rearing. B2–B5: generalized tonic and clonic seizure. B6: rigid posture after clonic convulsion.
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
The rats were killed by decapitation. The brains were quickly removed and dissected on ice into the striatum, amygdala, hypothalamus, frontal cortex, hippocampus and pyriform and entorhinal cortex according to the method of Glowinski and Iversen w6x with slight modifications w9,17x. Each brain region was weighed, homogenized by sonication in 2.0 N acetic acid, boiled for 10 min and centrifuged at 15 000 rpm for 30 min. Aliquots of the supernatant were lyophilized and stored at y208C until the assay. Each extract was then subjected to radioimmunoassay for each peptide. The measured immunoreactive values for peptides were expressed in ngrg wet weight Žw.wt... Authentic peptides and antibodies against NPY and CRF were purchased from Peninsula Labs., Belmont, CA, USA, and the radioactive tracers; 125 I-labeled NPY and 125 I-labeled CRF from Amersham Japan. The antiserum against NPY shows cross-reactivity with peptide YY which is not present in the rat brain but none with other neuropeptides. The antiserum against CRF shows no cross-reactivity with other neuropeptides. The measurement of SRIF was described previously w10,17x. Antiserum against SRIF crossreacted 50% with synthetic somatostatin-28 at a molar basis. In the separate experiment, gel-filtration profiles of rat brain extract revealed that the contribution of a peak co-eluting with somatostatin-28 was less than 10% as compared to that with somatostatin-14. The bound tracer was separated from the free tracer by double-antibody
257
method. All comparisons were performed within the same assay. Statistical analyses were performed by SPSS program ŽSPSS Japan Inc... The statistical significance of differences in peptide concentrations between different groups of rats was evaluated by 2-way ANOVA, 1-way ANOVA and multiple comparison ŽDuncan’s method.. Significant differences were taken as those with a P value less than 0.05. Fig. 2 shows IR-NPY contents in each brain region in the four groups. IR-NPY contents of 2-month IGERs were increased significantly only in the hippocampus, in comparison with those of 2-month controls Ž P - 0.05.. In 8-month IGERs, marked increments of IR-NPY contents were observed in the striatum, hippocampus, and pyriform and entorhinal cortex Ž P - 0.01.. Fig. 3 shows the contents of IR-SRIF and CRF in each brain region. Brain contents of IR-SRIF and CRF in both 2- and 8-month IGERs failed to show significant difference in comparison with those in the corresponding controls. The effect of aging was found in both peptides in several brain regions: IR-SRIF was increased and IR-CRF decreased along with aging Ž P - 0.01.. IGER provides a novel rat mutant with a unique combination of spontaneous GTCS, hippocampal microdysgenesis and enhanced excitability in the hippocampus as evidenced by depth electroencephalography. Although the
Fig. 2. IR-NPY contents Žngrg w.wt.. in the brain regions of F-1 controls at 2 months ŽF-1 young., IGERs at 2 months ŽIGER young., F-1 controls at 8 months ŽF-1 old. and IGERs at 8 months ŽIGER old.. Each column shows the value as mean" S.D. A 2-way ANOVA indicated a significant interactions between strain and age in the striatum, hippocampus and pyriform and entorhinal cortex. Duncan’s multiple comparison tests indicated statistically significant differences between IGERs at 8 months and all other groups in these regions Ž ) ) P - 0.01., and between IGERs at 2 months and F-1 controls at 2 months in the hippocampus Ž ) P - 0.05..
258
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
Fig. 3. IR-SRIF and IR-CRF contents Žngrg w.wt.. in the brain regions of F-1 controls at 2 months ŽF-1 young., IGERs at 2 months ŽIGER young., F-1 controls at 8 months ŽF-1 old. and IGERs at 8 months ŽIGER old.; see legend of Fig. 2 for cross-hatching. Data are shown as mean" S.D. A 2-way ANOVA found the main effect of age to be significant Ž ) ) P - 0.01. and no interactions in all regions except pyriform and entorhinal cortex ŽIR-SRIF. and in all regions except hypothalamus ŽIR-CRF..
original strain of this new inbred epileptic mutant was previously thought to be a Wistar rat strain, genome analysis using 24 microsatellite markers revealed that more than 16 loci Ž67%. in this mutant had alleles different from those in Wistar rats. Therefore, this epileptic strain is no longer considered a substrain of the Wistar rat and now the original strain is undefined. Because the first filial generation ŽF-1. between male epileptic rats and female Wistar rats neither showed obvious convulsive seizures nor microdysgenesis in the hippocampal formation w1x, agematched male rats of F-1 served as controls. The biochemical changes observed in IGER as compared to F-1 rats may thus contribute to the neuronal processes underlying the development of spontaneous GTCS. Increased contents of IR-NPY were found only in the hippocampus of 2-month IGERs without seizures, while these changes extended to the striatum, pyriform and entorhinal cortex as well as hippocampus, less prominently to amygdala, in 8-month IGERs with repetitive seizures. The microdysgenesis observed in the hippocampus of IGER may imply the presence of a deficit of the geneŽs. regulating the proliferation andror migration of the neurons during the developmental stage w1x. Although depth electroencephalography at the earlier stage of development is not available, initial spike-wave bursts always generate in
the hippocampus, proceeding to the neocortex when recorded at the later stage in this model w1x. Therefore, these electrographic findings may suggest that the genetically determined abnormality in the hippocampus provides the origin of epileptogenesis, in which the elevated NPY may have some causative roles. Along with aging, IGER developed repetitive GTCS and the histochemical studies have shown the astrogliosis in the cerebral cortex, amygdala and hippocampus as well as the mossy fiber sprouting in the DG in 8-month IGER w1x. It may be likely that the development of these histological changes parallels change of NPY contents in the hippocampus and related regions. In kainate-treated rats, Marksteiner et al. w12x reported that NPY progressively accumulated in the hilus and CA3 and, at later intervals, extended to the supragranular molecular layer of the DG indicating sprouting of mossy fibers, and the authors emphasized the important role of NPY in the development of limbic seizures. It was also described that a selective antibody raised against NPY infused bilaterally into the dorsal hippocampal CA3rDG of kainate-treated rats significantly reduced the number of animals with GTCS w23x. Recently, a mutant mice in which the NPY gene was disrupted in embryonic stem cells was developed, and these mice lacking NPY mRNA in the brain were reported
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
to be seizure susceptible w5x. Although this may imply a causative relationship of NPY with abnormal excitability in the central nervous system, the proposed suppressive action of NPY appears contradictory to the present results that the enhanced synthesis of NPY in the brain induces seizure vulnerability. However, NPY is known to affect synaptic transmission differently depending on the brain region. Whereas NPY exerts an inhibitory influence on the Schaffer collateral-CA1 synapsis w3x, the peptide facilitates NMDA-induced excitation on CA3 pyramidal cells w16x or inhibits depolarization-induced increases in intracellular Ca2q concentrations in granule cells in the DG w15x. Furthermore, NPY may play a different role in different regions. Hypothalamic NPY will be important in the feeding behavior, while the same peptide may be crucial to epileptogenesis in the hippocampus. In IGER, hypothalamic NPY was found to be at the same level as in F-1 controls. It will be of interest to determine whether changes in NPY expression induced by antibodies or antisense nucleotides alter the development of GTCS in this genetic model of limbic seizures. In contrast to the extensively studied findings on the participation of NPY and SRIF in the kindling models, the changes of neuropeptides in genetic models of epilepsy have rarely been investigated. The spontaneously epileptic rat ŽSER., a mutant homozygous for both zitter and tremor genes, exhibits absence-like seizures and GTCS without external stimulation from 7 to 8 weeks of age w7x. We reported previously that the robust increases of IR-NPY were observed in the striatum, amygdala, hippocampus and mesolimbic systems including the pyriform and entorhinal cortex except hypothalamus in homozygous compared to heterozygous SER w19x. Since homozygous SER with GTCS and heterozygous SER without GTCS are genetically close, the observed changes in brain NPY contents have been proposed to reflect the difference in the phenotypical manifestation of GTCS w19x. Regardless of the strain difference, these data are strikingly similar to those in IGER. Thus, we indicate the possibility that the increased NPY contents in the hippocampus and related structures reflect the abnormal excitability, resulting in enhanced seizure susceptibility. It has been reported that hippocampal CA3 neurons in SER become abnormally excitable in conjunction with the development of epileptic seizures w8x. IR-NPY was found to be increased in the striatum besides the hippocampus and related limbic structures in 8-month IGER. As the striatum is far from the locus underlying seizure susceptibility, this finding seems not to be readily explained. However, it might be of interest that the similar increase of NPY contents in the striatum was also found in homozygous SER as compared to heterozygous SER w19x. We failed to detect the difference of IR-SRIF and IR-CRF between IGERs and controls, while there was found the effect of aging in both peptides in several brain regions. SRIF has been frequently implicated in pathologi-
259
cal aging such as in Alzheimer’s disease w2x; however, the previous reports are still controversial as to age-related changes of SRIF in the rat brain w11,22x. The possible roles of these neuropeptides in relation to the epileptogenesis versus aging processes might deserve further investigations because the frequency of GTCS in both IGER and SER is known to steadily increase along with age.
Acknowledgements This study was supported by the Research Grant Ž7A-1. for Nervous and Mental Disorders to N.K. from the Ministry of Health and Welfare, and the Grant-in-Aid for Scientific Research Žc.-867104 to N.K. from the Ministry of Education, Science, Sports and Culture, Japan.
References w1x S. Amano, N. Ihara, S. Uemura, M. Yokoyama, M. Ikeda, T. Serikawa, M. Sasahara, H. Kataoka, Y. Hayase, F. Hazama, Development of a novel rat mutant with spontaneous limbic-like seizures, Am. J. Pathol. 149 Ž1996. 329–336. w2x J.R. Atack, M.F. Beal, C. May, J.A. Kaye, M.F. Mazurek, A.D. Kay, S.I. Rapoport, Cerebrospinal fluid somatostatin and neuropeptide Y. Concentrations in aging and in dementia of the Alzheimer type with and without extrapyramidal signs, Arch. Neurol. 45 Ž1988. 269–274. w3x W.F. Colmers, K. Lukowiak, Q.J. Pittman, Neuropeptide Y action in the rat hippocampal slice: site and mechanism of presynaptic inhibition, J. Neurosci. 8 Ž1988. 3827–3837. w4x C.L. Ehlers, S.J. Henriksen, M. Wang, J. Rivier, W. Vale, F.E. Bloom, Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats, Brain Res. 278 Ž1983. 332–336. w5x J.C. Erickson, K.E. Clegg, R.D. Palmiter, Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y, Nature 381 Ž1996. 415–421. w6x J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain: I. The disposition of w3Hxnorepinephrine, w3Hxdopamine and w3Hxdopa in various regions of the brain, J. Neurochem. 13 Ž1966. 655–669. w7x T. Inui, T. Yamamura, H. Yuasa, Y. Kawai, A. Okaniwa, T. Serikawa, J. Yamada, The spontaneously epileptic rat ŽSER., a zitter ) tremor double mutant rat: histopathological findings in the central nervous system, Brain Res. 517 Ž1990. 123–133. w8x K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats ŽSER., a double mutant, Exp. Neurol. 119 Ž1993. 287–290. w9x N. Kato, T. Higuchi, H.G. Friesen, J.A. Wada, Changes of immunoreactive somatostatin and beta-endorphin content in rat brain after amygdaloid kindling, Life Sci. 32 Ž1983. 2415–2422. w10x N. Kato, V.C. Sundmark, L. Van Middlesworth, V. Havlicek, H.G. Friesen, Immunoreactive somatostatin and beta-endorphin content in the brain of mature rats after neonatal exposure to propylthiouracil, Endocrinology 110 Ž1982. 1851–1855. w11x C. Kowalski, J. Micheau, R. Corder, R. Gaillard, B. Conte Devolx, Age-related changes in corticotropin-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and beta-endorphin in specific rat brain areas, Brain Res. 582 Ž1992. 38–46. w12x J. Marksteiner, M. Ortler, R. Bellmann, G. Sperk, Neuropeptide Y
260
w13x
w14x
w15x
w16x
w17x
w18x
Y. Takahashi et al.r Brain Research 776 (1997) 255–260 biosynthesis is markedly induced in mossy fibers during temporal lobe epilepsy of the rat, Neurosci. Lett. 112 Ž1990. 143–148. J. Marksteiner, G. Sperk, Concomitant increase of somatostatin, neuropeptide Y and glutamate decarboxylase in the frontal cortex of rats with decreased seizure threshold, Neuroscience 26 Ž1988. 379– 385. J. Marksteiner, G. Sperk, D. Maas, Differential increases in brain levels of neuropeptide Y and vasoactive intestinal polypeptide after kainic acid-induced seizures in the rat, Naunyn. Schmiedebergs Arch. Pharmacol. 339 Ž1989. 173–177. A.R. McQuiston, J.J. Petrozzino, J.A. Connor, W.F. Colmers, Neuropeptide Y1 receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells, J. Neurosci. 16 Ž1996. 1422–1429. F.P. Monnet, A. Fournier, G. Debonnel, C. de Montigny, Neuropeptide Y potentiates selectively the N-methyl-D-aspartate response in the rat CA3 dorsal hippocampus: I. Involvement of an atypical neuropeptide Y receptor, J. Pharmacol. Exp. Ther. 263 Ž1992. 1212–1218. S. Nagaki, N. Kato, Y. Minatogawa, T. Higuchi, Effects of anticonvulsants and gamma-aminobutyric acid ŽGABA.-mimetic drugs on immunoreactive somatostatin and GABA contents in the rat brain, Life Sci. 46 Ž1990. 1587–1595. A. Pitkanen, M.F. Beal, J. Sirvio, K.J. Swartz, P.T. Mannisto, P.J.
w19x
w20x
w21x
w22x
w23x
w24x
Riekkinen, Somatostatin, neuropeptide Y, GABA and cholinergic enzymes in brain of pentylenetetrazol-kindled rats, Neuropeptides 14 Ž1989. 197–207. M. Sadamatsu, H. Kanai, A. Masui, T. Serikawa, J. Yamada, M. Sasa, N. Kato, Altered brain contents of neuropeptides in spontaneously epileptic rats ŽSER. and tremor rats with absence seizures, Life Sci. 57 Ž1995. 523–531. C. Schwarzer, J.M. Williamson, E.W. Lothman, A. Vezzani, G. Sperk, Somatostatin, neuropeptide Y, neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy, Neuroscience 69 Ž1995. 831–845. C. Stenfors, P. Bjellerup, A.A. Mathe, E. Theodorsson, Concurrent analysis of neuropeptides and biogenic amines in brain tissue of rats treated with electroconvulsive stimuli, Brain Res. 698 Ž1995. 39–45. J.W. Unger, Y. Schmidt, Neuropeptide Y and somatostatin in the neocortex of young and aging rats: response to nucleus basalis lesions, J. Chem. Neuroanat. 7 Ž1994. 25–34. A. Vezzani, G. Civenni, M. Rizzi, A. Monno, S. Messali, R. Samanin, Enhanced neuropeptide Y release in the hippocampus is associated with chronic seizure susceptibility in kainic acid treated rats, Brain Res. 660 Ž1994. 138–143. S.R. Weiss, R.M. Post, P.W. Gold, G. Chrousos, T.L. Sullivan, D. Walker, A. Pert, CRF-induced seizures and behavior: interaction with amygdala kindling, Brain Res. 372 Ž1986. 345–351.
Short communication
Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor Ž CRF. in the brain of a novel epileptic mutant rat, Ihara’s genetically epileptic rat Ž IGER. Yoshinari Takahashi a
a, )
, Miyuki Sadamatsu a , Hirohiko Kanai a , Akira Masui a , Shigeru Amano b , Nobuo Ihara c , Nobumasa Kato a
Department of Psychiatry, Shiga UniÕersity of Medical Science, Seta Tsukinowacho, Otsu 520-21, Japan b Department of Pathology, Shiga UniÕersity of Medical Science, Otsu 520-21, Japan c Institute of ICR Research, Tanabe 610-03, Japan Accepted 2 September 1997
Abstract Ihara’s genetically epileptic rat ŽIGER. is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures. In the present study, the possible changes of three neuropeptides, neuropeptide Y ŽNPY., somatostatin ŽSRIF. and corticotropinreleasing factor ŽCRF., in the brains of IGER were investigated. Increased contents of immunoreactive ŽIR. NPY were found only in the hippocampus of 2-month IGERs before developing convulsive seizures, while similar increases of IR-NPY were discovered in the striatum and pyriform and entorhinal cortex as well as hippocampus in 8-month IGERs with repetitive seizures. There were no significant differences in the brain contents of IR-SRIF and IR-CRF between IGERs and the controls at both ages. These findings indicate an enhanced rate of NPY synthesis in this experimental model of epilepsy which may play a critical role in the development of epileptogenesis. q 1997 Elsevier Science B.V. Keywords: Ihara’s genetically epileptic rat; Neuropeptide Y; Somatostatin; Corticotropin-releasing factor; Epilepsy
Ihara’s genetically epileptic rat ŽIGER. is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures ŽFig. 1. w1x. Spontaneous generalized convulsions develop in almost all male rats starting from 4 to 5 months of age without any external stimuli, and the frequency of the seizures increased with aging. The seizures usually begin with face and head myoclonus, followed by rearing and generalized tonic and clonic seizures ŽGTCS.. Microdysgenesis, such as abnormal neuronal clustering, neuronal disarrangement or interruption of pyramidal neurons in the hippocampal formation, was found in the young rats without GTCS, indicating this microdysgenesis to be genetically determined. The mossy fiber sprouting in the dentate gyrus ŽDG. was obvious in the hippocampus of aged IGER after repetitive GTCS. Electrographic recording during GTCS demonstrated that sustained spike discharges emerged at the hippocampus and then propagated to the neocortex w1x.
)
Corresponding author. Fax: q81 Ž775. 439698; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 1 1 9 - 0
Recently, changes in several neuropeptides have been found in the brains of rats with seizure susceptibility. A possible causative role of neuropeptide Y ŽNPY. in animal epilepsy has especially received a considerable interest in numerous recent publications w12–14,19–21,23x. The kainate-induced limbic seizures w14x or electrically induced status epilepticus w20x have been reported to cause a longlasting elevation of immunoreactive ŽIR. NPY, especially noted in the hippocampus. In the chronic stage after kainate-induced seizures and pentylenetetrazol ŽPTZ. kindling, pronounced increases of NPY immunoreactivity and prepro-NPY mRNA have been found in the DG including hippocampal mossy fibers and their sprouting w12x. It is thus suggested that enhanced biosynthesis of NPY in the hippocampus may contribute to the elevated seizure susceptibility in these animal models of epilepsy. The finding that amygdaloid kindling resulted in the elevation of IR-somatostatin ŽSRIF. contents in several brain regions w9x was further confirmed with audiogenic seizure-susceptible rats w10x and PTZ kindled rats w18x. Corticotropin-releasing factor ŽCRF. was originally identified as a hypothalamic stimulator of corticotropin secretion
256
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
and subsequently shown to produce a potent behavioral activation and excitation of hippocampal pyramidal cells w24x. In addition, it was reported that the intraventricular injection of CRF induced spontaneous seizure activity in rats similar to amygdaloid kindling w4x. In the present study, the possible changes of these three neuropeptides, NPY, SRIF and CRF, were investigated in the brains of IGERs at two different ages: 2 months of age without spontaneous seizures and 8 months of age with repetitive seizures. IGERs were bred in the Department of Pathology,
Shiga University of Medical Science, as reported previously w1x. Seven male IGERs at 2 months of age Žbefore seizure development. and 4 male IGERs at 8 months of age Žafter seizures developed. were used in the present study. Male IGER and female Wistar rats were mixbred ŽF-1., and seven male offspring at 2 months and four male offspring at 8 months served as controls. There was no difference in body weight between young IGERs Ž245 " 15 g; mean " SD. and young controls Ž227 " 19 g., and between old IGERs Ž475 " 43 g. and old controls Ž463 " 29 g..
Fig. 1. A: male IGER at 8 months. B1: forelimb clonus with rearing. B2–B5: generalized tonic and clonic seizure. B6: rigid posture after clonic convulsion.
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
The rats were killed by decapitation. The brains were quickly removed and dissected on ice into the striatum, amygdala, hypothalamus, frontal cortex, hippocampus and pyriform and entorhinal cortex according to the method of Glowinski and Iversen w6x with slight modifications w9,17x. Each brain region was weighed, homogenized by sonication in 2.0 N acetic acid, boiled for 10 min and centrifuged at 15 000 rpm for 30 min. Aliquots of the supernatant were lyophilized and stored at y208C until the assay. Each extract was then subjected to radioimmunoassay for each peptide. The measured immunoreactive values for peptides were expressed in ngrg wet weight Žw.wt... Authentic peptides and antibodies against NPY and CRF were purchased from Peninsula Labs., Belmont, CA, USA, and the radioactive tracers; 125 I-labeled NPY and 125 I-labeled CRF from Amersham Japan. The antiserum against NPY shows cross-reactivity with peptide YY which is not present in the rat brain but none with other neuropeptides. The antiserum against CRF shows no cross-reactivity with other neuropeptides. The measurement of SRIF was described previously w10,17x. Antiserum against SRIF crossreacted 50% with synthetic somatostatin-28 at a molar basis. In the separate experiment, gel-filtration profiles of rat brain extract revealed that the contribution of a peak co-eluting with somatostatin-28 was less than 10% as compared to that with somatostatin-14. The bound tracer was separated from the free tracer by double-antibody
257
method. All comparisons were performed within the same assay. Statistical analyses were performed by SPSS program ŽSPSS Japan Inc... The statistical significance of differences in peptide concentrations between different groups of rats was evaluated by 2-way ANOVA, 1-way ANOVA and multiple comparison ŽDuncan’s method.. Significant differences were taken as those with a P value less than 0.05. Fig. 2 shows IR-NPY contents in each brain region in the four groups. IR-NPY contents of 2-month IGERs were increased significantly only in the hippocampus, in comparison with those of 2-month controls Ž P - 0.05.. In 8-month IGERs, marked increments of IR-NPY contents were observed in the striatum, hippocampus, and pyriform and entorhinal cortex Ž P - 0.01.. Fig. 3 shows the contents of IR-SRIF and CRF in each brain region. Brain contents of IR-SRIF and CRF in both 2- and 8-month IGERs failed to show significant difference in comparison with those in the corresponding controls. The effect of aging was found in both peptides in several brain regions: IR-SRIF was increased and IR-CRF decreased along with aging Ž P - 0.01.. IGER provides a novel rat mutant with a unique combination of spontaneous GTCS, hippocampal microdysgenesis and enhanced excitability in the hippocampus as evidenced by depth electroencephalography. Although the
Fig. 2. IR-NPY contents Žngrg w.wt.. in the brain regions of F-1 controls at 2 months ŽF-1 young., IGERs at 2 months ŽIGER young., F-1 controls at 8 months ŽF-1 old. and IGERs at 8 months ŽIGER old.. Each column shows the value as mean" S.D. A 2-way ANOVA indicated a significant interactions between strain and age in the striatum, hippocampus and pyriform and entorhinal cortex. Duncan’s multiple comparison tests indicated statistically significant differences between IGERs at 8 months and all other groups in these regions Ž ) ) P - 0.01., and between IGERs at 2 months and F-1 controls at 2 months in the hippocampus Ž ) P - 0.05..
258
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
Fig. 3. IR-SRIF and IR-CRF contents Žngrg w.wt.. in the brain regions of F-1 controls at 2 months ŽF-1 young., IGERs at 2 months ŽIGER young., F-1 controls at 8 months ŽF-1 old. and IGERs at 8 months ŽIGER old.; see legend of Fig. 2 for cross-hatching. Data are shown as mean" S.D. A 2-way ANOVA found the main effect of age to be significant Ž ) ) P - 0.01. and no interactions in all regions except pyriform and entorhinal cortex ŽIR-SRIF. and in all regions except hypothalamus ŽIR-CRF..
original strain of this new inbred epileptic mutant was previously thought to be a Wistar rat strain, genome analysis using 24 microsatellite markers revealed that more than 16 loci Ž67%. in this mutant had alleles different from those in Wistar rats. Therefore, this epileptic strain is no longer considered a substrain of the Wistar rat and now the original strain is undefined. Because the first filial generation ŽF-1. between male epileptic rats and female Wistar rats neither showed obvious convulsive seizures nor microdysgenesis in the hippocampal formation w1x, agematched male rats of F-1 served as controls. The biochemical changes observed in IGER as compared to F-1 rats may thus contribute to the neuronal processes underlying the development of spontaneous GTCS. Increased contents of IR-NPY were found only in the hippocampus of 2-month IGERs without seizures, while these changes extended to the striatum, pyriform and entorhinal cortex as well as hippocampus, less prominently to amygdala, in 8-month IGERs with repetitive seizures. The microdysgenesis observed in the hippocampus of IGER may imply the presence of a deficit of the geneŽs. regulating the proliferation andror migration of the neurons during the developmental stage w1x. Although depth electroencephalography at the earlier stage of development is not available, initial spike-wave bursts always generate in
the hippocampus, proceeding to the neocortex when recorded at the later stage in this model w1x. Therefore, these electrographic findings may suggest that the genetically determined abnormality in the hippocampus provides the origin of epileptogenesis, in which the elevated NPY may have some causative roles. Along with aging, IGER developed repetitive GTCS and the histochemical studies have shown the astrogliosis in the cerebral cortex, amygdala and hippocampus as well as the mossy fiber sprouting in the DG in 8-month IGER w1x. It may be likely that the development of these histological changes parallels change of NPY contents in the hippocampus and related regions. In kainate-treated rats, Marksteiner et al. w12x reported that NPY progressively accumulated in the hilus and CA3 and, at later intervals, extended to the supragranular molecular layer of the DG indicating sprouting of mossy fibers, and the authors emphasized the important role of NPY in the development of limbic seizures. It was also described that a selective antibody raised against NPY infused bilaterally into the dorsal hippocampal CA3rDG of kainate-treated rats significantly reduced the number of animals with GTCS w23x. Recently, a mutant mice in which the NPY gene was disrupted in embryonic stem cells was developed, and these mice lacking NPY mRNA in the brain were reported
Y. Takahashi et al.r Brain Research 776 (1997) 255–260
to be seizure susceptible w5x. Although this may imply a causative relationship of NPY with abnormal excitability in the central nervous system, the proposed suppressive action of NPY appears contradictory to the present results that the enhanced synthesis of NPY in the brain induces seizure vulnerability. However, NPY is known to affect synaptic transmission differently depending on the brain region. Whereas NPY exerts an inhibitory influence on the Schaffer collateral-CA1 synapsis w3x, the peptide facilitates NMDA-induced excitation on CA3 pyramidal cells w16x or inhibits depolarization-induced increases in intracellular Ca2q concentrations in granule cells in the DG w15x. Furthermore, NPY may play a different role in different regions. Hypothalamic NPY will be important in the feeding behavior, while the same peptide may be crucial to epileptogenesis in the hippocampus. In IGER, hypothalamic NPY was found to be at the same level as in F-1 controls. It will be of interest to determine whether changes in NPY expression induced by antibodies or antisense nucleotides alter the development of GTCS in this genetic model of limbic seizures. In contrast to the extensively studied findings on the participation of NPY and SRIF in the kindling models, the changes of neuropeptides in genetic models of epilepsy have rarely been investigated. The spontaneously epileptic rat ŽSER., a mutant homozygous for both zitter and tremor genes, exhibits absence-like seizures and GTCS without external stimulation from 7 to 8 weeks of age w7x. We reported previously that the robust increases of IR-NPY were observed in the striatum, amygdala, hippocampus and mesolimbic systems including the pyriform and entorhinal cortex except hypothalamus in homozygous compared to heterozygous SER w19x. Since homozygous SER with GTCS and heterozygous SER without GTCS are genetically close, the observed changes in brain NPY contents have been proposed to reflect the difference in the phenotypical manifestation of GTCS w19x. Regardless of the strain difference, these data are strikingly similar to those in IGER. Thus, we indicate the possibility that the increased NPY contents in the hippocampus and related structures reflect the abnormal excitability, resulting in enhanced seizure susceptibility. It has been reported that hippocampal CA3 neurons in SER become abnormally excitable in conjunction with the development of epileptic seizures w8x. IR-NPY was found to be increased in the striatum besides the hippocampus and related limbic structures in 8-month IGER. As the striatum is far from the locus underlying seizure susceptibility, this finding seems not to be readily explained. However, it might be of interest that the similar increase of NPY contents in the striatum was also found in homozygous SER as compared to heterozygous SER w19x. We failed to detect the difference of IR-SRIF and IR-CRF between IGERs and controls, while there was found the effect of aging in both peptides in several brain regions. SRIF has been frequently implicated in pathologi-
259
cal aging such as in Alzheimer’s disease w2x; however, the previous reports are still controversial as to age-related changes of SRIF in the rat brain w11,22x. The possible roles of these neuropeptides in relation to the epileptogenesis versus aging processes might deserve further investigations because the frequency of GTCS in both IGER and SER is known to steadily increase along with age.
Acknowledgements This study was supported by the Research Grant Ž7A-1. for Nervous and Mental Disorders to N.K. from the Ministry of Health and Welfare, and the Grant-in-Aid for Scientific Research Žc.-867104 to N.K. from the Ministry of Education, Science, Sports and Culture, Japan.
References w1x S. Amano, N. Ihara, S. Uemura, M. Yokoyama, M. Ikeda, T. Serikawa, M. Sasahara, H. Kataoka, Y. Hayase, F. Hazama, Development of a novel rat mutant with spontaneous limbic-like seizures, Am. J. Pathol. 149 Ž1996. 329–336. w2x J.R. Atack, M.F. Beal, C. May, J.A. Kaye, M.F. Mazurek, A.D. Kay, S.I. Rapoport, Cerebrospinal fluid somatostatin and neuropeptide Y. Concentrations in aging and in dementia of the Alzheimer type with and without extrapyramidal signs, Arch. Neurol. 45 Ž1988. 269–274. w3x W.F. Colmers, K. Lukowiak, Q.J. Pittman, Neuropeptide Y action in the rat hippocampal slice: site and mechanism of presynaptic inhibition, J. Neurosci. 8 Ž1988. 3827–3837. w4x C.L. Ehlers, S.J. Henriksen, M. Wang, J. Rivier, W. Vale, F.E. Bloom, Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats, Brain Res. 278 Ž1983. 332–336. w5x J.C. Erickson, K.E. Clegg, R.D. Palmiter, Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y, Nature 381 Ž1996. 415–421. w6x J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain: I. The disposition of w3Hxnorepinephrine, w3Hxdopamine and w3Hxdopa in various regions of the brain, J. Neurochem. 13 Ž1966. 655–669. w7x T. Inui, T. Yamamura, H. Yuasa, Y. Kawai, A. Okaniwa, T. Serikawa, J. Yamada, The spontaneously epileptic rat ŽSER., a zitter ) tremor double mutant rat: histopathological findings in the central nervous system, Brain Res. 517 Ž1990. 123–133. w8x K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats ŽSER., a double mutant, Exp. Neurol. 119 Ž1993. 287–290. w9x N. Kato, T. Higuchi, H.G. Friesen, J.A. Wada, Changes of immunoreactive somatostatin and beta-endorphin content in rat brain after amygdaloid kindling, Life Sci. 32 Ž1983. 2415–2422. w10x N. Kato, V.C. Sundmark, L. Van Middlesworth, V. Havlicek, H.G. Friesen, Immunoreactive somatostatin and beta-endorphin content in the brain of mature rats after neonatal exposure to propylthiouracil, Endocrinology 110 Ž1982. 1851–1855. w11x C. Kowalski, J. Micheau, R. Corder, R. Gaillard, B. Conte Devolx, Age-related changes in corticotropin-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and beta-endorphin in specific rat brain areas, Brain Res. 582 Ž1992. 38–46. w12x J. Marksteiner, M. Ortler, R. Bellmann, G. Sperk, Neuropeptide Y
260
w13x
w14x
w15x
w16x
w17x
w18x
Y. Takahashi et al.r Brain Research 776 (1997) 255–260 biosynthesis is markedly induced in mossy fibers during temporal lobe epilepsy of the rat, Neurosci. Lett. 112 Ž1990. 143–148. J. Marksteiner, G. Sperk, Concomitant increase of somatostatin, neuropeptide Y and glutamate decarboxylase in the frontal cortex of rats with decreased seizure threshold, Neuroscience 26 Ž1988. 379– 385. J. Marksteiner, G. Sperk, D. Maas, Differential increases in brain levels of neuropeptide Y and vasoactive intestinal polypeptide after kainic acid-induced seizures in the rat, Naunyn. Schmiedebergs Arch. Pharmacol. 339 Ž1989. 173–177. A.R. McQuiston, J.J. Petrozzino, J.A. Connor, W.F. Colmers, Neuropeptide Y1 receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells, J. Neurosci. 16 Ž1996. 1422–1429. F.P. Monnet, A. Fournier, G. Debonnel, C. de Montigny, Neuropeptide Y potentiates selectively the N-methyl-D-aspartate response in the rat CA3 dorsal hippocampus: I. Involvement of an atypical neuropeptide Y receptor, J. Pharmacol. Exp. Ther. 263 Ž1992. 1212–1218. S. Nagaki, N. Kato, Y. Minatogawa, T. Higuchi, Effects of anticonvulsants and gamma-aminobutyric acid ŽGABA.-mimetic drugs on immunoreactive somatostatin and GABA contents in the rat brain, Life Sci. 46 Ž1990. 1587–1595. A. Pitkanen, M.F. Beal, J. Sirvio, K.J. Swartz, P.T. Mannisto, P.J.
w19x
w20x
w21x
w22x
w23x
w24x
Riekkinen, Somatostatin, neuropeptide Y, GABA and cholinergic enzymes in brain of pentylenetetrazol-kindled rats, Neuropeptides 14 Ž1989. 197–207. M. Sadamatsu, H. Kanai, A. Masui, T. Serikawa, J. Yamada, M. Sasa, N. Kato, Altered brain contents of neuropeptides in spontaneously epileptic rats ŽSER. and tremor rats with absence seizures, Life Sci. 57 Ž1995. 523–531. C. Schwarzer, J.M. Williamson, E.W. Lothman, A. Vezzani, G. Sperk, Somatostatin, neuropeptide Y, neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy, Neuroscience 69 Ž1995. 831–845. C. Stenfors, P. Bjellerup, A.A. Mathe, E. Theodorsson, Concurrent analysis of neuropeptides and biogenic amines in brain tissue of rats treated with electroconvulsive stimuli, Brain Res. 698 Ž1995. 39–45. J.W. Unger, Y. Schmidt, Neuropeptide Y and somatostatin in the neocortex of young and aging rats: response to nucleus basalis lesions, J. Chem. Neuroanat. 7 Ž1994. 25–34. A. Vezzani, G. Civenni, M. Rizzi, A. Monno, S. Messali, R. Samanin, Enhanced neuropeptide Y release in the hippocampus is associated with chronic seizure susceptibility in kainic acid treated rats, Brain Res. 660 Ž1994. 138–143. S.R. Weiss, R.M. Post, P.W. Gold, G. Chrousos, T.L. Sullivan, D. Walker, A. Pert, CRF-induced seizures and behavior: interaction with amygdala kindling, Brain Res. 372 Ž1986. 345–351.