- Email: [email protected]
Differential effects of vigabatrin and zonisamide on the neuropeptide Y system in the hippocampus of seizure prone gerbil
Neuropeptides Neuropeptides 39 (2005) 507–513 www.elsevier.com/locate/npep
Differential effects of vigabatrin and zonisamide on the neuropeptide Y system in the hippocampus of seizure prone gerbil Sung-Eun Kwak a, Ji-Eun Kim a, Duk-Soo Kim a, Moo-Ho Won a, Hui-Chul Choi b, Yeong-In Kim c, Hong-Ki Song b, Soo-Young Choi d, Tae-Cheon Kang a,* a
Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Neurology, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Neurology, Kangnam St. MaryÕs Hospital, The Catholic University of Korea, Seoul 137-701, South Korea d Department of Biomedical Science, College of Natural Science, Hallym University, Chunchon 200-702, South Korea b
c
Received 13 May 2005; accepted 16 August 2005 Available online 27 September 2005
Abstract Changed neuropeptide Y (NPY) system in the hippocampus has been reported in various experimental epileptic models. However, there have been little data concerning the alteration in the NPY system in the epileptic hippocampus following treatment of anti-epileptic drugs (AEDs). In the present study, therefore, we performed analyses of effects of vigabatrin (VGB) and zonisamide (ZNS) treatment on the NPY system in the hippocampus of the seizure sensitive (SS) gerbils. In SS gerbil, NPY immunoreactivity in the hippocampus was lower than that in seizure resistant gerbil. Following VGB treatment, the number of NPY immunoreactive neurons and NPY mRNA expression were increased in the hilus and the hippocampus proper. In contrast, ZNS treatment markedly elevated only the density of NPY immunoreactive fibers in the dentate gyrus, not in the hippocampus proper, as compared with saline-treated animals. These patterns were observed in the dose-dependent manners. These findings suggest that AEDs treatments may distinctly affect the NPY system in the SS gerbil hippocampus. 2005 Elsevier Ltd. All rights reserved. Keywords: Neuropeptide Y; Hippocampus; Gerbil; Epilepsy; Zonisamide; Vigabatrin
1. Introduction Neuropeptide Y (NPY) is supposed as one of the endogenous anticonvulsive substances, since NPY coexists with c-aminobutyric acid (GABA) in neurons of the dentate gyrus and it inhibits the excitatory neuronal transmission (Colmers and Bahh, 2003). This suggestion is supported by a previous report demonstrating increase in seizure susceptibility in NPY-deficient mice (DePrato Primeaux et al., 2000). In addition, status * Corresponding author. Tel.: +82 33 248 2524; fax: +82 33 256 1614. E-mail addresses: [email protected] (S.-Y. Choi), tckang@ hallym.ac.kr (T.-C. Kang).
0143-4179/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2005.08.003
epilepticus induced by electrical stimulus has been reported to cause a long-lasting elevation of NPY immunoreactivity, especially noted in the hippocampus (Husum et al., 2002). It is thus suggested that the biosynthesis of NPY in the hippocampus may contribute to the seizure susceptibility in the animal models of epilepsy. On the other hand, the hypothesis of seizure activity is based on the impaired inhibitory transmission in the brain. Thus, the strategy of developing of anti-epileptic drug (AED) and the studies on epileptogenesis are focused on the metabolism of c-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system. Although anti-epileptic drugs (AEDs) affect various neurotransmitter systems in the
508
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
hippocampus (Kang et al., 2003a,b), little evidence supports the relationship between AEDs on the NPY system. Thus, the issue remains to be clarified as to whether AEDs may affect the NPY system in the hippocampus. In the present study, therefore, comparative analysis of the NPY system following two distinct AEDs, vigabatrin (VGB) and zonisamide (ZNS, 1,2benzisoxazole-3-methanesulfonamide), was conducted in Mongolian gerbil (one of genetic epilepsy models), since the deficiency of NPY in the hippocampus may be one of the factors in seizure activity in this animal (Kang et al., 2000a).
2. Materials and methods 2.1. Experimental animals and drug treatments This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) obtained from Experimental Animal Center, Hallym University, Chunchon, South Korea. Animals were housed at constant temperature (23 C) and relative humidity (60%) with a fixed 12 h light/dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Each animal was tested a minimum of three times as described by Paul et al. (1981). Only animals with a consistent stage 4 or 5 seizure score, according to the seizure severity rating scale of Loskota et al. (1974), were included in the present study as SS gerbils. Forty SS gerbils (about three-month old) were used in the present experiment. Animals were divided into five groups (n = 8, respectively), and each group was given below drug once a day for 1 week: (1) vigabatrin (VGB, c-vinyl-GABA, Sigma USA, 30 mg/kg, IP); (2) VGB (15 mg/kg, IP); (3) zonisamide (ZNS, Eisai Korea Inc., Korea, 30 mg/ kg, IP); (4) ZNS (15 mg/kg, IP); (5) saline. Eight SR gerbils were used as normal control. Prior to tissue processing, each animal was tested three times using the methods described above for checking behavioral effects (Kang et al., 2003d). 2.2. Tissue processing and immunohistochemistry One hours after the last injection, animals were anesthetized with ketamine, and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 lm and consecutive sections were collected in six-well plates
containing phosphate buffered saline (PBS). These freefloating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the rabbit anti-NPY antiserum (diluted 1:1, 500, Peninsula, USA) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing three times for 10 min with PBS, sections were incubated sequentially, in goat anti-rabbit IgG (Vector, USA) and streptavidin (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatin-coated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control resulted in the absence of immunoreactivity in any structures. 2.3. In situ hybridization For in situ hybridization study, the 28-mer antisense oligodeoxynucleotide probe (Sigma Genosys Australia Pty. Ltd) of the following sequence, complementary to nucleotides 195–222 of NPY cDNA (BC043012) was used to label NPY mRNA: 5 0 -GAG TAG TAT CTG GCC ATG TCC TCT GCT G-3 0 (Hwang et al., 2004). The antisense oligodexoynucleotide probe was labeled with biotin using the Fast-Tag oligonucleotide labeling kit (Vector, USA) by the manufacturerÕs protocol (http://www.vecterlabs.com). In situ hybridization was carried out by previous protocols (Kang et al., 2003c). In order to establish the specificity of the in situ hybridization, pre-treatment with RNase A was performed, which showed the absence of reactivity in any structure. 2.4. Quantitation of data and statistical analysis Cell counts were carried out with a computerized image analysis system (Leica image scale). Sections (15 sections per each animal) were viewed through a microscope connected via a CCD camera to a PC monitor. At a magnification of 25–50·, the hippocampal regions were outlined and their surface areas measured. NPY positive neurons were counted by clicking on the monitor, at a magnification of 100·. All NPY immunoreactive cells were counted regardless the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues. The estimated cell number (n) was the average of values from three adjacent sections. Since the nucleus size measurement was used to correct the potential sampling bias, the area measurement for each nucleus in the sample population was also measured at a magnification
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
of 200· and was reduced to a mean diameter [D = (average area of the nucleus/3.14) · 0.5 · 2]. The true estimate of cell number was then calculated by using Abercrombie correction method: N (per mm2) = n(T/ T + D)/A, where N is the true cell number, T is the section thickness and A is the measured area (mm2) of each hippocampal region (Geuna, 2000; Geisert et al., 2002). The relative density of NPY immunoreactive fibers was also assessed; () rare (see Fig. 2B2); (+) slight (see Fig. 2A2); (++) moderate (see Fig. 2A3); (+++) strong (see Fig. 2D3). All data obtained from the quantitative measurements were analyzed using one-way ANOVA to determine statistical significance. BonferroniÕs test
509
was used for post hoc comparisons. P values below either 0.01 or 0.05 were considered statistically significant (Kang et al., 2000a,b; Kwak et al., 2005).
3. Results In SR gerbils, NPY immunoreactivity was strongly observed in hilar neurons and in some nerve fibers. In SS gerbils, NPY immunoreactive neurons were weakly detected in the dentate gyrus, thus the number of NPY immunoreactive cells in SS gerbils (Figs. 1B and 2A, Table 1) was markedly lower than that in SR gerbils
Fig. 1. Showing horizontal sections of the gerbil hippocampus. In SR gerbils, NPY immunoreactivity is highly localized within the cell bodies and extends throughout their processes (A1–A4). In SS gerbils, NPY immunoreactivity is weakly detected in CA1 (B2) and CA2–3 (B3) regions (arrows). However, neurons in dentate hirus are nearly devoid of NPY immunoreactivity (B4). However, there is no difference of the density of NPY immunoreactive fibers in the hippocampus between SR and SS gerbils (arrowheads). The low dosage of VGB treatment (15 mg/kg) intensifies the NPY immunoreactivity in the neuronal cell bodies (C1–C4). Therefore, strong NPY immunoreactivity is detected in the CA2–3 interneurons as well as hilar neurons (C4). However, NPY immunoreactivity in the CA1 region is unaltered (C2), as compared with control animals (see B2). The NPY immunoreactivity in the hippocampus is more increased following high dosage of VGB treatment (30 mg/kg; D1–D4), as compared with the case of low dosage treatment (C1–C4). Thus, strong NPY immunoreactive neurons are also detected in the CA1 region (D2). Notice the reduction of the density of NPY immunoreactive fibers in the hippocampus, as compared with control animals (see A,B). Rectangles in panels 1 indicate the regions of panels 2–4; (panels 1) bar = 400 lm; (panels 2–4) bar = 50 lm.
510
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
Fig. 2. High magnification photographs of panels 2–4 in Fig. 1 (except SR gerbils). Panels A, B and C indicate the CA1 region, CA2–3 regions and the dentate gyrus, respectively. Bar = 25 lm.
Table 1 Profiles of NPY immunoreactivity in the gerbil hippocampus following drug treatments Saline (SR)
Saline (SS)
VGB (SS)
ZNS (SS)
15 mg/kg
30 mg/kg
15 mg/kg
30 mg/kg
Dentate gyrus
No DI
16.9 ± 2.04 ++
1.9 ± 1.04** ++
13.4 ± 3.98 /+
18.1 ± 8.04 /+
1. 3 ± 1.07** ++/+++
1.9 ± 1.12** +++
CA1
No DI
12.7 ± 2.64 +
13.9 ± 3.04 +
11.8 ± 2.84 /+
12.1 ± 1.98 /+
12.9 ± 2.32 +/++
13.2 ± 1.94 ++
CA2–3
No DI
14.9 ± 3.11 +
12.9 ± 4.07 +
12.4 ± 5.28 /+
15.7 ± 3.04 /+
13.1 ± 3.16 +/++
14.5 ± 2.04 ++
Abbreviations. No, number of NPY immunoreactive neurons (means ± SEM/mm2; significant differences from saline treated SR gerbil, *P < 0.05, **P < 0.01); DI, density of NPY immunoreactive fibers (, rare; +, weak; ++, moderate; +++, strong).
(Fig. 1A, Table 1). However, the density of NPY immunoreactive fibers in the SS gerbil hippocampus was similarly observed, as compared with SR gerbils.
Following both AEDs treatments, animals exhibited sedated behavior, and thus seemed somnolent and tended to lie down more so than the control animals.
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
Both VGB and ZNS treatments effectively reduced seizure score to 2.3–2.5 which was a lower than or the non-treated group. However, both AED treatments showed distinct effects on NPY immunoreacvity in the hippocampus. VGB treatment effectively intensified NPY immunoreactivity in the gerbil hippocampus. Therefore, the number of NPY immunoreactive neurons in the hilus was increased in the dose-dependent manners, as compared with saline-treated SS gerbils. Briefly, the low dosage of VGB treatment (15 mg/kg) intensifies NPY immunoreactivity in neuronal cell bodies, thus the number of NPY immunoreactive neurons in the hilar region was elevated. Strong NPY immunoreactivity is detected in CA2-3 interneurons. In this group, NPY immunoreactivity in the CA1 region is unaltered, as compared with control animals (Figs. 1C and 2B, Table 1). Following the high dosage of VGB treatment (30 mg/ kg), NPY immunoreactivity in the hippocampus is more increased, as compared with the case of the low dosage treatment. Thus, strong NPY immunoreactive neurons are also detected in the CA1 region (Figs. 1D and 2C,
511
and Table 1). However, the density of NPY immunoreactive fibers was reduced by the low and the high dosage of VGB treatment (Fig. 2A–C, Table 1). Following ZNS treatment, the density of NPY immunoreactive fibers was markedly elevated in the hippocampus, as compared with saline-treated animals. This elevated pattern was observed in the dose-dependent manners. However, ZNS treatment did not affect the population of NPY immunoreactive neurons (Figs. 2D and 2E, Table 1). The results of the present in situ hybridization study revealed that NPY mRNA expression level was reduced in the hippocampus of SS gerbils (Figs. 3B), as compared with SR gerbils (Figs. 3A). These results imply that in SS gerbils NPY expression may be down-regulated at transcription level. Following VGB treatment, NPY mRNA expression level was up-regulated in the hippocampus of SS gerbils, as compared with saline-treated SS gerbils. Similar to immunohistochemical data, the low dosage of VGB treatment (15 mg/kg) intensified NPY mRNA expression in CA2-3 interneurons and in hilar neurons
Fig. 3. The NPY mRNA expression patterns in the gerbil hippocampus. NPY mRNA expression level in the hippocampus of SR gerbil is higher than that of SS gerbil. Following VGB treatment, NPY mRNA expression is markedly elevated in the SS gerbil hippocampus, as compared with saline-treated SS animals (see B). This elevated pattern is observed in the dose-dependent manners (A, 15 mg/kg; B, 30 mg/kg). Bar = 50 lm.
512
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
(Fig. 3C). The high dosage of VGB treatment (30 mg/ kg) also increased NPY mRNA expression in CA1 interneurons (Fig. 3D). Following ZNS treatment, NPY mRNA expression in the SS gerbil hippocampus was unaltered, as compared with control SS gerbil (data not shown).
4. Discussion The major findings in the present study are that VGB and ZNS distinctly affected the NPY system in the gerbil hippocampus. The exact mechanisms or the biological reasons of these findings cannot be certified in this study. However, our findings may provide the first morphological piece of in vivo evidence that to the best of our knowledge has not been previously reported, which supports the differential pharmacological properties of AEDs in the NPY system in the epileptic hippocampus. The effects of neurotransmissions on the NPY system have been still controversial. Midgley et al. (1992, 1993) reported that phencyclidine (PCP) and MK-801, antagonists of N-methyl-D-aspartate (NMDA) receptor, reduced NPY level. In contrast, Fukui et al. (1995, 1996) reported that PCP treatment enhanced NPY synthesis. In addition, the enhancement of GABAergic inhibitory transmission following VGB treatment (Armijo et al., 1992; Gram et al., 1988; Loscher and Horstermann, 1994; Preece and Cerdan, 1996; Qume and Fowler, 1996) did not affect NPY system, whereas co-treatment of VGB with PCP effectively blocked reduction of NPY level induced by PCP (Midgley et al., 1992, 1993). Furthermore, NPY and GABA in nerve terminals have complementary actions in modulating neurotransmission (Parker et al., 1998). In the present study, VGB treatment effectively increased the number of NPY immunoreactive neurons in hippocampal neurons in dose-dependent manner. In addition, VGB treatment elevated NPY mRNA expression accompanied by the reduction in the density of NPY immunoreactive nerve fibers. These findings indicate that VGB treatment may increase NPY synthesis and its release from nerve terminals in the gerbil hippocampal interneurons. Unlike VGB, ZNS inhibits excitatory glutamatergic transmission, presumably by reducing glutamate release (Okada et al., 1998; Owen et al., 1997). In the present study, ZNS treatment markedly increased the density of NPY immunoreactive fibers in the hippocampus, whereas the number of NPY immunoreactive neurons and NPY mRNA expression were unaltered. With respect to the case of VGB treatment in the present study, these findings imply that accumulated NPY immunoreactivity in nerve fibers may indicate the inhibition of NPY release from nerve fibers. Taken together, our findings indicate that VGB and ZNS may reversely act on the NPY system in the gerbil hippocampus. However,
effects of ZNS and VGB on the NPY system may not be related to anti-epileptic effects of these drugs, since administration of each drug effectively inhibited seizure activity in SS gerbils. What is the biological meaning of the alterations in the NPY system following VGB or ZNS treatment? Interestingly, ZNS has an anti-obesity properties (Wilding, 2004; Bays, 2004), whereas bodyweight gain (obesity) is a common and frequent adverse effect associated with the use of VGB (Jallon and Picard, 2001). In fact, NPY receptor antagonists are believed as anti-obesity substances (Gehlert, 1999; Gehlert and Hipskind, 1997). Furthermore, enhanced GABA-mediated neurotransmission may increase appetite for carbohydrates and reduce energy expenditure (Jallon and Picard, 2001). Therefore, it is considerable that the distinct responses of the NPY system following ZNS or VGB treatment may be indirectly or directly related to additional or undesired effects of these AEDs. To understand the exact mechanisms of both AEDs concerning obesity, further researches are needed. In conclusion, the present data demonstrate that VGB and ZNS distinctly affected the NPY system in the SS gerbil hippocampus. These findings suggest that AEDs may regulate the NPY system in the epileptic hippocampus, and may be related to additional or side effects of AEDs. Acknowledgments The research presented was supported by the Basic Research Program of the Korea Science and Engineering Foundation Nos. R01-2002-000-00008-0, R01-2005000-10004-0 and M103KV010019-03K2201-01910; and by Hallym University. References Armijo, J.A., Arteaga, R., Valdizan, E.M., Herranz, J.L., 1992. Coadministration of vigabatrin and valproate in children with refractory epilepsy. Clin. Neuropharmacol. 15, 459–469. Bays, H.E., 2004. Current and investigational antiobesity agents and obesity therapeutic treatment targets. Obes. Res. 12, 1197–1211. Colmers, W.F., El Bahh, B., 2003. Neuropeptide Y and epilepsy. Epilepsy Curr. 2, 53–58. DePrato Primeaux, S., Holmes, P.V., Martin, R.J., Dean, R.G., Edwards, G.L., 2000. Experimentally induced attenuation of neuropeptide-Y gene expression in transgenic mice increases mortality rate following seizures. Neurosci. Lett. 287, 61–64. Fukui, K., Kawashima, Y., Iizumi, H., Utsumi, H., Nakajima, T., 1995. Immunohistochemical alterations in neuropeptide Y-positive nerve elements in rat cerebral cortex following acute phencyclidine treatment. Neuroreport 6, 626–628. Fukui, K., Kawashima, Y., Iizumi, H., Utsumi, H., Nakajima, T., 1996. The effects of acute phencyclidine treatment on neuropeptide Y (NPY) neuronal system in the rat arcuate nucleus studied by immunocytochemistry and in situ hybridization. J. Neural Transm. 103, 385–390.
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513 Gehlert, D.R., 1999. Role of hypothalamic neuropeptide Y in feeding and obesity. Neuropeptides 33, 329–338. Gehlert, D.R., Hipskind, P.A., 1997. Neuropeptide Y receptor antagonists in obesity. Expert Opin. Inves. Drugs 6, 1827–1838. Geisert Jr., E.E., Williams, R.W., Geisert, G.R., Fan, L., Asbury, A.M., Maecker, H.T., Deng, J., Levy, S., 2002. Increased brain size and glial cell number in CD81-null mice. J. Comp. Neurol. 453, 22–32. Geuna, S., 2000. Appreciating the difference between design-based and model-based sampling strategies in quantitative morphology of the nervous system. J. Comp. Neurol. 427, 333–339. Gram, L., Larsson, O.M., Johnsen, A.H., Schousboe, A., 1988. Effects of valproate, vigabatrin and aminooxyacetic acid on release of endogenous and exogenous GABA from cultured neurons. Epilepsy Res. 2, 87–95. Husum, H., Gruber, S.H., Bolwig, T.G., Mathe, A.A., 2002. Extracellular levels of NPY in the dorsal hippocampus of freely moving rats are markedly elevated following a single electroconvulsive stimulation, irrespective of anticonvulsive Y1 receptor blockade. Neuropeptides 36, 363–369. Hwang, B.H., Suzuki, R., Lumeng, L., Li, T.K., McBride, W.J., 2004. Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats. Neuropeptides 38, 359–368. Jallon, p., Picard, F., 2001. Bodyweight gain and anticonvulsants: a comparative review. Drug Saf. 24, 969–978. Kang, T.C., Park, S.H., Park, S.K., Lee, J.C., Jo, S.M., Do, S.G., Suh, J.G., Oh, Y.S., Lee, J.Y., Won, M.H., 2000a. The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil. Brain Res. 870, 179–184. Kang, T.C., Park, S.K., Do, S.G., Suh, J.G., Jo, S.M., Oh, Y.S., Jeong, Y.G., Won, M.H., 2000b. The over-expression of somatostatin in the gerbil entorhinal cortex induced by seizure. Brain Res. 882, 55–61. Kang, T.C., Park, S.K., Hwang, I.K., An, S.J., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2003a. The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation. Neurochem. Int. 42, 239–249. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Bae, J.C., Won, M.H., 2003b. Changed vesicular GABA transporter immunoreactivity in the gerbil hippocampus following spontaneous seizure and vigabatrin administration. Neurosci. Lett. 335, 207–211. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Won, M.H., 2003c. P2X2 and P2X4 receptor expression is regulated by a GABA(A) receptor-mediated mechanism in the gerbil hippocampus. Mol. Brain Res. 116, 168–175.
513
Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Choi, S.Y., Kwon, O.S., Won, M.H., 2003d. Effect of vigabatrin on glutamate dehydrogenase in the hippocampus of seizure prone gerbils. Neurosci. Lett. 340, 115–118. Kwak, S.E., Kim, J.E., Kim, D.S., Jung, J.Y., Won, M.H., Kwon, O.S., Choi, S.Y., Kang, T.C., 2005. Effects of GABAergic transmissions on the immunoreactivities of calcium binding proteins in the gerbil hippocampus. J. Comp. Neurol. 485, 153–164. Loscher, W., Horstermann, D., 1994. Differential effects of vigabatrin, gamma-acetylenic GABA, aminooxyacetic acid, and valproate on levels of various amino acids in rat brain regions and plasma. Naunyn-SchmiedebergÕs Arch. Pharmacol. 349, 270–278. Loskota, W.J., Lomax, P., Rich, S.T., 1974. The gerbil as a model for the study of the epilepsies. Seizure patterns and ontogenesis. Epilepsia 15, 109–119. Midgley, L.P., Bush, L.G., Gibb, J.W., Hanson, G.R., 1992. Characterization of phencyclidine-induced effects on neuropeptide Y systems in the rat caudate-putamen. Brain Res. 593, 89–96. Midgley, L.P., Bush, L.G., Gibb, J.W., Hanson, G.R., 1993. Differential regulation of neuropeptide Y systems in limbic structures of the rat. J. Pharmacol. Exp. Therap. 267, 707–713. Okada, M., Kawata, Y., Mizuno, K., Wada, K., Kondo, T., Kaneko, S., 1998. Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode. Brit. J. Pharmacol. 124, 1277–1285. Owen, A.J., Ijaz, S., Miyashita, H., Wishart, T., Howlett, W., Shuaib, A., 1997. Zonisamide as a neuroprotective agent in an adult gerbil model of global forebrain ischemia: a histological, in vivo microdialysis and behavioral study. Brain Res. 770, 115–122. Parker, D., Soderberg, C., Zotova, E., Shupliakov, O., Langel, U., Bartfai, T., Larhammar, D., Brodin, L., Grillner, S., 1998. Colocalized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission. Eur. J. Neurosci. 10, 2856–2870. Paul, L.A., Fried, I., Watanabe, K., Forsythe, A.B., Scheibel, A.B., 1981. Structural correlates of seizure behavior in the mongolian gerbil. Science 213, 924–926. Preece, N.E., Cerdan, S., 1996. Metabolic precursors and compartmentation of cerebral GABA in vigabatrin-treated rats. J. Neurochem. 67, 1718–1725. Qume, M., Fowler, L.J., 1996. Effects of chronic oral treatment with GABA-transaminase inhibitors on the GABA system in brain, liver, kidney, and plasma of the rat. Biochem. Pharmacol. 52, 1355–1363. Wilding, J., 2004. Clinical evaluation of anti-obesity drugs. Curr. Drug Targets 5, 325–332.
Differential effects of vigabatrin and zonisamide on the neuropeptide Y system in the hippocampus of seizure prone gerbil Sung-Eun Kwak a, Ji-Eun Kim a, Duk-Soo Kim a, Moo-Ho Won a, Hui-Chul Choi b, Yeong-In Kim c, Hong-Ki Song b, Soo-Young Choi d, Tae-Cheon Kang a,* a
Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Neurology, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Neurology, Kangnam St. MaryÕs Hospital, The Catholic University of Korea, Seoul 137-701, South Korea d Department of Biomedical Science, College of Natural Science, Hallym University, Chunchon 200-702, South Korea b
c
Received 13 May 2005; accepted 16 August 2005 Available online 27 September 2005
Abstract Changed neuropeptide Y (NPY) system in the hippocampus has been reported in various experimental epileptic models. However, there have been little data concerning the alteration in the NPY system in the epileptic hippocampus following treatment of anti-epileptic drugs (AEDs). In the present study, therefore, we performed analyses of effects of vigabatrin (VGB) and zonisamide (ZNS) treatment on the NPY system in the hippocampus of the seizure sensitive (SS) gerbils. In SS gerbil, NPY immunoreactivity in the hippocampus was lower than that in seizure resistant gerbil. Following VGB treatment, the number of NPY immunoreactive neurons and NPY mRNA expression were increased in the hilus and the hippocampus proper. In contrast, ZNS treatment markedly elevated only the density of NPY immunoreactive fibers in the dentate gyrus, not in the hippocampus proper, as compared with saline-treated animals. These patterns were observed in the dose-dependent manners. These findings suggest that AEDs treatments may distinctly affect the NPY system in the SS gerbil hippocampus. 2005 Elsevier Ltd. All rights reserved. Keywords: Neuropeptide Y; Hippocampus; Gerbil; Epilepsy; Zonisamide; Vigabatrin
1. Introduction Neuropeptide Y (NPY) is supposed as one of the endogenous anticonvulsive substances, since NPY coexists with c-aminobutyric acid (GABA) in neurons of the dentate gyrus and it inhibits the excitatory neuronal transmission (Colmers and Bahh, 2003). This suggestion is supported by a previous report demonstrating increase in seizure susceptibility in NPY-deficient mice (DePrato Primeaux et al., 2000). In addition, status * Corresponding author. Tel.: +82 33 248 2524; fax: +82 33 256 1614. E-mail addresses: [email protected] (S.-Y. Choi), tckang@ hallym.ac.kr (T.-C. Kang).
0143-4179/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2005.08.003
epilepticus induced by electrical stimulus has been reported to cause a long-lasting elevation of NPY immunoreactivity, especially noted in the hippocampus (Husum et al., 2002). It is thus suggested that the biosynthesis of NPY in the hippocampus may contribute to the seizure susceptibility in the animal models of epilepsy. On the other hand, the hypothesis of seizure activity is based on the impaired inhibitory transmission in the brain. Thus, the strategy of developing of anti-epileptic drug (AED) and the studies on epileptogenesis are focused on the metabolism of c-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system. Although anti-epileptic drugs (AEDs) affect various neurotransmitter systems in the
508
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
hippocampus (Kang et al., 2003a,b), little evidence supports the relationship between AEDs on the NPY system. Thus, the issue remains to be clarified as to whether AEDs may affect the NPY system in the hippocampus. In the present study, therefore, comparative analysis of the NPY system following two distinct AEDs, vigabatrin (VGB) and zonisamide (ZNS, 1,2benzisoxazole-3-methanesulfonamide), was conducted in Mongolian gerbil (one of genetic epilepsy models), since the deficiency of NPY in the hippocampus may be one of the factors in seizure activity in this animal (Kang et al., 2000a).
2. Materials and methods 2.1. Experimental animals and drug treatments This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) obtained from Experimental Animal Center, Hallym University, Chunchon, South Korea. Animals were housed at constant temperature (23 C) and relative humidity (60%) with a fixed 12 h light/dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Each animal was tested a minimum of three times as described by Paul et al. (1981). Only animals with a consistent stage 4 or 5 seizure score, according to the seizure severity rating scale of Loskota et al. (1974), were included in the present study as SS gerbils. Forty SS gerbils (about three-month old) were used in the present experiment. Animals were divided into five groups (n = 8, respectively), and each group was given below drug once a day for 1 week: (1) vigabatrin (VGB, c-vinyl-GABA, Sigma USA, 30 mg/kg, IP); (2) VGB (15 mg/kg, IP); (3) zonisamide (ZNS, Eisai Korea Inc., Korea, 30 mg/ kg, IP); (4) ZNS (15 mg/kg, IP); (5) saline. Eight SR gerbils were used as normal control. Prior to tissue processing, each animal was tested three times using the methods described above for checking behavioral effects (Kang et al., 2003d). 2.2. Tissue processing and immunohistochemistry One hours after the last injection, animals were anesthetized with ketamine, and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 lm and consecutive sections were collected in six-well plates
containing phosphate buffered saline (PBS). These freefloating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the rabbit anti-NPY antiserum (diluted 1:1, 500, Peninsula, USA) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing three times for 10 min with PBS, sections were incubated sequentially, in goat anti-rabbit IgG (Vector, USA) and streptavidin (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatin-coated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control resulted in the absence of immunoreactivity in any structures. 2.3. In situ hybridization For in situ hybridization study, the 28-mer antisense oligodeoxynucleotide probe (Sigma Genosys Australia Pty. Ltd) of the following sequence, complementary to nucleotides 195–222 of NPY cDNA (BC043012) was used to label NPY mRNA: 5 0 -GAG TAG TAT CTG GCC ATG TCC TCT GCT G-3 0 (Hwang et al., 2004). The antisense oligodexoynucleotide probe was labeled with biotin using the Fast-Tag oligonucleotide labeling kit (Vector, USA) by the manufacturerÕs protocol (http://www.vecterlabs.com). In situ hybridization was carried out by previous protocols (Kang et al., 2003c). In order to establish the specificity of the in situ hybridization, pre-treatment with RNase A was performed, which showed the absence of reactivity in any structure. 2.4. Quantitation of data and statistical analysis Cell counts were carried out with a computerized image analysis system (Leica image scale). Sections (15 sections per each animal) were viewed through a microscope connected via a CCD camera to a PC monitor. At a magnification of 25–50·, the hippocampal regions were outlined and their surface areas measured. NPY positive neurons were counted by clicking on the monitor, at a magnification of 100·. All NPY immunoreactive cells were counted regardless the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues. The estimated cell number (n) was the average of values from three adjacent sections. Since the nucleus size measurement was used to correct the potential sampling bias, the area measurement for each nucleus in the sample population was also measured at a magnification
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
of 200· and was reduced to a mean diameter [D = (average area of the nucleus/3.14) · 0.5 · 2]. The true estimate of cell number was then calculated by using Abercrombie correction method: N (per mm2) = n(T/ T + D)/A, where N is the true cell number, T is the section thickness and A is the measured area (mm2) of each hippocampal region (Geuna, 2000; Geisert et al., 2002). The relative density of NPY immunoreactive fibers was also assessed; () rare (see Fig. 2B2); (+) slight (see Fig. 2A2); (++) moderate (see Fig. 2A3); (+++) strong (see Fig. 2D3). All data obtained from the quantitative measurements were analyzed using one-way ANOVA to determine statistical significance. BonferroniÕs test
509
was used for post hoc comparisons. P values below either 0.01 or 0.05 were considered statistically significant (Kang et al., 2000a,b; Kwak et al., 2005).
3. Results In SR gerbils, NPY immunoreactivity was strongly observed in hilar neurons and in some nerve fibers. In SS gerbils, NPY immunoreactive neurons were weakly detected in the dentate gyrus, thus the number of NPY immunoreactive cells in SS gerbils (Figs. 1B and 2A, Table 1) was markedly lower than that in SR gerbils
Fig. 1. Showing horizontal sections of the gerbil hippocampus. In SR gerbils, NPY immunoreactivity is highly localized within the cell bodies and extends throughout their processes (A1–A4). In SS gerbils, NPY immunoreactivity is weakly detected in CA1 (B2) and CA2–3 (B3) regions (arrows). However, neurons in dentate hirus are nearly devoid of NPY immunoreactivity (B4). However, there is no difference of the density of NPY immunoreactive fibers in the hippocampus between SR and SS gerbils (arrowheads). The low dosage of VGB treatment (15 mg/kg) intensifies the NPY immunoreactivity in the neuronal cell bodies (C1–C4). Therefore, strong NPY immunoreactivity is detected in the CA2–3 interneurons as well as hilar neurons (C4). However, NPY immunoreactivity in the CA1 region is unaltered (C2), as compared with control animals (see B2). The NPY immunoreactivity in the hippocampus is more increased following high dosage of VGB treatment (30 mg/kg; D1–D4), as compared with the case of low dosage treatment (C1–C4). Thus, strong NPY immunoreactive neurons are also detected in the CA1 region (D2). Notice the reduction of the density of NPY immunoreactive fibers in the hippocampus, as compared with control animals (see A,B). Rectangles in panels 1 indicate the regions of panels 2–4; (panels 1) bar = 400 lm; (panels 2–4) bar = 50 lm.
510
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
Fig. 2. High magnification photographs of panels 2–4 in Fig. 1 (except SR gerbils). Panels A, B and C indicate the CA1 region, CA2–3 regions and the dentate gyrus, respectively. Bar = 25 lm.
Table 1 Profiles of NPY immunoreactivity in the gerbil hippocampus following drug treatments Saline (SR)
Saline (SS)
VGB (SS)
ZNS (SS)
15 mg/kg
30 mg/kg
15 mg/kg
30 mg/kg
Dentate gyrus
No DI
16.9 ± 2.04 ++
1.9 ± 1.04** ++
13.4 ± 3.98 /+
18.1 ± 8.04 /+
1. 3 ± 1.07** ++/+++
1.9 ± 1.12** +++
CA1
No DI
12.7 ± 2.64 +
13.9 ± 3.04 +
11.8 ± 2.84 /+
12.1 ± 1.98 /+
12.9 ± 2.32 +/++
13.2 ± 1.94 ++
CA2–3
No DI
14.9 ± 3.11 +
12.9 ± 4.07 +
12.4 ± 5.28 /+
15.7 ± 3.04 /+
13.1 ± 3.16 +/++
14.5 ± 2.04 ++
Abbreviations. No, number of NPY immunoreactive neurons (means ± SEM/mm2; significant differences from saline treated SR gerbil, *P < 0.05, **P < 0.01); DI, density of NPY immunoreactive fibers (, rare; +, weak; ++, moderate; +++, strong).
(Fig. 1A, Table 1). However, the density of NPY immunoreactive fibers in the SS gerbil hippocampus was similarly observed, as compared with SR gerbils.
Following both AEDs treatments, animals exhibited sedated behavior, and thus seemed somnolent and tended to lie down more so than the control animals.
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
Both VGB and ZNS treatments effectively reduced seizure score to 2.3–2.5 which was a lower than or the non-treated group. However, both AED treatments showed distinct effects on NPY immunoreacvity in the hippocampus. VGB treatment effectively intensified NPY immunoreactivity in the gerbil hippocampus. Therefore, the number of NPY immunoreactive neurons in the hilus was increased in the dose-dependent manners, as compared with saline-treated SS gerbils. Briefly, the low dosage of VGB treatment (15 mg/kg) intensifies NPY immunoreactivity in neuronal cell bodies, thus the number of NPY immunoreactive neurons in the hilar region was elevated. Strong NPY immunoreactivity is detected in CA2-3 interneurons. In this group, NPY immunoreactivity in the CA1 region is unaltered, as compared with control animals (Figs. 1C and 2B, Table 1). Following the high dosage of VGB treatment (30 mg/ kg), NPY immunoreactivity in the hippocampus is more increased, as compared with the case of the low dosage treatment. Thus, strong NPY immunoreactive neurons are also detected in the CA1 region (Figs. 1D and 2C,
511
and Table 1). However, the density of NPY immunoreactive fibers was reduced by the low and the high dosage of VGB treatment (Fig. 2A–C, Table 1). Following ZNS treatment, the density of NPY immunoreactive fibers was markedly elevated in the hippocampus, as compared with saline-treated animals. This elevated pattern was observed in the dose-dependent manners. However, ZNS treatment did not affect the population of NPY immunoreactive neurons (Figs. 2D and 2E, Table 1). The results of the present in situ hybridization study revealed that NPY mRNA expression level was reduced in the hippocampus of SS gerbils (Figs. 3B), as compared with SR gerbils (Figs. 3A). These results imply that in SS gerbils NPY expression may be down-regulated at transcription level. Following VGB treatment, NPY mRNA expression level was up-regulated in the hippocampus of SS gerbils, as compared with saline-treated SS gerbils. Similar to immunohistochemical data, the low dosage of VGB treatment (15 mg/kg) intensified NPY mRNA expression in CA2-3 interneurons and in hilar neurons
Fig. 3. The NPY mRNA expression patterns in the gerbil hippocampus. NPY mRNA expression level in the hippocampus of SR gerbil is higher than that of SS gerbil. Following VGB treatment, NPY mRNA expression is markedly elevated in the SS gerbil hippocampus, as compared with saline-treated SS animals (see B). This elevated pattern is observed in the dose-dependent manners (A, 15 mg/kg; B, 30 mg/kg). Bar = 50 lm.
512
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513
(Fig. 3C). The high dosage of VGB treatment (30 mg/ kg) also increased NPY mRNA expression in CA1 interneurons (Fig. 3D). Following ZNS treatment, NPY mRNA expression in the SS gerbil hippocampus was unaltered, as compared with control SS gerbil (data not shown).
4. Discussion The major findings in the present study are that VGB and ZNS distinctly affected the NPY system in the gerbil hippocampus. The exact mechanisms or the biological reasons of these findings cannot be certified in this study. However, our findings may provide the first morphological piece of in vivo evidence that to the best of our knowledge has not been previously reported, which supports the differential pharmacological properties of AEDs in the NPY system in the epileptic hippocampus. The effects of neurotransmissions on the NPY system have been still controversial. Midgley et al. (1992, 1993) reported that phencyclidine (PCP) and MK-801, antagonists of N-methyl-D-aspartate (NMDA) receptor, reduced NPY level. In contrast, Fukui et al. (1995, 1996) reported that PCP treatment enhanced NPY synthesis. In addition, the enhancement of GABAergic inhibitory transmission following VGB treatment (Armijo et al., 1992; Gram et al., 1988; Loscher and Horstermann, 1994; Preece and Cerdan, 1996; Qume and Fowler, 1996) did not affect NPY system, whereas co-treatment of VGB with PCP effectively blocked reduction of NPY level induced by PCP (Midgley et al., 1992, 1993). Furthermore, NPY and GABA in nerve terminals have complementary actions in modulating neurotransmission (Parker et al., 1998). In the present study, VGB treatment effectively increased the number of NPY immunoreactive neurons in hippocampal neurons in dose-dependent manner. In addition, VGB treatment elevated NPY mRNA expression accompanied by the reduction in the density of NPY immunoreactive nerve fibers. These findings indicate that VGB treatment may increase NPY synthesis and its release from nerve terminals in the gerbil hippocampal interneurons. Unlike VGB, ZNS inhibits excitatory glutamatergic transmission, presumably by reducing glutamate release (Okada et al., 1998; Owen et al., 1997). In the present study, ZNS treatment markedly increased the density of NPY immunoreactive fibers in the hippocampus, whereas the number of NPY immunoreactive neurons and NPY mRNA expression were unaltered. With respect to the case of VGB treatment in the present study, these findings imply that accumulated NPY immunoreactivity in nerve fibers may indicate the inhibition of NPY release from nerve fibers. Taken together, our findings indicate that VGB and ZNS may reversely act on the NPY system in the gerbil hippocampus. However,
effects of ZNS and VGB on the NPY system may not be related to anti-epileptic effects of these drugs, since administration of each drug effectively inhibited seizure activity in SS gerbils. What is the biological meaning of the alterations in the NPY system following VGB or ZNS treatment? Interestingly, ZNS has an anti-obesity properties (Wilding, 2004; Bays, 2004), whereas bodyweight gain (obesity) is a common and frequent adverse effect associated with the use of VGB (Jallon and Picard, 2001). In fact, NPY receptor antagonists are believed as anti-obesity substances (Gehlert, 1999; Gehlert and Hipskind, 1997). Furthermore, enhanced GABA-mediated neurotransmission may increase appetite for carbohydrates and reduce energy expenditure (Jallon and Picard, 2001). Therefore, it is considerable that the distinct responses of the NPY system following ZNS or VGB treatment may be indirectly or directly related to additional or undesired effects of these AEDs. To understand the exact mechanisms of both AEDs concerning obesity, further researches are needed. In conclusion, the present data demonstrate that VGB and ZNS distinctly affected the NPY system in the SS gerbil hippocampus. These findings suggest that AEDs may regulate the NPY system in the epileptic hippocampus, and may be related to additional or side effects of AEDs. Acknowledgments The research presented was supported by the Basic Research Program of the Korea Science and Engineering Foundation Nos. R01-2002-000-00008-0, R01-2005000-10004-0 and M103KV010019-03K2201-01910; and by Hallym University. References Armijo, J.A., Arteaga, R., Valdizan, E.M., Herranz, J.L., 1992. Coadministration of vigabatrin and valproate in children with refractory epilepsy. Clin. Neuropharmacol. 15, 459–469. Bays, H.E., 2004. Current and investigational antiobesity agents and obesity therapeutic treatment targets. Obes. Res. 12, 1197–1211. Colmers, W.F., El Bahh, B., 2003. Neuropeptide Y and epilepsy. Epilepsy Curr. 2, 53–58. DePrato Primeaux, S., Holmes, P.V., Martin, R.J., Dean, R.G., Edwards, G.L., 2000. Experimentally induced attenuation of neuropeptide-Y gene expression in transgenic mice increases mortality rate following seizures. Neurosci. Lett. 287, 61–64. Fukui, K., Kawashima, Y., Iizumi, H., Utsumi, H., Nakajima, T., 1995. Immunohistochemical alterations in neuropeptide Y-positive nerve elements in rat cerebral cortex following acute phencyclidine treatment. Neuroreport 6, 626–628. Fukui, K., Kawashima, Y., Iizumi, H., Utsumi, H., Nakajima, T., 1996. The effects of acute phencyclidine treatment on neuropeptide Y (NPY) neuronal system in the rat arcuate nucleus studied by immunocytochemistry and in situ hybridization. J. Neural Transm. 103, 385–390.
S.-E. Kwak et al. / Neuropeptides 39 (2005) 507–513 Gehlert, D.R., 1999. Role of hypothalamic neuropeptide Y in feeding and obesity. Neuropeptides 33, 329–338. Gehlert, D.R., Hipskind, P.A., 1997. Neuropeptide Y receptor antagonists in obesity. Expert Opin. Inves. Drugs 6, 1827–1838. Geisert Jr., E.E., Williams, R.W., Geisert, G.R., Fan, L., Asbury, A.M., Maecker, H.T., Deng, J., Levy, S., 2002. Increased brain size and glial cell number in CD81-null mice. J. Comp. Neurol. 453, 22–32. Geuna, S., 2000. Appreciating the difference between design-based and model-based sampling strategies in quantitative morphology of the nervous system. J. Comp. Neurol. 427, 333–339. Gram, L., Larsson, O.M., Johnsen, A.H., Schousboe, A., 1988. Effects of valproate, vigabatrin and aminooxyacetic acid on release of endogenous and exogenous GABA from cultured neurons. Epilepsy Res. 2, 87–95. Husum, H., Gruber, S.H., Bolwig, T.G., Mathe, A.A., 2002. Extracellular levels of NPY in the dorsal hippocampus of freely moving rats are markedly elevated following a single electroconvulsive stimulation, irrespective of anticonvulsive Y1 receptor blockade. Neuropeptides 36, 363–369. Hwang, B.H., Suzuki, R., Lumeng, L., Li, T.K., McBride, W.J., 2004. Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats. Neuropeptides 38, 359–368. Jallon, p., Picard, F., 2001. Bodyweight gain and anticonvulsants: a comparative review. Drug Saf. 24, 969–978. Kang, T.C., Park, S.H., Park, S.K., Lee, J.C., Jo, S.M., Do, S.G., Suh, J.G., Oh, Y.S., Lee, J.Y., Won, M.H., 2000a. The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil. Brain Res. 870, 179–184. Kang, T.C., Park, S.K., Do, S.G., Suh, J.G., Jo, S.M., Oh, Y.S., Jeong, Y.G., Won, M.H., 2000b. The over-expression of somatostatin in the gerbil entorhinal cortex induced by seizure. Brain Res. 882, 55–61. Kang, T.C., Park, S.K., Hwang, I.K., An, S.J., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2003a. The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation. Neurochem. Int. 42, 239–249. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Bae, J.C., Won, M.H., 2003b. Changed vesicular GABA transporter immunoreactivity in the gerbil hippocampus following spontaneous seizure and vigabatrin administration. Neurosci. Lett. 335, 207–211. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Won, M.H., 2003c. P2X2 and P2X4 receptor expression is regulated by a GABA(A) receptor-mediated mechanism in the gerbil hippocampus. Mol. Brain Res. 116, 168–175.
513
Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Choi, S.Y., Kwon, O.S., Won, M.H., 2003d. Effect of vigabatrin on glutamate dehydrogenase in the hippocampus of seizure prone gerbils. Neurosci. Lett. 340, 115–118. Kwak, S.E., Kim, J.E., Kim, D.S., Jung, J.Y., Won, M.H., Kwon, O.S., Choi, S.Y., Kang, T.C., 2005. Effects of GABAergic transmissions on the immunoreactivities of calcium binding proteins in the gerbil hippocampus. J. Comp. Neurol. 485, 153–164. Loscher, W., Horstermann, D., 1994. Differential effects of vigabatrin, gamma-acetylenic GABA, aminooxyacetic acid, and valproate on levels of various amino acids in rat brain regions and plasma. Naunyn-SchmiedebergÕs Arch. Pharmacol. 349, 270–278. Loskota, W.J., Lomax, P., Rich, S.T., 1974. The gerbil as a model for the study of the epilepsies. Seizure patterns and ontogenesis. Epilepsia 15, 109–119. Midgley, L.P., Bush, L.G., Gibb, J.W., Hanson, G.R., 1992. Characterization of phencyclidine-induced effects on neuropeptide Y systems in the rat caudate-putamen. Brain Res. 593, 89–96. Midgley, L.P., Bush, L.G., Gibb, J.W., Hanson, G.R., 1993. Differential regulation of neuropeptide Y systems in limbic structures of the rat. J. Pharmacol. Exp. Therap. 267, 707–713. Okada, M., Kawata, Y., Mizuno, K., Wada, K., Kondo, T., Kaneko, S., 1998. Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode. Brit. J. Pharmacol. 124, 1277–1285. Owen, A.J., Ijaz, S., Miyashita, H., Wishart, T., Howlett, W., Shuaib, A., 1997. Zonisamide as a neuroprotective agent in an adult gerbil model of global forebrain ischemia: a histological, in vivo microdialysis and behavioral study. Brain Res. 770, 115–122. Parker, D., Soderberg, C., Zotova, E., Shupliakov, O., Langel, U., Bartfai, T., Larhammar, D., Brodin, L., Grillner, S., 1998. Colocalized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission. Eur. J. Neurosci. 10, 2856–2870. Paul, L.A., Fried, I., Watanabe, K., Forsythe, A.B., Scheibel, A.B., 1981. Structural correlates of seizure behavior in the mongolian gerbil. Science 213, 924–926. Preece, N.E., Cerdan, S., 1996. Metabolic precursors and compartmentation of cerebral GABA in vigabatrin-treated rats. J. Neurochem. 67, 1718–1725. Qume, M., Fowler, L.J., 1996. Effects of chronic oral treatment with GABA-transaminase inhibitors on the GABA system in brain, liver, kidney, and plasma of the rat. Biochem. Pharmacol. 52, 1355–1363. Wilding, J., 2004. Clinical evaluation of anti-obesity drugs. Curr. Drug Targets 5, 325–332.