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Valproic acid reduces enhanced vesicular glutamate transporter immunoreactivities in the dentate gyrus of the seizure prone gerbil
Neuropharmacology 49 (2005) 912e921 www.elsevier.com/locate/neuropharm
Valproic acid reduces enhanced vesicular glutamate transporter immunoreactivities in the dentate gyrus of the seizure prone gerbil T.-C. Kang a,*, D.-S. Kim a, S.-E. Kwak a, J.-E. Kim a, D.W. Kim b, J.H. Kang c, M.H. Won a, O.-S. Kwon c, S.-Y. Choi b,* a
Department of Anatomy, College of Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea Department of Biomedical Science, College of Life Science, Hallym University, Chunchon 200-702, South Korea c Department of Biochemistry, College of Natural Science, Kyungpook National University, Taegu 702-702, South Korea b
Received 22 April 2005; received in revised form 27 July 2005; accepted 11 August 2005
Abstract To elucidate the relationship between glutamatergic current and vesicular glutamate transporter (VGLUT) expressions, we performed the comparative analyses of evoked potentials and VGLUT immunoreactivities in the dentate gyrus, and its response to antiepileptic drug treatments in a gerbil model. The EPSP slope that could be evoked in seizure sensitive (SS) gerbils was significantly greater than in seizure resistant (SR) gerbils. There was also a strong trend towards the larger population spike amplitude in SS gerbils. In addition, VGLUT immunoreactivities were markedly enhanced in the dentate gyrus of SS gerbils, as compared with the SR gerbils. Following valproic acid (VPA, 30 mg/kg), the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR and of SS gerbils, although this dosage of VPA had no effect in low stimulus currents in SS gerbils. Vigabatrin (VGB) and low dosage of VPA treatment did not affect the evoked responses. Similarly, VPA treatment reduced enhanced VGLUT immunoreactivities in the dentate gyrus of SS gerbils, whilst VGB did not. These findings suggest that up-regulation of VGLUT immunoreactivities may be related to the hyperexcitability of granule cells in SS gerbils, and altered VGLUT immunoreactivity in the dentate gyrus may be independent of GABAergic transmission. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Epilepsy; Hippocampus; Synaptic vesicle; Valproic acid; Vesicular glutamate transporter; Vigabatrin
Abbreviations: VGLUT, vesicular glutamate transporter; SS, seizure sensitive; SR, seizure resistant; VPA, valproic acid; GABA, g-aminobutyric acid; GAD, glutamic acid decarboxylase; GAT-1, GABA transporter; VGAT, vesicular GABA transport; NMDA, N-methyl-D-aspartate receptor; AEDs, antiepileptic drugs; VGB, vigabatrin; ACSF, artificial cerebrospinal fluid; EPSP, excitatory postsynaptic potential; PBS, phosphate buffered saline; DAB, 3, 3#-diaminobenzidine. * Corresponding authors. Tel.: C82 33 248 2524; fax: C82 33 256 1614 (T.-C. Kang); tel.: C82 33 248 2112; fax: C82 33 241 1463 (S.-Y. Choi). E-mail addresses: [email protected] (T.-C. Kang), sychoi@hallym. ac.kr (S.-Y. Choi). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.08.007
1. Introduction Epilepsy is a chronic condition characterized by the presence of spontaneous episodes of abnormal neuronal discharges. Reduced granule cell inhibition is considered the most common cause of temporal lobe epilepsy, since human patients and experimental animals with temporal lobe epilepsy display inhibitory interneuron loss (Margerison and Corsellis, 1996; De Lanerolle et al., 1989; Wittner et al., 2001). Recently, this general concept concerning epilepsy has been challenged, since
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many previous studies that evaluated tissue from patients or models of temporal lobe epilepsy found evidence of normal or enhanced granule cell inhibition, despite inhibitory interneuron loss. The fact is that the paired-pulse inhibition of granule cells is enhanced in most models of temporal lobe epilepsy (Tuff et al., 1983; Hass et al., 1996; Buckmaster and Dudek, 1997). The Mongolian gerbil presents a model for investigating directly the hypothesis that epileptogenesis is characterized by an alteration in the regulation of g-aminobutyric acid (GABA) metabolism, because epileptogenesis in the gerbil has been linked to abnormalities in the GABAergic system; e.g. mismatched glutamic acid decarboxylase (GAD) 65/GAD 67 ratio (Kang et al., 2001b), over-expressions of GABA shunt enzymes (Kang et al., 2003b) and neuronal GABA transporter (GAT-1) (Kang et al., 2001a), and to malfunctions of the vesicular GABA transport (VGAT) system (Kang et al., 2003a,c). These findings indicate a reduction in inhibitory neurotransmission in the dentate gyrus. On the other hand, Ribak and Khan (1987) reported the termination of seizure activity in seizure prone gerbils with bilateral lesions of the perforant path. These observations led us to speculate upon the possible existence of enhanced feedback/feedforward glutamatergic excitation to the dentate gyrus of gerbils, although no differences in the N-methyl-D-aspartate receptor (NMDA) system were found between seizure prone and normal gerbils (Suh et al., 2001). Glutamate has to be loaded into synaptic vesicles by proton-dependent transporters before its exocytotic release (Ozkan and Ueda, 1998; Reimer et al., 1998; Erickson and Varoqui, 2000; Gasnier, 2000). NaC-dependent inorganic phosphate transporter has now been unambiguously established as vesicular glutamate transporter (Hisano et al., 1997; Lee et al., 1999; Aihara et al., 2000; Bellocchio et al., 2000; Takamori et al., 2000, 2001; Bai et al., 2001; Herzog et al., 2001). However, little evidence supports the relationship between epilepsy and the functions of VGLUT. Thus, the issue remains to be clarified as to whether seizure activity in the hippocampus of gerbils may be related with VGLUT expressions, and whether antiepileptic drugs (AEDs) may affect excitability of granule cells or VGLUT expressions. Therefore, to elucidate the roles of glutamatergic systems in epilepsy, the comparative analysis of vesicular glutamate transporter (VGLUT) immunoreactivities and of the effects of two different AEDs, valproic acid (VPA) and vigabatin (VGB), on these expressions was conducted in a gerbil model. This is because these AEDs have distinct pharmacological profiles: VPA mainly acts as a NaC channel blocker (Johannessen et al., 2001), whereas VGB enhances GABA-mediated inhibition via irreversibly inhibiting GABA transaminase (GABA-T), which is a degradative enzyme of GABA (Macdonald and Kelly, 1993; Czuczwar and Patsalos, 2001; Kang et al., 2003d).
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2. Materials and methods 2.1. Experimental animals These studies utilized the progeny of male Mongolian gerbil (Meriones unguiculatus) weighing 80e88 g obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The 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. The procedures involving animals and their care were conducted in conformity with the institutional guidelines and in compliance with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 8523, 1985). Each animal was stimulated by vigorous stroking on the back with a pencil as described by Paul et al. (1981) and tested a minimum of three times. According to the seizure severity rating scale of Loskota et al. (1974) and Lee et al. (1987), seizures were classified; grade 1, a brief pause in normal activity accompanied by vibrissae twitches and retraction of the pinnae; grade 2, motor arrest with twitching of the vibrissae and retraction of the pinnae; grade 3, motor arrest with myoclonic jerks; grade 4, clonic-tonic seizures; grade 5, clonic-tonic seizures with body rollover. Only animals with a consistent stage 4 or 5 seizure score were included in the present study as seizure-sensitive (SS) gerbils. Seizure-resistant (SR) gerbils never demonstrated the seizure activity, thus they were assigned seizure severity score of 0 (Kang et al., 2001a,b; 2003aec). Both SR and SS gerbils were given the drug detailed below once a day for 1 week, respectively: (1) VGB (Sigma USA, 15 or 30 mg/kg, i.p., n Z 10, respectively); (2) VPA (Sigma USA, 10, 20, 30 mg/kg, i.p., n Z 10, respectively); (3) VGB (15 mg/kg) C VPA (30 mg/kg) (i.p., n Z 10); (4) VGB (30 mg/kg) C VPA (30 mg/kg) (i.p., n Z 10); (5) saline (n Z 10). 2.2. In vivo extracellular recording After 2 h after the last drug injection, all animals were anesthetized (urethane, 1.5 g/kg, i.p.) and placed in stereotaxic frames. Holes were drilled through the skull for introducing electrodes. The coordinates (in mm) referenced to bregma were as follows. For the recording electrode (to the dentate gyrus): ÿ3.3 anterior-posterior, 2.5 lateral. For the stimulating electrode (to the angular bundle): ÿ5.0 anterior-posterior, 4.3 lateral. Electrode depths were determined by optimizing the evoked response (Buckmaster et al., 1996, 2000). Glass microelectrodes (microfilament capillary 1.2 outer diameter; 5e10 MU) filled with artificial cerebrospinal fluid (ACSF, in mM; NaCl 126, KCl 5, CaCl2 2, MgCl2 2, NaH2PO4 1.25, NaHCO3 26, D-glucose 10, pH 7.2)
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present study were performed under the same circumstances and in parallel.
and bipolar tungsten stimulating electrodes were used. The reference electrode was placed in the cerebellum. The stimuli were applied as DC square pulses at 0.1 Hz with 150-ms constant current stimuli. Stimulus intensity range was set at 10e1000 mA. The characteristic response of the dentate granule cells to perforant path stimulation consisted of a positive-going field population spike. The population spike amplitude is proportional to the number of granule cells discharging an action potential (Andersen et al., 1971). The excitatory postsynaptic potential (EPSP) slope (25e75% of the initial rising phase) was also measured, since the EPSP slope is indicative of the total amount of excitatory transmitter (mainly glutamate) released by the perforant path volley at many synaptic sites (Lømo, 1971). Signals were recorded with DAM 80 differential amplifier (World Precision Instruments, USA) and data were digitized and analyzed on MacChart 5 (AD Instruments, Australia). Thereafter data were analyzed using the evoked-potential module in MacChart 5. For comparison, the data obtained from three to seven responses were averaged to minimize the variability between animals and each sampling of the population spike and the EPSP slope.
In three animals in each group, the hippocampus on each brain slice was removed and then homogenized in 10 mM PB containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF. After centrifugation, the protein concentrations in the supernatants were determined by using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 3 mg of total protein were boiled with an equal volume of 2! SDS sample buffer and boiled for 3 min, and then, each mixture was loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher & Schuell, USA). To reduced background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, sequentially incubated with primary antisera (1:10,000), peroxidase conjugated goat anti-guinea pig IgG (Sigma, USA), and then with ECL kit (Amersham, USA).
2.3. Anatomy
2.5. Quantitation of data and statistical analysis
After extracellular recording, seven animals in each group were perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer. 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 C and consecutive sections were collected in six-well plates containing phosphate buffered saline (PBS). These free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the guinea pig anti-VGLUT1 or VGLUT2 (diluted 1: 1000, Chemicon, 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-guinea pig IgG (Vector, USA) and ABC complex (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 3,3#-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on the gelatincoated 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. All experimental procedures in the
Sections (15 sections per each animal) were viewed through a microscope connected via a CCD camera to a PC monitor (n Z 7 per group). At a magnification of 25e50!, the region was outlined on a monitor and its area determined. Images of immunoreactivities in the hippocampal complex were also captured using an Applescanner. The brightness and contrast of each image file were calibrated using Adobe Photoshop version 2.4.1, and then analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. All data obtained from image analysis and extracellular recording were analyzed using a paired Student t-test to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. A P value below either 0.01 or 0.05 was considered statistically significant.
2.4. Western blot
3. Results 3.1. Extracellular recording Characteristic responses of the dentate gyrus to stimulation of the perforant path can be seen in Figs. 1e3. There was no difference of the threshold for evoking a population spike between SR and SS gerbils. However, there was a strong trend towards the larger population spike amplitude (by current O200 mA) in SS gerbils (14.1e17.3 mV, P ! 0.01) as compared with SR gerbils
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Fig. 1. The relationship between perforant path stimulation current and dentate granule cell responses in SR (B) and SS () gerbils before and after VPA (30 mg/kg) administration. (A) The stimulation currents O200 mA evoke larger population spike amplitude in SS gerbils than in SR gerbils though no difference of the threshold for evoking a population spike between SR and SS gerbils. (B) The EPSP slopes in SS gerbils are significantly greater than those in SR gerbils, when the stimulation currents are O50 mA. (C),(D) VPA (30 mg/kg) treatment reduces both population spike amplitude and the EPSP slope in the dentate gyrus of SR gerbils. (E),(F) VPA has no effect on them in low stimulus currents (10e100 mA) in SS gerbils, although VPA treatment shows the similar effects on the responses to O100 mA stimulation currents. **P ! 0.01.
(7.2e13.3 mV) (Fig. 1A). The EPSP slope (by current O50 mA) that could be evoked in SS gerbils (3.1e 6.7 mV/ms, P ! 0.01) was also significantly greater than in SR gerbils (1.7e3.3 mV/ms) (Fig. 1B). Following the VPA (30 mg/kg) treatment, however, the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR (population spike amplitude, 4.1e13.3 to 0e8.1 mV; EPSP slope, 0.9e3.3 to 0e 1.6 mV/ms, P ! 0.01, Fig. 1CeD) and of SS gerbils (population spike amplitude, 5.1e17.3 to 3e12.1 mV; EPSP slope 1.2e6.7 to 0.9e4.1 mV/ms, P ! 0.05,
Fig. 1EeF), although this dosage of VPA had no effect on them in low stimulus currents (10e100 mA) in SS gerbils. The mixture treatments of VGB (15 or 30 mg/kg) and VPA (30 mg/kg) also showed the similar effect on the population spike amplitude and the EPSP slope to VPA treatment (30 mg/kg). VGB alone or low dosage of VPA treatment did not affect the responses (Figs. 2 and 3). 3.2. Anatomy and Western blot In the dentate gyrus of SR gerbils, VGLUT1 immunoreactivity was strongly detected in all regions except
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Fig. 2. The effects of VGB and mixture of VGB C VPA on granule cell responses in SS gerbils. (A),(B) VGB (15 or 30 mg/kg) treatment shows no effect on the population spike amplitude and the EPSP slope in the granule cells in SS gerbils. (C),(D) The effect of VPA on the responses to the stimulation currents is unaltered with VGB co-treatment.
in the granule cell layer. Thus, VGLUT1 immunoreactivity was mainly detected in neuropils, not in perikarya (Fig. 4A). Unlike VGLUT1, VGLUT2 immunoreactivity was detected in nerve terminals in the granule cell layer of the dentate gyrus. VGLUT2 immunoreactivity was also observed in the outer two-thirds of the molecular layer of the dentate gyrus (Fig. 5A). This localization pattern of VGLUT immunoreactivities is consistent with that in rats (Bellocchio et al., 2000; Hisano et al., 2000). Surprisingly, in the dentate gyrus of SS gerbils (Figs. 4B and 5B), VGLUT immunoreactivities were markedly enhanced, as compared with the SR gerbils. Strong VGLUT1 immunoreactivity was found predominantly in the molecular layer of the dentate gyrus. In addition, an increase in VGLUT2 immunoreactivity was detected in nerve terminals within the granule cell layer. Elevated VGLUT2 immunoreactivity was also observed in the molecular layer of the dentate gyrus. These intensified VGLUT immunoreactivities were obviously detected in the septal side rather than in the temporal side. To investigate the pharmacological profiles of VGLUT, we administered two anti-epileptic drugs (AEDs), VPA or VGB, to gerbils in vivo. After VPA or VGB treatment, all animals exhibited sedated behavior, i.e., they seemed somnolent, and tended to lie down to assume a squatting posture without movement, more
so than saline treated animals. However, the responses of VGLUT immunoreactivity to drug treatment were significantly different. VPA (30 mg/kg) treatment significantly decreased both VGLUT immunoreactivities in the hippocampus of SS gerbils, as compared with saline treated SS gerbils (Figs. 4D and 5D). In contrast VGLUT immunoreactivities were unaffected by any dosage of VGB treatment (Figs. 4C and 5C) or low dosage (10 and 20 mg/kg) of VPA treatments (data not shown). These effects of two AEDs on the VGLUT immunoreactivities were similarly detected in the hippocampus of SR gerbils (data not shown). The results of the western blot study were consistent with those of immunohistochemical data (Fig. 6).
4. Discussion The epileptogenesis of gerbils has been unclear, although various neurotransmitter systems in the hippocampus of SS gerbils are different from those of SR gerbils (Kang et al., 2000, 2001a,b, 2003aec). In particular, reduced granule cell inhibition alone is insufficient to cause epilepsy of gerbils, because seizure-predisposed SS gerbils (before the onset of behaviorally detectable seizures) show the same pattern of paired-pulse inhibition abnormality as adult SS gerbils
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Fig. 3. Representative traces of population spikes evoked by stimulus 500 mA apart in SR and SS gerbils before and after treatment with VGB, VPA and VGB C VPA. Note only a single population spike is evoked by stimulation. VPA treatment and VGA C VPA result in a reduction in the amount of the population spike amplitude, as compared with saline treated control animals. However, treatment with VGB alone does not affect the population spike amplitude in SS gerbils.
(Buckmaster et al., 2000). Therefore, it is likely that the enhanced excitatory circuits in the dentate gyrus may contribute to epilepsy in this animal, since many reports have suggested that epilepsy may be, at least in part, a result of abnormal glutamatergic synaptic transmission (Li et al., 2004; Conn and Pin, 1997; Attwell et al., 1998a,b). In the present study, the EPSP slope and the population spike amplitude evoked in SS gerbils were significantly greater than in SR gerbils. Based on the electrophysiological properties of granule cells, these changed EPSP profiles are indicative of alterations in the total amount of excitatory transmitter release by the perforant path volley at many synaptic sites. This is because the extracellular EPSP represents the sum of a large number of individual EPSPs generated in the dendrites of a population of granule cells (Lømo, 1971). In fact, the kainate induced rat epilepsy model (Sokal and Large, 2001) shows that EPSP slope and the population spike amplitude are elevated as compared with control animals. Therefore, our findings
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indicate that excitatory input to granule cells in the hippocampus of SS gerbils may be higher than in SR gerbils. On the other hand, granule cells receive excitatory inputs from entorhinal cortex via the outer two-thirds of the molecular layer and from associational/commissural pathways via the inner one-third of the molecular layer (Wyss et al., 1979). These two pathways play an important role in feedback/feedforward excitatory projection in the dentate gyrus (Seress and Ribak, 1984; Sloviter et al., 2003). In the present study, VGLUT immunoreactivities in the molecular layer of the dentate gyrus were significantly enhanced in SS gerbils versus SR gerbils. These findings indicate that that the feedback/forward excitatory projection in the dentate gyrus of SS gerbils may be enhanced, and these elevated excitatory transmissions may cause seizure activity generation or spreading in the gerbil. This is because the expression of synaptic vesicle transporter can regulate the phenotype and quantal size, and that endogenous vesicle transporter expression is rate-limiting, whereby an increased number of vesicular transporters would maintain a higher fill rate (Pothos et al., 2000; Leslie et al., 2001; Pawlu et al., 2004). Furthermore, VGLUT expression reflects storage capacity for intracellular glutamate (Israel et al., 2001), and over-expression of VGLUT in Drosophila neurons leads to an increase in increased glutamate release (Daniels et al., 2004). Therefore, our morphological data indicate that the up-regulation of VGLUT immunoreactivities may represent strong excitatory input to the dentate gyrus in SS gerbils, as compared with SR gerbils. This hyperexcitatory input may evoke prolonged depolarizations and more action potentials in granule cells. VPA effectively reduces glutamate transmission and its presynaptic release in vivo and in vitro (Attwell et al., 1998a; Cunningham et al., 2003). Similarly, in the present study, VPA treatment (30 mg/kg) dramatically reduced VGLUT expressions, the EPSP slope and the population spike amplitude in the dentate gyri of SS and SR gerbils. These findings are in agreement with a previous study (Cunningham et al., 2003) concerning the effect of VPA on glutamate transmission. Furthermore, it is noteworthy that the effect of VPA treatment on the population spike and the EPSP slope in SS gerbils was less effective than that in SR gerbil, particularly in low stimulus current. These findings are further evidence supporting that up-regulation of VGLUT immunoreactivities may be closely related to the hyperexcitability of granule cells or the low threshold of seizure in SS gerbils. However, VGB treatment did not affect the evoked responses and VGLUT immunoreactivities in the dentate gyrus, although both VPA and VGB enhance GABA-mediated inhibition (Macdonald and Kelly, 1993; Czuczwar and Patsalos, 2001; Kang et al., 2003d). In addition, the effect of
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Fig. 4. VGLUT1 immunoreactivity in the hippocampus. In SR gerbils (A), VGLUT1 immunoreactivity is detected in all hippocampal regions except in pyramidal cell layers and in the granule cell layer (GL) of the dentate gyrus (A1e2). In SS gerbils (B), higher VGLUT1 immunoreactivity is observed in the inner (arrowheads) /outer (arrows) one-third of the molecular layer of the dentate gyrus, where VGLUT1 immunoreactivity is weakly detected in SR gerbils. Although VGB treatment (30 mg/kg) shows no effect on VGLUT1 immunoreactivity (C), VPA treatment (30 mg/kg) markedly decreases VGLUT1 immunoreactivity in the dentate gyrus, as compared with SS gerbils (D). Arrows and arrowhead indicate the outer or inner one-third of the molecular layer of the dentate gyrus, respectively. GL, granule cell layer. Scale bars Z 250 mm (AeD) and 35 mm (A1e2, B1e2, C1e 2 and D1e2).
VPA on the EPSP was unaffected by the mixture of various dosages of VGB. These findings indicate that the effect of VPA on the EPSP may not be related to GABAergic actions, and altered VGLUT
immunoreactivity in the dentate gyrus may be independent to GABAergic transmission unlike vesicular GABA transporter (Kang et al., 2003a,c). What are the mechanisms of VPA acting on glutamate
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Fig. 5. VGLUT2 immunoreactivity in the hippocampus. In SR gerbils (A), VGLUT2 immunoreactivity is detected in nerve terminals in the pyramidal cell layer of the hippocampus, in the granule cell layer (GL) and in the outer two-thirds of the molecular layer of the dentate gyrus. In SS gerbils (B), VGLUT2 immunoreactivity in the dentate gyrus is higher than that in SR gerbils. VGLUT2 immunoreactivity in SS gerbils is also unaffected by VGB treatment (C). Similar to VGLUT1, VPA treatment significantly reduces VGLUT2 immunoreactivity in the dentate gyrus of SS gerbils (D). Arrowheads indicate the inner one-third of the molecular layer of the dentate gyrus. GL, granule cell layer. Scale bars Z 250 mm (AeD) and 35 mm (A1e2, B1e2, C1e2 and D1e2).
transmission? Although the exact reasons cannot be explained in the present study, it should be noted that VPA causes inhibition of the TCA cycle activity, and this effect reduces neuronal excitability through modulation of NaC channels in mice brain in vivo
(Johannessen et al., 2001). Since the prolonged reduction in ATP synthesis by VPA may affect HC-ATPase activity that provides coupling glutamate transport via VGLUT (Aihara et al., 2000; Bellocchio et al., 2000), it is likely that NaC channel blockade by VPA may
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Fig. 6. The results of western blot (A) and the densitometric analysis (B) of VGLUT immunoreactivities in the dentate gyrus. In SS gerbil, the amounts of VGLUT immunoreactive bands are higher than those in SR gerbils. These elevated VGLUT immunoreactivities are decreased following VPA (30 mg/kg), not VGB treatment (30 mg/kg). Significant differences from SR gerbils, **P ! 0.01.
reduce excitatory current via reduction in VGLUT-mediated glutamate release. This hypothesis is supported by the similar effect of riluzole, a presynaptic glutamate release inhibitor, on the EPSP slope and the population spike amplitude in the gerbil hippocampus (unpublished data). To understand the precise roles of VPA in altered VGLUT expression further studies are needed. In conclusion, we observed elevations of VGLUT immunoreactivities, the EPSP slope and the population spike amplitude were elevated within the dentate gyrus of SS gerbils and their subsequent reduction induced by VPA treatment. These findings suggest that in SS gerbils the enhancement of excitatory neurotransmissions accompanied by the inadequate inhibitory input to granule cells may be related to epilepsy in this animal.
Acknowledgments The research presented was supported by the Basic Research Program of the Korea Science & Engineering Foundation number R01-2002-000-00008-0, R01-2005000-10004-0, by Ministry of Science and Technology number M103KV010019-03K2201-01910, and by Hallym University.
References Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., Takeda, J., 2000. Molecular cloning of a novel brain-type NaC-dependent inorganic phosphate cotransporter. J. Neurochem. 74, 2622e2625. Andersen, P., Holmqvist, B., Voorhoeve, P.E., 1971. Unit analysis of hippocampal population spikes. Exp. Brain Res. 208e221. Attwell, P.J., Koumentaki, A., Abdul-Ghani, A.S., Croucher, M.J., Bradford, H.F., 1998a. Specific group II metabotropic glutamate receptor activation inhibits the development of kindled epilepsy in rats. Brain Res. 787, 286e291. Attwell, P.J., Singh-Kent, N., Jane, D.E., Croucher, M.J., Bradford, H.F., 1998b. Anticonvulsant and glutamate release-
inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2S, 2#R, 3#R)-2-(2#, 3#-dicarboxycyclopropyl)glycine (DCG-IV). Brain Res. 805, 138e143. Bai, L., Xu, H., Collins, J.F., Ghishan, F.K., 2001. Molecular and functional analysis of a novel neuronal vesicular glutamate transporter. J. Biol. Chem. 276, 36734e36769. Bellocchio, E.E., Reimer, R.J., Fremeau, Jr., R.T., Edwards, R.H., 2000. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289, 957e960. Buckmaster, P.S., Dudek, F.E., 1997. Network properties of the dentate gyrus in epileptic rats with hilar neuron loss and granule cell axon reorganization. J. Neurophysiol. 77, 2685e2696. Buckmaster, P.S., Jongen-Relo, A.L., Davari, S.B., Wong, E.H., 2000. Testing the disinhibition hypothesis of epileptogenesis in vivo and during spontaneous seizures. J. Neurosci. 20, 6232e6240. Buckmaster, P.S., Tam, E., Schwartzkroin, P.A., 1996. Electrophysiological correlates of seizure sensitivity in the dentate gyrus of epileptic juvenile and adult gerbils. J. Neurophysiol. 76, 2169e2180. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205e237. Cunningham, M.O., Woodhall, G.L., Jones, R.S., 2003. Valproate modifies spontaneous excitation and inhibition at cortical synapses in vitro. Neuropharmacology 45, 907e917. Czuczwar, S.J., Patsalos, P.N., 2001. The new generation of GABA enhancers. Potential in the treatment of epilepsy. CNS Drugs 15, 339e350. Daniels, R.W., Collins, C.A., Gelfand, M.V., Dant, J., Brooks, E.S., Krantz, D.E., Diantonio, A., 2004. Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content. J. Neurosci. 24, 10466e10474. De Lanerolle, N.C., Kim, J.H., Robbins, R.J., Spencer, D.D., 1989. Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy. Brain Res. 495, 387e395. Erickson, J.D., Varoqui, H., 2000. Molecular analysis of vesicular amine transporter function and targeting to secretory organelles. FASEB J 14, 2450e2458. Gasnier, B., 2000. The loading of neurotransmitters into synaptic vesicles. Biochimie. 82, 327e337. Herzog, E., Bellenchi, G.C., Gras, C., Bernard, V., Ravassard, P., Bedet, C., Gasnier, B., Giros, B., El Mestikawy, S., 2001. The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons. J. Neurosci. 21, RC181. Hisano, S., Haga, H., Li, Z., Tatsumi, S., Miyamoto, K.I., Takeda, E., Fukui, Y., 1997. Immunohistochemical and RT-PCR detection of NaC-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain. Brain Res. 772, 149e155.
T.-C. Kang et al. / Neuropharmacology 49 (2005) 912e921 Hisano, S., Hoshi, K., Ikeda, Y., Maruyama, D., Kanemoto, M., Ichijo, H., Kojima, I., Takeda, J., Nogami, H., 2000. Regional expression of a gene encoding a neuron-specific NaC-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain. Brain Res. Mol. Brain Res. 83, 34e43. Israel, M., Tomasi, M., Bostel, S., Meunier, F.M., 2001. Cellular resistance to Evans blue toxicity involves an up-regulation of a phosphate transporter implicated in vesicular glutamate storage. J. Neurochem. 78, 658e663. Johannessen, C.U., Petersen, D., Fonnum, F., Hassel, B., 2001. The acute effect of valproate on cerebral energy metabolism in mice. Epilepsy Res. 47, 247e256. 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., 2000. The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil. Brain Res. 870, 179e184. Kang, T.C., Kim, H.S., Seo, M.O., Park, S.K., Kwon, H.Y., Kang, J.H., Won, M.H., 2001a. The changes in the expressions of gamma-aminobutyric acid transporters in the gerbil hippocampal complex following spontaneous seizure. Neurosci. Lett. 310, 29e32. Kang, T.C., Kim, H.S., Seo, M.O., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2001b. The temporal alteration of GAD67/GAD65 ratio in the gerbil hippocampal complex following seizure. Brain Res. 920, 159e169. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Bae, J.C., Won, M.H., 2003a. Changed vesicular GABA transporter immunoreactivity in the gerbil hippocampus following spontaneous seizure and vigabatrin administration. Neurosci. Lett. 335, 207e211. 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., 2003b. The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation. Neurochem. Int 42, 239e249. Kang, T.C., Park, S.K., Hwang, I.K., An, S.J., Won, M.H., 2003c. Presynaptic GABAB receptor-mediated regulation of vesicular GABA transporter expression in the gerbil hippocampus. Neurosci. Lett. 346, 49e52. 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, 115e118. Lee, R.J., Hong, J.S., McGinty, J.F., Lomax, P., 1987. Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure-sensitive Mongolian gerbils. Brain Res. 401, 353e358. Lee, R.Y., Sawin, E.R., Chalfie, M., Horvitz, H.R., Avery, L., 1999. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in caenorhabditis elegans. J. Neurosci. 19, 159e167. Leslie, K.R., Nelson, S.B., Turrigiano, G.G., 2001. Postsynaptic depolarization scales quantal amplitude in cortical pyramidal neurons. J. Neurosci. 21, RC170. Li, Z.P., Zhang, X.Y., Lu, X., Zhong, M.K., Ji, Y.H., 2004. Dynamic release of amino acid transmitters induced by valproate in PTZ-kindled epileptic rat hippocampus. Neurochem. Int. 44, 263e270. Lømo, T., 1971. Patterns of activation in a monosynaptic cortical pathway: the perforant path input to the dentate area of the hippocampal formation. Exp. Brain Res. 12, 18e45.
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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, 109e119. Macdonald, R.L., Kelly, K.M., 1993. Antiepileptic drug mechanisms of action. Epilepsia 34, S1eS8. Margerison, J.H., Corsellis, J.A., 1996. Epilepsy and the temporal lobes. A clinical, electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Brain 89, 499e530. Ozkan, E.D., Ueda, T., 1998. Glutamate transport and storage in synaptic vesicles. Jpn. J. Pharmacol. 77, 1e10. 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, 924e926. Pawlu, C., DiAntonio, A., Heckmann, M., 2004. Postfusional control of quantal current shape. Neuron. 42, 607e618. Pothos, E.N., Larsen, K.E., Krantz, D.E., Liu, Y., Haycock, J.W., Setlik, W., Gershon, M.D., Edwards, R.H., Sulzer, D., 2000. Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size. J. Neurosci. 20, 7297e7306. Reimer, R.J., Fon, E.A., Edwards, R.H., 1998. Vesicular neurotransmitter transport and the presynaptic regulation of quantal size. Curr. Opin. Neurobiol. 8, 405e412. Ribak, C.E., Khan, S.U., 1987. The effects of knife cuts of hippocampal pathways on epileptic activity in the seizure-sensitive gerbil. Brain Res. 418, 146e151. Seress, L., Ribak, C.E., 1984. Direct commisural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition. J. Neurocytol. 13, 215e225. Sloviter, R.S., Zappone, C.A., Harvey, B.D., Bumanglag, A.V., Bender, R.A., Frotscher, M., 2003. ‘‘Dormant basket cell’’ hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat. J. Comp. Neurol. 459, 44e76. Sokal, D.M., Large, C.H., 2001. The effects of GABA(B) receptor activation on spontaneous and evoked activity in the dentate gyrus of kainic acid-treated rats. Neuropharmacology 40, 193e202. Suh, J.G., Ryoo, Z.W., Won, M.H., Oh, Y.S., Kang, T.C., 2001. Differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure. Brain Res. 904, 104e111. Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2000. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189e194. Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2001. Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2). J. Neurosci. 21, RC182. Tuff, L.P., Racine, R.J., Adamec, R., 1983. The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. I. Paired-pulse depression. Brain Res. 277, 79e90. Wittner, L., Magloczky, Z., Borhegyi, Z., Halasz, P., Toth, S., Eross, L., Szabo, Z., Freund, T.F., 2001. Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus. Neuroscience 108, 587e600. Wyss, J.M., Swanson, L.W., Cowan, W.M., 1979. A study of subcortical afferents to the hippocampal formation in the rat. Neuroscience 4, 463e476.
Valproic acid reduces enhanced vesicular glutamate transporter immunoreactivities in the dentate gyrus of the seizure prone gerbil T.-C. Kang a,*, D.-S. Kim a, S.-E. Kwak a, J.-E. Kim a, D.W. Kim b, J.H. Kang c, M.H. Won a, O.-S. Kwon c, S.-Y. Choi b,* a
Department of Anatomy, College of Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea Department of Biomedical Science, College of Life Science, Hallym University, Chunchon 200-702, South Korea c Department of Biochemistry, College of Natural Science, Kyungpook National University, Taegu 702-702, South Korea b
Received 22 April 2005; received in revised form 27 July 2005; accepted 11 August 2005
Abstract To elucidate the relationship between glutamatergic current and vesicular glutamate transporter (VGLUT) expressions, we performed the comparative analyses of evoked potentials and VGLUT immunoreactivities in the dentate gyrus, and its response to antiepileptic drug treatments in a gerbil model. The EPSP slope that could be evoked in seizure sensitive (SS) gerbils was significantly greater than in seizure resistant (SR) gerbils. There was also a strong trend towards the larger population spike amplitude in SS gerbils. In addition, VGLUT immunoreactivities were markedly enhanced in the dentate gyrus of SS gerbils, as compared with the SR gerbils. Following valproic acid (VPA, 30 mg/kg), the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR and of SS gerbils, although this dosage of VPA had no effect in low stimulus currents in SS gerbils. Vigabatrin (VGB) and low dosage of VPA treatment did not affect the evoked responses. Similarly, VPA treatment reduced enhanced VGLUT immunoreactivities in the dentate gyrus of SS gerbils, whilst VGB did not. These findings suggest that up-regulation of VGLUT immunoreactivities may be related to the hyperexcitability of granule cells in SS gerbils, and altered VGLUT immunoreactivity in the dentate gyrus may be independent of GABAergic transmission. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Epilepsy; Hippocampus; Synaptic vesicle; Valproic acid; Vesicular glutamate transporter; Vigabatrin
Abbreviations: VGLUT, vesicular glutamate transporter; SS, seizure sensitive; SR, seizure resistant; VPA, valproic acid; GABA, g-aminobutyric acid; GAD, glutamic acid decarboxylase; GAT-1, GABA transporter; VGAT, vesicular GABA transport; NMDA, N-methyl-D-aspartate receptor; AEDs, antiepileptic drugs; VGB, vigabatrin; ACSF, artificial cerebrospinal fluid; EPSP, excitatory postsynaptic potential; PBS, phosphate buffered saline; DAB, 3, 3#-diaminobenzidine. * Corresponding authors. Tel.: C82 33 248 2524; fax: C82 33 256 1614 (T.-C. Kang); tel.: C82 33 248 2112; fax: C82 33 241 1463 (S.-Y. Choi). E-mail addresses: [email protected] (T.-C. Kang), sychoi@hallym. ac.kr (S.-Y. Choi). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.08.007
1. Introduction Epilepsy is a chronic condition characterized by the presence of spontaneous episodes of abnormal neuronal discharges. Reduced granule cell inhibition is considered the most common cause of temporal lobe epilepsy, since human patients and experimental animals with temporal lobe epilepsy display inhibitory interneuron loss (Margerison and Corsellis, 1996; De Lanerolle et al., 1989; Wittner et al., 2001). Recently, this general concept concerning epilepsy has been challenged, since
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many previous studies that evaluated tissue from patients or models of temporal lobe epilepsy found evidence of normal or enhanced granule cell inhibition, despite inhibitory interneuron loss. The fact is that the paired-pulse inhibition of granule cells is enhanced in most models of temporal lobe epilepsy (Tuff et al., 1983; Hass et al., 1996; Buckmaster and Dudek, 1997). The Mongolian gerbil presents a model for investigating directly the hypothesis that epileptogenesis is characterized by an alteration in the regulation of g-aminobutyric acid (GABA) metabolism, because epileptogenesis in the gerbil has been linked to abnormalities in the GABAergic system; e.g. mismatched glutamic acid decarboxylase (GAD) 65/GAD 67 ratio (Kang et al., 2001b), over-expressions of GABA shunt enzymes (Kang et al., 2003b) and neuronal GABA transporter (GAT-1) (Kang et al., 2001a), and to malfunctions of the vesicular GABA transport (VGAT) system (Kang et al., 2003a,c). These findings indicate a reduction in inhibitory neurotransmission in the dentate gyrus. On the other hand, Ribak and Khan (1987) reported the termination of seizure activity in seizure prone gerbils with bilateral lesions of the perforant path. These observations led us to speculate upon the possible existence of enhanced feedback/feedforward glutamatergic excitation to the dentate gyrus of gerbils, although no differences in the N-methyl-D-aspartate receptor (NMDA) system were found between seizure prone and normal gerbils (Suh et al., 2001). Glutamate has to be loaded into synaptic vesicles by proton-dependent transporters before its exocytotic release (Ozkan and Ueda, 1998; Reimer et al., 1998; Erickson and Varoqui, 2000; Gasnier, 2000). NaC-dependent inorganic phosphate transporter has now been unambiguously established as vesicular glutamate transporter (Hisano et al., 1997; Lee et al., 1999; Aihara et al., 2000; Bellocchio et al., 2000; Takamori et al., 2000, 2001; Bai et al., 2001; Herzog et al., 2001). However, little evidence supports the relationship between epilepsy and the functions of VGLUT. Thus, the issue remains to be clarified as to whether seizure activity in the hippocampus of gerbils may be related with VGLUT expressions, and whether antiepileptic drugs (AEDs) may affect excitability of granule cells or VGLUT expressions. Therefore, to elucidate the roles of glutamatergic systems in epilepsy, the comparative analysis of vesicular glutamate transporter (VGLUT) immunoreactivities and of the effects of two different AEDs, valproic acid (VPA) and vigabatin (VGB), on these expressions was conducted in a gerbil model. This is because these AEDs have distinct pharmacological profiles: VPA mainly acts as a NaC channel blocker (Johannessen et al., 2001), whereas VGB enhances GABA-mediated inhibition via irreversibly inhibiting GABA transaminase (GABA-T), which is a degradative enzyme of GABA (Macdonald and Kelly, 1993; Czuczwar and Patsalos, 2001; Kang et al., 2003d).
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2. Materials and methods 2.1. Experimental animals These studies utilized the progeny of male Mongolian gerbil (Meriones unguiculatus) weighing 80e88 g obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The 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. The procedures involving animals and their care were conducted in conformity with the institutional guidelines and in compliance with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 8523, 1985). Each animal was stimulated by vigorous stroking on the back with a pencil as described by Paul et al. (1981) and tested a minimum of three times. According to the seizure severity rating scale of Loskota et al. (1974) and Lee et al. (1987), seizures were classified; grade 1, a brief pause in normal activity accompanied by vibrissae twitches and retraction of the pinnae; grade 2, motor arrest with twitching of the vibrissae and retraction of the pinnae; grade 3, motor arrest with myoclonic jerks; grade 4, clonic-tonic seizures; grade 5, clonic-tonic seizures with body rollover. Only animals with a consistent stage 4 or 5 seizure score were included in the present study as seizure-sensitive (SS) gerbils. Seizure-resistant (SR) gerbils never demonstrated the seizure activity, thus they were assigned seizure severity score of 0 (Kang et al., 2001a,b; 2003aec). Both SR and SS gerbils were given the drug detailed below once a day for 1 week, respectively: (1) VGB (Sigma USA, 15 or 30 mg/kg, i.p., n Z 10, respectively); (2) VPA (Sigma USA, 10, 20, 30 mg/kg, i.p., n Z 10, respectively); (3) VGB (15 mg/kg) C VPA (30 mg/kg) (i.p., n Z 10); (4) VGB (30 mg/kg) C VPA (30 mg/kg) (i.p., n Z 10); (5) saline (n Z 10). 2.2. In vivo extracellular recording After 2 h after the last drug injection, all animals were anesthetized (urethane, 1.5 g/kg, i.p.) and placed in stereotaxic frames. Holes were drilled through the skull for introducing electrodes. The coordinates (in mm) referenced to bregma were as follows. For the recording electrode (to the dentate gyrus): ÿ3.3 anterior-posterior, 2.5 lateral. For the stimulating electrode (to the angular bundle): ÿ5.0 anterior-posterior, 4.3 lateral. Electrode depths were determined by optimizing the evoked response (Buckmaster et al., 1996, 2000). Glass microelectrodes (microfilament capillary 1.2 outer diameter; 5e10 MU) filled with artificial cerebrospinal fluid (ACSF, in mM; NaCl 126, KCl 5, CaCl2 2, MgCl2 2, NaH2PO4 1.25, NaHCO3 26, D-glucose 10, pH 7.2)
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present study were performed under the same circumstances and in parallel.
and bipolar tungsten stimulating electrodes were used. The reference electrode was placed in the cerebellum. The stimuli were applied as DC square pulses at 0.1 Hz with 150-ms constant current stimuli. Stimulus intensity range was set at 10e1000 mA. The characteristic response of the dentate granule cells to perforant path stimulation consisted of a positive-going field population spike. The population spike amplitude is proportional to the number of granule cells discharging an action potential (Andersen et al., 1971). The excitatory postsynaptic potential (EPSP) slope (25e75% of the initial rising phase) was also measured, since the EPSP slope is indicative of the total amount of excitatory transmitter (mainly glutamate) released by the perforant path volley at many synaptic sites (Lømo, 1971). Signals were recorded with DAM 80 differential amplifier (World Precision Instruments, USA) and data were digitized and analyzed on MacChart 5 (AD Instruments, Australia). Thereafter data were analyzed using the evoked-potential module in MacChart 5. For comparison, the data obtained from three to seven responses were averaged to minimize the variability between animals and each sampling of the population spike and the EPSP slope.
In three animals in each group, the hippocampus on each brain slice was removed and then homogenized in 10 mM PB containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF. After centrifugation, the protein concentrations in the supernatants were determined by using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 3 mg of total protein were boiled with an equal volume of 2! SDS sample buffer and boiled for 3 min, and then, each mixture was loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher & Schuell, USA). To reduced background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, sequentially incubated with primary antisera (1:10,000), peroxidase conjugated goat anti-guinea pig IgG (Sigma, USA), and then with ECL kit (Amersham, USA).
2.3. Anatomy
2.5. Quantitation of data and statistical analysis
After extracellular recording, seven animals in each group were perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer. 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 C and consecutive sections were collected in six-well plates containing phosphate buffered saline (PBS). These free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the guinea pig anti-VGLUT1 or VGLUT2 (diluted 1: 1000, Chemicon, 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-guinea pig IgG (Vector, USA) and ABC complex (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 3,3#-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on the gelatincoated 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. All experimental procedures in the
Sections (15 sections per each animal) were viewed through a microscope connected via a CCD camera to a PC monitor (n Z 7 per group). At a magnification of 25e50!, the region was outlined on a monitor and its area determined. Images of immunoreactivities in the hippocampal complex were also captured using an Applescanner. The brightness and contrast of each image file were calibrated using Adobe Photoshop version 2.4.1, and then analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. All data obtained from image analysis and extracellular recording were analyzed using a paired Student t-test to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. A P value below either 0.01 or 0.05 was considered statistically significant.
2.4. Western blot
3. Results 3.1. Extracellular recording Characteristic responses of the dentate gyrus to stimulation of the perforant path can be seen in Figs. 1e3. There was no difference of the threshold for evoking a population spike between SR and SS gerbils. However, there was a strong trend towards the larger population spike amplitude (by current O200 mA) in SS gerbils (14.1e17.3 mV, P ! 0.01) as compared with SR gerbils
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Fig. 1. The relationship between perforant path stimulation current and dentate granule cell responses in SR (B) and SS () gerbils before and after VPA (30 mg/kg) administration. (A) The stimulation currents O200 mA evoke larger population spike amplitude in SS gerbils than in SR gerbils though no difference of the threshold for evoking a population spike between SR and SS gerbils. (B) The EPSP slopes in SS gerbils are significantly greater than those in SR gerbils, when the stimulation currents are O50 mA. (C),(D) VPA (30 mg/kg) treatment reduces both population spike amplitude and the EPSP slope in the dentate gyrus of SR gerbils. (E),(F) VPA has no effect on them in low stimulus currents (10e100 mA) in SS gerbils, although VPA treatment shows the similar effects on the responses to O100 mA stimulation currents. **P ! 0.01.
(7.2e13.3 mV) (Fig. 1A). The EPSP slope (by current O50 mA) that could be evoked in SS gerbils (3.1e 6.7 mV/ms, P ! 0.01) was also significantly greater than in SR gerbils (1.7e3.3 mV/ms) (Fig. 1B). Following the VPA (30 mg/kg) treatment, however, the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR (population spike amplitude, 4.1e13.3 to 0e8.1 mV; EPSP slope, 0.9e3.3 to 0e 1.6 mV/ms, P ! 0.01, Fig. 1CeD) and of SS gerbils (population spike amplitude, 5.1e17.3 to 3e12.1 mV; EPSP slope 1.2e6.7 to 0.9e4.1 mV/ms, P ! 0.05,
Fig. 1EeF), although this dosage of VPA had no effect on them in low stimulus currents (10e100 mA) in SS gerbils. The mixture treatments of VGB (15 or 30 mg/kg) and VPA (30 mg/kg) also showed the similar effect on the population spike amplitude and the EPSP slope to VPA treatment (30 mg/kg). VGB alone or low dosage of VPA treatment did not affect the responses (Figs. 2 and 3). 3.2. Anatomy and Western blot In the dentate gyrus of SR gerbils, VGLUT1 immunoreactivity was strongly detected in all regions except
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Fig. 2. The effects of VGB and mixture of VGB C VPA on granule cell responses in SS gerbils. (A),(B) VGB (15 or 30 mg/kg) treatment shows no effect on the population spike amplitude and the EPSP slope in the granule cells in SS gerbils. (C),(D) The effect of VPA on the responses to the stimulation currents is unaltered with VGB co-treatment.
in the granule cell layer. Thus, VGLUT1 immunoreactivity was mainly detected in neuropils, not in perikarya (Fig. 4A). Unlike VGLUT1, VGLUT2 immunoreactivity was detected in nerve terminals in the granule cell layer of the dentate gyrus. VGLUT2 immunoreactivity was also observed in the outer two-thirds of the molecular layer of the dentate gyrus (Fig. 5A). This localization pattern of VGLUT immunoreactivities is consistent with that in rats (Bellocchio et al., 2000; Hisano et al., 2000). Surprisingly, in the dentate gyrus of SS gerbils (Figs. 4B and 5B), VGLUT immunoreactivities were markedly enhanced, as compared with the SR gerbils. Strong VGLUT1 immunoreactivity was found predominantly in the molecular layer of the dentate gyrus. In addition, an increase in VGLUT2 immunoreactivity was detected in nerve terminals within the granule cell layer. Elevated VGLUT2 immunoreactivity was also observed in the molecular layer of the dentate gyrus. These intensified VGLUT immunoreactivities were obviously detected in the septal side rather than in the temporal side. To investigate the pharmacological profiles of VGLUT, we administered two anti-epileptic drugs (AEDs), VPA or VGB, to gerbils in vivo. After VPA or VGB treatment, all animals exhibited sedated behavior, i.e., they seemed somnolent, and tended to lie down to assume a squatting posture without movement, more
so than saline treated animals. However, the responses of VGLUT immunoreactivity to drug treatment were significantly different. VPA (30 mg/kg) treatment significantly decreased both VGLUT immunoreactivities in the hippocampus of SS gerbils, as compared with saline treated SS gerbils (Figs. 4D and 5D). In contrast VGLUT immunoreactivities were unaffected by any dosage of VGB treatment (Figs. 4C and 5C) or low dosage (10 and 20 mg/kg) of VPA treatments (data not shown). These effects of two AEDs on the VGLUT immunoreactivities were similarly detected in the hippocampus of SR gerbils (data not shown). The results of the western blot study were consistent with those of immunohistochemical data (Fig. 6).
4. Discussion The epileptogenesis of gerbils has been unclear, although various neurotransmitter systems in the hippocampus of SS gerbils are different from those of SR gerbils (Kang et al., 2000, 2001a,b, 2003aec). In particular, reduced granule cell inhibition alone is insufficient to cause epilepsy of gerbils, because seizure-predisposed SS gerbils (before the onset of behaviorally detectable seizures) show the same pattern of paired-pulse inhibition abnormality as adult SS gerbils
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Fig. 3. Representative traces of population spikes evoked by stimulus 500 mA apart in SR and SS gerbils before and after treatment with VGB, VPA and VGB C VPA. Note only a single population spike is evoked by stimulation. VPA treatment and VGA C VPA result in a reduction in the amount of the population spike amplitude, as compared with saline treated control animals. However, treatment with VGB alone does not affect the population spike amplitude in SS gerbils.
(Buckmaster et al., 2000). Therefore, it is likely that the enhanced excitatory circuits in the dentate gyrus may contribute to epilepsy in this animal, since many reports have suggested that epilepsy may be, at least in part, a result of abnormal glutamatergic synaptic transmission (Li et al., 2004; Conn and Pin, 1997; Attwell et al., 1998a,b). In the present study, the EPSP slope and the population spike amplitude evoked in SS gerbils were significantly greater than in SR gerbils. Based on the electrophysiological properties of granule cells, these changed EPSP profiles are indicative of alterations in the total amount of excitatory transmitter release by the perforant path volley at many synaptic sites. This is because the extracellular EPSP represents the sum of a large number of individual EPSPs generated in the dendrites of a population of granule cells (Lømo, 1971). In fact, the kainate induced rat epilepsy model (Sokal and Large, 2001) shows that EPSP slope and the population spike amplitude are elevated as compared with control animals. Therefore, our findings
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indicate that excitatory input to granule cells in the hippocampus of SS gerbils may be higher than in SR gerbils. On the other hand, granule cells receive excitatory inputs from entorhinal cortex via the outer two-thirds of the molecular layer and from associational/commissural pathways via the inner one-third of the molecular layer (Wyss et al., 1979). These two pathways play an important role in feedback/feedforward excitatory projection in the dentate gyrus (Seress and Ribak, 1984; Sloviter et al., 2003). In the present study, VGLUT immunoreactivities in the molecular layer of the dentate gyrus were significantly enhanced in SS gerbils versus SR gerbils. These findings indicate that that the feedback/forward excitatory projection in the dentate gyrus of SS gerbils may be enhanced, and these elevated excitatory transmissions may cause seizure activity generation or spreading in the gerbil. This is because the expression of synaptic vesicle transporter can regulate the phenotype and quantal size, and that endogenous vesicle transporter expression is rate-limiting, whereby an increased number of vesicular transporters would maintain a higher fill rate (Pothos et al., 2000; Leslie et al., 2001; Pawlu et al., 2004). Furthermore, VGLUT expression reflects storage capacity for intracellular glutamate (Israel et al., 2001), and over-expression of VGLUT in Drosophila neurons leads to an increase in increased glutamate release (Daniels et al., 2004). Therefore, our morphological data indicate that the up-regulation of VGLUT immunoreactivities may represent strong excitatory input to the dentate gyrus in SS gerbils, as compared with SR gerbils. This hyperexcitatory input may evoke prolonged depolarizations and more action potentials in granule cells. VPA effectively reduces glutamate transmission and its presynaptic release in vivo and in vitro (Attwell et al., 1998a; Cunningham et al., 2003). Similarly, in the present study, VPA treatment (30 mg/kg) dramatically reduced VGLUT expressions, the EPSP slope and the population spike amplitude in the dentate gyri of SS and SR gerbils. These findings are in agreement with a previous study (Cunningham et al., 2003) concerning the effect of VPA on glutamate transmission. Furthermore, it is noteworthy that the effect of VPA treatment on the population spike and the EPSP slope in SS gerbils was less effective than that in SR gerbil, particularly in low stimulus current. These findings are further evidence supporting that up-regulation of VGLUT immunoreactivities may be closely related to the hyperexcitability of granule cells or the low threshold of seizure in SS gerbils. However, VGB treatment did not affect the evoked responses and VGLUT immunoreactivities in the dentate gyrus, although both VPA and VGB enhance GABA-mediated inhibition (Macdonald and Kelly, 1993; Czuczwar and Patsalos, 2001; Kang et al., 2003d). In addition, the effect of
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Fig. 4. VGLUT1 immunoreactivity in the hippocampus. In SR gerbils (A), VGLUT1 immunoreactivity is detected in all hippocampal regions except in pyramidal cell layers and in the granule cell layer (GL) of the dentate gyrus (A1e2). In SS gerbils (B), higher VGLUT1 immunoreactivity is observed in the inner (arrowheads) /outer (arrows) one-third of the molecular layer of the dentate gyrus, where VGLUT1 immunoreactivity is weakly detected in SR gerbils. Although VGB treatment (30 mg/kg) shows no effect on VGLUT1 immunoreactivity (C), VPA treatment (30 mg/kg) markedly decreases VGLUT1 immunoreactivity in the dentate gyrus, as compared with SS gerbils (D). Arrows and arrowhead indicate the outer or inner one-third of the molecular layer of the dentate gyrus, respectively. GL, granule cell layer. Scale bars Z 250 mm (AeD) and 35 mm (A1e2, B1e2, C1e 2 and D1e2).
VPA on the EPSP was unaffected by the mixture of various dosages of VGB. These findings indicate that the effect of VPA on the EPSP may not be related to GABAergic actions, and altered VGLUT
immunoreactivity in the dentate gyrus may be independent to GABAergic transmission unlike vesicular GABA transporter (Kang et al., 2003a,c). What are the mechanisms of VPA acting on glutamate
T.-C. Kang et al. / Neuropharmacology 49 (2005) 912e921
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Fig. 5. VGLUT2 immunoreactivity in the hippocampus. In SR gerbils (A), VGLUT2 immunoreactivity is detected in nerve terminals in the pyramidal cell layer of the hippocampus, in the granule cell layer (GL) and in the outer two-thirds of the molecular layer of the dentate gyrus. In SS gerbils (B), VGLUT2 immunoreactivity in the dentate gyrus is higher than that in SR gerbils. VGLUT2 immunoreactivity in SS gerbils is also unaffected by VGB treatment (C). Similar to VGLUT1, VPA treatment significantly reduces VGLUT2 immunoreactivity in the dentate gyrus of SS gerbils (D). Arrowheads indicate the inner one-third of the molecular layer of the dentate gyrus. GL, granule cell layer. Scale bars Z 250 mm (AeD) and 35 mm (A1e2, B1e2, C1e2 and D1e2).
transmission? Although the exact reasons cannot be explained in the present study, it should be noted that VPA causes inhibition of the TCA cycle activity, and this effect reduces neuronal excitability through modulation of NaC channels in mice brain in vivo
(Johannessen et al., 2001). Since the prolonged reduction in ATP synthesis by VPA may affect HC-ATPase activity that provides coupling glutamate transport via VGLUT (Aihara et al., 2000; Bellocchio et al., 2000), it is likely that NaC channel blockade by VPA may
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Fig. 6. The results of western blot (A) and the densitometric analysis (B) of VGLUT immunoreactivities in the dentate gyrus. In SS gerbil, the amounts of VGLUT immunoreactive bands are higher than those in SR gerbils. These elevated VGLUT immunoreactivities are decreased following VPA (30 mg/kg), not VGB treatment (30 mg/kg). Significant differences from SR gerbils, **P ! 0.01.
reduce excitatory current via reduction in VGLUT-mediated glutamate release. This hypothesis is supported by the similar effect of riluzole, a presynaptic glutamate release inhibitor, on the EPSP slope and the population spike amplitude in the gerbil hippocampus (unpublished data). To understand the precise roles of VPA in altered VGLUT expression further studies are needed. In conclusion, we observed elevations of VGLUT immunoreactivities, the EPSP slope and the population spike amplitude were elevated within the dentate gyrus of SS gerbils and their subsequent reduction induced by VPA treatment. These findings suggest that in SS gerbils the enhancement of excitatory neurotransmissions accompanied by the inadequate inhibitory input to granule cells may be related to epilepsy in this animal.
Acknowledgments The research presented was supported by the Basic Research Program of the Korea Science & Engineering Foundation number R01-2002-000-00008-0, R01-2005000-10004-0, by Ministry of Science and Technology number M103KV010019-03K2201-01910, and by Hallym University.
References Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., Takeda, J., 2000. Molecular cloning of a novel brain-type NaC-dependent inorganic phosphate cotransporter. J. Neurochem. 74, 2622e2625. Andersen, P., Holmqvist, B., Voorhoeve, P.E., 1971. Unit analysis of hippocampal population spikes. Exp. Brain Res. 208e221. Attwell, P.J., Koumentaki, A., Abdul-Ghani, A.S., Croucher, M.J., Bradford, H.F., 1998a. Specific group II metabotropic glutamate receptor activation inhibits the development of kindled epilepsy in rats. Brain Res. 787, 286e291. Attwell, P.J., Singh-Kent, N., Jane, D.E., Croucher, M.J., Bradford, H.F., 1998b. Anticonvulsant and glutamate release-
inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2S, 2#R, 3#R)-2-(2#, 3#-dicarboxycyclopropyl)glycine (DCG-IV). Brain Res. 805, 138e143. Bai, L., Xu, H., Collins, J.F., Ghishan, F.K., 2001. Molecular and functional analysis of a novel neuronal vesicular glutamate transporter. J. Biol. Chem. 276, 36734e36769. Bellocchio, E.E., Reimer, R.J., Fremeau, Jr., R.T., Edwards, R.H., 2000. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289, 957e960. Buckmaster, P.S., Dudek, F.E., 1997. Network properties of the dentate gyrus in epileptic rats with hilar neuron loss and granule cell axon reorganization. J. Neurophysiol. 77, 2685e2696. Buckmaster, P.S., Jongen-Relo, A.L., Davari, S.B., Wong, E.H., 2000. Testing the disinhibition hypothesis of epileptogenesis in vivo and during spontaneous seizures. J. Neurosci. 20, 6232e6240. Buckmaster, P.S., Tam, E., Schwartzkroin, P.A., 1996. Electrophysiological correlates of seizure sensitivity in the dentate gyrus of epileptic juvenile and adult gerbils. J. Neurophysiol. 76, 2169e2180. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205e237. Cunningham, M.O., Woodhall, G.L., Jones, R.S., 2003. Valproate modifies spontaneous excitation and inhibition at cortical synapses in vitro. Neuropharmacology 45, 907e917. Czuczwar, S.J., Patsalos, P.N., 2001. The new generation of GABA enhancers. Potential in the treatment of epilepsy. CNS Drugs 15, 339e350. Daniels, R.W., Collins, C.A., Gelfand, M.V., Dant, J., Brooks, E.S., Krantz, D.E., Diantonio, A., 2004. Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content. J. Neurosci. 24, 10466e10474. De Lanerolle, N.C., Kim, J.H., Robbins, R.J., Spencer, D.D., 1989. Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy. Brain Res. 495, 387e395. Erickson, J.D., Varoqui, H., 2000. Molecular analysis of vesicular amine transporter function and targeting to secretory organelles. FASEB J 14, 2450e2458. Gasnier, B., 2000. The loading of neurotransmitters into synaptic vesicles. Biochimie. 82, 327e337. Herzog, E., Bellenchi, G.C., Gras, C., Bernard, V., Ravassard, P., Bedet, C., Gasnier, B., Giros, B., El Mestikawy, S., 2001. The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons. J. Neurosci. 21, RC181. Hisano, S., Haga, H., Li, Z., Tatsumi, S., Miyamoto, K.I., Takeda, E., Fukui, Y., 1997. Immunohistochemical and RT-PCR detection of NaC-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain. Brain Res. 772, 149e155.
T.-C. Kang et al. / Neuropharmacology 49 (2005) 912e921 Hisano, S., Hoshi, K., Ikeda, Y., Maruyama, D., Kanemoto, M., Ichijo, H., Kojima, I., Takeda, J., Nogami, H., 2000. Regional expression of a gene encoding a neuron-specific NaC-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain. Brain Res. Mol. Brain Res. 83, 34e43. Israel, M., Tomasi, M., Bostel, S., Meunier, F.M., 2001. Cellular resistance to Evans blue toxicity involves an up-regulation of a phosphate transporter implicated in vesicular glutamate storage. J. Neurochem. 78, 658e663. Johannessen, C.U., Petersen, D., Fonnum, F., Hassel, B., 2001. The acute effect of valproate on cerebral energy metabolism in mice. Epilepsy Res. 47, 247e256. 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., 2000. The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil. Brain Res. 870, 179e184. Kang, T.C., Kim, H.S., Seo, M.O., Park, S.K., Kwon, H.Y., Kang, J.H., Won, M.H., 2001a. The changes in the expressions of gamma-aminobutyric acid transporters in the gerbil hippocampal complex following spontaneous seizure. Neurosci. Lett. 310, 29e32. Kang, T.C., Kim, H.S., Seo, M.O., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2001b. The temporal alteration of GAD67/GAD65 ratio in the gerbil hippocampal complex following seizure. Brain Res. 920, 159e169. Kang, T.C., An, S.J., Park, S.K., Hwang, I.K., Bae, J.C., Won, M.H., 2003a. Changed vesicular GABA transporter immunoreactivity in the gerbil hippocampus following spontaneous seizure and vigabatrin administration. Neurosci. Lett. 335, 207e211. 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., 2003b. The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation. Neurochem. Int 42, 239e249. Kang, T.C., Park, S.K., Hwang, I.K., An, S.J., Won, M.H., 2003c. Presynaptic GABAB receptor-mediated regulation of vesicular GABA transporter expression in the gerbil hippocampus. Neurosci. Lett. 346, 49e52. 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, 115e118. Lee, R.J., Hong, J.S., McGinty, J.F., Lomax, P., 1987. Increased enkephalin and dynorphin immunoreactivity in the hippocampus of seizure-sensitive Mongolian gerbils. Brain Res. 401, 353e358. Lee, R.Y., Sawin, E.R., Chalfie, M., Horvitz, H.R., Avery, L., 1999. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in caenorhabditis elegans. J. Neurosci. 19, 159e167. Leslie, K.R., Nelson, S.B., Turrigiano, G.G., 2001. Postsynaptic depolarization scales quantal amplitude in cortical pyramidal neurons. J. Neurosci. 21, RC170. Li, Z.P., Zhang, X.Y., Lu, X., Zhong, M.K., Ji, Y.H., 2004. Dynamic release of amino acid transmitters induced by valproate in PTZ-kindled epileptic rat hippocampus. Neurochem. Int. 44, 263e270. Lømo, T., 1971. Patterns of activation in a monosynaptic cortical pathway: the perforant path input to the dentate area of the hippocampal formation. Exp. Brain Res. 12, 18e45.
921
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, 109e119. Macdonald, R.L., Kelly, K.M., 1993. Antiepileptic drug mechanisms of action. Epilepsia 34, S1eS8. Margerison, J.H., Corsellis, J.A., 1996. Epilepsy and the temporal lobes. A clinical, electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Brain 89, 499e530. Ozkan, E.D., Ueda, T., 1998. Glutamate transport and storage in synaptic vesicles. Jpn. J. Pharmacol. 77, 1e10. 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, 924e926. Pawlu, C., DiAntonio, A., Heckmann, M., 2004. Postfusional control of quantal current shape. Neuron. 42, 607e618. Pothos, E.N., Larsen, K.E., Krantz, D.E., Liu, Y., Haycock, J.W., Setlik, W., Gershon, M.D., Edwards, R.H., Sulzer, D., 2000. Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size. J. Neurosci. 20, 7297e7306. Reimer, R.J., Fon, E.A., Edwards, R.H., 1998. Vesicular neurotransmitter transport and the presynaptic regulation of quantal size. Curr. Opin. Neurobiol. 8, 405e412. Ribak, C.E., Khan, S.U., 1987. The effects of knife cuts of hippocampal pathways on epileptic activity in the seizure-sensitive gerbil. Brain Res. 418, 146e151. Seress, L., Ribak, C.E., 1984. Direct commisural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition. J. Neurocytol. 13, 215e225. Sloviter, R.S., Zappone, C.A., Harvey, B.D., Bumanglag, A.V., Bender, R.A., Frotscher, M., 2003. ‘‘Dormant basket cell’’ hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat. J. Comp. Neurol. 459, 44e76. Sokal, D.M., Large, C.H., 2001. The effects of GABA(B) receptor activation on spontaneous and evoked activity in the dentate gyrus of kainic acid-treated rats. Neuropharmacology 40, 193e202. Suh, J.G., Ryoo, Z.W., Won, M.H., Oh, Y.S., Kang, T.C., 2001. Differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure. Brain Res. 904, 104e111. Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2000. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189e194. Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2001. Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2). J. Neurosci. 21, RC182. Tuff, L.P., Racine, R.J., Adamec, R., 1983. The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. I. Paired-pulse depression. Brain Res. 277, 79e90. Wittner, L., Magloczky, Z., Borhegyi, Z., Halasz, P., Toth, S., Eross, L., Szabo, Z., Freund, T.F., 2001. Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus. Neuroscience 108, 587e600. Wyss, J.M., Swanson, L.W., Cowan, W.M., 1979. A study of subcortical afferents to the hippocampal formation in the rat. Neuroscience 4, 463e476.