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Article |
Correspondence to Hiroshi Ohno: ohno{at}rcai.riken.jp
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Abstract |
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Abbreviations used in this paper: AP, adaptor protein; EC, entorhinal cortex; ES, embryonic stem; GABA,
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Introduction |
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Mutations in the ß3A subunit of ubiquitous AP-3A have been identified in patients suffering from the Hermansky-Pudlak syndrome (HPS), in which the function and/or biogenesis of lysosomes and lysosome-related organelles such as melanosomes and platelet dense granules are impaired (Dell'Angelica et al., 1999; Swank et al., 2000). As a result, the HPS patients suffer from such symptoms as abnormal secretion of lysosomal enzymes, pigmentation defect, and prolonged bleeding time. Pearl mice, one of the HPS model mutants, also bear a mutation in the ß3A gene and share the same phenotypes with HPS patients (Feng et al., 1999). Another HPS model, mocha mice, has mutations in the common subunit (Kantheti et al., 1998). As a result, in addition to the phenotypes seen in pearl mice and HPS patients, mocha mice suffer from neurological disorders, such as abnormal electrocorticogram, the recording of electrical activity from cerebral cortex, and inner ear disorders (deafness and balance problem; Rolfsen and Erway, 1984; Noebels and Sidman, 1989; Kantheti et al., 1998). It is possible that these dysfunctions are due to the absence of AP-3B in mocha mice, although little is known about the role of AP-3B in vivo.
To investigate the physiological role of AP-3B, we generated µ3B-deficient mice using the gene targeting technique. Morphological analyses indicated that AP-3B is involved in the biogenesis of synaptic vesicles in vivo. Biochemical and electrophysiological studies corroborated the dysfunction of -aminobutyric acid (GABA) ergic synaptic transmission in µ3B/ mice. Consequently, the µ3B/ mice suffered from spontaneous recurrent epileptic seizures. These findings suggest that AP-3B is responsible for efficient synaptic transmission, particularly the inhibitory one, by regulating the formation and function of a subset of synaptic vesicles.
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Results |
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Seizure susceptibility of µ3B/Neo mice
The frequency of birth of µ3B/Neo mice was in accordance with Mendelian expectations. The mice were fertile and survived for at least more than one year. Although the µ3B/
Neo mice appeared normal, some adult mice exhibited spontaneous epileptic seizures upon presentation of such stimuli as positional change (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200405032/DC1). More than half of the mice suffered from seizures at the age of 15 wk or over. In addition, the electrocorticogram revealed that all of the µ3B/
Neo mice tested showed an abnormal epileptic pattern, namely, interictal spikes, which was never observed in wild-type mice (Fig. 2 A). These observations prompted us to test the seizure susceptibility of µ3B/
Neo mice.
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We further examined the seizure susceptibility of µ3B/Neo mice by electrical kindling, an established model for experimental seizure (Goddard, 1967). Generalized seizure (class 5) was observed in wild-type mice after 1012 stimulations (Fig. 2 C). Notably, all the µ3B/
Neo mice tested reached class 5 within the first two stimulations. Consistently, the afterdischarge, electrical activity recorded after stimulation, evoked by the first kindling stimulation lasted much longer in µ3B/
Neo mice than in wild-type mice (Fig. 2 D; average of the duration of afterdischarge: 7.0 ± 1.3 s in wild-type mice (n = 4) vs. 14.1 ± 4.1 s in µ3B/
Neo mice (n = 4), P < 0.05, t test). Furthermore, the seizure phenotype of µ3B/
Neo mice subjected to kindling stimulation was different from that of wild-type mice, but identical to the spontaneous seizure displayed by µ3B/
Neo mice (unpublished data). Therefore, it is likely that the generalized seizure in µ3B/
Neo mice evoked by kindling stimulation is due to intrinsic epileptogenesis rather than acquired one by the kindling. Together, these results demonstrate that µ3B/
Neo mice have higher seizure susceptibility than wild-type mice.
Morphological abnormalities in excitatory and inhibitory presynaptic terminals of µ3B/Neo mice
There was no difference in brain weight between wild-type and µ3B/Neo mice at any of the postnatal developmental phases. Conventional histological examination including Nissl as well as hematoxylin and eosin staining revealed no abnormality in the overall structure of the brain from µ3B/
Neo mice (unpublished data). Immunohistochemistry showed no astrogliosis in the hippocampus, indicative of neuronal degeneration, suggesting that apparent neuronal cell death does not occur in µ3B/
Neo mice (unpublished data).
We next performed ultrastructural examination of the hippocampus. The number of synaptic vesicles per unit area was decreased in µ3B/Neo mice. The density of synaptic vesicles in excitatory terminals was lower in µ3B/
Neo mice than in wild-type mice at the age of 416 wk (Fig. 3, A, B, and E). The density of synaptic vesicles in inhibitory terminals was also lower in µ3B/
Neo mice than in wild-type mice at the age of 2, 6, and 8 wk (Fig. 3, C, D, and F). In addition, the diameter of the synaptic vesicles in inhibitory synaptic terminals in µ3B/
Neo mice was evidently smaller than that in wild-type mice (Fig. 3, G and H). Thus, these results indicate that AP-3B is involved in the biogenesis of synaptic vesicles in hippocampus in vivo.
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We next examined whether µ3B deficiency affected the localization of the synaptic vesicle proteins including VGAT. Immunohistochemical staining revealed that the distribution of VGAT in the hippocampus from µ3B/Neo mice is comparable to that from wild-type mice (Fig. S1, A and I, available at http://www.jcb.org/cgi/content/full/jcb.200405032/DC1). In cultured hippocampal neurons, VGAT was observed only in axon, where it colocalized with synaptophysin in µ3B-deficient as well as wild-type neurons at 3 d in vitro (Fig. S1, B and J), suggesting that VGAT was targeted to the axon properly in µ3B-deficient neuron. At 14 d in vitro, VGAT was colocalized with both synaptophysin (Fig. S1, C, D, K, and L) and synaptotagmin (Fig. S1, G and O) at synaptic boutons in wild-type and µ3B/
Neo neurons. VGLUT1 was also colocalized with both synaptophysin (Fig. S1, E, F, M, and N) and synaptotagmin (not depicted) in neurons from both genotypes. Localization of VAMP2 was also normal in µ3B/
Neo neuron (Fig. S1, H and P). These results indicate that there is no obvious abnormality in the localization of synaptic vesicle proteins including VGAT in µ3B/
Neo neurons.
Synaptic potentiation is enhanced in µ3B/Neo mice through reduced GABAergic synaptic inhibition
It is well established that the threshold for the induction of long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampal CA1 region is regulated by GABAA receptor-mediated inhibitory synaptic inputs that are activated by afferent fiber stimulation for LTP induction (Wigstrom and Gustafsson, 1983): disinhibition by the blockade of GABAA receptor facilitates LTP induction. To test whether GABAergic synaptic inhibition is impaired in µ3B/Neo mice, we examined the effect of picrotoxin (PTX; 100 µM), a GABAA receptor antagonist, on LTP induction. LTP induced by standard conditioning (100 Hz for 1 s) in µ3B/
Neo mice was intact either in the presence (P > 0.05; Fig. 5 A) or in the absence (P > 0.05; Fig. 5 B) of PTX. However, when weak conditioning (100 Hz for 200 ms) was applied in the absence of PTX (Fig. 5 D), LTP was not induced in wild-type mice (99.3 ± 1.4% of baseline), whereas stable potentiation was induced in µ3B/
Neo mice (117.7 ± 1.7% of baseline; P < 0.05). This difference disappeared when PTX was present (P > 0.05; Fig. 5 C), indicating that the phenotype observed in Fig. 5 D was dependent on GABAA receptor-mediated synaptic transmission. Thus, it is conceivable that when a weaker tetanus is used, the influence of inhibition is relatively stronger and LTP induction is suppressed in wild-type mice, whereas LTP is induced in µ3B/
Neo mice because the inhibition is weaker. These results suggest impaired GABAergic synaptic transmission in µ3B/
Neo mice, and are consistent with the reduced GABA release from presynaptic terminals in µ3B/
Neo mice.
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Discussion |
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µ3B/Neo mice were observed to suffer from spontaneous recurrent epileptic seizures. They were also more susceptible to drug-induced seizures than their wild-type counterpart. The kindling procedure revealed that µ3B/
Neo mice rapidly reached class 5, or generalized seizure. Judging from the observations of the seizure phenotype, however, we surmise that the generalized seizure in µ3B/
Neo mice evoked by kindling stimulation is due to intrinsic epileptogenesis rather than induction by the kindling. It is considered that the suppression by inhibitory neurons of the propagation of kindlingstimulation-induced hyperexcitability prevents the development of behavioral seizures in wild-type mice at the early stages of kindling (Sato et al., 1990). Therefore, the generalized seizure in µ3B/
Neo mice induced at the early stages of kindling further suggests a disorder in the inhibitory neurons in the mice.
AP-3A deficiency in mammals, such as HPS patients and pearl mice, was reported to result in the dysfunction of lysosomes and lysosome-related organelles (Dell'Angelica et al., 1999; Swank et al., 2000). In contrast to AP-3A, however, the function of AP-3B has remained unknown. Mocha mice lacking both AP-3A and AP-3B show neurological phenotypes including abnormal electrocorticogram and inner ear disorders such as deafness and balance problem, in addition to the phenotype seen in AP-3A deficiency (Noebels and Sidman, 1989; Kantheti et al., 1998). Therefore, the neurological disorder observed in mocha mice has been predicted to be due to the AP-3B deficiency. Contrary to the prediction, however, µ3B/Neo mice exhibited neither deafness nor balance problem. The inner ear disorder has been attributed to an insufficiency of heavy metals such as zinc and/or manganese (Rolfsen and Erway, 1984). Kantheti et al. (1998) reported the lack of zinc as well as ZnT-3, a zinc transporter localized in synaptic vesicles, in the brain of mocha mice. By contrast, we observed neither any apparent differences in zinc staining nor the immunolocalization of ZnT-3 in the hippocampus of µ3B/
Neo mice as well as pearl mice, consistent with the lack of inner ear symptoms in these mice (unpublished data). These observations suggest that the inner ear phenotype as well as the mislocalization of ZnT-3 only appears when both AP-3A and AP-3B are deficient in mocha mice.
Functional as well as ultrastructural analyses corroborate the impaired inhibitory synaptic transmission in the absence of µ3B. The K+-evoked release of GABA was decreased significantly in µ3B/Neo mice compared with wild-type mice, whereas that of glutamate was not affected. Electrophysiological experiments demonstrated that, in µ3B/
Neo mice, LTP was induced under the condition that synaptic potentiation was not induced because of the GABAergic synaptic inhibition in wild-type mice. Furthermore, optical recording experiments demonstrated that the neuronal excitability in EC propagated to the CA1 region in the µ3B-deficient condition (8-wk-old µ3B/
Neo mice), which was not observed in the normal condition. The TA pathway can enhance via direct excitatory projection, or suppress via indirect GABAergic interneuron-associated projection, the excitability of CA1 pyramidal cells (Heinemann et al., 2000; Remondes and Schuman, 2002). Together, the results suggest the impairment of GABAergic synaptic inhibition and are consistent with the impairment of GABA release in µ3B/
Neo mice.
Considering that AP-3B is likely expressed in virtually all neurons, the apparent difference in phenotype between excitatory and inhibitory neurons is not fully understood. Similar phenotypic differences between excitatory and inhibitory neurons were observed in several mice deficient in synaptic vesicle proteins (Augustin et al., 1999; Terada et al., 1999; Schoch et al., 2002). Excitatory and inhibitory neurons may exhibit different dependences on these molecules as well as AP-3B to exert their functions. It is also possible that an inhibitory-synapsespecific cargo protein of AP-3B may be responsible for the difference. We surmise that VGAT is an obvious cargo candidate. The amount of VGAT was decreased in the hippocampus of µ3B/Neo mice. It has been shown that unc-47, a VGAT mutant of C. elegans, displays an impairment in GABAergic neurotransmission (McIntire et al., 1997). Therefore, it is conceivable that the impairment of GABAergic synaptic function in µ3B/
Neo mice is, at least in part, due to the reduction of the hippocampal VGAT proteins. Notably, we have identified a potential di-leucine signal, one of the well-characterized sorting signals recognized by AP complexes (Bonifacino and Traub, 2003), in the cytoplasmic tail of VGAT (unpublished data). Thus, AP-3B may play a role in the inclusion of VGAT into synaptic vesicles. However, no reduction of VGAT was detected in the whole brain of µ3B/
Neo mice. One of the possible explanations is that the reduction was apparent in the µ3B/
Neo hippocampus, where the expression level of the µ3B is among the highest and more neurons may depend on µ3B for the targeting of VGAT to synaptic vesicles. Further studies are conducted to address this issue.
In conclusion, the present work has demonstrated the critical role of AP-3B in functional synaptic transmission, particularly the inhibitory one. µ3B deficiency caused an impairment of GABA release possibly because of the reduction of VGAT, which is reflected by the decreased threshold of LTP induction and the abnormal propagation of neuronal excitability in the hippocampus. As a result, µ3B-deficient mice suffered from recurrent epileptic seizure. µ3B/Neo mice may serve as a novel animal model of epilepsy, one of the most common neurological disorders, to benefit epileptic patients in the future.
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Materials and methods |
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Generation of µ3B-deficient mice
A genomic DNA clone containing the µ3B gene was isolated from a mouse 129SV/J genomic library (Stratagene). The targeting vector consisted of a 3-kb EcoRI genomic fragment, EGFP cDNA (CLONTECH Laboratories, Inc.), the Neo gene flanked with two loxP sites (H. Gu, Columbia University, New York, NY; Nishimura et al., 2002) at both ends, a 1.5-kb SalIBamHI genomic fragment, and the HSV-tk gene in pBluescript II SK(+) (Stratagene), as depicted in Fig. 1. EGFP cDNA was placed in frame immediately after the start codon of the µ3B gene so that EGFP was transcribed under the control of µ3B promoter activity. The targeting vector was transfected into ES cells by electroporation. Homologous recombination was confirmed by Southern blotting using both probes S (a PCR product) and L (an EcoRISsp I 0.5-kb fragment). µ3B/ mice were generated essentially as described previously (Ohno et al., 1993). We obtained µ3B/ mice lacking the Neo gene (µ3B/Neo mice) by crossing them with Cre-transgenic mice (Sakai and Miyazaki, 1997). Deletion of the Neo gene was confirmed by Southern blotting using both probe L (Fig. 1 A) and Neo probe (not depicted). We further backcrossed the µ3B/
Neo mice with C57BL/6 mice to at least seven generations to establish µ3B/
Neo mice with the C57BL/6 background. RT-PCR was performed using sense (5'-atgctggacaatgggttcccc-3') and antisense (5'-aattgtagggttctcatcggg-3') primers for µ3B, and sense (5'-caccggcctctccaccatg-3') and antisense (5'-gtgttctgctggtagtggtcg-3') primers for EGFP. All experiments were conducted using littermate or age-matched C57BL/6 mice as control.
Immunoblotting
Whole brains or hippocampi from mice with both genotypes were homogenized in lysis buffer containing 320 mM sucrose and 10 mM Hepes, pH 7.4, with protease inhibitors (Roche Molecular Biochemicals). Synaptosomal and LP2 fraction was prepared as described previously (Huttner et al., 1983). The lysates were subjected to SDS-PAGE and immunoblotting using the following antibodies: anti adaptin (AP.6; American Type Culture Collection), anti-synaptotagmin and anti-VAMP2 (M. Takahashi, Kitazato University, Sagamihara, Japan), anti-synaptophysin (SY38; PROGEN), antirabphilin-3A and ß3B (BD Transduction Laboratory), anti-µ3 (J.S. Bonifacino, National Institutes of Health, Bethesda, MD), anti-Rab3A (Y. Takai, Osaka University, Suita, Japan), anti-GAPDH (CHEMICON International, Inc.), anti-VGLUT1 and anti-VGLUT2 (R.H. Edwards, University of California San Francisco, San Francisco, CA), anti-ZnT3 antibody (T. Palmiter, University of Washington, Seattle, WA), and anti-VGAT antibody (Miyazaki et al., 2003). Western blots were visualized with an ECL system (Super Signal; Pierce Chemical Co.). Chemiluminescence signals were detected by LAS-1000 plus (Fuji Photo Film) and quantified using Image Gause Software (Fuji Photo Film).
Electrocorticogram
Mice were anesthetized with pentobarbital (40 mg/kg, i.p.). Bipolar silver ball electrodes (diameter, 1.0 mm; distance, 3.5 mm) were stereotaxically implanted onto the epidural surface of the left primary motor and left primary somatosensory cortices according to the stereotaxic coordinates of Franklin and Paxinos (2001): 1.18 mm anterior to the bregma and 1.2 mm lateral to the midline, and 2.06 mm posterior to the bregma and 3.0 mm lateral to the midline, respectively. After convalescence for at least 2 wk, electrocorticogram was recorded with a continuous video-EEG monitoring system for freely moving mice.
Pharmacological analysis of seizure susceptibility
The experiment was performed essentially as described previously (Tecott et al., 1995). In brief, PTZ (Sigma-Aldrich) was infused into the tail vein of wild-type (4 wk old, n = 6; 8 wk old, n = 5) or µ3B/Neo (4 wk old, n = 4; 8 wk old, n = 5) mice at a constant rate (1.5 mg/min) and the time required by the animals to go through four stages as judged by their behavior was measured in a blind fashion. The stages are as follows: stage I, no movement; stage II, twitches of head and body; stage III, tonicclonic convulsion; and stage IV, extension and death.
Electrical kindling
Before the surgical procedures, the mice were anesthetized with pentobarbital (40 mg/kg, i.p.). A bipolar stimulation-recording electrode made of stainless steel was stereotaxically implanted into the left basolateral nucleus of the amygdala according to the stereotaxic coordinates of Franklin and Paxinos (2001): 1.94 mm posterior from the bregma, 2.9 mm lateral from the midline and 4.2 mm below the dura mater.
After a recovery period of 10 d, the mice were exposed to kindling stimulation of the left amygdala once daily, consisting of 2 s, 50 Hz biphasic square pulses at the intensity of the afterdischarge threshold. The development of behavioral seizures was classified according to the criteria of Racine (1972).
Morphological studies
Mice from both genotypes were anesthetized with pentobarbital sodium (50 mg/kg) and transcardially perfused with 4% PFA in 0.1 M phosphate buffer, pH 7.4, at 2, 4, 6, 8, and 16 wk old (n = 3, respectively). 4-µm-thick, paraffin-embedded sections were prepared and stained with hematoxylin and eosin and by the Klüver-Barrera method.
For immunohistochemistry, 50-µm-thick vibratome sections were immunostained using the avidinbiotinperoxidase complex method with a Vectastain avidinbiotinperoxidase complex kit (Vector Laboratories). The sections were incubated with rabbit antimouse GAD67 antibody (1:100; Yamada et al., 2001), rabbit anti-VGAT antibody (1:1,000; Miyazaki et al., 2003) and rabbit anti-GFAP antibody (1:500; DakoCytomation). The reaction was visualized with 0.02% 3,-3'-DAB tetrachloride and 0.005% H2O2 in 0.05 M Tris-HCl buffer, pH 7.6, for 10 min at RT.
For EM, the brains were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and 50-µm-thick vibratome sections were cut from the hippocampus. Inhibitory synaptic inputs to the hippocampus are known to converge onto the perisomatic region as well as the basal and apical dendrites of pyramidal cells (Freund and Buzsaki, 1996). Virtually all the synapses on pyramidal cell somata contained pleomorphic vesicles associated with symmetric membrane differentiations, characteristic of inhibitory synapses (Beaulieu and Colonnier, 1985; Freund and Buzsaki, 1996). In addition, nerve terminals immunoreactive for VGAT, a marker for inhibitory neurons (McIntire et al., 1997; Chaudhry et al., 1998), were numerous in the perisomatic region of the CA1 pyramidal cells. Based on these observations, the axon terminals containing pleomorphic vesicles and making symmetric synaptic contact with the pyramidal cell somata were considered to be inhibitory GABAergic terminals. Electron micrographs (x30,000) of asymmetric synaptic terminals in the stratum lacunosum-moleculare (excitatory terminals) and symmetric synaptic terminals that contact with pyramidal cell somata (inhibitory terminals) of the CA1 were taken from random positions. Morphometric analysis was performed on enlarged prints (x45,000). The synaptic boutons and vesicles were digitized using a Macintosh personal computer with a flat head scanner and an interactive pen display at a final magnification of 90,000. NIH Image 1.62 software was used to calculate the areas of synaptic boutons and the numbers and diameters of the synaptic vesicles. All morphometric analyses were performed without knowledge of the genotype by coding specimens.
Measurement of neurotransmitter level
The determination of neurotransmitter release from the hippocampal minislice was performed according to previous studies (Zhu et al., 2000; Okada et al., 2003). The levels of glutamate and GABA were determined by HPLC with fluorescence detection (Zhu et al., 2000; Okada et al., 2001). The excitation and emission fluorescence wavelengths were 340 and 445 nm, respectively. The mobile phase was 0.1 M phosphate buffer, pH 6.0, containing 20% methanol and the flow rate was 300 l/min (Zhu et al., 2000; Okada et al., 2003).
Electrophysiological analysis
Mutant and their littermate wild-type mice (male; 714 wk old) were deeply anesthetized with halothane and decapitated, and then the brains were removed. Hippocampal slices (400 µm thick) were cut with a vibratome tissue slicer and placed in a humidified interface-type holding chamber for at least 1 h. A single slice was then transferred to the recording chamber and submerged in a continuously perfusing medium that had been saturated with 95% O2/5%CO2. The medium contained 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 1.0 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose. All experiments were conducted at 2526°C. Field-potential recordings were made using a glass electrode (3 M NaCl) placed in the stratum radiatum of the CA1 region. An amplifier (model Axopatch 1D; Axon Instruments) was used and the signal was filtered at 1 kHz. Responses were digitized at 10 kHz, stored in a personal computer and analyzed using pClamp 8.1 (Axon Instruments). To evoke excitatory synaptic responses, a bipolar tungsten stimulating electrode was placed in the stratum radiatum, and Schaffer collateral/commissural fibers were stimulated at 0.1 Hz. All experiments were performed in a blind fashion. The data are expressed as means ± SEM. The t test was used for determining whether there was a significant difference (P < 0.05) in the mean between two sets of data.
Optical recording
The procedure for the preparation of brain slices (300 µm) including EC and ventral hippocampus was based on the experimental procedures described in a previous work (Okada et al., 2003). After stabilization, 100 mM di-4-ANEPPS (Molecular Probes), dissolved in 2.7% ethanol and 0.13% Cremophor EL, was added to the incubation medium for 30 min to stain the brain slice. The final concentration of di-4-ANEPPS in the incubation medium was 100 µM.
The MED probe was composed of transparent materials except the electrodes, thereby allowing the localization of the electrodes in the slice under a microscope. After staining, each slice was positioned on a MED probe (MED-P5155; Alpha MED Sciences Co.) such that the array of electrodes was consistent with regard to layers II, III, and IV. The imaging system used a high-speed fluorescence CCD camera (MiCAM01; BrainVision) and a fluorescence microscope (THT-aIII; BrainVision) consisting of an objective lens (PLANAPO x 1; Leica), a projection lens (PLANAPO x 1.6; Leica), a dichroic mirror (575 nm), and absorption (530 nm) and excitation (590 nm) filters (Tominaga et al., 2001; Okada et al., 2004).
During optical recording, the MED probe was superfused with artificial cerebrospinal fluid (in mM: NaCl 124, KCl 5, MgSO4 1.3, Na2HPO4 1.25, CaCl2 2.6, NaHCO3 22, glucose 10) bubbled with 95% O2 and 5% CO2 and maintained continuously at 35°C (Zhu et al., 2000; Okada et al., 2003). EC layers II, III, and IV were stimulated by three electrodes simultaneously at 5 s intervals. The stimulation intensity was adjusted to obtain a submaximal evoked field potential (100 µA). Each stimulus consisted of bipolar constant current pulses of 100 µsec duration. Stimulation patterns were designed using data acquisition software (Panasonic: MED conductor) and delivered through an isolator (BSI-2; Alpha MED Sciences Co.). These procedures of electrical stimulation were controlled using a computer running on Windows NT.
Online supplemental material
Fig. S1 depicts localization of synaptic vesicle proteins in the hippocampus of wild-type and µ3B/Neo mice. Video 1 shows spontaneous seizure of µ3B/
Neo mice. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200405032/DC1.
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Acknowledgments |
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This work was supported by Grants-in-Aid for Young Scientists (B) (to F. Nakatsu), Scientific Research (to H. Ohno, H. Kamiya, T. Manabe, and F. Mori), Scientific Research in Priority Areas (to H. Ohno), Protein 3000 Project (to H. Ohno) and Special Coordination Funds for the Promotion of Science and Technology (to T. Manabe) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, RISTEX, JST (Japan Science and Technology Agency; to T. Manabe), the Uehara Memorial Foundation (to H. Ohno), the Naito Foundation (to H. Ohno and T. Manabe), the Sumitomo Foundation (to T. Manabe), and the Terumo Life Science Foundation (to T. Manabe). We declare that we have no competing financial interests.
Submitted: 6 May 2004
Accepted: 24 August 2004
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