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• W2059713022 abstract "Voltage-dependent N-type Ca2+ channels play important roles in the regulation of diverse neuronal functions in the brain, but little is known about its role in social aggressive behaviors. Mice lacking the α1B subunit (Cav2.2) of N-type Ca2+ channels showed markedly enhanced aggressive behaviors to an intruder mouse in the resident-intruder test. The dorsal raphe nucleus (DRN), which contains serotonin neurons, is known to be involved in aggression in animals. We thus examined the DRN neurons in the Cav2.2-deficient (Cav2.2–/–) mice. Microinjection of ω-conotoxin GVIA, an N-type Ca2+ channel-specific blocker, into the DRN of wild type mice resulted in escalated aggression, mimicking the phenotypes of Cav2.2–/–. Electrophysiological analysis showed increased firing activity of serotonin neurons with a reduced inhibitory neurotransmission in the Cav2.2–/– DRN. Cav2.2–/– mice showed an elevated level of arginine vasopressin, an aggression-related hormone, in the cerebrospinal fluid. In addition, Cav2.2–/– mice showed an increase of serotonin in the hypothalamus. These results suggest that N-type Ca2+ channels at the DRN have a key role in the control of aggression. Voltage-dependent N-type Ca2+ channels play important roles in the regulation of diverse neuronal functions in the brain, but little is known about its role in social aggressive behaviors. Mice lacking the α1B subunit (Cav2.2) of N-type Ca2+ channels showed markedly enhanced aggressive behaviors to an intruder mouse in the resident-intruder test. The dorsal raphe nucleus (DRN), which contains serotonin neurons, is known to be involved in aggression in animals. We thus examined the DRN neurons in the Cav2.2-deficient (Cav2.2–/–) mice. Microinjection of ω-conotoxin GVIA, an N-type Ca2+ channel-specific blocker, into the DRN of wild type mice resulted in escalated aggression, mimicking the phenotypes of Cav2.2–/–. Electrophysiological analysis showed increased firing activity of serotonin neurons with a reduced inhibitory neurotransmission in the Cav2.2–/– DRN. Cav2.2–/– mice showed an elevated level of arginine vasopressin, an aggression-related hormone, in the cerebrospinal fluid. In addition, Cav2.2–/– mice showed an increase of serotonin in the hypothalamus. These results suggest that N-type Ca2+ channels at the DRN have a key role in the control of aggression. Voltage-dependent Ca2+ channels play important roles in the regulation of diverse neuronal functions, including neurotransmitter release, regulation of cell membrane excitability, and control of gene expression. Ca2+ influx via N-type Ca2+ channels has a crucial role in controlling the release of excitatory and inhibitory neurotransmitters at presynaptic terminals in central synapses (1Catterall W.A. Cell Calcium.. 1998; 24: 307-323Google Scholar). Recently, genetically engineered mice lacking N-type Ca2+ channel α1B subunit have been developed and used in experiments to clarify in vivo functions of N-type Ca2+ channels. Although previous analyses of Cav2.2-deficient (Cav2.2–/–) 4The abbreviations used are: Cav2.2, N-type Ca2+ channels α1B subunit; DRN, dorsal raphe nucleus; 5-HT, serotonin; AVP, arginine vasopressin; GABA, γ-aminobutyric acid; CSF, cerebrospinal fluid; IPSC, inhibitory postsynaptic current; EPSC, evoked postsynaptic current; HPLC, high pressure liquid chromatography; ADHD, attention deficit/hyperactivity disorder; 5-HIAA, 5-hydroxyindolacetic acid. mice have revealed physiological roles of N-type Ca2+ channels in various behaviors (2Kim C. Jun K. Lee T. Kim S.S. McEnery M.W. Chin H. Kim H.L. Park J.M. Kim D.K. Jung S.J. Kim J. Shin H.S. Mol. Cell Neurosci... 2001; 18: 235-245Google Scholar, 3Ino M. Yoshinaga T. Wakamori M. Miyamoto N. Takahashi E. Sonoda J. Kagaya T. Oki T. Nagasu T. Nishizawa Y. Tanaka I. Imoto K. Aizawa S. Koch S. Schwartz A. Niidome T. Sawada K. Mori Y. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 5323-5328Google Scholar, 4Beuckmann C.T. Sinton C.M. Miyamoto N. Ino M. Yanagisawa M. J. Neurosci... 2003; 23: 6793-6797Google Scholar, 5Newton P.M. Orr C.J. Wallace M.J. Kim C. Shin H.S. Messing R.O. J. Neurosci... 2004; 24: 9862-9869Google Scholar, 6Jeon D. Kim C. Yang Y.M. Rhim H. Yim E. Oh U. Shin H.S. Genes Brain Behav.. 2007; 6: 375-388Google Scholar), there was no study of the role of N-type Ca2+ channels in aggression. It is believed that most social animals possess neural mechanisms for the control of aggression, which is essential for maintenance of beneficial relationships among members in a community. Aggression is considered a complex social behavior influenced by both internal (e.g. hormones or genes) and external stimuli (e.g. drive, TV, and frustration etc.) (7de Waal F.B. Science.. 2000; 289: 586-590Google Scholar). Deficit of the control mechanisms for suppression of aggression might be implicated in the development of violence in various psychiatric disorders, such as attention deficit/hyperactivity disorder (ADHD) and personality disorders (8Fountoulakis K.N. Leucht S. Kaprinis G.S. Curr. Opin. Psychiatry.. 2008; 21: 84-92Google Scholar, 9Castellanos F.X. Elia J. Kruesi M.J. Gulotta C.S. Mefford I.N. Potter W.Z. Ritchie G.F. Rapoport J.L. Psychiatry Res... 1994; 52: 305-316Google Scholar). The modulation of aggression has been associated with a change in the central serotonin (5-HT) system at the dorsal raphe nucleus (DRN) (10Bannai M. Fish E.W. Faccidomo S. Miczek K.A. Psychopharmacology.. 2007; 193: 295-304Google Scholar, 11Mos J. Olivier B. Poth M. Van Oorschot R. Van Aken H. Eur. J. Pharmacol... 1993; 238: 411-415Google Scholar, 12Sijbesma H. Schipper J. de Kloet E.R. Mos J. van Aken H. Olivier B. Pharmacol. Biochem. Behav... 1991; 38: 447-458Google Scholar, 13van der Vegt B.J. Lieuwes N. van de Wall E.H. Kato K. Moya-Albiol L. Martinez-Sanchis S. de Boer S.F. Koolhaas J.M. Behav. Neurosci... 2003; 117: 667-674Google Scholar, 14Dahlstrom A. Fuxe K. Experientia.. 1964; 20: 398-399Google Scholar). In the DRN, the 5-HT neuronal activity showing a slow and regular firing pattern (15Allers K.A. Sharp T. Neuroscience.. 2003; 122: 193-204Google Scholar, 16Trulson M.E. Jacobs B.L. Brain Res... 1979; 163: 135-150Google Scholar, 17Vandermaelen C.P. Aghajanian G.K. Brain Res... 1983; 289: 109-119Google Scholar) is known to be influenced by γ-aminobutyric acid (GABA), 5-HT1A autoreceptor, and noradrenergic input (17Vandermaelen C.P. Aghajanian G.K. Brain Res... 1983; 289: 109-119Google Scholar). Furthermore, the modulation of these signals in the DRN affected aggression behaviors in animals (10Bannai M. Fish E.W. Faccidomo S. Miczek K.A. Psychopharmacology.. 2007; 193: 295-304Google Scholar, 11Mos J. Olivier B. Poth M. Van Oorschot R. Van Aken H. Eur. J. Pharmacol... 1993; 238: 411-415Google Scholar, 12Sijbesma H. Schipper J. de Kloet E.R. Mos J. van Aken H. Olivier B. Pharmacol. Biochem. Behav... 1991; 38: 447-458Google Scholar, 13van der Vegt B.J. Lieuwes N. van de Wall E.H. Kato K. Moya-Albiol L. Martinez-Sanchis S. de Boer S.F. Koolhaas J.M. Behav. Neurosci... 2003; 117: 667-674Google Scholar, 18Pudovkina O.L. Cremers T.I. Westerink B.H. Synapse.. 2003; 50: 77-82Google Scholar, 19Tao R. Auerbach S.B. Brain Res... 2003; 961: 109-120Google Scholar). It has been shown that the Cav2.2 is highly expressed in the DRN of adult rats (20Tanaka O. Sakagami H. Kondo H. Brain Res. Mol. Brain Res... 1995; 30: 1-16Google Scholar). However, the role of Cav2.2 in the DRN has not been examined, nor has the involvement of Cav2.2 in the regulation of aggression. Therefore, we have characterized aggression behaviors of Cav2.2–/– mice and investigated the properties of the Cav2.2–/– DRN neurons. Interestingly, the Cav2.2–/– mice exhibited an enhanced aggressive behavior and a reduced inhibitory transmission in the DRN. In addition, Cav2.2–/– mice showed an increased level of arginine vasopressin (AVP) in the cerebrospinal fluid (CSF), together with an increase of 5-HT in the hypothalamus. Our results suggest a possibility that the N-type Ca2+ channel plays a critical role in the suppression of aggressive behaviors. Animals—All of the animals were handled in accordance with the animal care and use guidelines of the Korea Institute of Science and Technology. The animals were housed in a temperature- and humidity-controlled environment with free access to food and water under a 12-h light/12-h dark cycle. Mice lacking the α1B subunit (Cav2.2) were established previously (2Kim C. Jun K. Lee T. Kim S.S. McEnery M.W. Chin H. Kim H.L. Park J.M. Kim D.K. Jung S.J. Kim J. Shin H.S. Mol. Cell Neurosci... 2001; 18: 235-245Google Scholar). For this study, homozygotes (Cav2.2–/–) in the F1 (C57BL/6J × 129S4/SvJae) background were produced by crossing heterozygous 129 co-isogenic mice (Cav2.2+/–) with heterozygous N12 C57BL/6J mice (Cav2.2+/–). The Cav2.2–/– male mice (10–15 weeks old) in the F1 background were used for behavioral tests, and wild type littermates served as control. All of the behavioral experiments were performed and videotaped by one investigator, and the tape was interpreted by another investigator blinded to the genotypes of the animals used in the experiments. Resident-Intruder Test—The test was based on a previously described protocol (21Chiavegatto S. Dawson V.L. Mamounas L.A. Koliatsos V.E. Dawson T.M. Nelson R.J. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 1277-1281Google Scholar). The isolation-induced resident-intruder aggression test was conducted by introducing a small, experimentally naïve male C57BL/6J mouse (5–6 weeks old) that had been housed in groups of five into a clear polycarbonate cage (27 × 21 × 17 cm) of a resident male mouse (10–15 weeks old) that had been housed in isolation for at least 4 weeks without a change of bedding for 7 days before testing. The behavior of the resident mouse was videotaped, and offensive aggression was measured by determining the latency to the first attack and total number of bite attacks by the isolated resident mouse during 15 min of exposure to the experimental naive male C57BL/6J intruder mouse. If an animal did not make a bite attack, the latency to the first attack was recorded as 900 s (test duration), and all the other attack scores were recorded as zero. Water Competition Test—The water competition test used was based on a previously described method (22Muehlenkamp F. Lucion A. Vogel W.H. Pharmacol. Biochem. Behav... 1995; 50: 671-674Google Scholar). Male mice of both genotypes of equal weight were paired and housed together in a same cage. After 6 days, the animals were deprived of water for 23 h. One water bottle was introduced with a shielded spout so that only one animal could drink at a time. The time (in seconds) of spout possession and water consumption were recorded for 2 min. The animal with the longest duration of water consumption and spout possession was considered to be the dominant animal. Intra-raphe Injection and Histology—Wild type male mice were anesthetized with Avertin® (2,2,2 tribromoethanol; 0.2 ml/10 g of 20 mg/ml solution), and a 26-gauge guide cannular (Plastics One) for microinjection of the N-type Ca2+ channel blocker, ω-conotoxin GVIA (ω-GVIA), was stereotaxically placed into the DRN. The cannular was implanted at anterior-posterior = –4.5 mm from the bregma; lateral = ±1.2 mm; ventral = –4.0 mm; tilted 22.5 degree, as calculated from the mouse brain atlas (23Kang S.J. Cho S.H. Park K. Yi J. Yoo S.J. Shin K.S. Mol. Cells.. 2008; 25: 124-130Google Scholar). After 10 days, the animals were injected either with vehicle (0.9% NaCl containing 1 mg/ml cytochrome C) alone or with ω-GVIA (9 ng/0.2 μl/5 min, Alomone) into the DRN 1 h before the resident-intruder test. After the experiments, the mice were deeply anesthetized by injection of Avertin®, and the brains were removed, fixed in 4% paraformaldehyde at 4 °C for at least 48 h, and stored in a 30% sucrose solution until being sliced on a freezing microtome (30-μm-thick sections) (23Kang S.J. Cho S.H. Park K. Yi J. Yoo S.J. Shin K.S. Mol. Cells.. 2008; 25: 124-130Google Scholar). The brains were lightly stained with cresyl violet for histological verification of needle placement. All of the injection sites included in the statistical analyses were referenced to the mouse brain atlas (24Paxinos G. Franklin K.B.J. The Mouse Brain in Stereotaxic Coordinates.2nd Ed. Academic Press, San Diego2001Google Scholar) and illustrated in Fig. 2. Extracellular Recording of 5-HT Neurons—Mice (8–12 weeks old) were decapitated, and coronal 400-μm brain stem slices containing the DRN were prepared in oxygenated cold artificial CSF (124 mm NaCl, 3.5 mm KCl, 1.25 mm NaH2PO4, 2 mm CaCl2, 1.3 mm MgSO4, 26 mm NaHCO3, and 10 mm glucose, pH 7.4). After incubation for 1 h at room temperature, a single slice was submerged in a recording chamber and continuously superfused with oxygenated artificial CSF (34 °C; 95% O2/5% CO2) (25Jeon D. Song I. Guido W. Kim K. Kim E. Oh U. Shin H.S. J. Biol. Chem... 2008; 283: 12093-12101Google Scholar). Firing was recorded in the DRN using a borosilicate glass microelectrode (8–10 MΩ) filled with 2 m NaCl and evoked by adding the α1-adrenoreceptor agonist phenylephrine (3 μm) (17Vandermaelen C.P. Aghajanian G.K. Brain Res... 1983; 289: 109-119Google Scholar). The cells were identified as putative 5-HT neurons according to the following criteria: biphasic action potentials, long spike width (>2 ms), slow and regular pattern of discharges, and reversible inhibition by 5-HT (100 μm) (15Allers K.A. Sharp T. Neuroscience.. 2003; 122: 193-204Google Scholar, 17Vandermaelen C.P. Aghajanian G.K. Brain Res... 1983; 289: 109-119Google Scholar). Signals were amplified 10,000-fold and filtered at 300∼3000 Hz by a Cyberamp-402 amplifier (Axon Instruments), digitized at a 10-kHz sampling rate, and stored in a computer (Digidata; Axon Instruments). The number of spikes was quantified using Mini-Analysis software (Synaptosoft). Whole Cell Patch Clamp Recording of 5-HT Neurons—Mice (2.5–3.5 weeks old) were decapitated, and brain stem slices (250∼300 μm) containing the DRN were prepared as described above for extracellular recordings, and the methods of patch clamp recording were described in our previous report (25Jeon D. Song I. Guido W. Kim K. Kim E. Oh U. Shin H.S. J. Biol. Chem... 2008; 283: 12093-12101Google Scholar). Pipettes (3–4 MΩ) were filled with the internal solution for patch clamp recording (140 mm KCl, 0.1 mm CaCl2, 4.6 mm MgCl2, 0.1 mm EGTA, 10 mm HEPES, 4 mm Na-ATP, and 0.4 mm Na-GTP, adjusted to pH 7.3 with KOH). The DRNs were visually identified by their position along the midline in the slice and by their morphology. Although the staining of 5-HT or tryptophan hydroxylase is needed exactly to identify a 5-HT neuron, cells, at least, that meet the electrophysiological criteria of a 5-HT neuron were recorded (supplemental data). For IPSC and EPSC measurement, the cells were voltage-clamped at –60 mV. GABA receptor-mediated sIPSC and eIPSC was isolated and recorded in the presence of 6-cyano-7-nitroquinoxaline-2.3-dione (10 μm, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist), dl-2-amino-5-phosphonovaleric acid (50 μm, N-methyl-d-aspartate receptor antagonist), and CGP 55845 (5 μm, a selective GABAB receptor antagonist). sEPSC and eEPSC were measured in the presence of bicuculline (10 μm, GABAA receptor antagonist) and CGP 55845. For the eIPSC and eEPSC experiments, the current was evoked within DRN by a bipolar tungsten electrode. The signals were filtered at 2 kHz, digitized at 5 kHz sampling rate, and stored in a computer (Digidata; Axon Instrument). The data was analyzed by Minianlysis software (Synaptosoft Inc.). Radioimmunoassay of AVP—To determine the basal levels of CSF AVP hormones, individually housed male mice were left undisturbed for 4 weeks and anesthetized with Avertin® in the light phase. Collection of CSF samples was based on what was described by DeMattos et al. (26DeMattos R.B. Bales K.R. Parsadanian M. O'Dell M.A. Foss E.M. Paul S.M. Holtzman D.M. J. Neurochem... 2002; 81: 229-236Google Scholar). Clear CSF samples were drawn (10–15 μl/animal) from the cisterna magna using a glass capillary to avoid blood contamination of CSF. All of the samples were centrifuged for at 3000 × g for 15 min at 4 °C. The clear supernatants were stored at –70 °C until the assay. The AVP levels were measured in 10-μl samples of CSF using a radioimmunoassay kit (Peninsula Laboratories) according to the manufacturer's protocols. Ventricular Infusion of a V1a Receptor Antagonist—Male mice were anesthetized with Avertin®, and an osmotic pump (flow rate, 0.25 μl/h; ALZET) for infusion of AVP antagonist was stereotaxically placed with its tip into a lateral ventricle region. The stereotaxic coordinates for the microinfusion were anterior-posterior = –0.22 mm from the bregma; lateral = ±1.0 mm; ventral = –2.8 mm. The animals were infused with either vehicle (phosphate-buffered saline) or the selective V1a receptor antagonist d((CH2) 15,Tyr(Me)2,Arg8)-vasopressin (AVP-8, 10 ng/μl; Bachem) into the lateral ventricle using the pump according to the manufacturer's protocols. After 10 days, the resident-intruder test was performed. Analysis of Monoamines by High Pressure Liquid Chromatography (HPLC)—The adult brain was rapidly removed and dissected on ice as previously described (27Iversen L.L. Glowinski J. J. Neurochem... 1966; 13: 671-682Google Scholar). The hypothalamus was immediately homogenized in 0.1 m perchloric acid with a Tissue Tearer (Biospec) at 30,000 rpm for 30 s. The homogenates (5% w/v) were centrifuged at 48,000 × g for 20 min at 4 °C, and the supernatants were collected and stored at –70 °C until analysis. The endogenous levels of norepinephrine, dopamine, 5-HT, and their metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid (5-HIAA) were determined by reverse phase HPLC with an ICA-3063 electrochemical detector (Toa). Briefly, 10-μl portions of the samples were separated using a C18 reversed-phase column (octadecyl silica; 4.6 mm × 150 mm; NaKalai, Japan) and separated using a mobile phase of 85:15 0.5 m KH2PO4, pH 3.2, containing 2.5 mm 1-octane sulfate and 10-μm EDTA/methanol delivered at 0.9 ml/min. The oxidation potential of the detector was fixed at 750 mV using a glass carbon working electrode versus an Ag/AgCl reference electrode. The peak areas of the internal standard (dihydrobenzylamine) were used to quantify the sample peaks. The values obtained were expressed as μg/g of tissue wet weight. Statistical Analyses—All of the values are presented as the means ± S.E. of the means (S.E.), and statistical analysis was performed using Student's t test. The values were considered statistically different at p < 0.05. Enhanced Aggression in Cav2.2–/– Mice—To quantify the aggressive behaviors of Cav2.2–/– male mice, we performed the resident-intruder test (21Chiavegatto S. Dawson V.L. Mamounas L.A. Koliatsos V.E. Dawson T.M. Nelson R.J. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 1277-1281Google Scholar). As shown in Fig. 1 (A and B), Cav2.2–/– mice showed a significantly shortened latency to the first attack on the intruder mouse and an increased number of attacks compared with wild type male littermates, showing that Cav2.2–/– mice are significantly more aggressive than the wild type mice. Because dominant males are more aggressive than subordinate males in intermale conflicts to maintain their social position (28Raleigh M.J. McGuire M.T. Brammer G.L. Pollack D.B. Yuwiler A. Brain Res... 1991; 559: 181-190Google Scholar), we next performed the water competition test (22Muehlenkamp F. Lucion A. Vogel W.H. Pharmacol. Biochem. Behav... 1995; 50: 671-674Google Scholar). As shown in Fig. 1C, Cav2.2–/– mice displayed a longer duration of water consumption than the wild type. There was no significant difference in the duration of water consumption over a 2-min test period between the two genotypes, when they were separately housed (data not shown). These results demonstrate that Cav2.2–/– mice are dominant over the wild type mice in social interactions. Intra-raphe Injection of an N-type Ca2+ Channel Blocker—To examine whether the inactivation of N-type Ca2+ channels in the DRN is responsible for the increased aggression of Cav2.2–/– mice, we focally injected an N-type Ca2+ channel blocker, ω-conotoxin GVIA (ω-GVIA), into the DRN of wild type male mice according to the procedures described under “Experimental Procedures.” The mice microinjected with ω-GVIA showed symptoms mimicking the phenotype of the Cav2.2–/– mice: a significantly reduced latency to the first attack and an increased number of bite attacks on the intruder compared with the saline-treated control mice (Fig. 2, C and D). Histological analyses confirmed that injection placements were correctly located in the dorsal raphe (Fig. 2B). An example of the cannular tract and injection site is shown in Fig. 2A. These results indicate that the highly aggressive behaviors of Cav2.2–/– mice was primarily due to a lack of N-type Ca2+ channels in the DRN and also suggest that a developmental anomaly is not a factor in the expression of the aggression phenotype. Increased Activities of 5-HT Neurons in Cav2.2–/– Mice—To define the alterations in the DRN that resulted from the mutation and were responsible for the aggression phenotype, we examined the physiological properties of the DRN in slices. First, we recorded extracellularly the single-unit activities from the putative 5-HT neurons, identified according to the criteria described in literature (15Allers K.A. Sharp T. Neuroscience.. 2003; 122: 193-204Google Scholar, 17Vandermaelen C.P. Aghajanian G.K. Brain Res... 1983; 289: 109-119Google Scholar), in the DRN slices of the wild type and Cav2.2–/– mice. Firing was induced by adding the α1-adrenoreceptor agonist phenylephrine (3 μm). The results show that 5-HT neurons of the Cav2.2–/– DRN fired more frequently than those of the wild type mice (wild type, 1.66 ± 0.16 Hz; Cav2.2–/– mice, 2.85 ± 0.23 Hz; p < 0.01; Student's t test) (Fig. 3, A and B). For further analysis of 5-HT neurons in the DRN of the Cav2.2–/– mice, we next tried to identify 5-HT neurons, based on electrophysiological criteria as described under “Experimental Procedures” and supplemental data and measured their activity by whole cell patch clamp recordings with the procedures. We analyzed individual action potential shapes in the current clamp recordings. There was no difference in the amplitude or duration of action potential or in the amplitude of after hyperpolarization between the wild type and Cav2.2–/– mice (supplemental Fig. S1 and Table S1). However, when we slightly depolarized membrane potential of the cells by an intracellular current injection of +20 pA, we consistently observed that the 5-HT neurons in the Cav2.2–/– DRN fired more frequently than those of the wild type mice (wild type, 2.48 ± 0.27 Hz; Cav2.2–/–, 3.57 ± 0.36 Hz; p < 0.05; Student's t test) (Fig. 4, A–C, before bicuculline). These results were consistent with the extracellular single-unit recordings as described above (Fig. 3). The activity of 5-HT neurons can be highly modulated by input from the GABAergic interneurons (19Tao R. Auerbach S.B. Brain Res... 2003; 961: 109-120Google Scholar). To test whether the increased firing rate of 5-HT neurons in the Cav2.2–/– DRN was due to a decreased GABA inhibition, we recorded neuronal activities from identified 5-HT neurons, based on electrophysiological criteria, in the presence of bicuculline, a GABAA receptor antagonist. The application of bicuculline (10 μm) increased the firing rates in both Cav2.2–/– and the wild type to a similar level (the wild type with bicuculline, 4.01 ± 0.51 Hz; Cav2.2–/– with bicuculline, 4.56 ± 0.39 Hz; p = 0.48; Student's t test) (Fig. 4C). However, the magnitude of the increase of the firing rate was significantly greater (p < 0.01; Student's t test) in the wild type (161.56 ± 7.55%) than in the Cav2.2–/– (131.10 ± 4.75%), reflecting the already increased level of firing in the mutant, presumably because of decreased inhibition (Fig. 4D). These results suggested a possibility that the increased firing rate in the 5-HT neurons of Cav2.2–/– mice was due to an impaired GABA transmission in the DRN. Decreased GABA Release in the Cav2.2–/– DRN—To define further how the inhibitory transmission is altered in the Cav2.2–/– DRN, we measured spontaneous and evoked inhibitory postsynaptic currents (sIPSC and eIPSC, respectively) from identified 5-HT neurons, based on electrophysiological criteria, in DRN slices under a voltage-clamp configuration. We found that the frequency of sIPSC in Cav2.2–/– neurons was significantly reduced compared with that of wild type neurons (wild type, 3.17 ± 0.68 Hz; Cav2.2–/–, 1.05 ± 0.48 Hz; p < 0.05; Fig. 5B), without any significant change in the amplitude (wild type, 46.84 ± 14.42 pA; Cav2.2–/–, 41.74 ± 3.94 pA; p = 0.85; Fig. 5C). Furthermore, this reduction of sIPSC in the Cav2.2–/– was mimicked in the wild type slices by an N-type channel blocker; when ω-GVIA (1 μm) was added to the recording solution of the wild type slice (n = 5), the frequency of the sIPSC was reduced to the values displayed in the Cav2.2–/– (wild type with ω-GVIA, 1.18 ± 0.45 Hz; Fig. 5B), without any change in the amplitude (wild type with ω-GVIA, 42.04 ± 11.40 pA; Fig. 5C). Next, we recorded the eIPSC in 5-HT neurons following a focal stimulation within the DRN. The maximum amplitude of eIPSC in the Cav2.2–/– was lower than that in the wild type (wild type, 172.89 ± 20.53 pA; Cav2.2–/–, 32.69 ± 5.92 pA; p < 0.0001; Fig. 5D). Furthermore, application of ω-GVIA to the wild type slice significantly reduced the amplitude of eIPSC, by 75% (Fig. 5E). These results on sIPSC and eIPSC indicate that presynaptic GABA release is impaired with normal postsynaptic GABA responses in the Cav2.2–/– DRN and suggest that N-type Ca2+ channels have a crucial role in the release of GABA in the DRN. In contrast to IPSC, there was no difference in the frequency or amplitude of the spontaneous excitatory postsynaptic current (sEPSC) between the two genotypes (supplemental Fig. S2, B and C). The maximum amplitude of evoked excitatory postsynaptic current (eEPSC) in the Cav2.2–/– was also similar to that of the wild type (supplemental Fig. S2, D and E). Thus, these results indicate that excitatory transmissions into 5-HT neurons were not altered in the Cav2.2–/– DRN. Increased Levels of CSF AVP and Hypothalamic 5-HT/5-HIAA in Cav2.2–/–—Central AVP is known to play an important role in the regulation of aggressive behaviors. In particular, an increased level of CSF AVP has been consistently implicated in enhanced aggressive behaviors in various species including humans (29Ferris C.F. Potegal M. Physiol. Behav... 1988; 44: 235-239Google Scholar, 30Haller J. Makara G.B. Barna I. Kovacs K. Nagy J. Vecsernyes M. J. Neuroendocrinol... 1996; 8: 361-365Google Scholar, 31Coccaro E.F. Kavoussi R.J. Hauger R.L. Cooper T.B. Ferris C.F. Arch. Gen. Psychiatry.. 1998; 55: 708-714Google Scholar). We thus tried to measure AVP levels in the CSF of the Cav2.2–/– mice and found that the Cav2.2–/– mice have a significantly higher level of AVP in the CSF compared with wild type mice (Fig. 6A). We then examined whether the increased level of AVP is related to the enhanced aggression of Cav2.2–/– mice. It has been demonstrated that microinjection of the AVP receptor (V1a) antagonist d(CH2)5,Tyr(Me)AVP into the anterior hypothalamus of the golden hamster rapidly inhibits aggression (29Ferris C.F. Potegal M. Physiol. Behav... 1988; 44: 235-239Google Scholar, 32Ferris C.F. Melloni Jr., R.H. Koppel G. Perry K.W. Fuller R.W. Delville Y. J. Neurosci... 1997; 17: 4331-4340Google Scholar). In our experiments, microinfusion of an AVP receptor V1a antagonist, d((CH2)51,Tyr(Me)2, Arg8)-vasopressin (AVP-8, 10 ng/μl, 0.25 μl/hr), into the lateral ventricle using an osmotic pump reduced the level of aggressiveness of Cav2.2–/– mice (Fig. 6, B and C). However, the same dose of the antagonist did not affect the aggression behavior of wild type mice (Fig. 6, B and C). These results suggest that the elevation of AVP in the CSF is at least in part involved in the enhanced aggressive behaviors of Cav2.2–/– mice. A large number of studies have revealed that AVP is produced in the hypothalamus, including the paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, and suprachiasmatic nucleus, in addition to other brain areas including bed nucleus of the stria terminalis, medial amygdale, and lateral septum (32Ferris C.F. Melloni Jr., R.H. Koppel G. Perry K.W. Fuller R.W. Delville Y. J. Neurosci... 1997; 17: 4331-4340Google Scholar, 33Davis E.S. Marler C.A. Neuroscience.. 2004; 127: 611-624Google Scholar, 34DeVries G.J. Buijs R.M. Van Leeuwen F.W. Caffe A.R. Swaab D.F. J. Comp. Neurol... 1985; 233: 236-254Google Scholar, 35Delville Y. De Vries G.J. Ferris C.F. Brain Behav. Evol... 2000; 55: 53-76Google Scholar). Moreover, those AVP neurons in the hypothalamus are innervated by serotonergic neurons originating in the dorsal and median raphe nucleus (36Sawchenko P.E. Swanson L.W. Steinbusch H.W. Verhofstad A.A. Brain Res... 1983; 277: 355-360Google Scholar, 37Larsen P.J. Hay-Schmidt A. Vrang N. Mikkelsen J.D. Neuroscience.. 1996; 70: 963-988Google Scholar). Therefore, we next attempted to measure the amount of monoamines including 5-HT in the hypothalamus of the Cav2.2–/– mice using HPLC. The results revealed that the levels of 5-HT and 5-HIAA were significantly elevated in the Cav2.2–/– hypothalamus compared with those of the wild type (Fig. 6D), indicating increased 5-HT release in the Cav2.2–/– hypothalamus. However, there was no significant difference in the levels of other monoamines between the two genotypes. In this study, we examined aggressive b" @default.
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• W2059713022 title "Deletion of N-type Ca2+ Channel Cav2.2 Results in Hyperaggressive Behaviors in Mice" @default.
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