Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M800816200 on March 13, 2008

J. Biol. Chem., Vol. 283, Issue 18, 12093-12101, May 2, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/18/12093    most recent
M800816200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, D.
Right arrow Articles by Shin, H.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, D.
Right arrow Articles by Shin, H.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Ablation of Ca2+ Channel β3 Subunit Leads to Enhanced N-Methyl-D-aspartate Receptor-dependent Long Term Potentiation and Improved Long Term Memory*Formula

Daejong Jeon{ddagger}§, Inseon Song{ddagger}, William Guido, Karam Kim||, Eunjoon Kim||, Uhtaek Oh§1, and Hee-Sup Shin{ddagger}2

From the {ddagger}Center for Neural Science, Korea Institute of Science and Technology, Seoul 136-791, Korea, §National Creative Research Initiative Center for Sensory Research, Seoul National University, College of Pharmacy, Seoul 151-742, Korea, the Department of Anatomy and Neurobiology, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298, and ||National Creative Research Initiative Center for Synaptogenesis, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea

Received for publication, January 31, 2008 , and in revised form, March 12, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The β subunits of voltage-dependent Ca2+ channels (VDCCs) have marked effects on the properties of the pore-forming {alpha}1 subunits of VDCCs, including surface expression of channel complexes and modification of voltage-dependent kinetics. Among the four different β subunits, the β3 subunit (Cavβ3) is abundantly expressed in the hippocampus. However, the role of Cavβ3 in hippocampal physiology and function in vivo has never been examined. Here, we investigated Cavβ3-deficient mice for hippocampus-dependent learning and memory and synaptic plasticity at hippocampal CA3-CA1 synapses. Interestingly, the mutant mice exhibited enhanced performance in several hippocampus-dependent learning and memory tasks. However, electrophysiological studies revealed no alteration in the Ca2+ current density, the frequency and amplitude of miniature excitatory postsynaptic currents, and the basal synaptic transmission in the mutant hippocampus. On the other hand, however, N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic currents and NMDAR-dependent long term potentiation were significantly increased in the mutant. Protein blot analysis showed a slight increase in the level of NMDAR-2B in the mutant hippocampus. Our results suggest a possibility that, unrelated to VDCCs regulation, Cavβ3 negatively regulates the NMDAR activity in the hippocampus and thus activity-dependent synaptic plasticity and cognitive behaviors in the mouse.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-dependent Ca2+ channels (VDCCs)3 play important roles in the regulation of diverse neuronal functions by mediating Ca2+ entry into cells. VDCCs have multiple subunit structures consisting of a major pore-forming subunit ({alpha}1) and several auxiliary subunits ({alpha}2{delta}, β, and {gamma}) (1, 2). VDCCs are classified into L-type (Cav1.1, Cav1.2, Cav1.3, and Cav1.4: {alpha}1S, {alpha}1C, {alpha}1D, and {alpha}1F, respectively), P/Q-type (Cav2.1: {alpha}1A), N-type (Cav2.2: {alpha}1B), R-type (Cav2.3: {alpha}1E), and T-type (Cav3.1, Cav3.2, and Cav3.3: {alpha}1G, {alpha}1H, and {alpha}1I, respectively) based on electrophysiological and pharmacological properties (3). Among the auxiliary subunits, the β subunits are entirely cytosolic, and they have marked effects on the properties of VDCCs {alpha}1 subunits, including trafficking of Ca2+ channel complexes to the plasma membrane, voltage dependence and activation/inactivation kinetics of Ca2+ currents (4, 5). Four β subunits (Cavβ1–4) have been cloned, and each Cavβ has distinctive properties (5), but their functional roles in the brain in vivo are still poorly understood.

Structurally, Cavβ has five different domains, with the two conserved domains sharing significant homology among the β subunits. The conserved domains were revealed as an Src homology 3 (SH3) domain and a guanylate kinase (GK) domain (6–9), and thus Cavβ is included in membrane-associated guanylate kinase family that has scaffolding functions. Interestingly, it has been suggested that Cavβ can bind to other molecules (10, 11). For example, Cavβ could directly interact with small G-proteins (Gem and Rem) and dynamin (12–14). In addition, recent studies have suggested that Cavβ can work without marked influence on VDCCs. For example, regulation of gene transcription by a direct interaction between a short splice variant of Cavβ4 and a nuclear protein was shown in the cochlea (15). Cavβ3 was also shown to regulate insulin secretion by acting on the intracellular Ca2+ store, whereas Ca2+ currents of VDCCs were not affected (16). This study suggests that Cavβ can function as a multifunctional protein.

Of the Cavβ subunits, Cavβ3 is highly expressed in the brain, especially in the hippocampus (17). It was shown that {alpha}1 subunits of N- and L-type VDCCs were preferentially associated with Cavβ3 in the hippocampus (18–20). In addition, N- and L-type VDCCs have been strongly implicated in activity-dependent long lasting synaptic changes, such as LTP, as well as in learning and memory (21, 22). Therefore, we examined the Cavβ3-deficient mice (23) for hippocampus-dependent learning and memory and synaptic plasticity. Interestingly, long term memory and NMDAR-dependent LTP were increased in the Cavβ3-deficient mice, whereas there was no significant change in Ca2+ currents. Furthermore, the mutant mice showed increased NMDAR-mediated synaptic responses and an increased NR2B level in the hippocampus. These results reveal Ca2+ channel-independent functions of Cavβ3 in the hippocampus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—The generation of mice lacking Cavβ3 was described in our previous study (23). Cavβ3 heterozygous (Cavβ3+/–) mice were backcrossed into two inbred backgrounds, C57BL/6J and 129S4/SvJae, each over 18 generations. Cavβ3 wild-type (Cavβ3+/+) and Cavβ3-deficient (Cavβ3–/–) mice used for analysis were obtained from interbreeding Cavβ3+/– mice of the two backgrounds. Animal care and handling were carried out according to the institutional guidelines. The mice were maintained with free access to food and water under a 12:12-h light/dark cycle. Behavioral experiments were performed on 8–12-week-old mice. All experiments were performed in a blind manner with respect to the genotype.

Contextual and Cued Fear Conditioning—The fear conditioning was carried out as described in our previous study (24). A fear-conditioning shock chamber (19 x 20 x 33 cm) containing a stainless steel rod floor (5 mm diameter, spaced 1 cm apart) and a monitor was used (WinLinc behavioral experimental control software, Coulbourn Instruments). For conditioning, mice were placed in the fear-conditioning apparatus chamber for 2 min, and then a 28-s acoustic conditioned stimulus (CS) was delivered. Following the CS, a 0.5-mA shock of unconditioned stimulus was immediately applied to the floor grid for 2 s. This protocol was performed twice at 60-s interval. To assess contextual learning, the animals were placed back into the training context 24 h after training, and then freezing behavior was observed for 4 min. To assess cued learning, the animals were placed in a different context (a novel chamber, odor, floor, and visual cues) 24 h after training, and their behaviors were monitored for 5 min. During the last 3 min of this test, animals were exposed to the tone. Fear response was quantified by measuring the length of the time when the animal showed freezing behaviors, which was defined as lack of movements with a crouching position, except for respiratory movements (25). Foot-shock intensity was evaluated by placing naive animals in the conditioning chamber used for fear conditioning. Animals were subjected to a 1-s series of gradually increasing mild foot-shock amperage at 20-s intervals as follows: 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, and 0.6 mA. The shock intensity that evoked initial sensation responses (flinching and running), vocalization, and jumping was recorded for each mouse.

Novel Object Recognition Memory Task—The task was performed as described (24, 26, 27). The mice were individually habituated to an open-field box (40 x 40 x 40 cm) for 3 days. During the training trial, two objects were placed in the box, and animals were allowed to explore them for 5 min. A mouse was considered to be exploring the object when its head was facing the object within 1-inch distance. Following retention intervals (1 or 24 h), animals were placed back into the box with two objects in the same locations, but one of the familiar objects was replaced by a novel object, and mice were then allowed to explore the two objects for 5 min. The preference percentage, percentage of the time spent exploring the novel object over the total time spent exploring both objects, was used to quantitate the recognition memory.

Social Transmission of Food Preference Task—This task was performed as described previously (21, 28, 29), with slight modifications. "Demonstrator" mice were given a distinctively scented food (cinnamon or cocoa) for 2 h and then immediately allowed to interact with "observer" mice for 30 min. Either 1 or 24 h later, observers were given a choice between two scented foods: either the same scented food that the demonstrators had eaten (cued) or another distinctively scented food (non-cued). Half of the observers in each genotype was subjected to interaction with the demonstrators that had eaten cinnamon as cued food and the other half with those that had eaten cocoa as cued food to control for the possibility of food preference bias.

Whole-cell Patch Clamp Recording on Acutely Isolated CA1 Pyramidal Neurons and on Hippocampal Slices—All experiments were performed on 2–3-week-old mice. Preparation of and recording from hippocampal slices (400 µm thick) were as described in our previous study (21, 30). Hippocampal slices were prepared in oxygenated, cold ACSF (124 mM NaCl, 3.5 mM KCl, 1.25 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, and 10 mM glucose, pH 7.4). For the measurement of Ca2+ currents, acutely isolated CA1 pyramidal neurons were prepared from hippocampal slices, as described in our previous study (30). The recorded CA1 neurons were voltage-clamped at –60 mV using glass pipette electrodes (3–5 M{Omega} series resistance <20 M{Omega}) and the I-V curve was generated in a stepwise fashion: +10-mV increments from –60 to +40 mV. Internal pipette solution contained the following, 130 mM CsCl, 10 mM EGTA, 10 mM HEPES, 4 mM MgCl2, 4 mM MgATP, 0.3 mM Tris-GTP, 5 mM tetraethylammonium chloride, and was brought to pH 7.4 with NaOH. Extracellular solution contained the following, 25 mM tetraethylammonium chloride, 5 mM 4-aminopyridine, 20 mM HEPES, 3 mM KCl, 5 mM CaCl2, 2 mM MgCl2, 100 mM NaCl, 0.001 mM tetrodotoxin, and was brought to pH 7.4 with NaOH. For the measurement of after hyperpolarization (AHP) currents, visually guided CA1 pyramidal neurons in hippocampal slice were held at –55 mV, and currents were evoked by depolarizing voltage commands to 20 mV for 200 ms followed by a return to –55 mV for 10 s. During recording, the slices were superfused with ACSF at room temperature. Glass pipettes (3–5 M{Omega}) were filled with solution containing 140 mM KMeSO4, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 2 mM Mg-ATP, 0.4 mM Na2-GTP, and 0.02 mM EGTA (pH 7.3, 290 mosM). In addition, action potentials (APs) were triggered under current clamp mode by depolarizing current injection (from +30 to +90 pA), and the number of AP (from threshold to the peak) and AP durations (width at half-height) were measured. The internal solution for mEPSC (miniature excitatory postsynaptic currents) recording was filled with the following buffer, 135 mM potassium gluconate, 5 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 0.5 mM CaCl2, 5 mM Mg-ATP and 0.3 mM Na-GTP, and was brought to pH 7.4 with KOH. The experiment was performed in the presence of tetrodotoxin (1 µM) and bicuculline (10 µM, a GABA type a receptor antagonist). The recorded CA1 pyramidal neurons were voltage-clamped at –70 mV. The frequency and amplitude of mEPSCs were analyzed with MiniAnalysis (Synaptosoft) (21). For the measurement of AMPAR- and NMDAR-mediated synaptic currents in visually guided CA1 pyramidal neurons, pipettes (3–5 M{Omega}) were filled with the internal solution (130 mM cesium gluconate, 5 mM KCl, 0.1 mM CaCl2, 2.0 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 10 mM QX-314, 4 mM Na-ATP, and 0.4 mM Na-GTP, brought to pH 7.3 with CsOH). The currents were measured in the presence of bicuculline (10 µM) and CGP 55845 (5 µM, a GABA type B receptor antagonist). The synaptic currents were evoked by a bipolar tungsten electrode that was placed in the stratum radiatum. NMDAR- and AMPAR-mediated responses were discriminated based on their distinct kinetics and voltage dependence; the NMDAR-mediated currents were measured at +40 mV, 100 ms after the response onset, whereas the AMPAR-mediated currents were taken as the peak amplitude response recorded at –70 mV (31). D-AP5 (50 µM) blocked the late component of the currents recorded at +40 mV, whereas CNQX (10 µM), an AMPA receptor blocker, eliminated the currents recorded at –70 mV. Whole-cell patch clamp currents were recorded and digitized with a MultiClamp 700A amplifier and a Digidata 1320 or 1322A (Axon Instruments, CA), and acquired data were analyzed with the pCLAMP version 9.2 (Axon Instruments) and the Mini Analysis Program (Synaptosoft).

Extracellular Recording on Hippocampal Slices—Preparation of hippocampal slices and the method of field excitatory postsynaptic potentials (fEPSPs) recording have been described previously (21, 24). Hippocampal slices (400 µm) were prepared from 7–8-week-old mice, as described above. Slices were then placed in a warm, humidified (32 °C, 95% O2, 5% CO2) recording chamber containing oxygenated ACSF and maintained for 1.5 h prior to experiments. A bipolar stimulating electrode was placed in the stratum radiatum in the CA1 region, and extracellular field potentials were also recorded in the stratum radiatum using a glass microelectrode (borosilicate glass, 3–5 M{Omega}, filled with 3 M NaCl). Test responses were elicited at 0.033 Hz. Base-line stimulation was delivered at an intensity that evoked a response that was ~40% of the maximum evoked response. LTP was induced electrically by one of the following protocols: 1) 100-Hz LTP was induced for 100 ms, 300 ms, or 1 s; 2) 200-Hz LTP was induced by 10 trains of 200-ms stimulation at 200 Hz delivered every 5 s. LTD was elicited by paired-pulses low frequency stimulation (PP-LFS) (50-ms pulse interval at 1 Hz for 15 min). Drugs were added to the perfusion medium at least 30 min before recording.

Immunohistology and Western Blot—Immunostaining was performed as described previously (32, 33). Animals were anesthetized and perfused through the heart with 50 ml of cold saline and 50 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were then removed and were post-fixed overnight. Coronal sections containing hippocampus were stained with the following primary antibodies: anti-β3 subunit (anti-Cavβ3, Alomone Labs), anti-SMI-32, and anti-GAD. A biotinylated secondary antibody and the avidin/biotin system were used for each antibody followed by a 3,3'-diaminobenzidene reaction. Some of the DAB reactions incorporated a nickel intensification procedure. For gross morphology of the hippocampus, Nissl staining was used. For Western blot analysis, total hippocampal proteins were prepared as described previously (34). 25 µg of protein were loaded per lane and analyzed by SDS-PAGE followed by Western blotting. The following antibodies have been described previously: NMDAR 2A/2B (35) and GluR1/2 (34). The following fusion protein was used for the generation of the following polyclonal antibody: H6-rat NMDAR1 (amino acids 340–561; 1740 guinea pig). Antibody for {alpha}-tubulin was purchased from Sigma.

Statistical Analysis—All data are given as mean ± S.E. Two-way repeated ANOVA, one-way ANOVA, and Student's t test were used for statistical analyses. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Normal Gross Morphology of the Hippocampus in the Cavβ3–/– Mice—We first examined the cytoarchitectonic divisions in the brain of the Cavβ3–/– mice, especially in the hippocampus. The Cavβ3–/– mice exhibited normal hippocampal divisions, including CA1, CA2, CA3, and dentate gyrus. No expression of Cavβ3 was observed in the Cavβ3–/– hippocampus (Fig. 1A), whereas Cavβ3 was abundant in the wild-type hippocampus as was shown previously (17). The immunoreactivities and the expression patterns of SMI-32 (a neurofilament protein) (Fig. 1B) and GAD (GABA-synthesizing enzyme) (Fig. 1C) were normally observed in the hippocampus of the Cavβ3–/– mice as in the Cavβ3+/+ mice. In addition, Nissl staining of the coronal brain sections revealed no gross abnormalities in the hippocampus of the Cavβ3–/– mice (Fig. 1D).

Enhanced Contextual Fear Conditioning in the Cavβ3–/– Mice—Because Cavβ3 is highly expressed in the hippocampus and is known to be associated with N- or L-type VDCCs, which play important roles in hippocampus-dependent learning and memory in animals (17–19), we examined whether the deletion of Cavβ3 affected the animal's capacity for hippocampus-dependent learning and memory. First, we subjected the mice to the fear conditioning assay that is known to require the function of the hippocampus (36). The Cavβ3+/+ (n = 14) and Cavβ3–/– (n = 14) mice showed similar levels of freezing response during the training (Fig. 2A). In the contextual fear memory assay performed 24 h after the training, the Cavβ3–/– mice displayed more freezing behavior than the Cavβ3+/+ (F(1, 26) = 8.36, p < 0.01, two-way repeated ANOVA), indicating an enhanced long term memory of the Cavβ3–/– mice for contextual fear conditioning. A post hoc test (Scheffe's test) also revealed significant differences between the two genotypes during the 2nd (p < 0.05), the 3rd (p < 0.05), and the 4th min (p < 0.05) (Fig. 2B). On the other hand, no difference was observed between the two genotypes in the cued fear conditioning assay (Fig. 2C), indicating that the enhanced memory in the Cavβ3–/– mice is limited to the hippocampus-dependent fear conditioning. There was no significant difference in response to variable electric intensities between Cavβ3–/– (n = 7) and Cavβ3+/+ (n = 9) mice, indicating comparable reactivity or sensitivity to electric foot-shock of the two genotypes (Fig. 2D).


Figure 1
View larger version (121K):
[in this window]
[in a new window]

  		FIGURE 1.
Histological assessment of the hippocampus of the Cavβ3–/– mouse. A, strong immunoreactivity of Cavβ3 in the Cavβ3+/+ hippocampus indicates that it is highly expressed in the hippocampus, whereas there is no detectable signal in the Cavβ3–/–. Similar levels of SMI-32 (B) and GAD (C) immunoreactivity are observed in the hippocampus of Cavβ3+/+ and Cavβ3–/– brain. SMI-32 staining provides Golgi-like staining of neurons/axons, and GAD immunoreactivity is restricted to small interneurons around the pyramidal layer. D, normal gross morphology of the hippocampus revealed by Nissl staining in comparable hippocampal regions of Cavβ3+/+ and Cavβ3–/– mice. The scale bars equal 100 µm(A, B, and D) and 50 µm(C). Arrowheads indicate CA1, CA2, and CA3 region in order.

 
Enhanced Novel Object Recognition Memory in the Cavβ3–/– Mice—We next subjected the mice to the novel object recognition task that is based on the animal's ability to discriminate a novel object from a familiar one, which requires the hippocampus (37). We first assessed the amount of time spent by the animals exploring the two objects during the training trial, and we found that both of the genotypes, Cavβ3+/+ (n = 17) and Cavβ3–/– mice (n = 14), explored the two objects for equal time (Fig. 2E), which indicated no preference of the animals for either object. At a 1-h retention interval, when one of the familiar objects was replaced by a novel one, both Cavβ3+/+ (n = 8) and Cavβ3–/– mice (n = 7) exhibited increased preference for the novel object to the familiar one (F(1, 13) = 22.86, p < 0.001, two-way repeated ANOVA). No difference, however, was found between the two genotypes (F(1, 13) = 0.01, p = 0.96, one-way ANOVA) (+/+, 72.90 ± 4.27%; –/–, 73.34 ± 8.83%) (Fig. 2F). At the 24-h retention test, however, Cavβ3–/– mice (n = 7) showed increased preference for the novel object compared with Cavβ3+/+ (n = 9) (F(1, 14) = 36.14, p < 0.001, two-way repeated ANOVA, Scheffe's post hoc test, p < 0.01) (+/+, 62.68 ± 6.26%; –/–, 88.90 ± 3.23%) (Fig. 2F), indicating that the Cavβ3–/– mice have an enhanced performance in the object recognition memory task.

Enhanced Long Term Memory in the Social Transmission of Food Preference Task in the Cavβ3–/– Mice—Finally, we carried out the social transmission of food preference assay, another hippocampus-dependent memory task. This task exploits the tendency of mice to prefer food that they have recently smelled on the breath of other mice (demonstrator mice), and subsequently, this tests their ability to learn and remember the information transmitted by olfactory cues during social interactions. 1 h after social interactions with demonstrator mice, both Cavβ3+/+ (n = 7) and Cavβ3–/– (n = 6) mice preferred the "cued" food to the "non-cued" food, and there was no significant difference between the two genotypes (+/+, 83.70 ± 3.63%; –/–, 75.87 ± 7.34%, F(1, 11) = 0.72, p = 0.41, one-way ANOVA) (Fig. 2G). The amount of total food eaten was not different between genotypes during this task (Fig. 2H). These results indicate that the mice were not deficient in olfaction or social interactions.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 2.
Enhanced long term memory in hippocampus-dependent learning and memory tasks of Cavβ3–/– mice. A–D, fear conditioning. A, freezing behavior on the day of training in Cavβ3+/+ (n = 14) and Cavβ3–/– (n = 14). Solid line indicates the duration of CS (tone, 28 s), and the triangles indicate unconditioned stimulus (foot shock, 2 s). B, contextual fear conditioning 24 h after training. Cavβ3–/– mice displayed more freezing behavior than the Cavβ3+/+ for contextual fear conditioning. *, p < 0.05, Scheffe's post hoc test. C, cued fear conditioning 24 h after training. CS (tone) presentation is indicated by the solid line. D, responses to variable foot shock intensities. F, flinching; R, running; V, vocalization; J, jumping. E and F, novel object recognition task. E, mean exploratory preference during training in Cavβ3+/+ (n = 17) and Cavβ3–/– mice (n = 14). F, exploration to a novel object after each retention time. At 24 h retention, Cavβ3–/– mice show increased preference for the novel object compared with Cavβ3+/+. Dotted line indicates equal exploration of all objects. **, p < 0.01, Scheffe's post hoc test. G and H, social transmission of food preference task. G, at 1-h retention, both genotypes show a preference for the cued food, and there is no difference between genotypes (+/+, n = 7; –/–, n = 6). After 24 h, however, Cavβ3–/– mice (n = 10) exhibit more preference to cued food than Cavβ3+/+ mice (n = 10). *, p < 0.05, one-way ANOVA. H, total food eaten during each test time.

 
On the other hand, 24 h after interactions with demonstrator mice, Cavβ3–/– mice (n = 10) exhibited significantly increased preference for cued food compared with Cavβ3+/+ mice (n = 10) (+/+, 71.61 ± 4.72%; –/–, 88.62 ± 3.56%, F(1, 18) = 7.10, p < 0.05, one-way ANOVA) (Fig. 2G). There was no significant difference between genotypes in the amount of total food that was eaten (Fig. 2H). These results suggest that Cavβ3–/– mice displayed an enhanced memory in the social transmission of food preference task.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 3.
Normal Ca2+ currents in hippocampal CA1 pyramidal neurons of Cav β3–/– mice. A, representative Ca2+ current traces in Cavβ3+/+ and Cavβ3–/–. Ca2+ currents were elicited by voltage step depolarizations (+10 mV increments) from –60 mV to +40 mV. Scale bars, 200 pA and 50 ms. B, I-V curve of total Ca2+ currents. There are no differences in total current density between genotypes. The current density was estimated by dividing the peak amplitude by the cell capacitance (pA/pF). C, Ca2+ current divided by maximum values of Ca2+ current (I/Imax). D, {tau} value was obtained by fitting current traces evoked at 0 mV to a single exponential curve.

 
No Change in Ca2+ Currents in the Cavβ3–/– CA1 Pyramidal Neurons—Next we examined whether Ca2+ currents (ICa) are altered or not in the Cavβ3–/– neurons by whole-cell patch clamp recordings in CA1 pyramidal neurons. Total Ca2+ currents were activated by step depolarizations (+10-mV increments) from a holding potential of –60 mV (Fig. 3A). In CA1 neurons from both Cavβ3+/+ and Cavβ3–/– mice, Ca2+ currents reached their maximum amplitudes at ~0 mV (Fig. 3B). Unlike previous studies that showed a reduced Ca2+ current density in Cavβ3–/– neurons (superior cervical ganglion neurons (23), dorsal root ganglion neurons (38), and olfactory sensory neurons (39)), there was no significant difference in the Ca2+ current density between Cavβ3+/+ and Cavβ3–/– CA1 pyramidal neurons (+/+, 35.46 ± 2.94 pA/pF, n = 18, at 0 mV; –/–, 34.80 ± 3.06 pA/pF, n = 21, p = 0.88, Student's t test) (Fig. 3B). Furthermore, there was no difference in the Ca2+ current divided by maximum values of the Ca2+ current (I/Imax) (Fig. 3C), and in the time constant ({tau}) of Ca2+ current decay (+/+, 82.70 ± 9.75 ms; –/–, 77.70 ± 12.58 ms, p = 0.76, Student's t test) (Fig. 3D), indicating no changes in voltage dependence and inactivating kinetics in the Cavβ3–/– CA1 neurons.

Normal Intrinsic Firing Properties and AHP Currents in the Cavβ3–/–—As a close coupling was reported by co-immunoprecipitation between Cavβ3 and N- or L-type VDCCs in hippocampal neurons (18–20), we measured N- or L-type VDCCs-mediated cellular properties in CA1 neurons. Ca2+ influx through N- or L-type VDCCs is known to be linked to the functions of Ca2+-activated K+ channels that are involved in shaping of APs, including the duration of AP and after hyperpolarization (AHP), and thus can modulate firing properties (40). First we produced AP discharges by a depolarizing current injection under the current clamp mode (Fig. 4A). The CA1 pyramidal neurons of Cavβ3+/+ (n = 8) and Cavβ3–/– (n = 13) displayed very similar firing patterns. No significant difference was observed in the interspike intervals (Fig. 4C), the number (Fig. 4B) and duration (Fig. 4D) of APs. To directly assess the functions of Ca2+-activated K+ channels, we recorded AHP currents. Again, there was no difference in the AHP current between the Cavβ3+/+ (n = 8) and the Cavβ3–/– (n = 9) (+/+, 131.60 ± 15.86 pA; –/–, 112.16 ± 15.42 pA, p = 0.41, Student's t test) (Fig. 4E). These results show that the Cavβ3–/– mutation did not affect intrinsic firing behaviors of hippocampal CA1 neurons.


Figure 4
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 4.
Normal firing properties and normal AHP currents of Cavβ3–/– CA1 pyramidal neurons. A, action potentials are generated by increasing depolarizing current injections from +30 to 90 pA. Scale bars, 15 mV and 500 ms. B, number of APs evoked at different current injections. C and D, there are no significant differences in the interspike intervals (C) and the duration (half-width) (D) of APs between genotypes during a 90-pA current injection. E, voltage clamp recordings of AHP current after a 200-ms depolarization pulse to +20 mV. Bar graph indicates the amplitudes of AHP current measured at 30 ms after pulse-offset. Scale bars, 40 pA and 100 ms.

 
Normal Basal Synaptic Transmission and Short Term Plasticity in the Cavβ3–/– Mice—We then examined the basal synaptic function at hippocampal CA3-CA1 synapses in the Cavβ3–/– mice. In mEPSCs (Fig. 5A), Cavβ3–/– mice showed frequencies and amplitudes similar to those of Cavβ3+/+ mice (Fig. 5B). In addition, fEPSPs were recorded from the CA1 area of the hippocampus in response to stimulations of Schaffer collateral fibers. As illustrated in Fig. 5C, the input-output relation of synaptic transmission was not altered in the Cavβ3–/– mice (+/+, n = 10; –/–, n = 12). We next studied the effect of the Cavβ3 mutation on paired-pulse facilitation (PPF), a presynaptic form of short term plasticity. PPF is a transient enhancement of neurotransmitter release induced by two closely spaced stimuli. This increase in release is usually attributed to intracellular Ca2+ concentration in the presynaptic terminal following the first stimulus (41, 42). There were no significant differences in all tested interpulse intervals between the Cavβ3+/+ (n = 7) and the Cavβ3–/– (n = 9) (Fig. 5D). Taken together, these results indicate that the Cavβ3 mutation had no significant effect upon the basal synaptic function and the presynaptic short term plasticity in hippocampal CA3-CA1 synapses.


Figure 5
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 5.
Basal synaptic functions at hippocampal CA3-CA1 synapses in the Cavβ3–/–. A, representative sample traces of mEPSCs from Cavβ3+/+ and Cavβ3–/– mice. Scale bars, 20 pA and 500 ms. B, frequencies (left) and amplitudes (right) of mEPSCs in CA1 pyramidal neurons were not statistically different between the two genotypes. C, slope of fEPSPs elicited by a given presynaptic fiber volley. The input-output relationship of basal synaptic transmission is not altered in Cavβ3–/– mice. D, normal PPF in the Cavβ3–/–. Traces are responses at 50-ms interpulse interval.

 
Enhanced NMDAR-dependent LTP in the Cavβ3–/– Mice—We then investigated the mutant mice for activity-dependent long lasting synaptic changes, such as LTP and LTD, a cellular model of learning and memory (43). We tried to induce LTP by several different stimulation protocols. LTP was induced by 100-Hz (300 ms and 1 s) or 200-Hz tetanic stimulations. As shown in Fig. 6A, an administration of tetanus at 100 Hz for 1 s elicited a significantly increased potentiation in the Cavβ3–/– compared with that in the Cavβ3+/+ (–/–, 169.47 ± 7.33% of base line at 60 min, n = 9; +/+, 144.75 ± 6.10%, n = 9, p < 0.05, Student's t test). With a 200-Hz tetanic stimulation, the Cavβ3–/– also exhibited more robust potentiation than Cavβ3+/+ (–/–, 231.92 ± 15.72% of base line at 60 min, n = 10; +/+, 181.74 ± 17.58%, n = 8, p < 0.05, Student's t test) (Fig. 6B). Even at short 100-Hz stimulations for 300 ms, enhanced LTP in the Cavβ3–/– was also observed (–/–, 139.31 ± 7.35% of base line at 60 min, n = 10; +/+, 114.70 ± 8.03%, n = 8, p < 0.05, Student's t test) (Fig. 6C). However, in the presence of D-AP5, a specific NMDAR inhibitor, the enhancement of LTP in the Cavβ3–/– disappeared under the same stimulation condition, and a similar level of potentiation was induced in the two genotypes (Fig. 6, D and E). Together, these results indicate that the increased potentiation in the Cavβ3–/– is NMDAR-dependent LTP. No significant difference was noted between the two genotypes in LTD that was induced by PP-LFS (Fig. 6F).


Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 6.
Enhanced LTP in the Cavβ3–/–. A, LTP elicited by tetanus at 100 Hz for 1 s. LTP of Cavβ3–/– is significantly higher than that of Cavβ3+/+ mice (p < 0.05). B, LTP elicited by stimulations at 200 Hz. Cavβ3–/– mice also showed enhanced LTP (p < 0.05). C, LTP elicited by tetanus at 100 Hz for 300 ms. LTP of Cavβ3–/– is significantly higher than that of Cavβ3+/+ mice (p < 0.05). D and E, in the presence of D-AP5 (50 µM), LTP elicited by stimulations at 100 Hz (D) 200 Hz (E). There is no significant difference between genotypes. F, LTD was induced by PP-LFS.

 
Increased NMDAR-mediated Synaptic Currents and NR2B Levels in the Cavβ3–/– Mice—NMDAR is known to play a crucial role in LTP, as well as learning and memory (43–46). Therefore, we examined the possibility that changes in the synaptic responses mediated by NMDAR might underlie the increased LTP in Cavβ3–/– mice. To evaluate this possibility, we first measured the NMDAR-mediated fEPSPs by adding CNQX (10 µM), an AMPA receptor blocker, to the buffer with reduced Mg2+ concentration (0.1 mM). A significant difference was noted between the Cavβ3+/+ and the Cavβ3–/– in these NMDAR-mediated field responses; the Cavβ3–/– (n = 13) exhibited higher NMDAR-mediated fEPSPs than the Cavβ3+/+ (n = 12) (F(1, 23) = 5.52, p < 0.05, two-way repeated ANOVA) (Fig. 7A). To assess this finding more directly, we measured the excitatory postsynaptic currents (EPSCs) evoked by stimulations at Schaffer collateral axons under the whole-cell voltage clamp conditions in CA1 neurons. It was found that there was no significant difference in the amplitude of AMPAR-mediated EPSCs at –70 mV between the two genotypes (Fig. 7B, left). However, a significant difference was noted in the NMDAR/AMPAR amplitude ratio between Cavβ3+/+ (n = 15, 0.28 ± 0.04 at +40 mV) and Cavβ3–/– (n = 13, 0.47 ± 0.06 at +40 mV) (p < 0.05, Student t test) (Fig. 7B, right). Together, these results indicate that NMDAR-mediated responses are increased in Cavβ3–/– mice.


Figure 7
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 7.
Increased NMDAR-mediated responses and increased NR2B levels in the hippocampus of Cavβ3–/– mice. A, NMDAR-mediated synaptic potentials in the presence of CNQX (10 µM) and reduced Mg2+ (0.1 mM). The Cavβ3–/– shows higher NMDAR-mediated fEPSPs than Cavβ3+/+ (F(1, 23) = 5.52, p < 0.05, two-way repeated ANOVA). B, NMDAR- and AMPAR-mediated EPSCs recorded under voltage clamp. Representative traces of EPSCs evoked at –70 mV and +40 mV in Cavβ3+/+ and Cavβ3–/–. Scale bars, 0.1 nA and 50 ms. There are no differences in AMPAR-mediated EPSCs at –70 mV between genotypes (left bar graph), but the ratio of NMDAR/AMPAR response in the Cavβ3–/– is higher than that of the Cavβ3+/+ (right bar graph). NMDAR-mediated responses were taken from the amplitude of currents at +40 mV, 100 ms after EPSCs onset, whereas the AMPAR-mediated responses were taken as the peak amplitude of EPSCs recorded at –70 mV. *, p < 0.05. C, Western blot analysis. The relative levels of glutamate receptors in hippocampal proteins of Cavβ3–/– mice. The NR2B level of Cavβ3–/– mice was relatively increased, whereas other glutamate receptors did not change. The equal amount of protein loading was confirmed by normalizing against the amount of tubulin. *, p < 0.05.

 
In an effort to obtain some clue for the mechanism underlying the increased NMDAR responses in the Cavβ3–/– mice, we quantified the levels of NMDAR subunits by Western blot analysis. It was found that the protein level of NR2B subunit in the hippocampus of the Cavβ3–/– mice (n = 3, 1.14 ± 0.05, normalized to Cavβ3+/+ values) was slightly increased relative to that of the Cavβ3+/+ mice (n = 3) (p < 0.05, Student's t test) (Fig. 7C). There were no significant changes in the levels of other glutamate receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we analyzed the Cavβ3-deficient mice with respect to their capacity for learning/memory and synaptic plasticity. Although there was no change in VDCCs currents and basal synaptic transmission, we found that the deletion of Cavβ3 caused an increase of NR2B expression and NMDAR activities, including currents and LTP, in the hippocampus and an enhanced capacity for learning and memory. This study demonstrates a previously unidentified outcome of the deletion of Cavβ3 in the adult brain.

Yet the Cavβ subunits of VDCCs have been known to be associated with VDCCs and regulate Ca2+ influx through VDCCs by modulating the properties of VDCCs {alpha}1 subunits, including trafficking of channel complexes to the plasma membrane, Ca2+ current densities, and voltage-dependent activation or inactivation (4, 5). Of the Cavβ subtypes, the Cavβ3 is the predominant form in the brain (17), and its role in several neurons has been revealed by studies carried out using mice lacking the Cavβ3. In superior cervical ganglion neurons, the Cavβ3–/– showed reduced N- and L-type Ca2+ currents relative to the Cavβ3+/+ and shifting of voltage-dependent activation in P/Q-type Ca2+ currents (23). In dorsal root ganglion neurons, the Cavβ3–/– mice showed a reduced expression of N-type VDCCs and functional alterations of Ca2+ currents, which was thought to be involved in the reduced pain responses of the Cavβ3–/– mice (38). In olfactory sensory neurons, the Cavβ3–/– mice also exhibited decreased protein expressions and Ca2+ currents of L-type and N-type VDCCs, leading to increased olfactory neuronal activities (39). These reduced expressions of proteins or Ca2+ currents of VDCCs might be considered to mostly result from deficiency in trafficking of channel complexes to the plasma membrane.

However, although the Cavβ3 is known to be highly expressed in the hippocampus (17) and has been shown to associate with 42% of the {alpha}1 subunits of L-type VDCCs in the hippocampus (18), we could not observe a change in nifedipine-sensitive L-type Ca2+ currents in hippocampal CA1 pyramidal neurons of the Cavβ3–/– mice (supplemental Fig. 1). In addition, there were no clear differences in the patterns of the immunohistological labeling for the {alpha}1C (Cav1.2) and the {alpha}1D (Cav1.3) subunits of L-type VDCCs in the hippocampus, between the two genotypes (supplemental Fig. 2). Furthermore, although Cavβ3 in the brain was shown to associate with about 52% {alpha}1B subunit of N-type VDCCs that play a crucial role in neurotransmitter release at hippocampal CA3-CA1 synapses (19–21, 47, 48), the basal synaptic transmission, including mEPSCs, was not altered at hippocampal CA3-CA1 synapses of the Cavβ3–/– mice. Therefore, some compensation by other Cavβ isotypes might have occurred for the deletion of Cavβ3 in the hippocampus of the Cavβ3–/– mice, as was reported in olfactory sensory neurons of the Cavβ3–/– mice (39).

Instead, however, we found an increased LTP at hippocampal CA3-CA1 synapses in the Cavβ3–/– mice. The induction of LTP by a tetanic stimulation at 100 Hz is known to be dependent on NMDAR, and 200-Hz LTP requires both NMDAR and L-type VDCCs at hippocampal CA3-CA1 synapses (49). When NMDAR was blocked by D-AP5, the enhancement in 100-Hz and 200-Hz LTP of the Cavβ3–/– mice was obliterated. This indicates that the increased potentiation in the Cavβ3–/– is of the NMDAR-dependent component in LTP, rather than L-type VDCC-dependent. The increased LTP and long term memory in the Cavβ3–/– mice could be analogous to other cases where an alteration of NMDAR-mediated synaptic responses resulting from the increased levels of NR2B was shown (45, 46).

Although the Ca2+ currents and mEPSCs were measured from 2- to 3-week-old mice, basal synaptic transmission and LTP were recorded in 7- to 8-week-old mice. Thus, no alteration in Ca2+ currents of at least N- and L-type VDCCs could be expected in the adult Cavβ3–/– mice, because they showed normal responses in basal synaptic transmission and NMDA-independent LTP, in which N- and L-type VDCCs have a crucial role, respectively (21, 22, 47–49).

Our results suggest a possibility that Cavβ3 can be a multifunctional protein as was shown for other Cavβ isotypes. The studies of crystal structures revealed that Cavβ subunits belong to membrane-associated guanylate kinase family that has scaffolding functions, suggesting that the Cavβ can play a role in scaffolding multiple signaling pathways by protein-protein interactions through SH3 and GK domains (6, 8, 9). Recently, it was suggested that the Cavβ could directly interact with other proteins, and furthermore it could function without marked influences on the property of VDCCs (10, 11, 50). The physiological unbinding of the Cavβ from the VDCCs complex has already been demonstrated from the inactivation heterogeneity of VDCCs and reversibility of the interaction with {alpha}1 subunits (51, 52). It was reported that Cavβ could directly bind to Gem and Rem, small G-proteins that have a GTPase activity, and this interaction inhibited the surface expression and the activity of VDCCs (12, 13). In addition, it was also shown that Cavβ could promote endocytosis of VDCCs by interaction with dynamin (14). A short splice variant of Cavβ4 could directly interact with CHCB2, a nuclear protein, and then translocate into the nucleus for the subsequent regulation of gene transcription in the cochlea (15). In this study, it was found that the Cavβ could function independently from VDCCs without marked influences on the surface expression and voltage-dependent properties of VDCCs. Furthermore, inositol 1,4,5-trisphosphate-mediated signaling was enhanced in Cavβ3-deficient pancreatic β cells, whereas Ca2+ currents of VDCCs were not affected (16). Similarly, Cavβ were found to internalize Shaker K+ channels by association with dynamin (14). These activities of Cavβ are considered to be completely independent of VDCCs regulation, and thus indicate that Cavβ can function as a multifunctional protein by interactions with other proteins. In this light, it might be possible that the Cavβ3 can directly or indirectly associate with NR2B.

Although our results showed a modest increase of NR2B in the mutant, it is not clear whether this increase can totally explain how the NMDAR activities are enhanced. In the meantime, it was discovered that the C-terminal tail region of Cav1.3 L-type VDCC bound to the SH3 domain of Shank, a postsynaptic scaffolding protein (53–55). Shank is also known to associate with GKAP-PSD95-NR2B through postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain (56). One of the binding sites of Cavβ is the C-terminal tail region of {alpha}1 subunits of VDCCs (6, 8, 9, 57). In this light, the removal of Cavβ3 might have an influence on the interaction of VDCCs and their partners and then could lead to an alteration in the NMDAR activity. Alternatively, we cannot rule out the possibility that a compensatory increase of other Cavβ isotypes or other developmental compensation, which may have occurred in the Cavβ3–/– hippocampus, could also be linked to the alteration in the NMDAR activity. In addition, previously described behavioral alterations from the changes in dorsal root ganglion or olfactory neuronal activities in the Cavβ3–/– mice (38, 39) could contribute to the phenotypes shown in our results.

Initially, we started investigating the role of the Cavβ3 in synaptic transmission and hippocampus-dependent learning and memory because of its known relationship with N- or L-type VDCCs. Interestingly, we found that the ablation of Cavβ3 led to enhanced LTP and capacity for learning and memory in the animal. These phenotypes appear to be due to the increased NMDAR activity with increased NR2B levels in the Cavβ3–/– mice. Even though the precise mechanism of the enhancement of the NMDAR activity in the Cavβ3–/– mice is not yet completely understood, our experiments may reveal a potentially novel function of Cavβ3, unrelated to a role associated with VDCCs. Further studies of the relationship, including direct or indirect protein-protein interactions, between Cavβ3 and NMDAR will be needed to confirm this role of Cavβ3 in the adult brain.


   FOOTNOTES
 
* This work was supported by the National Honor Scientist Program of Korea, grants from Korea Institute of Science and Technology, the National Creative Research Initiatives of the Ministry of Science and Technology of Korea, and Virginia Commonwealth University Medical Center Grant NEI EY12716. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence may be addressed. Tel.: 82-2-880-7854; Fax: 82-2-872-0596; E-mail: utoh{at}plaza.snu.ac.kr. 2 To whom correspondence may be addressed. Tel.: 82-2-958-6931; Fax: 82-2-958-6937; E-mail: shin{at}kist.re.kr.

3 The abbreviations used are: VDCC, voltage-dependent Ca2+ channel; Cavβ3, Ca2+ channels β3 subunit; EPSC, excitatory postsynaptic current; mEPSCs, miniature EPSC; LTP, long term potentiation; NMDAR, N-methyl-D-aspartate receptor; AMPA, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA receptor; CS, conditioned stimulus; GAD, glutamate decarboxylase; AHP, after hyperpolarization; fEPSPs, field excitatory postsynaptic potentials; SH3, Src homology 3; GK, guanylate kinase; ANOVA, analysis of variance; GABA, {gamma}-aminobutyric acid; PPF, paired-pulse facilitation; M{Omega}, megohm; LTD, long term depression; AP, action potential; PP-LFS, paired-pulses low frequency stimulation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Minjeong Sun, Seungeun Lee, and Sangwoo Kim for help in behavioral experiments and Jeremy Mills and Erick Green for help with histology.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hofmann, F., Lacinova, L., and Klugbauer, N. (1999) Rev. Physiol. Biochem. Pharmacol. 139, 33–87[Medline] [Order article via Infotrieve]
  2. Catterall, W. A. (2000) Neuron 26, 13–25[CrossRef][Medline] [Order article via Infotrieve]
  3. Ertel, E. A., Campbell, K. P., Harpold, M. M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T. P., Tanabe, T., Birnbaumer, L., Tsien, R. W., and Catterall, W. A. (2000) Neuron 25, 533–535[CrossRef][Medline] [Order article via Infotrieve]
  4. Berrow, N. S., Campbell, V., Fitzgerald, E. M., Brickley, K., and Dolphin, A. C. (1995) J. Physiol. (Lond.) 482, 481–491[Abstract/Free Full Text]
  5. Dolphin, A. C. (2003) J. Bioenerg. Biomembr. 35, 599–620[CrossRef][Medline] [Order article via Infotrieve]
  6. Chen, Y. H., Li, M. H., Zhang, Y., He, L. L., Yamada, Y., Fitzmaurice, A., Shen, Y., Zhang, H., Tong, L., and Yang, J. (2004) Nature 429, 675–680[CrossRef][Medline] [Order article via Infotrieve]
  7. Hanlon, M. R., Berrow, N. S., Dolphin, A. C., and Wallace, B. A. (1999) FEBS Lett. 445, 366–370[CrossRef][Medline] [Order article via Infotrieve]
  8. Opatowsky, Y., Chen, C. C., Campbell, K. P., and Hirsch, J. A. (2004) Neuron 42, 387–399[CrossRef][Medline] [Order article via Infotrieve]
  9. Van Petegem, F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Nature 429, 671–675[CrossRef][Medline] [Order article via Infotrieve]
  10. Hidalgo, P., and Neely, A. (2007) Cell Calcium 42, 389–396[CrossRef][Medline] [Order article via Infotrieve]
  11. Rousset, M., Cens, T., and Charnet, P. (2005) Sci. STKE 2005, PE11
  12. Beguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001) Nature 411, 701–706[CrossRef][Medline] [Order article via Infotrieve]
  13. Finlin, B. S., Crump, S. M., Satin, J., and Andres, D. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14469–14474[Abstract/Free Full Text]
  14. Gonzalez-Gutierrez, G., Miranda-Laferte, E., Neely, A., and Hidalgo, P. (2007) J. Biol. Chem. 282, 2156–2162[Abstract/Free Full Text]
  15. Hibino, H., Pironkova, R., Onwumere, O., Rousset, M., Charnet, P., Hudspeth, A. J., and Lesage, F. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 307–312[Abstract/Free Full Text]
  16. Berggren, P. O., Yang, S. N., Murakami, M., Efanov, A. M., Uhles, S., Kohler, M., Moede, T., Fernstrom, A., Appelskog, I. B., Aspinwall, C. A., Zaitsev, S. V., Larsson, O., de Vargas, L. M., Fecher-Trost, C., Weissgerber, P., Ludwig, A., Leibiger, B., Juntti-Berggren, L., Barker, C. J., Gromada, J., Freichel, M., Leibiger, I. B., and Flockerzi, V. (2004) Cell 119, 273–284[CrossRef][Medline] [Order article via Infotrieve]
  17. Ludwig, A., Flockerzi, V., and Hofmann, F. (1997) J. Neurosci. 17, 1339–1349[Abstract/Free Full Text]
  18. Pichler, M., Cassidy, T. N., Reimer, D., Haase, H., Kraus, R., Ostler, D., and Striessnig, J. (1997) J. Biol. Chem. 272, 13877–13882[Abstract/Free Full Text]
  19. Scott, V. E., De Waard, M., Liu, H., Gurnett, C. A., Venzke, D. P., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 3207–3212[Abstract/Free Full Text]
  20. Vance, C. L., Begg, C. M., Lee, W. L., Haase, H., Copeland, T. D., and McEnery, M. W. (1998) J. Biol. Chem. 273, 14495–14502[Abstract/Free Full Text]
  21. Jeon, D., Kim, C., Yang, Y. M., Rhim, H., Yim, E., Oh, U., and Shin, H. S. (2007) Genes Brain Behav. 6, 375–388[CrossRef][Medline] [Order article via Infotrieve]
  22. Moosmang, S., Haider, N., Klugbauer, N., Adelsberger, H., Langwieser, N., Muller, J., Stiess, M., Marais, E., Schulla, V., Lacinova, L., Goebbels, S., Nave, K. A., Storm, D. R., Hofmann, F., and Kleppisch, T. (2005) J. Neurosci. 25, 9883–9892[Abstract/Free Full Text]
  23. Namkung, Y., Smith, S. M., Lee, S. B., Skrypnyk, N. V., Kim, H. L., Chin, H., Scheller, R. H., Tsien, R. W., and Shin, H. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12010–12015[Abstract/Free Full Text]
  24. Jeon, D., Yang, Y. M., Jeong, M. J., Philipson, K. D., Rhim, H., and Shin, H. S. (2003) Neuron 38, 965–976[CrossRef][Medline] [Order article via Infotrieve]
  25. Lu, Y. M., Jia, Z., Janus, C., Henderson, J. T., Gerlai, R., Wojtowicz, J. M., and Roder, J. C. (1997) J. Neurosci. 17, 5196–5205[Abstract/Free Full Text]
  26. Mansuy, I. M., Winder, D. G., Moallem, T. M., Osman, M., Mayford, M., Hawkins, R. D., and Kandel, E. R. (1998) Neuron 21, 257–265[Medline] [Order article via Infotrieve]
  27. Podhorna, J., and Brown, R. E. (2002) Genes Brain Behav. 1, 96–110[CrossRef][Medline] [Order article via Infotrieve]
  28. Bunsey, M., and Eichenbaum, H. (1995) Hippocampus 5, 546–556[CrossRef][Medline] [Order article via Infotrieve]
  29. Kogan, J. H., Frankland, P. W., Blendy, J. A., Coblentz, J., Marowitz, Z., Schutz, G., and Silva, A. J. (1997) Curr. Biol. 7, 1–11[Medline] [Order article via Infotrieve]
  30. Song, I., Kim, D., Choi, S., Sun, M., Kim, Y., and Shin, H. S. (2004) J. Neurosci. 24, 5249–5257[Abstract/Free Full Text]
  31. Saura, C. A., Choi, S. Y., Beglopoulos, V., Malkani, S., Zhang, D., Shankaranarayana Rao, B. S., Chattarji, S., Kelleher, R. J., III, Kandel, E. R., Duff, K., Kirkwood, A., and Shen, J. (2004) Neuron 42, 23–36[CrossRef][Medline] [Order article via Infotrieve]
  32. Jeon, D., Chu, K., Jung, K. H., Kim, M., Yoon, B. W., Lee, C. J., Oh, U., and Shin, H. S. (2007) Cell Calcium, in press
  33. Kang, Y. S., Kong, J. H., Park, W. M., Kwon, O. J., Lee, J. E., Kim, S. Y., and Jeon, C. J. (2002) Mol. Cells 14, 361–366[Medline] [Order article via Infotrieve]
  34. Elias, G. M., Funke, L., Stein, V., Grant, S. G., Bredt, D. S., and Nicoll, R. A. (2006) Neuron 52, 307–320[CrossRef][Medline] [Order article via Infotrieve]
  35. Sheng, M., Cummings, J., Roldan, L. A., Jan, Y. N., and Jan, L. Y. (1994) Nature 368, 144–147[CrossRef][Medline] [Order article via Infotrieve]
  36. Phillips, R. G., and LeDoux, J. E. (1992) Behav. Neurosci. 106, 274–285[CrossRef][Medline] [Order article via Infotrieve]
  37. Vnek, N., and Rothblat, L. A. (1996) J. Neurosci. 16, 2780–2787[Abstract/Free Full Text]
  38. Murakami, M., Fleischmann, B., De Felipe, C., Freichel, M., Trost, C., Ludwig, A., Wissenbach, U., Schwegler, H., Hofmann, F., Hescheler, J., Flockerzi, V., and Cavalie, A. (2002) J. Biol. Chem. 277, 40342–40351[Abstract/Free Full Text]
  39. Shiraiwa, T., Kashiwayanagi, M., Iijima, T., and Murakami, M. (2007) Biochem. Biophys. Res. Commun. 355, 1019–1024[CrossRef][Medline] [Order article via Infotrieve]
  40. Faber, E. S., and Sah, P. (2003) Neuroscientist 9, 181–194[Abstract/Free Full Text]
  41. Regehr, W. G., Delaney, K. R., and Tank, D. W. (1994) J. Neurosci. 14, 523–537[Abstract]
  42. Zucker, R. S. (1999) Curr. Opin. Neurobiol. 9, 305–313[CrossRef][Medline] [Order article via Infotrieve]
  43. Bliss, T. V., and Collingridge, G. L. (1993) Nature 361, 31–39[CrossRef][Medline] [Order article via Infotrieve]
  44. McGaugh, J. L. (2000) Science 287, 248–251[Abstract/Free Full Text]
  45. Tang, Y. P., Shimizu, E., Dube, G. R., Rampon, C., Kerchner, G. A., Zhuo, M., Liu, G., and Tsien, J. Z. (1999) Nature 401, 63–69[CrossRef][Medline] [Order article via Infotrieve]
  46. Tsien, J. Z., Huerta, P. T., and Tonegawa, S. (1996) Cell 87, 1327–1338[CrossRef][Medline] [Order article via Infotrieve]
  47. Dunlap, K., Luebke, J. I., and Turner, T. J. (1995) Trends Neurosci. 18, 89–98[CrossRef][Medline] [Order article via Infotrieve]
  48. Wu, L. G., and Saggau, P. (1997) Trends Neurosci. 20, 204–212[CrossRef][Medline] [Order article via Infotrieve]
  49. Cavus, I., and Teyler, T. (1996) J. Neurophysiol. 76, 3038–3047[Abstract/Free Full Text]
  50. Dolphin, A. C. (2006) Br. J. Pharmacol. 147, S56–S62
  51. Bichet, D., Lecomte, C., Sabatier, J. M., Felix, R., and De Waard, M. (2000) Biochem. Biophys. Res. Commun. 277, 729–735[CrossRef][Medline] [Order article via Infotrieve]
  52. Restituito, S., Cens, T., Rousset, M., and Charnet, P. (2001) Biophys. J. 81, 89–96[Medline] [Order article via Infotrieve]
  53. Olson, P. A., Tkatch, T., Hernandez-Lopez, S., Ulrich, S., Ilijic, E., Mugnaini, E., Zhang, H., Bezprozvanny, I., and Surmeier, D. J. (2005) J. Neurosci. 25, 1050–1062[Abstract/Free Full Text]
  54. Sheng, M., and Kim, E. (2000) J. Cell Sci. 113, 1851–1856[Abstract]
  55. Zhang, H., Maximov, A., Fu, Y., Xu, F., Tang, T. S., Tkatch, T., Surmeier, D. J., and Bezprozvanny, I. (2005) J. Neurosci. 25, 1037–1049[Abstract/Free Full Text]
  56. Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, C., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569–582[CrossRef][Medline] [Order article via Infotrieve]
  57. Walker, D., and De Waard, M. (1998) Trends Neurosci. 21, 148–154[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. Kim, D. Jeon, Y.-H. Kim, C. J. Lee, H. Kim, and H.-S. Shin
Deletion of N-type Ca2+ Channel Cav2.2 Results in Hyperaggressive Behaviors in Mice
J. Biol. Chem., January 30, 2009; 284(5): 2738 - 2745.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/18/12093    most recent
M800816200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeon, D.
Right arrow Articles by Shin, H.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeon, D.
Right arrow Articles by Shin, H.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement