Haloperidol Induces Calcium Ion Influx Via L-Type Calcium Channels ...

1 downloads 0 Views 1MB Size Report
Haloperidol Induces Calcium Influx and Renders Hippocampal Cells More Susceptible to Oxidative Stress. N-, and R-types. The LVA calcium channels, ...
Mol. Cells, Vol. 22, No. 1, pp. 51-57

Molecules and Cells ©KSMCB 2006

Haloperidol Induces Calcium Ion Influx Via L-Type Calcium Channels in Hippocampal HN33 Cells and Renders the Neurons More Susceptible to Oxidative Stress Hyeon Soo Kim, Sanatombi Yumkham, Jang Hyun Choi, Eung-Kyun Kim, Yong Sik Kim1, Sung Ho Ryu, and Pann-Ghill Suh* Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea; 1 Department of Psychiatry and Behavioral Sciences and Institute of Human Behavioral Medicine, Seoul National University Hospital, Seoul 110-799, Korea. (Received March 9, 2006; Accepted June 14, 2006)

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of proapoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate. Keywords: Calcium; Haloperidol; Hippocampus; L-Type Calcium Channel; Oxidative Stress; Verapamil.

Introduction Haloperidol is used extensively in the treatment of several neuropsychiatric disorders. Although this treatment has a high rate of success, it also induces extrapyramidal side effects (EPS) such as tardive dyskinesia (TD), seriously limiting its use as an antipsychotic agent. The precise * To whom correspondence should be addressed. Tel: 82-54-279-2293; Fax: 82-54-279-0645 E-mail: [email protected]

mechanism responsible for these side effects is unclear, but it is evident that they are associated with the application of antipsychotic drugs. In some studies, neurotoxicity has been tentatively implicated in neuroleptic-induced EPS and TD (Gil-ad et al., 2001). Chronic blockade of dopamine D2 receptors in the striatum results in persistently enhanced release of glutamate, which kills striatal neurons (Tseng and Lin-Shiau, 2003). L-type calcium channel inhibitors have been reported to reduce haloperidol-induced TD (Naidu and Kulkarni, 2001), suggesting that calcium ions are involved in haloperidol-induced neurotoxicity. Control of intracellular free calcium ion concentration is a prerequisite for cell survival, because of the essential role of calcium ions in intracellular processes in neurons (Choi, 1988; Sattler and Tymianski, 2001; Siesjo, 1989; Thayer et al., 2002; Torreano and Cohan, 2003). Intracellular calcium ion release and extracellular calcium ion influx is regulated by immensely complex sets of signals. Therefore, understanding the manner in which calcium ions regulate diverse cellular processes constitutes one of the primary goals of neuronal science. Neurons are known to be exquisitely sensitive to the increase in the level of intracellular calcium ions. Within neurons, several signals, including ischemia (Fern, 1998), beta-bungarotoxin (Turrone et al., 2002), and corticosterone (Roy and Sapolsky, 2003; Takahashi et al., 2002), are mediated via calcium ion influx through both voltage-and receptor gated ion channels. Calcium channels are traditionally classified into two subtypes; high-voltage activated (HVA) and low-voltage activated (LVA) subtypes (Bean, 1989; Tsien et al., 1988). The HVA calcium channels initially activate at relatively depolarized potentials. This class includes the L-, P/Q-,

52

Haloperidol Induces Calcium Influx and Renders Hippocampal Cells More Susceptible to Oxidative Stress

N-, and R-types. The LVA calcium channels, commonly referred to as T-type, have a lower range of activation and inactivation. Although some reports have asserted that certain neuroleptics may also interact with calcium channels (Choi, 1988), the significance of these interactions is yet to be established. In order to elucidate the mechanisms underlying the neurotoxic effects associated with haloperidol, we have analyzed its effect on cytosolic calcium ion concentrations. Here, we show that haloperidol can induce calcium ion influx via L-type calcium channels, and that it renders neurons more susceptible to oxidative stress.

Materials and Methods Cell culture and reagents We used the immortalized hippocampal cell line HN33 derived from fusion of primary neurons from the hippocampuses of 21-day postnatal mice with N18TG2 neuroblastoma cells. They exhibit the morphological, immunological, and electrophysiological characteristics typical of hippocampal neurons in culture. The HN33 cells express at least two of the neurofilament triplet proteins, supporting their classification as neuron. They were grown at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). They were plated at 1 × 105 cells per well onto uncoated, 24-well plastic culture dishes in DMEM supplemented with 10% (vol/vol) FBS (Life Technologies, USA), maintained at 37°C in a humidified atmosphere containing 95% air/5% CO2 and grown to 90% confluence. Haloperidol, BAPTAAM [1,2-Bis-(2-aminophenoxy) ethane-N,N,N,N′-tetra acetic acid tetrakis (acetoxymethyl ester)], nifedipine, verapamil and MTT [3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetra-zolium bromide] were purchased from Sigma-Aldrich (USA). Anti-Bax, anti-Bcl, anti-actin antibodies were from New England BioLabs (USA). Fluo3-AM, and DCF-DA (2′, 7′-dichlorofluore-scin diacetate) were bought from Molecular Probes (USA). MTT assay MTT assays were used as a crude measure of cell viability. MTT is converted by metabolically active cells into a colored water-insoluble formazan salt. HN33 cells were seeded at a density of 5 × 104/ml in 96-well plates, and allowed to grow for 24 h. The growth medium was replaced with serum-free medium for 24 hours prior to treatment. Subsequently, MTT reagents (7.5 mg/ml in PBS) were added to the cells (10 μl/well), and the culture was incubated for 30 minutes at 37°C. The reaction was then stopped by adding acidified Triton buffer [0.1 M HCl, 10% (v/v) Triton X-100; 50 μl/well], and the tetrazolium crystals were dissolved by mixing for 20 min on a plate shaker at room temperature. The samples were then read with a plate reader (Bio-Rad 450, USA) at 595 nm test wavelength and 650 nm reference wavelength. The results shown are representative of experiments repeated at least in triplicate. Calcium measurement by confocal microscopy Calcium ion

concentration was measured by confocal microscopy (Zeiss LSM 510 Meta, Germany) with the calcium-sensitive indicator, Fluo-3 AM. In brief, HN33 cells were loaded with 5 μM Fluo3AM in normal incubation medium at room temperature for 45 min. After washing with the same medium, the cells were incubated for 15 min in the absence of Fluo-3 AM to completely deesterify the dye. The culture dishes were then placed onto a thermostatted stage on an inverted confocal microscope and observed with the ×20 objective. Excitation of Fluo-3 AM was provided by the 488-nm line of an argon laser, and the emission range was 515 nm. Immunoblotting analysis HN33 cells were grown in 6-well plates. At 60−70% confluence, they were serum-starved for 24 h before treatment at 37°C with the selected agents. The medium was then aspirated, and the cells were washed twice in ice-cold PBS and lysed in 100 μl of lysis buffer (0.5% deoxycholate, 0.1% SDS, 1% Nonidet P-40, 150 mM NaCl and 50 mM TrisHCl, pH 8) containing proteinase inhibitors (0.5 μM aprotinin, 1 μM phenylmethylsulfonyl fluoride, 1 μM leupeptin) (SigmaAldrich, USA). The supernatants were sonicated briefly, heated at 95°C for 5 min, centrifuged for 5 min, run on SDS-PAGE (8−16%) gradient gels, and transferred to polyvinylidene difluoride membranes. The blots were then incubated overnight at 4°C with primary antibodies, and washed 6 times in Trisbuffered saline/0.1% Tween 20 before probing with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were visualized by ECL (Amersham Biosciences, UK). In some cases, the blots were stripped and reprobed with other antibodies. Detection of ROS HN33 cells were grown to confluence in 35mm dishes. They were loaded with 25 mM DCF-DA (a ROSsensitive dye) for 40 min, and incubated with hydrogen peroxide (100 μM) alone or with a combination of hydrogen peroxide and the indicated agents. Fluorescence was then detected by confocal microscope at excitation wavelength 488 nm and emission wavelength 520 nm. Sets of three dishes were examined by confocal microscopy with similar results, and representative experiments are shown in the corresponding photomicrographs. Morphological changes Morphological changes were observed following the application of the indicated agents. After 24 h of incubation in DMEM (10% FBS), we replaced the medium with 1% FBS containing DMEM. Then, the various agents were applied for 6 h. Morphology was assessed using transmission images from a confocal microscope with the ×20 objective. Tunnel assay Apoptosis was detected by labeling fragmented DNA with ApopTag peroxides (KIT S7101, Intergen Company, USA), according to the manufacturer’s instructions. After fixation, the cells were incubated with terminal deoxynucleotidyl transferase (TdT) in the presence of nucleotides. TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3′-OH ends of double- or single-stranded DNA. The cells

Hyeon Soo Kim et al.

A

B

53

C

D

Fig. 1. Haloperidol induces calcium influx via L-type calcium channel in HN33 cells. A. The effect of haloperidol on calcium influx. Calcium responses were measured after haloperidol (10 μM) treatment in the presence of extracellular calcium. To detect the calcium signal, Fluo-3 AM was added to the culture medium for 45 min. The dishes were then transferred to an inverted confocal microscope, and observed with the ×20 objective. B. The effect of extracellular calcium on haloperidol-induced calcium ion influx. The calcium response was determined after haloperidol treatment in the absence of extracellular calcium. C. The effect of KCl (50 mM) on calcium influx. Calcium responses were measured after KCl treatment in the presence of extracellular calcium. D. The effect of the indicated concentrations of verapamil on haloperidol-induced calcium ion influx. Verapamil was added for 30 min prior to haloperidol treatment, and calcium signals assessed in the presence of extracellular calcium.

were then incubated with buffer and anti-digoxigenin peroxidase, rinsed and stained with AEC-chromogen. They were then counterstained with propidium iodide (PI) (50 μg/ml). Cells treated in the same way in the absence of Tdt served as negative controls. The apoptotic index is defined as the percentage of apoptotic cells among the total number of cells in a sample. Data analysis Data are expressed as the means ± S.E.M. Statistical analyses were conducted using SigmaStat (SPSS Inc., USA). Differences were considered significant at p < 0.05. Differences between basal and stimulated conditions were assessed via ANOVA, with Holm-Sidak comparisons.

Results Haloperidol induces calcium ion influx via L-type calcium channels in HN33 cells In order to determine the role of haloperidol in neurotoxicity, we tested whether it (10 µM) affected intracellular calcium ion concentrations in HN33 hippocampal neuron cells. The addition of haloperidol increased intracellular calcium ion concentrations in the presence of extracellular calcium (Fig. 1A). No increase was observed in the absence of extracellular calcium (Fig. 1B). Potassium chloride (50 mM) induces

calcium ion influx via L-type calcium channels (Rathi et al., 2004) and was found to increase intracellular calcium ion concentration in our system (Fig. 1C). To determine whether calcium channels were involved, we pre-treated the cells with verapamil (10 µM), a specific L-type calcium channel blocker, prior to haloperidol treatment. This inhibited the haloperidol-induced calcium ion increase in a dose-dependent manner (Fig. 1D). These results indicate that haloperidol induces calcium ion influx via L-type calcium channels. Haloperidol-induced calcium influx affects on cytotoxic calcium release To understand the effect of the calcium ion influx induced by haloperidol, we pre-treated the cells with haloperidol (10 µM) and measured calcium concentration in response to oxidative stress. In order to induce oxidative stress, we applied 100 µM of hydrogen peroxide to the cells. Oxidative stress caused an increase in calcium ion concentration in cells pretreated with haloperidol or potassium chloride (50 mM), but not in untreated cells. In order to confirm this result, we pretreated the cells with verapamil (10 µM) prior to exposure to haloperidol. Calcium ion levels did not increase in cells receiving verapamil treatment. This suggests that the haloperidol-mediated calcium ion influx may induce cyto-

54

Haloperidol Induces Calcium Influx and Renders Hippocampal Cells More Susceptible to Oxidative Stress

A

Fig. 2. Haloperidol-induced calcium influx affects on cytotoxic calcium release. The effect of haloperidol-induced calcium ion influx on hydrogen peroxide-induced cytotoxic calcium release. Hydrogen peroxide (100 μM) was applied to the cells under the indicated experimental conditions. Subsequent to hydrogen peroxide treatment, we measured the concentrations of intracellular calcium. To detect the calcium signals, Fluo-3 AM was added to the culture medium for 45 minutes. The dishes were then transferred to an inverted confocal microscope, and observed with the ×20 objective. Values are expressed as the means ± SEM of three separate experiments. p < 0.001.

B

toxic calcium release in response to oxidative stress. Haloperidol-induced calcium influx renders neuron more susceptible to oxidative stress In order to gain insight into the role of calcium ions in neuronal toxicity, we measured cell viability assay to determine whether haloperidol-mediated calcium influx affected neuronal fate. Oxidative stress (100 µM H2O2) alone caused a decrease in neuronal viability by approximately 5%. This decrease was exacerbated to about 40% in cells pretreated with haloperidol (10 µM) or potassium chloride (50 mM). Consistent with the results of Fig. 2, this decrease was attenuated when the cells were pre-treated with verapamil (Fig. 3A). Accordingly, the morphological damage caused by exposure to oxidative stress that is normally associated with haloperidol or potassium chloride pre-treatment was not apparent in the presence of verapamil (Fig. 3B). On the other hand, cells could not be rescued by treatment of BAPTA-AM (10 µM), an intracellular calcium chelator. These findings show that haloperidol-induced calcium influx determines the fate of the neuronal cells. Haloperidol-induced calcium influx increases the levels of ROS and Bax in respond to oxidative stress To understand the signal pathways activated by haloperidolinduced calcium ion influx, we pre-treated the cells with haloperidol (10 µM) prior to hydrogen peroxide (100 µM), and assessed levels of ROS. ROS is known to generate signals harmful to the neurons. Oxidative stress-induced

Fig. 3. Haloperidol-induced calcium influx renders neurons more susceptible to oxidative stress. A. The effect of haloperidol-induced calcium influx on cell viability. Cells were pretreated with verapamil (10 μM) or BAPTA-AM (10 μM) for 30 min prior to haloperidol (10 μM) treatment, and were then coincubated for 15 min. Hydrogen peroxide (100 μM) was then added under various experimental conditions. Values are expressed as percentages of absorbance (MTT) with regard to controls. Data are means ± SEM of three separate experiments, each performed in quadruplicate. p < 0.001. B. The effect of haloperidol-induced calcium influx on cell morphology. Cells were plated in 12-well plates. Verapamil and BAPTA-AM were added for 30 min prior to haloperidol treatment, followed by hydrogen peroxide. The cells were then cultured for an additional 6 h. Cell morphology was visualized by confocal microscopy with the ×20 objective. Scale bar = 20 μm.

ROS levels in cells exposed to haloperidol (10 µM) were significantly rescued by verapamil treatment, but not by exposure to BAPTA-AM (10 µM) (Fig. 4A). ROS also increased in potassium chloride-pretreated cells in response to oxidative stress. These results supported the hypothesis that calcium ion influx plays a vital role in oxidative stress-induced ROS generation. In order to ascertain whether the up-regulation of ROS levels was correlated with neuronal toxicity, we performed Western blots to determine whether ROS affected the expression of the apoptosis-related genes, Bax and Bcl. Pro-apoptotic

Hyeon Soo Kim et al.

55

A

B

C

Fig. 4. Haloperidol-induced calcium ion influx increases ROS and Bax levels in response to oxidative stress. A. The effect of haloperidolinduced calcium ion influx on ROS generation. To detect ROS, the cells were pre-incubated for 45 minutes with DCF-DA in culture medium. This is followed by hydrogen peroxide and the levels of ROS were measured in the indicated experimental conditions using the 488 nm laser line of argon. The red pseudocolor image indicates high concentration and green color, low concentration. B. The effect of haloperidol-induced calcium ion influx on Bax expression. Cell lysates were prepared under conditions identical to those of Fig. 4A, 20 μg samples of lysates were used for SDS-PAGE and immunoblotting with anti-Bax and anti-Bcl antibodies. Anti-actin antibody was used as a quantitation control. C. The effect of haloperidol-induced calcium ion influx on apoptosis. Cells were exposed to oxidative stress under a variety of experimental conditions. After 24 h incubation, they were fixed in 4% buffered formaldehyde, and Tunnel assays conducted to stain apoptotic cells (brown: upper panel). Propidium iodide (50 μg/ml) was used to stain nuclei (red: lower panel). The stained and non-stained cells were counted in triplicate samples in three independent experiments. Values are means ± SEM of three separate experiments. p < 0.001. Scale bar = 50 μm.

Bax expression increased in response to oxidative stress in cells pre-treated with haloperidol or with potassium chloride, and this was inhibited by verapamil, but not by BAPTA-AM (Fig. 4B). However, anti-apoptotic Bcl expression decreased after induction of oxidative stress in the haloperidol pre-treated cells, and this was again pre-

vented by verapamil (Fig. 4B). This inhibitory effect of verapamil on oxidative stress-induced apoptosis in haloperidol-pre-treated cells was confirmed by Tunnel assays (Fig. 4C). These results together indicate that calcium influx due to haloperidol exposure augments oxidative stress-induced neuronal apoptosis.

56

Haloperidol Induces Calcium Influx and Renders Hippocampal Cells More Susceptible to Oxidative Stress

A

B

Fig. 5. Mibefradil has no effect on haloperidol-induced calcium ion influx. A. The effect of mibefradil on haloperidol-induced calcium ion influx. After changing to serum-free conditions, the HN33 cells were pre-incubated with haloperidol in the presence of either nifedipine (5 μM), a specific L-type calcium channel blocker, or mibefradil (1 μM), a specific T-type calcium channel blocker. After exposure to hydrogen peroxide (100 μM), calcium levels were monitored. B. The effect of mibefradil on haloperidol-induced ROS generation. After changing to serumfree conditions, the HN33 cells were pre-treated with haloperidol in the presence of either nifedipine or mibefradil. After expose to hydrogen peroxide, ROS levels were monitored.

Mibefradil has no effect on haloperidol-induced calcium influx To rule out the involvement of other calcium channels, we pre-treated the cells with haloperidol (10 µM) prior to hydrogen peroxide exposure in the presence of mibefradil (1 µM), a T-type calcium channel blocker and evaluated the extent of calcium ion release. Hydrogen peroxide-mediated calcium influx was not affected. However, another L-type calcium channels blocker, nifedipine (5 µM), again blocked hydrogen peroxide induced-calcium influx as well as ROS generation (Figs. 5A and 5B). These results demonstrate that haloperidol induces calcium influx, specifically via L-type calcium channel.

Discussion The major results of this study are that haloperidol induces calcium influx via L-type calcium channels, and that it renders neuron more susceptible to oxidative stress, thereby indicating that this calcium ion influx affects neuronal fate. The hippocampus is used for rapid and automatic encoding of arbitrary conjunctions of existing cortical information. Previous research involving MRI scans has shown that hippocampal volume tends to fall in cases of chronic schizophrenia (Anderson et al., 2002). Increasing evidence also suggests that hippocampal atrophy is cen-

tral to the origin of a range of neuropsychiatric disorders. While no single theory accounts for adequately for the underlying mechanism, the hippocampus is generally considered to be centrally involved. To be sure, questions remain regarding the reasons for the high susceptibility of hippocampus to the effects of neuroleptics. We detected no calcium increase in the haloperidol-treated cells in the presence of L-type calcium channels inhibitors or extracellular calcium chelators. This indicates that no significant quantities of calcium were released from the intracellular stores of calcium. Moreover, the near-complete protection from oxidative stress manifested in these cells clearly indicates that extracellular calcium constitutes a key regulator of cell viability. Calcium ion influence neuronal homeostasis at many levels including phosphorylation and protein kinase C activity (Cullen and Lockyer, 2002). Neurons are also known to be exquisitely sensitive to the duration, amplitude, and localization of transient increases in intracellular calcium ion concentration. Calcium influx is known to be mediated, at least in part, via voltage-gated channels, but its significance with regard to cell survival or death remains uncertain. The results of the present study show that calcium influx followed by haloperidol treatment renders neurons more sensitive to oxidative stress. Our results thus indicate that haloperidol-triggered calcium influx plays a crucial role in the pathogenesis of neuroleptic-induced neurotoxicity. In our experiment, we used a somewhat higher concentration (10 µM) of haloperidol than the therapeutically effective plasma concentration (10−50 nM). A study using hippocampal HT22 cells to investigate the molecular pathways leading to the oxidative neurotoxicity of haloperidol, showed that cell viability was reduced by approximately 40% at a concentration of 50 µM, 20% at 10 µM, and almost 100% at 100 µM haloperidol (Post et al., 1998). Considering the fact that the therapeutically effective concentration of haloperidol is low, this result suggests that somewhat higher concentrations are required to detect the effect of haloperidol in the cell culture system. There are two possible causes for this. First, the environmental differences between cell culture and in vivo conditions. Second, artifacts of the immortalized cell line. Certain neuroleptics are thought to interact with calcium channels. Moreover, the therapeutic efficacy of some of these agents relies on inhibition of T-type calcium channels (Gomora et al., 2001; Santi et al., 2002). However, the selectivity of these agents for different calcium channel subtypes is yet to be explained. The results of the present study demonstrate that the calcium channel subtypes play different roles in regulating neuronal function. Questions remain regarding the manner in which haloperidol mediates calcium-influx via the L-type calcium channels. One possibility is that it reduces the activation threshold of the channel. This could be tested by

Hyeon Soo Kim et al.

patch-clamp experiments. Till recently, it was thought that repeated treatments with neuroleptics were associated with biochemical and morphological alterations as well as upregulation of D2mediated changes in neuronal function. However, a study showed that short-term inhibition of D2-like receptors induced fiber and terminal degeneration and significantly decreased tyrosine hydroxylase and brain-derived neurotrophic factor immunoreactivity (Meredith et al., 2004). This indicates that short-term treatment with D2 antagonists is sufficient to induce changes in the biochemical and morphological profiles. In conclusion, we have attempted to characterize the effects of haloperidol on levels of intracellular calcium ions. Our data demonstrate that haloperidol induces calcium ion influx via L-type calcium channels, rendering the neurons more susceptible to oxidative stress. Further studies will be necessary to determine the physiological significance of the L-type calcium channels for haloperidolrelated symptoms, including extrapyramidal symptoms.

Acknowledgment This study was supported by the Next Generation New Technology Development Program (10027891) of the Ministry of Commerce, Industry and Energy (MOCIE).

References Anderson, J. E., Wible, C. G., McCarley, R. W., Jakab, M., Kasai, K., et al. (2002) An MRI study of temporal lobe abnormalities and negative symptoms in chronic schizophrenia. Schizophr. Res. 58, 123−134. Bean, B. P. (1988) Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51, 367−384. Choi, D. W. (1988) Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 11, 465−469. Cullen, P. J. and Lockyer, P. J. (2002) Integration of calcium and ras signaling. Nat. Rev. Mol. Cell. Biol. 3, 339−348. Fern, R. (1988) Intracellular calcium and cell death during ischemia in neonatal rat white matter astrocytes in situ. J. Neurosci. 18, 7232−7243. Gil-ad, I., Shtaif, B., Shiloh, R., and Weizman, A. (2001) Evaluation of the neurotoxic activity of typical and atypical neuroleptics: relevance to iatrogenic extrapyramidal symptoms. Cell. Mol. Neurobiol. 21, 705−716. Gomora, J. C., Daud, A. N., Weiergraber, M., and Perez-Reyes, E. (2001) Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol. Pharmacol. 60, 1121− 1132.

57

Meredith, G. E., Switzer, R. C. 3rd., and Napier, T. C. (2004) Short-term D2 receptor blockade induces synaptic degeneration, reduces levels of tyrosine hydroxylase and brainderived neurotrophic factor, and enhances D2-mediated firing in the ventral pallidum. Brain Res. 995, 14−22. Naidu, P. S. and Kulkarni, S. K. (2001) Excitatory mechanisms in neuroleptic-induced vacuous chewing movements (VCMs): possible involvement of calcium and nitric oxide. Behav. Pharmacol. 12, 209−216. Post, A., Holsboer, F., and Behl, C. (1998) Induction of NFkappaB activity during haloperidol-induced oxidative toxicity in clonal hippocampal cells: suppression of NF-kappaB and neuroprotection by antioxidants. J. Neurosci. 18, 8236− 8246. Rathi, S. S., Saini, H. K., Xu, Y. J., and Dhalla, N. S. (2004) Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes. Mol. Cell. Biochem. 263, 151−162. Roy, M. and Sapolsky, R. M. (2003) The exacerbation of hippocampal excitotoxicity by glucocorticoids is not mediated by apoptosis. Neuroendocrinology 77, 24−31. Santi, C. M., Cayabyab, F. S., Sutton, K. G., McRory, J. E., Mezeyova, J., et al. (2002) Differential inhibition of T-type calcium channels by neuroleptics. J. Neurosci. 22, 396−403. Sattler, R. and Tymianski, M. (2001) Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Mol. Neurobiol. 24, 107−129. Siesjo, B. K. (1989) Calcium and cell death. Magnesium 8, 223−237. Siesjo, B. K., Bengtsson, F., Grampp, W., and Theander, S. (1989) Calcium, excitotoxins, and neuronal death in the brain. Ann. N. Y. Acad. Sci. 568, 234−251. Takahashi, T., Kimoto, T., Tanabe, N., Hattori, T. A., Yasumatsu, N., et al. (2002) Corticosterone acutely prolonged NMDA receptor-mediated calcium elevation in cultured rat hippocampal neurons. J. Neurochem. 83, 1441−1451. Thayer, S. A., Usachev, Y. M., and Pottorf, W. J. (2002) Modulating Ca2+ clearance from neurons. Front. Biosci. 7, d1255− 1279. Torreano, P. J. and Cohan, C. S. (2003) Calcium and voltage dependent inactivation of sodium calcium currents limits calcium influx in helisoma neuron. J. Neurobiol. 54, 439−456. Tseng, W. P. and Lin-Shiau, S. Y. (2003) Neuronal death signaling by beta-bungarotoxin through activation of the N-methylD-aspartate (NMDA) receptor L-type calcium channel. Biochem. Pharmacol. 65, 131−142. Tsien, R. W., Lipscombe, D., Madison, D. V., Bley, K. R., and Fox, A. P. (1988) Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci. 11, 431−438. Turrone, P., Remington, G., and Nobrega, J. N. (2002) The vacuous chewing movement (VCM) model of tardive dyskinesia revisited: is there a relationship to dopamine D (2) receptor occupancy? Neurosci. Biobehav. Rev. 26, 361−380.