Skip to main content
NCBI home page
VA Author Manuscripts logoLink to VA Author Manuscripts
. Author manuscript; available in PMC: 2020 Nov 5.
Published in final edited form as: Psychopharmacology (Berl). 2020 Jul 24;237(11):3303–3314. doi: 10.1007/s00213-020-05613-w

Multiple sources of internal calcium stores mediate ethanol-induced presynaptic inhibitory GABA release in the central nucleus of the amygdala in mice

Qiang Li 1, Rebecca C Klein 1, Scott D Moore 1
PMCID: PMC7644111  NIHMSID: NIHMS1632593  PMID: 32705289

Abstract

Rationale

It has been demonstrated that ethanol can enhance GABA release in various brain regions via presynaptic mechanisms. However, the presynaptic action of ethanol on inhibitory GABA release is still not well understood.

Objectives

Since calcium is required for neurotransmitter release from presynaptic terminals, the purpose of this study was to investigate the role of both internal and external calcium signaling in ethanol-induced enhancement of GABA release within the central amygdala nucleus (CeA) in acute brain slice preparations.

Methods

Whole-cell patch clamp electrophysiology was used to record miniature GABAA receptor-mediated inhibitory postsynaptic currents (mIPSCs) from CeA neurons. Ethanol-enhanced mIPSCs were recorded in the presence of antagonists that regulate internal and external calcium mediated processes.

Results

Bath applied ethanol dose-dependently increased the mean frequency of mIPSCs without altering mIPSC amplitude. Ethanol-induced increases in mIPSC frequency were antagonized by dantrolene, 2-APB, and the endoplasmic reticulum calcium pump (SERCA) antagonists thapsigargin and cyclopiazonic acid (CPA). Blocking calcium release from mitochondria or via exocytosis with ruthenium red also attenuated mIPSCs while frequency was not altered in the presence of a non-selective calcium channel blocker cadmium. The L-type calcium blocker nifedipine, but not its analogue nimodipine, blocked ethanol induced enhancement in CeA neurons.

Conclusions

These results demonstrate ethanol-induced presynaptic release of GABA is mediated by internal calcium stores and by disrupting neurotransmitter exocytosis within the CeA, a critical brain area involved in drugs of abuse and alcohol addiction.

Keywords: Alcohol, Presynaptic Release, GABA, Internal Calcium Stores, Inhibitory Postsynaptic Currents, Amygdala

Introduction

Presynaptic neurotransmitter release in the form of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) serves as one of essential processes critical for transmitting information between neurons in a local neuronal circuit within the brain. Such a fundamental physiological process not only influences how a biological signal is processed and transmitted, it also serves a critical regulatory locus whereby both the fidelity and the efficiency of the synapses could be readily modulated by various physiological and pharmacological chemicals including drugs of abuse (Gipson et al. 2014; Lovinger and Roberto 2013; Luscher and Malenka 2011).

As one of key anatomical structures in the limbic system responsible for essential biological processes leading to drug addiction including alcohol dependence (Kang-Park et al. 2013; Li et al. 2014; Varodayan et al. 2017a; Varodayan et al. 2016), the central nucleus of the amygdala (CeA) contains heterogenous projecting neurons of which 90% release GABA as the main synaptic neurotransmitter and plays an essential role in the initiation and the development alcohol dependence. Acute effects of alcohol are usually found to strengthen inhibitory synaptic transmission mediated via GABAA receptors (Lovinger and Roberto 2013). However, how ethanol affects the synaptic transmission is still not well understood.

Ethanol-induced enhancement in inhibitory postsynaptic transmission has been observed in both cortical and subcortical regions including the hippocampus (Li et al. 2006; Yan et al. 2010) and the amygdala (Gilpin et al. 2015; Herman and Roberto 2016; Kang-Park et al. 2013; Li et al. 2014; Varodayan et al. 2017a). Our recent study indicated that certain ionic channels such as presynaptic BK channels could also mediate ethanol-induced enhancement in presynaptic GABA release via ethanol inhibition of presynaptic BK channels in rat CeA (Li et al. 2014). Furthermore, ethanol inhibition of whole calcium currents isolated from rat CeA neurons strongly suggest that calcium channels may play a critical role in mediating synaptic function following acute (Li et al. 2014) and chronic exposure to ethanol (Varodayan et al. 2017a).

A number of studies suggest that spontaneous miniature synaptic neurotransmitter release depends upon presynaptic internal calcium stores and the exhaustion of internal calcium stores by blocking calcium reuptake remarkably reduces excitatory and inhibitory neurons transmitter release (Collin et al. 2005; Ghatpande et al. 2006; Le Magueresse and Cherubini 2007; Simkus and Stricker 2002). For example, the effects of ethanol-induced spontaneous GABA release in cerebellum Purkinje neurons could be affected when various biological processes leading to calcium release from presynaptic internal stores were disrupted pharmacologically (Kelm et al. 2007).

Building upon these findings, we employed whole-cell patch clamp technique coupling with pharmacological approaches in brain slices to investigate the role of presynaptic internal and external calcium sources in mediating ethanol-induced GABA release in mouse CeA. We found that ethanol dose-dependently facilitates inhibitory neurotransmitter release in CeA neurons and that this release is dependent upon intracellular rather than extracellular calcium signals.

Materials and methods

Detailed materials and methods are described in the Supplemental Information. Briefly, brain slices containing CeA were prepared from 60 day old male mice (C57BL/6J) and GABAA receptor-mediated mIPSCs were isolated using whole-cell voltage clamp techniques. Various chemical compounds were tested for their effects on ethanol-induced increase in mIPSCs. Statistical analyses were performed as appropriate. Animal protocols were approved by the Institutional Animal Care and Use Committees of both Duke University and the Durham VA Medical Center.

Results

Isolation of mIPSCs in CeA neurons was achieved by blockade of action-potential dependent sIPSCs with 1.0 μM TTX (Supplemental Figure S1). Subsequent addition of ethanol into the bath solution induced a concentration-dependent increase in mIPSC frequency without altering amplitude, indicating a presynaptic action of ethanol on GABA release (Supplemental Figure S2).

Ethanol induced-increase in mIPSCs frequency does not require external calcium influx

Presynaptic calcium homeostasis is mainly regulated by two calcium sources: external calcium influx and internal calcium stores. We first assessed the external calcium influx on ethanol-induced enhancement of CeA mIPSC frequency in the presence of cadmium chloride (CdCl2), a nonspecific calcium channel blocker. Both 50 mM and 100 mM ethanol significantly increased the frequency of mIPSCs in the presence of 200 μΜ CdCl2 bath applied in three mice as illustrated in Figure 1A. Ethanol shifted the cumulative distribution of inter-event intervals of mIPSCs to the left, indicating that ethanol significantly (K-S test, p<0.001) shortened the inter-event intervals of mIPSCs frequency (Figure 1B). Interestingly, bath application of ethanol resulted in an increase in mIPSCs frequency in the presence of CdCl2 (black bars in Figure-1C) than those slices not treated with CdCl2 (grey bars in Figure 1C), suggesting that there could be a change in the sensitivity of mIPSCs to ethanol while calcium channels are blocked. Additionally, we found that ethanol continued to facilitate mIPSC frequency in three mice even in Ca++-free solution as shown in Figure 1D. Again, although ethanol increased mIPSC frequency under both conditions, the mean amplitudes of mIPSCs were unaffected as summarized in Figure 1E.

Figure 1.

Figure 1.

Open in a new tab

Ethanol-induced increase in the frequency of mIPSCs does not depend on external calcium flux. A. Ethanol (50 and 100 mM) still increases mIPSC frequency in the presence of CdCl2. B. Cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. Effect of ethanol on mIPSC frequency in the absence (gray bars) and presence of CdCl2 (black bars) (n=7–8 neurons in each group). Note. Ethanol still causes a strong increase in mIPSC frequency in CeA neurons treated with CdCl2. D. Effect of ethanol on mIPSC frequency in Ca++-free solution (n=7 neurons in each group). E. Effect of ethanol on the amplitude of mIPSCs in the presence of CdCl2 or Ca++- free solution.

Ethanol induced-increase in mIPSC frequency is occluded after the depletion of internal calcium stores

We next explored whether calcium released from internal stores served as a major calcium source in the presynaptic terminals (Augustine et al. 2003; Collin et al. 2005), and that such release could be responsible for the action of ethanol on mIPSCs as reported in rat cerebellum Purkinje neurons (Kelm et al. 2007) or in cultured spinal cord neurons (Mariqueo et al. 2014). Since the sources of the stored calcium consist of the mitochondria, the endoplasmic reticulum and other uncharacterized mechanisms (Hirasawa and Pittman 2003), we used selective pharmacological compounds to block each of these intracellular calcium store pathways and to assess their roles of facilitating action of ethanol on GABA release from presynaptic terminals as described below.

We first blocked calcium release from intracellular stores through ryanodine receptor (RYR) channels with dantrolene which has been shown to inhibit the elevations of neuronal Ca2+ evoked by N-methyl-D-aspartate, glutamate, or potassium depolarization in cultured rat frontal cortical neurons (Hayashi et al. 1997) in a total of 10 mice. Due to a slow onset required to establish the full effect of the drug, dantrolene was added into the perfusion solution at least 20 minutes prior to ethanol. Dantrolene (10 μΜ) alone did not alter baseline mIPSC frequency: [1.76 ± 0.34 (control, n=8) vs.2.38 ± 0.37 Hz (10 minutes after dantrolene, n=8), paired t-test, p>0.05)] and 2.01 ± 0.13 Hz (20 minutes after dantrolene, n=6, paired t-test, p>0.05) compared to control. Although the frequency of mIPSCs showed a tendency to increase with a higher concentration of dantrolene (100 μΜ), there was no significant change in mean mIPSCs frequency before and after treatment with dantrolene [1.60 ± 0.36 (control) vs. 2.91 ± 0.83Hz, n=4, paired t-test, p>0.05]. The amplitude of mIPSCs was unaffected by dantrolene (10 μΜ or 100 μΜ). Figure 2A shows the representative traces of mIPSCs recorded before (upper traces) and after ethanol 50mM (middle traces) and 100mM (lower traces) in slices treated with 10 μΜ dantrolene. Ethanol failed to enhance mIPSC frequency (F(2,17)=0.19788, p>0.05) and did not change the cumulative distribution of inter-event intervals of mIPSCs in this CeA neuron (Figure 2B, K-S test, p>0.05). Since ethanol still caused a slight increase in mIPSCs frequency in two cells as shown in a scatter plot of Figure-2C, a higher concentration of dantrolene (100μΜ) was applied in a subset of CeA neurons. Again, ethanol (50 mM, n=4 and 100 mM, n=4) still exerted no effect on mIPSC frequency (one-way ANOVA, F(2,9)=0.797, p>0.05). Ethanol-induced enhancements in mIPSC frequency was occluded in the presence of dantrolene as shown in Figure 2D. Furthermore, in the presence of dantrolene of 10 and 100 μM, 50 mM (Figure 2E, one-way ANOVA, F(3, 28)=0.65397, p>0.05) and 100 mM (Figure-4F, one-way ANOVA, F(2, 9)=1.09125, p>0.05) ethanol did not change mIPSCs amplitudes.

Figure 2.

Figure 2.

Open in a new tab

Blockade of ryanodine receptor channels with dantrolene (Dan) prevents ethanol-induced increase in the frequency of mIPSCs. A. Ethanol (50 and 100 mM) failed to cause an increase in mIPSC frequency in the presence of Dan. B. No change in cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. A scatter of plot showing effect of 100 mM (n=6), but not 50mM (n=7), ethanol still causes an increase in mIPSC frequency in the presence of Dan (10 μM) in two CeA cells. D. Effect of ethanol on mIPSC frequency in the presence of Dan (10 and 100 μM) (n=6–7 neurons in each group). E. Effect of ethanol on mIPSC amplitude in the presence of Dan (10 μM). C. Effect of ethanol on mIPSC amplitude in the presence of Dan (100μM).

Figure 4.

Figure 4.

Open in a new tab

Blockade of sarcoplasmic/endoplasmic reticulum Ca++ ATPase (SERCA) with non-competitive blocker cyclopiazonic acid (CPA) prevents ethanol-induced increases in the frequency of mIPSCs. A. Ethanol (50 and 100 mM) failed to cause an increase in mIPSC frequency in the presence of CPA (20 μM) B. No change in cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. Effect of ethanol on mIPSC frequency in the presence of CPA (20 μM). D. Effect of ethanol on mIPSC amplitude in the presence of CPA (20 μM).

The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pathway is another source of internal calcium critical for intracellular Ca2+ signaling. To determine if this pathway plays a role in presynaptic release, the effects of ethanol on mIPSC activity were determined in the presence of the specific SERCA blocker thapsigargin in three mice. Bath applied thapsigargin (2–5 μΜ) for 10–20 minutes showed a tendency to increase mIPSC frequency from a baseline of 2.27 ± 0.47 Hz to 3.73 ± 0.94 Hz after 10 minutes (n=6) and to 2.72 ± 0.51 after 20 minutes (n=4) (paired t-test, p>0.05). Thapsigargin alone did not change the mIPSC amplitudes [−39.18 ± 4.17 pA (baseline) vs. −38.46 ± 3.0 pA, n=6 (10 minutes) and −39.35 ± 3.67 pA, n=4 (20 minutes), paired t-test, p>0.05]. Thapsigargin (5 μΜ) significantly prevented ethanol-induced enhancement of mIPSC frequency but had no effect on the amplitudes (Figure 3A). A K-S test further revealed that mIPSC frequency was unaffected (K-S test, p>0.05, Figure 3B). In all CeA neurons tested, thapsigargin (2–5 μΜ) inhibited increases in mIPSC frequency induced by ethanol (50 mM (n=10) and 100 mM (n=8)) but had no effect on amplitudes of mIPSCs (Figures 3C and 3D, respectively).

Figure 3.

Figure 3.

Open in a new tab

Blockade of sarcoplasmic/endoplasmic reticulum Ca++ ATPase (SERCA) with thapsigargin (TG) prevents ethanol-induced increases in the frequency of mIPSCs. A. Ethanol (50 and 100 mM) failed to cause an increase in mIPSC frequency in the presence of TG (5 μM) B. No change in cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. Effect of ethanol on mIPSC frequency in the presence of TG (2–5 μM) (n=8 neurons in each). D. Effect of ethanol on mIPSC amplitude in the presence of TG (2–5 μM).

We further confirmed this finding by applying cyclopiazonic acid (CPA), a non-competitive inhibitor of SERCA in 4 mice. Data presented in Figure 4 show that blockade of SERCA with CPA (20 μΜ) also prevented the stimulatory action of ethanol (50 mM, n=8) and 100 mM, n=4) on CeA mIPSCs (Figure 4, one-way ANOVA, F(2, 17)=0.49502., p>0.05). Similarly, ethanol did not alter mIPSC amplitudes (Figure 4D, F(2, 17)=0.40708, p>0.05).

In addition to SERCAs, the IP3Rs are considered to be a critical modulatory pathway regulating release of calcium from internal stores. We next assessed whether IP3Rs also participate in ethanol-induced GABA release. To this end, we bath applied 42 μM diphenylboric acid 2-amino-ethyl ester (2-APB), an IP3R receptor antagonist, to the slices obtained from 3 mice. Ethanol (50 and 100 mM) did not facilitate CeA mIPSCs frequency in slices pretreated with 2-APB (42 μM) (Figure 5A and 5B (K-S test, p>0.05). Action of 2-APB on ethanol induced increase in mIPSCs were tested in 5 CeA neurons and the results were summarized in Figure 5C. No significant difference in neither mean frequency (Figure 5C, one-way ANOVA F(2,11)=0.07673, p>0.05) nor amplitude (Figure 5D, one-way ANOVA F(2,11)=0.36311, p>0.05)) of mIPSCs before and after bath administration of ethanol (50 mM and 100 mM) was observed.

Figure 5.

Figure 5.

Open in a new tab

Blockade of the IP3R pathway with 2-APB prevents ethanol-induced increases in the frequency of mIPSCs. A. Ethanol (50 and 100 mM) failed to cause an increase in mIPSC frequency in the presence of 2-APB (42 μM). B. No change in cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. Effect of ethanol on mIPSC frequency in the presence of 2-APB (42 μM). D. Effect of ethanol on mIPSCs amplitude in the presence of 2-APB (42 μM).

Inhibition of mitochondrial calcium release and exocytosis of GABA prevents ethanol induced-increase in mIPSCs frequencies

Thus far, our findings provide evidence that calcium released from internal stores plays a critical role in mediating ethanol-induced enhancement in presynaptic GABA release and that depletion of internal calcium stores disrupts the stimulatory action of ethanol on GABA release in CeA neurons. Since calcium released from mitochondria or processes leading to exocytosis of neurotransmitters also regulate presynaptic GABA release in central neurons (Bardo et al. 2002; Sciancalepore et al. 1998), including rat basolateral amygdala neurons (Koyama et al. 1999), ethanol could also potentially affect this pathway in promoting presynaptic GABA release. To address this, we tested whether ruthenium red affected ethanol-induced facilitation of mIPSC frequency. Since this compound blocks mitochondrial Ca++ transport and facilitates release of GABA via exocytosis mechanisms by binding to cell membranes (Glitsch et al. 2002; Koyama et al. 1999; Trudeau et al. 1996), we hypothesized that if ethanol utilizes both pathways to facilitate GABA release, the depletion of mitochondria calcium and exocytosis of GABA with ruthenium red will attenuate ethanol-induced GABA release. Bath application of ruthenium red alone in a total of 3 mice caused a robust and dose-dependent increase in mIPSCs frequency but left the mean amplitude of mIPSCs unchanged (Figure 6). Treatment with 1μM ruthenium red caused a persistent increase in mIPSCs frequency (Figure 6A middle traces) while a high concentration of 100 μM induced an increase in mIPSC frequency so robust that the events become indistinguishable (Figure 6B, bottom traces). Thus, slices were pre-treated with 1 μM ruthenium red in order to test the effect on ethanol-enhanced mIPSCs. In the presence of 1 μM ruthenium red, addition of ethanol (50 and 100 mM) did not result in a significant increase in mIPSC frequency but with a tendency to slightly decrease and the cumulative distribution curves of the mIPSC frequency were not different before and after ethanol (K-S test, p>0.05, Figure 6C). The overall effect of ethanol on mIPSC frequency (Figure 6D, n=5 (50 mM) and n=4 (100 mM), one-way ANOVA F(2,11)=0.95658, p>0.05)) and the amplitude (Figure-6E, n=5 (50 mM) and n=4 (100 mM), one-way ANOVA F(2,11)=1.28479, p>0.05) in the presence of ruthenium red are shown and ethanol no longer exerts its stimulatory effect on mIPSC frequency after calcium released from the mitochondria and exocytosis were disrupted by ruthenium red. Taken together, our novel findings strongly indicate that ethanol could affect these previous uncharacterized pathways involving GABA release in mouse CeA.

Figure 6.

Figure 6.

Open in a new tab

Depletion of mitochondria calcium release with ruthenium red (RR) prevents ethanol-induced increases in mIPSC frequency. A. Bath application of RR (1 and 100 μM) causes dose-dependent increases in mIPSC frequency by depleting mitochondria calcium release. B. Ethanol (50 and 100 mM) failed to cause an further increase in mIPSC frequency in the presence of RR. C. No change in cumulative distribution of inter-event intervals of mIPSCs following ethanol application. C. Effect of ethanol on mIPSC frequency in the presence of RR (1 μM) (n=6 neurons in each). D. Effect of ethanol on mIPSC amplitude in the presence of RR (1 μM).

Ethanol induced-increase in mIPSCs frequency is independent of L-type calcium channels

As indicated in a recent report, ethanol failed to increase in sIPSCs in CeA neurons from naive rats, but not from those rats exposed to chronic ethanol after L-type calcium channels were blocked with nifedipine (10 μm), a specific dihydropyridine calcium blocker for L-type calcium channels (Varodayan et al. 2017a). Moreover, nifedipine could modulate neurotransmitter release in a L-type calcium channel independent manner in magnocellular neurons in the supraoptic nucleus, the paraventricular nucleus, suprachiasmatic nucleus and nucleus accumbens (Hirasawa and Pittman 2003). These findings prompted us to question if ethanol could also modulate this calcium channel-independent pathway in order to facilitate ethanol-induced enhancement in GABA release in the CeA.

We first determined if nifedipine increase mIPSCs frequency of CeA neurons by bath application of 10μM nifedipine in the dark (see supplemental method) in five mice. As shown in Figure-7A, nifedipine (10μM) alone caused a significant increase in mIPSCs frequency but had no effect on amplitude. In this CeA neuron, for instance, bath applied nifedipine for 10 minutes caused the frequency to increase from 2.09 to 2.91Hz of control, but the amplitude was unchanged. Nifedipine caused a significant shortening of the inter-event interval of mIPSCs (K-S test, p<0.001, n=8, Figure-7B). There was a significant increase in the mean mIPSC frequency (paired t-test, p<0.001,n=8, Figure-7C) but not in the mean mIPSCs amplitude (paired t-test, p>0.05, n=8, Figure-7D). In order to determine if such argumentation in mIPSC frequency of CeA neurons was unique to nifedipine, another dihydropyridine with a chemical structure similar to nifedipine, nimodipine was chosen to determine whether nifedipine’s effects on GABA release in our preparation could be reproduced. Unexpectedly, bath application of nimodipine (50μM) for over 20 minutes did not cause a significant increase in mean mIPSCs frequency. As showed in Figure-7E, nimodipine (50μM) had no apparent effect on mIPSCs frequency, and no effect on the cumulative distribution of inter-event of mIPSCs (,K-S test, p>0.05,n=7, Figure-7F) was observed. Effects on neither the mean frequency (paired t-test, p>0.05, n=7, Figure-7G) nor amplitude of mIPSCs (paired t-test, p>0.05,n=7, Figure-7H) were detected.

Figure-7.

Figure-7.

Open in a new tab

Effects of L-type calcium channel blockers on presynaptic GABA releases. A. Traces showing the enhancement effect of nifedipine (10μM) on mIPSCs frequency. B. A cumulative distribution of inter-event intervals of mIPSCs following nifedipine (K-S, p<0.001). C. Nifedipine caused a significant increase in the mean mIPSC frequency (paired t-test, p<0.001). D. Nifedipine has no effect on the mean mIPSCs amplitude (paired t-test, p>0.05). E. Traces showing the effect of nimodipine (50μM) on mIPSCs frequency. F. A cumulative distribution of inter-event intervals of mIPSCs following nimodipine (K-S, p>0.05). G. Nimodipine has no effect on the mean mIPSC frequency (paired t-test, p>0.05). H. Nimodipine has no effect on the mean mIPSCs amplitude (paired t-test, p>0.05).

As shown in Figure 7, nifedipine, but not its structural analog nimodipine, significantly increases mIPSC frequency in CeA neurons, suggesting that although the amygdala expresses multiple types of high-voltage activated calcium channels including L-type calcium channels in rats (Foehring and Scroggs 1994; Li et al. 2014; Viana and Hille 1996; Yu and Shinnick-Gallagher 1997) and mice (Davis and Bauer 2012), nifedipine apparently exerts its stimulatory effect on neurotransmitter release unrelated to its inhibition of L-type calcium channels in CeA neurons. We further explored if ethanol could also utilize this unique L-type calcium channel independent pathway, which was used by nifedipine, to facilitate GABA release. Indeed, ethanol failed to cause an increase in mIPSC frequency in the presence of nifedipine. We have tested the effect of ethanol on mIPSCs at 50 mM (n=7 neurons) and 100 mM (n=6 neurons) and the typical traces of mIPSCs during nifedipine and its co-application with ethanol from a CeA neuron were shown in Figure 8A. After 10 minutes of incubation with 10 μΜ nifedipine, 50 mM ethanol was bath-applied for 10 minutes and then 100mM for another 10 minutes. This CeA cell failed to respond to ethanol with an increase in mIPSC frequency. A cumulative distribution of the inter-event intervals of mIPSCs further indicated that ethanol did not change the frequency distribution (K-S test, p>0.05, Figure 8B). Overall, in the presence of nifedipine, ethanol had no effect on the mean mIPSC frequency (Figure-8C, 50 mM, n=7 and 100 mM, n=6, one-way ANOVA, F(2,18)=1.64899, p>0.05) or amplitude (Figure-7D one-way ANOVA, F(2,18)=1.44987, p>0.05). In contrast, ethanol (50 and 100 mM) still enhanced mIPSC frequency in the presence of nimodipine (50 μM), as shown in Figure 8E and 8F. In all CeA neurons tested in five mice, bath application of ethanol (50 mM (n=10) and 100 mM (n=7)) continue to facilitate mean mIPSCs frequency (Figure 8G, one-way ANOVA, F(2,24)=7.70409, p<0.05), with no effect on mean amplitude of mIPSCs (Figure 8H, one-way ANOVA, F(2,24)=0.06562, p>0.05) in CeA cells treated with nimodipine.

Figure 8.

Figure 8.

Open in a new tab

Nifedipine prevents ethanol-induced enhancement in mIPSCs independent of L-type calcium channel. A. Ethanol (50 and 100 mM) failed to cause an increase in mIPSCs frequency in the presence of nifedipine (10μM). B. No changes in the cumulative distribution of inter-event intervals of mIPSCs following co-application of nifedipine and ethanol (K-S test, p<0.05). C. No significant change mIPSC frequency following ethanol application. D. No changes in mIPSC amplitude in the presence of nifedipine and ethanol. E. Bath application of nimodipine (10 μM) failed to prevent ethanol-induced increase in mIPSC frequency. F. A significant change in cumulative distribution of inter-event intervals of mIPSCs following co-application of nimodipine and ethanol (50 and 100 mM) (K-S test, p>0.05). G. A significant change in mean mIPSCs frequency following ethanol application. H. No changes in mIPSC amplitude in the presence of nimodipine and ethanol.

Discussion

The primary purpose of this study was to determine the role of internal and external sources of calcium in mediating ethanol-induced augmentation of GABA release from presynaptic terminals in CeA neurons in mice. We found that ethanol-induced enhancement in GABA release does not depend on trans-membrane calcium influx but could readily be disrupted by inhibition/reduction of calcium from internal calcium stores via different pathways. In addition, we have also identified previously uncharacterized and novel molecular pathways through which ethanol, serving as a strong secretagogue, stimulates inhibitory GABA release from presynaptic terminals located within the CeA.

Neurons in the central nucleus of the amygdala are primarily GABAergic, and GABAergic transmission within the amygdala is regulated by neuromodulators and neuropeptides in rodents and humans (Ehrlich et al. 2009). Ethanol enhances mIPSCs via presynaptic GABA release from CeA neurons in mice in a similar way observed in a variety of brain regions through which the effect of ethanol is mediated by multiple molecular pathways (Criado and Morales 2000; Criswell et al. 2008; Kelm et al. 2011; Rinker et al. 2017). For example, in the present study an increase in the frequency of mIPSCs in CeA neurons was consistently observed in the absence of extracellular calcium or in the presence of the non-selective calcium channel blocker CdCl2, but it has been reported that effect of ethanol on mIPSCs of CeA neuron in rats appears to be prevented in a low calcium extracelllar environment(Varodayan et al. 2017b). The discrepancy between these findings is unclear but it could be related to the difference in animals used In addition, mIPSCS recorded from CeA neurons in the presence of TTX virtually abolished the contribution of voltage-gated calcium channels (VGCCs) to synaptic release since these low- or high-VGCCs usually activate after the cell membrane potential is positive to −70 mV or even −20 mV (Tsien et al. 1988). Under these experimental conditions, the contributions of calcium influx as an essentially required step to promote ethanol-induced increase in mIPSCs frequency were eliminated. However, ethanol–induced enhancement in mIPSCs frequency is virtually prevented when an optimal internal calcium homeostasis was disrupted by blocking a number of molecular pathways leading to calcium release from internal calcium stores. Our present findings indicate that depletion of calcium from internal calcium stores via the blockage of intracellular calcium release from the endoplasmic reticulum (ER) via the ryanodine receptors with dantrolene or by disrupting reuptake of calcium via the SERCA pathways with either thapsigargin or CPA all prevent ethanol-induced increase in GABA release. Dantrolene has been shown to inhibit of intracellular calcium concentrations induced by KCl or NMDA in cultured frontal cortical neurons in rats (Hayashi et al. 1997), thus providing neuroprotection against neurotoxicity induced by a rise of intracellular calcium following ischemia (Nakayama et al. 2002). This finding suggests that, in our experimental conditions, dantrolene could prevent the enhancement in mIPSC frequency induced by ethanol via its inhibition of calcium release from intracellular calcium stores (Mody and MacDonald 1995). In addition, occlusion of calcium release from the SERCA with thapsigargin effectively blocks calcium pumps of the ER and abolished an ethanol-induced enhancement in mIPSC frequency. This leads to depletion of the internal calcium store, especially in its late stage of bath application (Mizerna et al. 2007). This action was further confirmed by bath application of non-competitive SERCA blocker CPA in which ethanol also failed to increase mIPSC frequency. As a result, these actions could effectively diminish the availability of intracellular calcium in the intracellular milieu for ethanol. Our results are consistent with an observation by Takahashi et al. in which thapsigargin blocks hydrogen peroxide (H2O2)-induced increases in mIPSCs in spinal cord substantia geletinosa neurons (Takahashi et al. 2007). In addition, an inhibition of the IP3R pathway with 2-APB also prevents the enhancement of mIPSC frequency induced by ethanol. Taken together, these results strongly indicate that maintaining optimal intracellular calcium homeostasis is a key determinant for actions of ethanol in regulating presynaptic neurotransmitter release. Moreover, the fact that ethanol enhances the frequency of mIPSCs but has no obvious effects on amplitude points to its action on presynaptic machinery involved in the neurotransmitter release process. Postsynaptic neurons filled with the intracellular calcium chelator BAPTA does not affect ethanol-induced enhancement of mIPSC frequency in cerebellum Purkinje neurons (Kelm et al. 2007). Combining this finding with our results, we postulate that internal calcium stores in presynaptic sites are the primary mediator responsible for ethanol actions on GABA release in the CeA.

Our results are in agreement with those of Kelm et al. (Kelm et al. 2007), who showed that internal calcium stores are required for ethanol to facilitate GABA release from presynaptic terminals in Purkinje neurons in cerebellum slices and in mechanically dissociated Purkinje neurons in which inhibition of presynaptic calcium store by the compounds known to interfere with calcium signaling originating from internal calcium stores prevent the ethanol-induced enhancement in mIPSC frequency. In addition, our results are also consistent with those observed in mouse spinal cord slice neurons in which ethanol-induced increases in glycinergic synaptic currents is also dependent on internal calcium (Mariqueo et al. 2014).

In addition to release of calcium from well documented sources of internal calcium stores via several pathways such as inositol 1,4,5-trisphophate (IP3) sensitive, ryanodine-sensitive calcium release and the SERCA, calcium release or uptake from mitochondria represent a novel mechanism whereby ethanol could also promote GABA release in CeA neurons. By taking advantage of its inhibition of release of calcium from mitochondria and facilitation of exocytosis of neurotransmitters including GABA in central synapses (Bae et al. 2003; Yuan and Atchison 2016), ruthenium red, a mitochondria calcium transport blocker, interferes with this pathway and appears to be participating in the facilitation of GABA release induced by ethanol. Despite that there seems to be some debate as to the selectivity of ruthenium red on mitochondria, with some reports suggesting RR demonstrates some RyR/SERCA antagonism at doses relevant to the results in this study (e.g. ~5μM) (Zima et al. 2008), the modest effects of dantrolene, thapsigargin, etc. on mIPSC frequency in contrast to observations with RR seem to suggest specific (or at least non-IP3/SERCA/etc) effects of RR on mitochondrial calcium. This is a novel target pathway for further delineation of ethanol action at central synapses. As shown in Figure 6, CeA neurons treated with ruthenium red resulted in a strong and robust increase in mIPSC frequency that failed to respond to further ethanol exposure. This could be due to the depletion of intracellular calcium following strong release or a direct calcium independent action on the secretory apparatus via exocytosis (Trudeau et al. 1996). Our results indicate that central synapses respond to ruthenium red with a strong increase in mIPSC frequency following calcium release from mitochondria but not from presynaptic calcium stores (Lim et al. 2003), thus affecting ethanol action on mIPSC frequency in a way similar to those observed in the presence of dantrolene or thapsigargin. In hippocampal neurons, ruthenium red also facilitates miniature GABAergic currents independent of internal calcium stores (Sciancalepore et al. 1998). As a result, the depletion of mitochondria calcium prevents promotion of GABA release induced by ethanol. Therefore, this internal calcium store independent pathway represents a new cellular mechanism involved in central neurotransmitter release (Trudeau et al. 1996) to further investigate ethanol actions at central synapses. It should also be noted that IP3R antagonism but not blocking the RyR pathway could prevent ethanol-induced enhancement of action-potential dependent sIPSCs frequencies in CeA of rats(Varodayan et al. 2017a).

Another novel observation from our present study is that there could be previously unknown and uncharacterized molecular pathways which are also independent of both internal calcium stores and L-type calcium channels through which ethanol facilitates GABA release from presynaptic terminals. As reported previously, nifedipine, a L-type voltage dependent calcium channel blocker, could trigger excitatory and inhibitory neurotransmitter release in the central synapses and this effect is largely independent of its classical inhibition of L-type calcium channels but due to a direct action on the release process as suggested (Hirasawa and Pittman 2003). In addition, such action was not duplicated by another L-type voltage dependent calcium channel, nimodipine. This result was also supported by the findings that ethanol is still able to increase mIPSC frequency in the presence of non-specific calcium blocker CdCl2 in this and another study (Kelm et al. 2007). Moreover, in our present study, we further revealed that the L-type calcium blocker nifedipine, but not its chemical structure analogue nimodipne, increases GABA release in CeA neurons and this uncharacterized action affects ethanol-induced enhancement in GABA release. In spinal cord substantia geletinosa neurons, bath application of nifedipine reduces H2O2-induced increases in mIPSCs (Takahashi et al. 2007) However, in that study, blocking the H2O2- induced increase in mIPSCs by nifedipine was considered to be related to its unexpected blockade of an IP3R pathway linking to calcium-induced calcium release. The results obtained from our nimodipine study indicated that nifedipine appeared unlikely to work in the same manner because nimodipine does not produce the same effect. Hence, nifedipine did increase the frequency of mIPSCs in CeA neurons but this effect was not related to its inhibition of L-type calcium channels in CeA neurons. This novel finding strongly suggests that ethanol could promote GABA release by a direct action on the release process which is independent of L-type calcium channels. It has been known that L-type voltage calcium channels are widely expressed in the amygdala and participate in alcohol dependence (Uhrig et al. 2016) and fear extinction which could directly relate to alcohol use (Davis and Bauer 2012). In addition, calcium channels in the central amygdala nucleus of rats were inhibited by acute ethanol (Li et al. 2014). A recent study by Varodayan et al. reported that disruption of L-type voltage dependent calcium channels with nefidipine in CeA neurons in rats could subsequently disrupt GABA release (Varodayan et al. 2017a). Although it has been shown that nifedipine could increase neurotransmitter release in the hypothalamic neurons in rats (Hirasawa and Pittman 2003), it is still unknown if CeA neurons respond to nifedipine differently between mice and rats. Overall, our finding clearly extends our knowledge of the action of ethanol on synaptic GABA release by including this previously uncharacterized synaptic signaling which is also involved in the regulation of neurotransmitter release in the presence of ethanol.

It is worth pointing our that the CeA includes a number of discriminable GABAergic cell subtypes that participate differently within the intra-regional microcircuit(Herman et al. 2016) and alcohol appears to exert its more potent blocking action on VGCC on those GABAergic cells specifically located in the medial CeA of rats(Varodayan et al. 2017b). In this present study, no discriminable effort was attempted to record CeA neurons that were specifically located in the given subregions, it should therefore, be caution if the aforementioned mechanism is common to all GABAergic neurons in the CeA, and a further study should specifically focus on the GABAergic cells located in the different subregions of the CeA.

Taken together, these results suggest that central synapses within the central nucleus of the amygdala in mice utilize multiple sources of calcium signaling to affect ethanol-induced GABA release. By mobilizing calcium and disrupting internal calcium homeostasis, acute ethanol could regulate central neurotransmitter release in regulating acute anxiolytic properties of CeA neurons, which is the part of alcohol addiction and dependence.

Supplementary Material

figures 1

Acknowledgments

Funding

This work was funded by a VA Merit Review grant to SDM (I01BX001271) and a Duke University Pilot grant to QL.

Footnotes

Disclosures

The authors have no disclosures or conflicts of interest to report.

References

  1. Augustine GJ, Santamaria F, Tanaka K (2003) Local calcium signaling in neurons. Neuron 40: 331–46. [DOI] [PubMed] [Google Scholar]
  2. Bae JH, Park JW, Kwon TK (2003) Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochemical and biophysical research communications 303: 1073–9. [DOI] [PubMed] [Google Scholar]
  3. Bardo S, Robertson B, Stephens GJ (2002) Presynaptic internal Ca2+ stores contribute to inhibitory neurotransmitter release onto mouse cerebellar Purkinje cells. British journal of pharmacology 137: 529–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collin T, Marty A, Llano I (2005) Presynaptic calcium stores and synaptic transmission. Current opinion in neurobiology 15: 275–81. [DOI] [PubMed] [Google Scholar]
  5. Criado JR, Morales M (2000) Acute ethanol induction of c-Fos immunoreactivity in pre-pro-enkephalin expressing neurons of the central nucleus of the amygdala. Brain research 861: 173–7. [DOI] [PubMed] [Google Scholar]
  6. Criswell HE, Ming Z, Kelm MK, Breese GR (2008) Brain regional differences in the effect of ethanol on GABA release from presynaptic terminals. The Journal of pharmacology and experimental therapeutics 326: 596–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis SE, Bauer EP (2012) L-Type Voltage-Gated Calcium Channels in the Basolateral Amygdala Are Necessary for Fear Extinction. The Journal of Neuroscience 32: 13582–13586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehrlich I, Humeau Y, Grenier F, Ciocchi S, Herry C, Lüthi A (2009) Amygdala Inhibitory Circuits and the Control of Fear Memory. Neuron 62: 757–771. [DOI] [PubMed] [Google Scholar]
  9. Foehring RC, Scroggs RS (1994) Multiple high-threshold calcium currents in acutely isolated rat amygdaloid pyramidal cells. Journal of neurophysiology 71: 433–6. [DOI] [PubMed] [Google Scholar]
  10. Ghatpande AS, Sivaraaman K, Vijayaraghavan S (2006) Store calcium mediates cholinergic effects on mIPSCs in the rat main olfactory bulb. Journal of neurophysiology 95: 1345–55. [DOI] [PubMed] [Google Scholar]
  11. Gilpin NW, Herman MA, Roberto M (2015) The central amygdala as an integrative hub for anxiety and alcohol use disorders. Biological psychiatry 77: 859–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gipson CD, Kupchik YM, Kalivas PW (2014) Rapid, transient synaptic plasticity in addiction. Neuropharmacology 76 Pt B: 276–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glitsch MD, Bakowski D, Parekh AB (2002) Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake. The EMBO journal 21: 6744–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayashi T, Kagaya A, Takebayashi M, Oyamada T, Inagaki M, Tawara Y, Yokota N, Horiguchi J, Su TP, Yamawaki S (1997) Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons. Journal of Neural Transmission 104: 811–824. [DOI] [PubMed] [Google Scholar]
  15. Herman MA, Contet C, Roberto M (2016) A Functional Switch in Tonic GABA Currents Alters the Output of Central Amygdala Corticotropin Releasing Factor Receptor-1 Neurons Following Chronic Ethanol Exposure. J Neurosci 36: 10729–10741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Herman MA, Roberto M (2016) Cell-type-specific tonic GABA signaling in the rat central amygdala is selectively altered by acute and chronic ethanol. Addict Biol 21: 72–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirasawa M, Pittman QJ (2003) Nifedipine facilitates neurotransmitter release independently of calcium channels. Proceedings of the National Academy of Sciences 100: 6139–6144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kang-Park M, Kieffer BL, Roberts AJ, Siggins GR, Moore SD (2013) kappa-Opioid receptors in the central amygdala regulate ethanol actions at presynaptic GABAergic sites. J Pharmacol Exp Ther 346: 130–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kelm MK, Criswell HE, Breese GR (2007) Calcium release from presynaptic internal stores is required for ethanol to increase spontaneous gamma-aminobutyric acid release onto cerebellum Purkinje neurons. The Journal of pharmacology and experimental therapeutics 323: 356–64. [DOI] [PubMed] [Google Scholar]
  20. Kelm MK, Criswell HE, Breese GR (2011) Ethanol-enhanced GABA release: a focus on G protein-coupled receptors. Brain research reviews 65: 113–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koyama S, Kubo C, Rhee JS, Akaike N (1999) Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons. The Journal of physiology 518 (Pt 2): 525–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Le Magueresse C, Cherubini E (2007) Presynaptic calcium stores contribute to nicotine-elicited potentiation of evoked synaptic transmission at CA3-CA1 connections in the neonatal rat hippocampus. Hippocampus 17: 316–25. [DOI] [PubMed] [Google Scholar]
  23. Li Q, Madison R, Moore SD (2014) Presynaptic BK channels modulate ethanol-induced enhancement of GABAergic transmission in the rat central amygdala nucleus. J Neurosci 34: 13714–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li Q, Wilson WA, Swartzwelder HS (2006) Developmental differences in the sensitivity of spontaneous and miniature IPSCs to ethanol. Alcoholism, clinical and experimental research 30: 119–26. [DOI] [PubMed] [Google Scholar]
  25. Lim R, Oleskevich S, Few AP, Leao RN, Walmsley B (2003) Glycinergic mIPSCs in mouse and rat brainstem auditory nuclei: modulation by ruthenium red and the role of calcium stores. The Journal of physiology 546: 691–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lovinger DM, Roberto M (2013) Synaptic effects induced by alcohol. Current topics in behavioral neurosciences 13: 31–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luscher C, Malenka RC (2011) Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling. Neuron 69: 650–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mariqueo TA, Agurto A, Munoz B, San Martin L, Coronado C, Fernandez-Perez EJ, Murath P, Sanchez A, Homanics GE, Aguayo LG (2014) Effects of ethanol on glycinergic synaptic currents in mouse spinal cord neurons. Journal of neurophysiology 111: 1940–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mizerna EP, Fedulova SA, Veselovsky NS (2007) Effect of thapsigargin on inhibitory synaptic transmission between cultured neurons of the rat hippocampus. Neurophysiology 39: 327–329. [Google Scholar]
  30. Mody I, MacDonald JF (1995) NMDA receptor-dependent excitotoxicity: the role of intracellular Ca2+ release. Trends in pharmacological sciences 16: 356–9. [DOI] [PubMed] [Google Scholar]
  31. Nakayama R, Yano T, Ushijima K, Abe E, Terasaki H (2002) Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia. Anesthesiology 96: 705–10. [DOI] [PubMed] [Google Scholar]
  32. Rinker JA, Marshall SA, Mazzone CM, Lowery-Gionta EG, Gulati V, Pleil KE, Kash TL, Navarro M, Thiele TE (2017) Extended Amygdala to Ventral Tegmental Area Corticotropin-Releasing Factor Circuit Controls Binge Ethanol Intake. Biol Psychiatry 81: 930–940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sciancalepore M, Savic N, Gyori J, Cherubini E (1998) Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons. Journal of neurophysiology 80: 2316–22. [DOI] [PubMed] [Google Scholar]
  34. Simkus CR, Stricker C (2002) The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex. The Journal of physiology 545: 521–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takahashi A, Mikami M, Yang J (2007) Hydrogen peroxide increases GABAergic mIPSC through presynaptic release of calcium from IP3 receptor-sensitive stores in spinal cord substantia gelatinosa neurons. The European journal of neuroscience 25: 705–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Trudeau LE, Doyle RT, Emery DG, Haydon PG (1996) Calcium-independent activation of the secretory apparatus by ruthenium red in hippocampal neurons: a new tool to assess modulation of presynaptic function. The Journal of neuroscience : the official journal of the Society for Neuroscience 16: 46–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP (1988) Multiple types of neuronal calcium channels and their selective modulation. Trends in neurosciences 11: 431–8. [DOI] [PubMed] [Google Scholar]
  38. Uhrig S, Vandael D, Marcantoni A, Dedic N, Bilbao A, Vogt MA, Hirth N, Broccoli L, Bernardi RE, Schönig K, Gass P, Bartsch D, Spanagel R, Deussing JM, Sommer WH, Carbone E, Hansson AC (2016) Differential Roles for L-Type Calcium Channel Subtypes in Alcohol Dependence. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 42: 1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Varodayan FP, de Guglielmo G, Logrip ML, George O, Roberto M (2017a) Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms. The Journal of neuroscience : the official journal of the Society for Neuroscience 37: 4593–4603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Varodayan FP, Logrip ML, Roberto M (2017b) P/Q-type voltage-gated calcium channels mediate the ethanol and CRF sensitivity of central amygdala GABAergic synapses. Neuropharmacology 125: 197–206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Varodayan FP, Soni N, Bajo M, Luu G, Madamba SG, Schweitzer P, Parsons LH, Roberto M (2016) Chronic ethanol exposure decreases CB1 receptor function at GABAergic synapses in the rat central amygdala. Addiction biology 21: 788–801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Viana F, Hille B (1996) Modulation of High Voltage-Activated Calcium Channels by Somatostatin in Acutely Isolated Rat Amygdaloid Neurons. The Journal of Neuroscience 16: 6000–6011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yan H, Li Q, Madison R, Wilson WA, Swartzwelder HS (2010) Differential sensitivity of hippocampal interneurons to ethanol in adolescent and adult rats. J Pharmacol Exp Ther 335: 51–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yu B, Shinnick-Gallagher P (1997) Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. Journal of neurophysiology 77: 690–701. [DOI] [PubMed] [Google Scholar]
  45. Yuan Y, Atchison WD (2016) Multiple Sources of Ca2+ Contribute to Methylmercury-Induced Increased Frequency of Spontaneous Inhibitory Synaptic Responses in Cerebellar Slices of Rat. Toxicol Sci 150: 117–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zima AV, Picht E, Bers DM, Blatter LA (2008) Partial inhibition of sarcoplasmic reticulum ca release evokes long-lasting ca release events in ventricular myocytes: role of luminal ca in termination of ca release. Biophys J 94: 1867–79. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

figures 1
NIHMS1632593-supplement-figures_1.docx (2.1MB, docx)

RESOURCES