Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

doi:10.3389/fncel.2018.00521

. 2019 Jan 14:12:521.

doi: 10.3389/fncel.2018.00521. eCollection 2018.

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

Da-Yong Li^{1

2

3}, Yan-Hua Bing^{1

4}, Chun-Ping Chu¹, Xun Cui¹, Song-Biao Cui⁴, De-Lai Qiu^{1

2}, Li-Da Su⁵

Affiliations

¹ Key Laboratory of Cellular Function and Pharmacology of Jilin Province, Yanbian University, Yanji, China.
² Department of Physiology and Pathophysiology, College of Medicine, Yanbian University, Yanji, China.
³ College of Basic Courses, Zhejiang Shuren University, Hangzhou, China.
⁴ Department of Neurology, Affiliated Hospital of Yanbian University, Yanji, China.
⁵ Neuroscience Care Unit, Second Affiliated Hospital of Zhe-Jiang University School of Medicine, Hangzhou, China.

PMID: 30692916
PMCID: PMC6339896
DOI: 10.3389/fncel.2018.00521

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

Da-Yong Li et al. Front Cell Neurosci. 2019.

. 2019 Jan 14:12:521.

doi: 10.3389/fncel.2018.00521. eCollection 2018.

Authors

Da-Yong Li^{1

2

3}, Yan-Hua Bing^{1

4}, Chun-Ping Chu¹, Xun Cui¹, Song-Biao Cui⁴, De-Lai Qiu^{1

2}, Li-Da Su⁵

Affiliations

¹ Key Laboratory of Cellular Function and Pharmacology of Jilin Province, Yanbian University, Yanji, China.
² Department of Physiology and Pathophysiology, College of Medicine, Yanbian University, Yanji, China.
³ College of Basic Courses, Zhejiang Shuren University, Hangzhou, China.
⁴ Department of Neurology, Affiliated Hospital of Yanbian University, Yanji, China.
⁵ Neuroscience Care Unit, Second Affiliated Hospital of Zhe-Jiang University School of Medicine, Hangzhou, China.

PMID: 30692916
PMCID: PMC6339896
DOI: 10.3389/fncel.2018.00521

Abstract

The cerebellum is sensitive to ethanol (EtOH) consumption. Chronic EtOH consumption impairs motor learning by modulating the cerebellar circuitry synaptic transmission and long-term plasticity. Under in vitro conditions, acute EtOH inhibits both parallel fiber (PF) and climbing fiber (CF) long-term depression (LTD). However, thus far it has not been investigated how chronic EtOH consumption affects sensory stimulation-evoked LTD at the molecular layer interneurons (MLIs) to the Purkinje cell (PC) synapses (MLI-PC LTD) in the cerebellar cortex of living animals. In this study, we investigated the effect of chronic EtOH consumption on facial stimulation-evoked MLI-PC LTD, using an electrophysiological technique as well as pharmacological methods, in urethane-anesthetized mice. Our results showed that facial stimulation induced MLI-PC LTD in the control mice, but it could not be induced in mice with chronic EtOH consumption (0.8 g/kg; 28 days). Blocking the cannabinoid type 1 (CB1) receptor activity with AM-251, prevented MLI-PC LTD in the control mice, but revealed a nitric oxide (NO)-dependent long-term potentiation (LTP) of MLI-PC synaptic transmission (MLI-PC LTP) in the EtOH consumption mice. Notably, with the application of a NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP) alone prevented the induction of MLI-PC LTD, but a mixture of SNAP and AM-251 revealed an MLI-PC LTP in control mice. In contrast, inhibiting NO synthase (NOS) revealed the facial stimulation-induced MLI-PC LTD in EtOH consumption mice. These results indicate that long-term EtOH consumption can impair the sensory stimulation-induced MLI-PC LTD via the activation of a NO signaling pathway in the cerebellar cortex in vivo in mice. Our results suggest that the chronic EtOH exposure causes a deficit in the cerebellar motor learning function and may be involved in the impaired MLI-PC GABAergic synaptic plasticity.

Keywords: cerebellar purkinje cell; ethanol; in vivo cell-attached recording; molecular layer interneuron; nitric oxide; plasticity; sensory stimulation.

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Figures

**Figure 1**
Property of facial stimulation-evoked responses in cerebellar Purkinje cells (PCs). **(A)** Under cell-attached recording conditions (current-clamp mode; I = 0 pA), superposition of five sweeps shows a cerebellar PC in response to the tactile stimulation (bar, 10 ms) in artificialcerebrospinal fluid (ACSF). **(B)** The superposition of five sweeps shows the PC in response to the tactile stimulation (bar, 10 ms) in the presence of gabazine (50 μM). APs denote action potentials.

**Figure 2**
Facial stimulation at 1 Hz induces molecular layer interneuron-PC long-term depression (MLI-PC) LTD in the cerebellar cortex of control mice. **(A)** Representative cell-attached recording traces, showing air-puff stimulation (10 ms, 60 psi)-evoked responses in a cerebellar PC before (Pre) and after (post) delivering 1 Hz (240 pulses) stimulation. **(B)** Summary of the normalized positive component (P1) amplitude under control conditions (No stimulation, open circle; n = 8 mice) and delivery of 1 Hz facial stimulation (arrow head; filled circles; n = 8 mice). **(C)** Bar graph (n = 8 mice) showing the normalized amplitude of P1 before (Pre sti.) and after (Post sti.) delivery of 1 Hz stimulation and no stimulation (No sti). **(D)** Summary of data (n = 8 mice) showing the normalized pause of a simple spike firing before (Pre sti.) and after (Post sti.) delivery of 1 Hz stimulation and no stimulation (No sti.). Note that facial stimulation at 1 Hz induced MLI-PC LTD in mouse cerebellar cortex. Data points are mean ± SEM. ^#P < 0.05 vs. baseline (Pre-sti.).

**Figure 3**
Facial stimulation at 1 Hz failed to induce MLI-PC LTD in ethanol (EtOH) consumption mice. (A) Representative traces showing air-puff stimulation (10 ms, 60 psi)-evoked responses in a PC from an EtOH consumption mouse before (left) and after delivering 1 Hz stimulation. **(B)** Summary of the normalized P1 amplitude under delivery of 1 Hz facial stimulation (arrow head; n = 8 mice). **(C)** Bar graph (n = 8 mice) showing the normalized amplitude of P1 before (Pre) and after (Post) delivery of 1 Hz stimulation. (D) Summary of data (n = 8 mice) showing the normalized pause of a simple spike firing before (Pre) and after (Post) delivery of 1 Hz stimulation. Note that air-puff stimulation at 1 Hz failed to induce MLI-PC LTD in EtOH consumption mice. Data points are mean ± SEM.

**Figure 4**
Blocking cannabinoid type 1 (CB1) receptors activity, facial stimulation failed to induce MLI-PC LTD in control mice, but induced MLI-PC long-term potentiation (LTP) in EtOH consumption mice. **(A)** Representative cell-attached recording traces showing air-puff stimulation (10 ms, 60 psi)-evoked responses in a cerebellar PC from an EtOH consumption mouse before (left) and after delivering 1 Hz (240 pulses) stimulation in the presence of a CB1 antagonist, AM251 (5 μM). **(B)** Summary of the normalized P1 amplitude after delivery of 1 Hz facial stimulation (arrow head) in control mice (black; n = 7 mice) and EtOH consumption mice (Red; n = 7). **(C)** Bar graph (n = 7 mice) showing the normalized amplitude of P1 before (Pre EtOH) and after (Post, EtOH; Post control) delivery of 1 Hz stimulation in EtOH consumption and control mice. (D) Summary of data (n = 7 mice) showing the normalized pause of a simple spike firing before and after delivery of 1 Hz stimulation in EtOH consumption and control mice. Note that blocking by CB1 receptor activity, facial stimulation failed to induce MLI-PC LTD in control mice, but induced MLI-PC LTP in EtOH consumption mice.

**Figure 5**
Inhibition of nitric oxide synthase (NOS), facial stimulation induced MLI-PC LTD in EtOH consumption mice. **(A)** In the presence of the NOS inhibitor, representative traces showing air-puff stimulation (10 ms, 60 psi) evoked responses in a cerebellar PC from an EtOH consumption mouse before (Pre) and after (Post) delivering 1 Hz (240 pulses) stimulation. **(B)** Summary of the normalized P1 amplitude after delivery of 1 Hz facial stimulation (arrow head) in treatment with ACSF (blue) and the NOS inhibitor (purple; n = 7 mice). **(C)** Bar graph (n = 7 mice) showing the normalized amplitude of P1 before (Pre) and after (Post) delivery of 1 Hz stimulation. **(D)** Summary of data (n = 7 mice) showing the normalized pause of a simple spike firing before (Pre) and after (Post) delivery of 1 Hz stimulation. Note that at the inhibition of NOS, facial stimulation induced MLI-PC LTD, in EtOH consumption mice.

**Figure 6**
In the presence of NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP; 100 μM), facial stimulation failed to induce MLI-PC LTD in control mice. **(A)** In the presence of the NO donor, representative traces showing air-puff stimulation (10 ms, 60 psi) evoked responses in a cerebellar PC from a control mouse before and after delivering 1 Hz stimulation. **(B)** Summary of the normalized P1 amplitude after delivery of 1 Hz facial stimulation (arrow head) in the presence of the NO donor in control mice (n = 7 mice). **(C)** Bar graph (n = 7 mice) showing the normalized amplitude of P1 before (Pre) and after (Post) delivery of 1 Hz stimulation. **(D)** Summary of data (n = 7 mice) showing the normalized pause of a simple spike firing before (Pre) and after (Post) delivery of 1 Hz stimulation. Note that in the presence of the NO donor, facial stimulation failed to induce MLI-PC LTD in control mice.

**Figure 7**
In the presence of the NO donor, SNAP (100 μM) and the CB1 receptor blocker, AM-251 (5 μM), facial stimulation induced MLI-PC LTP in control mice. (A) In the presence of SNAP and AM-251, representative traces showing air-puff stimulation (10 ms, 60 psi) evoked responses a cerebellar PC from a control mouse before and after delivering 1 Hz stimulation. **(B)** Summary of the normalized P1 amplitude after delivery of 1 Hz facial stimulation (arrow head) in the presence of SNAP and AM-251 in control mice (n = 6 mice). **(C)** Bar graph (n = 6 mice) showing the normalized amplitude of P1 before (Pre) and after (Post) delivery of 1 Hz stimulation. **(D)** Summary of data (n = 6 mice) showing the normalized pause of a simple spike firing before (Pre) and after (Post) delivery of 1 Hz stimulation. Note that in the presence of SNAP and AM-251, facial stimulation induced MLI-PC LTP in control mice.

**Figure 8**
Inhibition of NOS and CB1 receptor activity, facial stimulation induced neither LTD nor LTP at MLI-PC synapses in EtOH consumption mice. (A) In the presence of the NOS inhibitor and the CB1 receptor blocker, representative traces showing air-puff stimulation (10 ms, 60 psi)-evoked responses in a cerebellar PC of EtOH consumption mice before (left) and after delivering 1 Hz stimulation. **(B)** Summary of the normalized P1 amplitude after delivery of 1 Hz facial stimulation (arrow head) in EtOH consumption mice (n = 7 mice). **(C)** Bar graph (n = 7 mice) showing the normalized amplitude of P1 before and after delivery of 1 Hz stimulation in EtOH consumption mice. **(D)** Summary of data (n = 7 mice) showing the normalized pause of a simple spike firing before and after delivery of 1 Hz stimulation in EtOH consumption. Note that at the inhibition of NOS and CB1 receptor activity, facial stimulation failed to induce MLI-PC LTD/LTP in EtOH consumption mice.

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References

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1. Bing Y. H., Wu M. C., Chu C. P., Qiu D. L. (2015). Facial stimulation induces long-term depression at cerebellar molecular layer interneuron-Purkinje cell synapses in vivo in mice. Front. Cell. Neurosci. 9:214. 10.3389/fncel.2015.00214 - DOI - PMC - PubMed
1. Botta P., de Souza F. M., Sangrey T., De Schutter E., Valenzuela C. F. (2010). Alcohol excites cerebellar Golgi cells by inhibiting the Na+/K+ ATPase. Neuropsychopharmacology 35, 1984–1996. 10.1038/npp.2010.76 - DOI - PMC - PubMed

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[1] Anggono V., Huganir R. L. (2012). Regulation of AMPA receptor trafficking and synaptic plasticity. Curr. Opin. Neurobiol. 22, 461–469. 10.1016/j.conb.2011.12.006 - DOI - PMC - PubMed

[2] Anggono V., Huganir R. L. (2012). Regulation of AMPA receptor trafficking and synaptic plasticity. Curr. Opin. Neurobiol. 22, 461–469. 10.1016/j.conb.2011.12.006 - DOI - PMC - PubMed

[3] Beierlein M., Regehr W. G. (2006). Local interneurons regulate synaptic strength by retrograde release of endocannabinoids. J. Neurosci. 26, 9935–9943. 10.1523/JNEUROSCI.0958-06.2006 - DOI - PMC - PubMed

[4] Beierlein M., Regehr W. G. (2006). Local interneurons regulate synaptic strength by retrograde release of endocannabinoids. J. Neurosci. 26, 9935–9943. 10.1523/JNEUROSCI.0958-06.2006 - DOI - PMC - PubMed

[5] Belmeguenai A., Botta P., Weber J. T., Carta M., De Ruiter M., De Zeeuw C. I., et al. . (2008). Alcohol impairs long-term depression at the cerebellar parallel fiber-Purkinje cell synapse. J. Neurophysiol. 100, 3167–3174. 10.1152/jn.90384.2008 - DOI - PMC - PubMed

[6] Belmeguenai A., Botta P., Weber J. T., Carta M., De Ruiter M., De Zeeuw C. I., et al. . (2008). Alcohol impairs long-term depression at the cerebellar parallel fiber-Purkinje cell synapse. J. Neurophysiol. 100, 3167–3174. 10.1152/jn.90384.2008 - DOI - PMC - PubMed

[7] Bing Y. H., Wu M. C., Chu C. P., Qiu D. L. (2015). Facial stimulation induces long-term depression at cerebellar molecular layer interneuron-Purkinje cell synapses in vivo in mice. Front. Cell. Neurosci. 9:214. 10.3389/fncel.2015.00214 - DOI - PMC - PubMed

[8] Bing Y. H., Wu M. C., Chu C. P., Qiu D. L. (2015). Facial stimulation induces long-term depression at cerebellar molecular layer interneuron-Purkinje cell synapses in vivo in mice. Front. Cell. Neurosci. 9:214. 10.3389/fncel.2015.00214 - DOI - PMC - PubMed

[9] Botta P., de Souza F. M., Sangrey T., De Schutter E., Valenzuela C. F. (2010). Alcohol excites cerebellar Golgi cells by inhibiting the Na+/K+ ATPase. Neuropsychopharmacology 35, 1984–1996. 10.1038/npp.2010.76 - DOI - PMC - PubMed

[10] Botta P., de Souza F. M., Sangrey T., De Schutter E., Valenzuela C. F. (2010). Alcohol excites cerebellar Golgi cells by inhibiting the Na+/K+ ATPase. Neuropsychopharmacology 35, 1984–1996. 10.1038/npp.2010.76 - DOI - PMC - PubMed

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Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

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Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

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Abstract

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