Benzodiazepine exposure induces transcriptional down-regulation of GABAA receptor α1 subunit gene via L-type voltage-gated calcium channel activation in rat cerebrocortical neurons

Journal Pre-proof BENZODIAZEPINE EXPOSURE INDUCES TRANSCRIPTIONAL DOWN-REGULATION OF GABAA RECEPTOR ␣1 SUBUNIT GENE VIA L-TYPE VOLTAGE-GATED CALCIUM CHANNEL ACTIVATION IN RAT CEREBROCORTICAL NEURONS Mar´ıa Florencia Foitzick, Nelsy Beatriz Medina, Luc´ıa Candela Iglesias Garc´ıa, Mar´ıa Clara Gravielle

PII:

S0304-3940(20)30071-9

DOI:

https://doi.org/10.1016/j.neulet.2020.134801

Reference:

NSL 134801

To appear in:

Neuroscience Letters

Received Date:

1 October 2019

Revised Date:

24 January 2020

Accepted Date:

30 January 2020

Please cite this article as: Foitzick MF, Medina NB, Garc´ıa LCI, Gravielle MC, BENZODIAZEPINE EXPOSURE INDUCES TRANSCRIPTIONAL DOWN-REGULATION OF GABAA RECEPTOR ␣1 SUBUNIT GENE VIA L-TYPE VOLTAGE-GATED CALCIUM CHANNEL ACTIVATION IN RAT CEREBROCORTICAL NEURONS, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134801

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BENZODIAZEPINE EXPOSURE INDUCES TRANSCRIPTIONAL DOWNREGULATION OF GABAA RECEPTOR α1 SUBUNIT GENE VIA L-TYPE VOLTAGE-GATED CALCIUM CHANNEL ACTIVATION IN RAT CEREBROCORTICAL NEURONS

María Florencia Foitzicka*, Nelsy Beatriz Medinaa*, Lucía Candela Iglesias García a and

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María Clara Gravielle a *

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Co-first authors

Instituto de Investigaciones Farmacológicas (ININFA). Facultad de Farmacia y

Corresponding author:

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Bioquímica. Universidad de Buenos Aires. CONICET. Buenos Aires, Argentina

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María Clara Gravielle, PhD Instituto de Investigaciones Farmacológicas (ININFA) Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. CONICET. Junín 956, C1113AAD Buenos Aires Argentina Phone: +54-11-5287-4524/25 E-mail: [email protected]

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Highlights

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-Sustained diazepam exposure induced uncoupling of GABA/benzodiazepine sites -Diazepam produced transcriptional repression of GABAA receptor α1 subunit gene -Diazepam-induced uncoupling and α1 subunit down-regulation were blocked by nifedipine.

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Abstract

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GABAA receptors are targets of different pharmacologically relevant drugs, such as

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barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines

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potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1-3, 5), β2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous

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and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their

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pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA

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receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated

calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

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List of abbreviations γ-aminobutyric acid

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(GABA) (BDNF)

flunitrazepam

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ethylenediaminetetraacetic acid

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calcium/calmodulin-dependent protein kinase II

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L-type voltage-gated calcium channel

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brain-derived neurotrophic factor

(CaMKII) (EDTA) (FNZ) (L-VGCC) (NRO)

phenylmethylsulfonyl fluoride

(PMSF)

phosphate buffered saline

(PBS)

polymerase chain reaction

(PCR)

sodium dodecyl sulfate

(SDS)

tris buffered saline

(TBS)

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nuclear run-on

horseradish peroxidase

(HRP)

standard error of the mean

(SEM)

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Keywords: diazepam, GABA, tolerance, uncoupling

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1. Introduction

Synaptic GABAergic transmission regulates brain function by establishing an

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appropriate balance between excitation and inhibition of the neuronal circuits. Synaptic

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inhibition is controlled by homeostatic mechanisms that involve structural changes in GABAergic synapses [9]. For example, neuronal activity controls accumulation of GABAA receptors at synaptic sites by regulation of receptor ubiquitination and subsequent

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proteosomal degradation [40], receptor insertion [39], and diffusion properties of the receptor [30].

Persistent activation of GABAA receptors by positive allosteric modulators, such as

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benzodiazepines, barbiturates and ethanol, induces adaptive changes that result in tolerance [43]. In particular, the clinical use of benzodiazepines has been limited by the development of tolerance to most of their pharmacological actions [11, 12, 45]. The understanding of the molecular basis of benzodiazepine tolerance has been challenging due to the complexity of this regulatory process. For instance, tolerance to each pharmacological effect of benzodiazepines exhibits a different temporal course, suggesting the existence of several

mechanisms [6, 11, 12, 45]. Moreover, different reports suggest that tolerance to a specific pharmacological effect depends on the activation of GABAA receptors containing certain α subunit subtypes [5, 44-46]. These results may indicate the coexistence of multiple adaptive alterations depending on the brain area and the GABAA receptor subtype involved. Neuronal activity modulates the strength of GABAergic transmission by different

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mechanisms that involve the influx of calcium through L-type voltage-gated calcium channels (L-VGCC). For example, activation of L-VGCC alters the turnover of GABAA

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receptor subunits by regulating the activity of the proteosome [38]. The activation of L-

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VGCC also regulates the insertion of GABAA receptors into the plasma membrane via the phosphorylation of the receptor by CaMKII [39]. On the other hand, prolonged exposure to

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GABAA receptor allosteric modulators regulates L-VGCC. In particular, sustained benzodiazepine treatment potentiated calcium currents through L-VGCC in rat

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hippocampal CA1 neurons [51] and in mouse cultured cerebrocortical neurons [21]. Moreover, the reduction in the GABAA receptor-mediated currents in rat hippocampal CA1

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neurons induced by chronic treatment with benzodiazepines is inhibited by prior injection of an inhibitor of L-VGCC [52]. In addition, it was recently reported that benzodiazepineinduced disruption of GABAergic synapses is mediated by calcium mobilization from

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intracellular compartments [31]. Together, these results suggest that the tolerance mechanism involves an increase in cytosolic calcium. Numerous reports indicate that chronic benzodiazepine treatment induces uncoupling

of the GABA and benzodiazepine sites, suggesting changes in GABAA receptor function [6]. This uncoupling is accompanied by selective alterations in GABAA receptor subunit

transcripts in specific brain regions [11, 12, 45]. The mechanism of uncoupling is still unclear. To dissect the molecular mechanism underlying the tolerance process, we investigate here the effect of a 48-h exposure of rat primary neuronal cultures from the cerebral cortex to diazepam. We demonstrated that diazepam-induced uncoupling is prevented in the

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presence of flumazenil, a benzodiazepine site antagonist, or picrotoxin, a GABAA receptor channel blocker, suggesting that uncoupling occurs through a benzodiazepine binding site-

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dependent mechanism that involves receptor activation. In addition, uncoupling was

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blocked by nifedipine, an inhibitor of L-VGCC. Diazepam treatment also produced a selective decrease in the mRNA and protein levels of the GABAA receptor α1 subunit,

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which was dependent on L-VGCC activation. This decrease was due to transcriptional down-regulation of α1 subunit gene. Together, our results suggest that persistent exposure

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of cortical neurons to benzodiazepines produces adaptive changes in the GABAA receptors

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that are dependent on the activation of L-VGCC.

2. Materials and methods

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2.1. Primary cultures

Fifteen pregnant rats (Sprague-Dawley) were purchased from the Facultad de

Farmacia y Bioquímica, Universidad de Buenos Aires. All the experiments were carried out in accordance with the National and International Guidelines (National Institute of Health Guide for the Care and Use of Laboratory Animals) on the ethical use of animals. All efforts were made to minimize the number of animals used and their suffering. This study

was approved by the Animal Use and Care Committee from the Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires (Res. 4662). Primary cultures were prepared from 18-day-old rat embryos as previously described [13]. Briefly, whole brains were removed, and cerebral cortices were dissected under a microscope and placed in ice-cold Hanks´ solution. Tissue was minced with a small pair of scissors, triturated with a serological pipette, and centrifuged for 5 min at 500 g. The resulting pellet was resuspended

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in 5 mL of plating medium (NeurobasalTM medium plus 10 % fetal bovine serum, 100

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units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM glutamine, Invitrogen, Carlsbad,

CA, USA) and triturated again with a serological pipette. The cell suspension was added to

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a final volume of plating medium and plated at a density of ¾ cortices per 100 mm culture

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dish coated with poly-L-lysine (0.1 mg/mL, Sigma-Aldrich, St Louis, MO, USA). Cultures were incubated at 37˚C in 5 % CO2, and after 1 h, the medium was aspirated and replaced

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with serum-free medium containing B27 serum-free supplement (Invitrogen).

2.2. Drug treatment

Cultures, 2-3 x 100 mm dishes/treatment group, containing 10 ml of medium, were treated on day 7 by adding a small volume (100 μL) of a concentrated drug stock or

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vehicle. All the drugs were dissolved in Me2SO. The final concentrations were: 25 μM diazepam (a gift from Roemmers, Buenos Aires, Argentina), 50 μM flumazenil (SigmaAldrich), 100 μM picrotoxin (Sigma-Aldrich) or 10 μM nifedipine (Sigma-Aldrich). The final concentration of Me2SO in the medium was 0.2 %. Cells were collected after 48 h of incubation.

2.3. Binding assay Cultures were washed once with ice-cold phosphate buffered saline (PBS), scraped from culture dishes, and centrifuged at 500 g for 5 min. The pellet was homogenized in 1 mM EDTA/1 mM PMSF (1 mL per dish) with 12 strokes using a glass Dounce

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homogenizer and dialyzed against 4 X 4 L of potassium phosphate buffer (pH 7.4)

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overnight at 4C.

Aliquots of homogenates (75-100 g protein) were incubated at a final volume of

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0.5 mL for 60 min at 0 C with 0.5 nM [3H]flunitrazepam ([3H]FNZ, Perkin Elmer Life

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Sciences, Waltham, MA, USA) alone or in the presence of 1 mM GABA (Sigma-Aldrich). Nonspecific binding was determined in the presence of 100 M diazepam and subtracted

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from total binding to yield specific binding. The reaction was stopped by the addition of 5 mL ice-cold PBS, and the aliquots were immediately vacuum filtered through glass fiber

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filters (Whatman GF/B). Filters were washed three times with 5 mL ice-cold PBS. Radioactivity retained on the filters was quantified with liquid scintillation spectrometry. The enhancement of [3H]FNZ binding by GABA was estimated as percentage of

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potentiation as follows:

% potentiation = [(specific binding in presence of GABA/specific binding in

absence of GABA)-1] x 100. Changes in the enhancement of [3H]FNZ binding by GABA were expressed as percentage of coupling and defined as follows:

% coupling = (% potentiation treated/% potentiation control) X 100.

2.4. Real-time PCR Cultures were washed with ice-cold PBS and total RNA was extracted using the

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RNeasy midi kit (Qiagen, Hilden, Germany). The primers (Tecnolab, Buenos Aires, Argentina) and the probe (TaqMan, Applied Biosystems, Foster city, CA, USA) were

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designed using the Primer Express software (Applied Biosystems). The sequences of the subunit primers were as follows: α1, 5´-CCCCGGCTTGGCAACTAT-3´ and 5´-

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TGTCTCAGGCTTGACTTCTTTCG-3´; α2, 5´- GACTGGGAGACAGCATTACTGAAG-

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3´ and 5´- TCTGAGACAGGGCCAAAACTG-3´; α3, 5´-

CACCATGACCACCTTGAGTATCA-3´ and 5´- CCGTCGCGTATGCCACTT-3´; α5, 5´-

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CAACATCACAATATTCACCAGGATCT-3´ and 5´-CCCAGG CCGCAGTCTGT-3´; 2, 5´- CCCTGCCCCTACCATTGATA-3´ and 5´- GGTGGCATTGTTCATTTGGAT-3´. The

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sequences of subunit probes were as follows: α1, 5´-TAAAAGTGCGACCATAGAA-3´; α2, 5´- CTCCACCAACATCTATG-3´; α3, 5´-TGCCAGAAACTCTTTAC-3´; α5, 5´CTCTTGGATGGCTATGAC-3´; 2, 5´- CGTCCCAGATCAGCA-3´. The ribosomal RNA probe and primers were purchased from Applied Biosystems. Quantitative one-step real-

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time PCR was performed in an Applied Biosystems 7500 real time PCR system using an AgPath IDTM one-step RT PCR kit (Ambion, Austin, TX, USA). The standard curves for relative quantification were generated with 0.5 to 60 ng of total RNA isolated from control cultures (treated with vehicle). The PCR reactions were performed in triplicate in a total volume of 25 μL containing AgPath IDTM master mixture, 250 nM of the subunit probes,

900 nM of the subunit primers, 50 nM of the 18S rRNA probe, and 50 nM of the 18S rRNA primers. The reaction conditions were 45°C for 10 min and 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 45 s. The relative amount of the subunit mRNAs was normalized to the relative amount of the 18S rRNA (internal control).

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2.5. Western blot

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Cells were washed with ice-cold PBS, scraped from the dishes, and centrifuged at 500 g for 5 min. The pellet was homogenized with ice-cold radioimmunoprecipitation assay

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(RIPA) lysis buffer containing a protease inhibitor cocktail (Roche Diagnostics, RischRotkreuz, Switzerland) and incubated at 4˚C for 20 min with rotation. The lysates were

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centrifuged for 5 min at 2,000 g. Proteins from the supernatant (approximately 40 μg) were

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separated on 10 % acrylamide gels and transferred to a nitrocellulose membrane. The blots were blocked for 2 h with 5 % nonfat dry milk in 20 mM tris buffered saline (TBS) buffer containing 0.1 % Tween-20. Blots were incubated with a rabbit antibody against α1

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(1:1,000 dilution, Cat. # MABN489, Millipore, Burlington, MA, USA) overnight at 4˚C in blocking solution. The protein subunit was detected by incubation with a secondary horseradish peroxidase (HRP)-conjugated antibody (1:2,000 dilution, Santa Cruz

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Biotechnology, Dallas, TX, USA) for 1.5 h at room temperature followed by enhanced chemiluminescence (ECL detection kit, Pierce, Rockford, IL, USA). The blots were stripped and reprobed by incubation with an anti-β-actin antibody (1:4,000 dilution, Cat. # A5441, Sigma-Aldrich) overnight at 4°C, followed by incubation with a secondary HRPconjugated antibody (1:2,000, Santa Cruz Biotechnology) for 1.5 h at room temperature.

The subunit signal was normalized to that of the actin to control for loading amount variability. Densitometry was performed with the NIH Image J program.

2.6. Nuclear run-on assays

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Transcriptional activity was measured by a nuclear run-on (NRO) protocol based on bromouridine immunocapture and real-time PCR that enables to quantify nascent

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transcription [35]. Cells were washed with ice-cold PBS, scraped from the dishes and

counted using a hemocytometer in order to equalize the number of nuclei between samples

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(4 million nuclei per sample). Cells were lysed with Igepal buffer (10 mM Tris-HCl, pH

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7.4, 10 mM NaCl, 3 mM MgCl2 and 0.5 % Igepal) and nuclear fractions were isolated. Transcription reactions were performed in the presence of BrUTP (Sigma-Aldrich) for 30

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min at 30 °C. At the end of this incubation, nuclear RNA was extracted using the MEGAclear transcription clean-up kit (Life Technologies, Carlsbad, CA, USA). Genomic

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DNA contamination was removed using the TURBO DNA-free kit (Life Technologies). Immunoprecipitation of bromouridylated NRO-RNA was performed using protein G Dynabeads ( Life Technologies) and anti-BrdU monoclonal antibody (2 μg per sample, Santa Cruz Biotechnology). Extraction of NRO-RNAs was performed using TRIzol reagent

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(Life Technologies) and quantification was carried out by reverse-transcription quantitative real-time PCR.

2.7. Statistical analysis

The results are expressed as the means ± SEM derived from independent cultures. Differences among treatments were analyzed by one-sample Student´s t test or one-way ANOVA followed by Tukey´s post hoc test as appropriate.

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3. Results

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benzodiazepine sites depends on L-VGCC activation

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3.1. Diazepam-induced reduction in the allosteric interactions between GABA and

To investigate the effect of continuous administration of benzodiazepines on the

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coupling between GABA and benzodiazepine sites, we exposed rat cultured cortical

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neurons to diazepam (25 μM) for 48 h. FNZ binding experiments were performed to measure the potentiation of benzodiazepine binding by GABA (Fig. 1A). Diazepam

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treatment induced a significant decrease in the GABA-stimulated [3H]FNZ binding (40 % uncoupling, p<0.05). Diazepam-induced uncoupling was prevented in the presence of flumazenil, a benzodiazepine site antagonist, or picrotoxin, a GABAA receptor channel blocker. These results suggest that uncoupling is mediated by a mechanism that involves

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the binding of diazepam to the specific benzodiazepine site and activation of the GABAA receptor. Uncoupling was also blocked by nifedipine, indicating that this process depends on the activation of L-VGCCs (Fig. 1B).

3.2 Benzodiazepine treatment decreases GABAA receptor α1 subunit levels

Since benzodiazepine stimulation of GABA responses is influenced by the subtype of GABAA receptor α subunit, we studied the effect of sustained diazepam exposure on the mRNA levels of α1, α2, α3 and α5 subunits, which are the α subunits present in benzodiazepine-sensitive receptors. In addition, we investigated the effect of diazepam treatment on the mRNA levels of 2 subunits, a subunit that is required in all

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benzodiazepine-sensitive GABAA receptors (Fig. 2). Quantitative real-time PCR assays demonstrated that diazepam produced a significant decrease in the transcript levels of the

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GABAA receptor α1 subunit (49 %, p<0.05) without changes in the α2, α3, α5 or 2

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subunits. This decrease was prevented in the presence of nifedipine (Fig. 3A).

The results from western blot experiments showed that the diazepam-induced

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decrease in the mRNA levels of α1 subunit was associated with a corresponding reduction in the α1 peptide levels (40 %, p<0.05), which was also inhibited by nifepidine (Fig. 3B

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and C).

3.3 Transcriptional regulation of GABAA receptor α1 subunit gene In order to investigate whether diazepam-induced down-regulation of GABAA

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receptor α1 subunit was due to a transcriptional repression mechanism, we studied the effect of the benzodiazepine on the transcriptional activity of the α1 gene. To this end, we performed NRO experiments to quantify bromouridine labeled nascent RNA molecules derived from nuclear isolates [35]. Results from these experiments indicated that the decrease in α1 steady-state mRNA levels produced by the diazepam treatment was due to a

reduction in nascent transcription (Fig. 4). This effect was prevented by co-incubation with nifedipine.

4. Discussion This work demonstrates that sustained benzodiazepine treatment induces

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uncoupling of GABA/benzodiazepine sites and down-regulation of GABAA receptor α1

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subunit transcription through a signaling pathway that involves activation of L-VGCCs.

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Since GABAA receptors mediate the majority of pharmacological actions of benzodiazepines, they should play an important role mediating the adaptive mechanisms of

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tolerance. On the other hand, the glutamatergic hypothesis of benzodiazepine tolerance suggests that adaptive changes in the excitatory transmission occur to counteract the

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enhanced inhibition induced by benzodiazepines. The involvement of glutamatergic mechanisms in the development of benzodiazepine tolerance remains unclear. Prolonged

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treatments with benzodiazepines have been shown to regulate the expression of NMDA and AMPA receptors [23, 43]. AMPA-receptor GluR-A subunit-deficient mice exhibit reduced benzodiazepine tolerance [1]. NMDA antagonists prevent the development of tolerance to

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the sedative but not to the anxiolytic effects of benzodiazepines, suggesting different mechanisms [2]. Uncoupling of GABA/benzodiazepine site interactions induced by longterm exposure of neuronal cultures to GABA was not inhibited by co-incubation with a NMDA antagonist [28]. To elucidate the molecular mechanism of tolerance, we studied the regulation of GABAA receptors in rat cultured cortical neurons under sustained exposure to diazepam.

Our results from binding experiments (Fig. 1) showed that a 48-h treatment of the cortical neurons with diazepam produced a significant decrease in the coupling between GABA and benzodiazepine sites. This uncoupling was prevented in the presence of flumazenil or picrotoxin (Fig. 1A), suggesting that it depends on diazepam binding to the benzodiazepine site and on GABAA receptor activation.

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Uncoupling has been reported to occur as a consequence of benzodiazepine treatments in vivo, in primary neuronal cultures, and in cell lines expressing recombinant

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GABAA receptors. Different reports suggest that benzodiazepine-induced uncoupling of

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GABA/benzodiazepine site interactions, measured by binding assays, correlates with a functional uncoupling. For example, a sustained exposure of primary brain cultures to

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flurazepam resulted in a decrease in the degree of potentiation of GABA-activated membrane currents by flurazepam [33]. In addition, a long-term in vivo treatment with

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flurazepam produced a reduction in the ability of zolpidem to enhance the decay constant of miniature inhibitory currents in hippocampus [42]. Finally, results from chloride uptake

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experiments demonstrated that both in vivo [29] and in vitro [18] treatments with benzodiazepines attenuate the ability of benzodiazepines to enhance GABA-induced 36Clinflux.

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Several lines of evidence suggest a link between uncoupling and the development of tolerance. In fact, a relationship between the magnitude of anticonvulsant tolerance induced by long-term benzodiazepine and the efficacy of benzodiazepines for uncoupling induction has been observed [15]. Moreover, the degree of uncoupling was correlated with the benzodiazepine efficacies [15]. On the other hand, the reversion of benzodiazepine-induced

uncoupling exhibited a time-course similar to that of tolerance to the locomotor-impairment effect [37, 41]. Although some studies suggested that uncoupling was the consequence of prolonged in vivo benzodiazepine treatments [7, 41], one report [17] showed that uncoupling was induced by a single diazepam injection with a peak 4-12 h later. Acute tolerance has also

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been detected after a single dose of benzodiazepines [8, 25, 49], further supporting a relationship between uncoupling and the development of tolerance. The reported time-

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course of uncoupling produced by in vitro benzodiazepine exposure is variable.

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Benzodiazepine treatment of chick brain neuronal cultures resulted in uncoupling with a t1/2 of 18 h [36]. In cell lines expressing recombinant GABAA receptors, benzodiazepine

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exposure induced uncoupling with t1/2 of 32 min in Ltk- fibroblast cells [22] and 3 h in WSS-1 kidney cells [48]. The rapid onset of uncoupling may indicate that this regulatory

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process is the initial event of an adaptive mechanism that leads to tolerance. Results from some studies suggest that prolonged benzodiazepine treatments induce

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alterations in GABAA receptor α subunit composition at synapses. A report from Jacob et al. [20] demonstrated that a 24 h-flurazepam treatment of hippocampal cultures produced a change in the GABAA receptor subunit combination at the plasma membrane due to a

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selective increase in the degradation of α2-containing receptors after endocytosis. Results from a recent work [27] revealed that a 12-24 h treatment with diazepam caused a reduction in the confinement of α2/2 GABAA receptors at synapses. Our results indicated that a 48htreatment with diazepam produced a selective decrease in both transcript and protein levels of the GABAA receptor α1 subunit, the most abundant and ubiquitous α subunit subtype (Fig. 2 and 3). Benzodiazepine-induced down-regulation of α1 subunit has been

demonstrated in a number of reports [14, 19, 26, 32], however, in some studies no alteration [3, 16, 50] or an increase [7, 34] in this subunit was observed. These discrepancies are probably due to differences in the treatment paradigms. In order to investigate the mechanism of benzodiazepine-induced reduction in GABAA receptor α1 subunit levels, we tested the effect of diazepam on the transcriptional

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treatment induced a transcriptional repression of α1 subunit gene.

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activity of α1 gene. Results from NRO experiments (Fig. 4) demonstrated that diazepam

During development, the expression of the different α subunit subtypes in rat cerebral

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cortex considerably changes [10, 24]. Since all the α subunit subtypes present in the mature cerebral cortex (α1-5) are already expressed at embryonic day 18 [24], the developmental

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stage in which we prepared the primary cultures, it is unlikely that α subunit subtypes other

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than α1 are regulated by long-term treatments with diazepam at adulthood. We demonstrated here (Fig. 1B) that diazepam-induced uncoupling is prevented by

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co-incubation with nifedipine, a L-VGCC inhibitor. Diazepam-induced decreases in α1 mRNA and subunit levels were the result of transcriptional repression of α1 gene by a mechanism dependent on L-VGCC activation (Fig. 3 and 4).These results may indicate that sustained diazepam treatment stimulates the calcium influx through L-VGCC, which

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activates a signaling pathway that leads to uncoupling and transcriptional repression of α1 subunit gene. Calcium influx across the plasma membrane results in the activation of different signaling molecules, such as calcium-sensitive adenylatecyclase, calcium/calmodulin-activated kinases, and Ras. These molecules, in turn, activate signaling cascades, which amplify the calcium signal and transport it to the nucleus [47].

Transcription of the different genes that encode the GABAA receptor subunits probably involves a complex system of regulatory controls that are not yet identified. A recent report suggests that the decrease in GABAA receptor α1 subunit expression produced by sustained benzodiazepine treatment is mediated by an epigenetic mechanism involving histone deacetylation at the α1 promoter [4]. Further experiments will be necessary to elucidate the specific signaling pathway involved in the diazepam-induced transcriptional

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repression of α1 subunit gene.

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5. Conclusions

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Our study indicates that sustained benzodiazepine treatment produces uncoupling and transcriptional down-regulation of GABAA receptor α1 subunit via activation of L-VGCC.

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The relationship between uncoupling and the decrease in α1 is unknown. Uncoupling may be the result of a change in GABAA receptor subunit composition or, alternatively,

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uncoupling and α1 down-regulation may represent independent adaptive receptor alterations. The understanding of the molecular basis of benzodiazepine tolerance may facilitate the development of new drugs that can maintain their efficacies during long

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treatments.

Conflict of interest: none.

Acknowledgements:

This study was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (PIP11220130100266).

Author Contributions:

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MFF, NBM and LCGI performed the experiments. MCG designed the experiments and

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wrote the manuscript.

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Figure Captions

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Fig. 1. Uncoupling of GABA/benzodiazepine site interactions is produced by sustained exposure to diazepam. (A) Cortical neurons were incubated for 48 h with vehicle (control), 25 μM diazepam (DZ), 50 μM flumazenil (Flum), 25 μM diazepam plus 50 μM flumazenil (DZ + Flum), 100 μM picrotoxin (Picro) or 25 μM diazepam plus 100 μM picrotoxin (DZ + Picro). (B) Cortical neurons were incubated for 48 h with vehicle (control), 25 μM diazepam (DZ), 10 μM nifedipine (Nifed), 25 μM diazepam plus 10 μM nifedipine (DZ + Nifed). (A) and (B) Coupling between GABA and benzodiazepine binding sites was measured as the potentiation of [3H]flunitrazepam (FNZ, 0.5 nM) binding by GABA (1 mM). Data are expressed as percentages of control values (defined as 100 %) and represent the mean ± SEM of 3-4 independent cultures. Significant differences: *DZ versus control (p< 0.05, one-sample Student´s t test); #DZ versus DZ + Flum, DZ versus DZ + Picro, DZ versus DZ + Nifed (p< 0.05, one-way ANOVA and Tukey´s post hoc test).

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Fig. 2. Effect of prolonged treatment with diazepam on the mRNA levels of α1, α2, α3, α4, α5 and 2 GABAA receptor subunits. Cortical neurons were treated for 48 h with vehicle (control) or 25 μM diazepam (DZ). Quantitative real-time PCR experiments were performed using total RNA. The results are expressed as percentage of control (defined as

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100 %). Data represent the mean ± SEM of 4-7 independent cultures. * Significantly different from control (p< 0.05, one-sample Student´s t test).

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Fig. 3. Down-regulation of α1 GABAA receptor subunit induced by prolonged diazepam treatment is dependent on L-VGCC activation. Cortical neurons were exposed to vehicle (control), 25 μM diazepam (DZ), 10 μM nifedipine (Nifed), or 25 μM diazepam plus 10 μM nifedipine (DZ + Nifed) for 48 h. (A) Quantitative real-time PCR experiments were performed using total RNA. (B) Representative western blot of protein homogenates from cultures. (C) Bar graph of the densitometry analyses of α1 subunit level normalized to β-actin expression. The results are expressed as percentage of control (defined as 100 %). Data represent the mean ± SEM of 4-12 independent cultures. * Significantly different from control (p< 0.05, one-sample Student´s t test). #DZ versus DZ + Nifed (p< 0.05, one-way ANOVA and Tukey´s post hoc test).

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Fig. 4 Diazepam down-regulation of GABAA receptor α1 subunit at the transcriptional level. Cortical neurons were incubated for 48 h with vehicle (control), 25 μM diazepam (DZ), 10 μM nifedipine (Nifed), 25 μM diazepam plus 10 μM nifedipine (DZ + Nifed). Transcriptional activity was measured by NRO and real-time PCR assays. The results are expressed as percentage of control (defined as 100 %). Data represent the mean ± SEM of 4 independent cultures. * Significantly different from control (p< 0.05, onesample Student´s t test). #DZ versus DZ + Nifed ( p< 0.05, one-way ANOVA and Tukey´s post hoc test).

Credit author statement:

María F. Foitzick, Nelsy B. Medina and Lucía C. Iglesias García: performed experiments

María F. Foitzick, Nelsy B. Medina and María C. Gravielle: designed the research María C. Gravielle: wrote the manuscript

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[6] [7]

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T. Aitta-Aho, O.Y. Vekovischeva, P.J. Neuvonen, E.R. Korpi, Reduced benzodiazepine tolerance, but increased flumazenil-precipitated withdrawal in AMPA-receptor GluR-A subunit-deficient mice, Pharmacol Biochem Behav 92 (2009) 283-290. C. Allison, J.A. Pratt, Neuroadaptive processes in GABAergic and glutamatergic systems in benzodiazepine dependence, Pharmacol Ther 98 (2003) 171-195. M.I. Arnot, M. Davies, I.L. Martin, A.N. Bateson, GABA(A) receptor gene expression in rat cortex: differential effects of two chronic diazepam treatment regimes, J Neurosci Res 64 (2001) 617-625. J. Auta, E. Gatta, J.M. Davis, S.C. Pandey, A. Guidotti, Potential role for histone deacetylation in chronic diazepam-induced downregulation of alpha1-GABAA receptor subunit expression, Pharmacology research & perspectives 6 (2018) e00416. J. Auta, F. Impagnatiello, B. Kadriu, A. Guidotti, E. Costa, Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem, Neuropharmacology 55 (2008) 148-153. A.N. Bateson, Basic Pharmacologic Mechanisms Involved in Benzodiazepine Tolerance and Withdrawal, Curr Pharmacol Des 8 (2002) 5-21. M.C. Ferreri, M.L. Gutierrez, M.C. Gravielle, Tolerance to the sedative and anxiolytic effects of diazepam is associated with different alterations of GABAA receptors in rat cerebral cortex, Neuroscience 310 (2015) 152-162. S.E. File, L.J. Wilks, P.S. Mabbutt, Withdrawal, tolerance and sensitization after a single dose of lorazepam, Pharmacol Biochem Behav 31 (1988) 937-940. C.E. Flores, P. Mendez, Shaping inhibition: activity dependent structural plasticity of GABAergic synapses, Front Cell Neurosci 8 (2014) 327. J. Fritschy, J. Paysan, A. Enna, H. Mohler, Switch in the expression of rat GABAAreceptor subtypes during postnatal development: an immunohistochemical study, J. Neurosci. 14 (1994) 5302-5324. M.C. Gravielle, Activation-induced regulation of GABAA receptors: Is there a link with the molecular basis of benzodiazepine tolerance?, Pharmacol Res 109 (2016) 92-100.

ur na

[1]

of

References

[9]

[10]

[11]

[18]

[19]

[20]

[21]

[22]

[23]

Jo

[24]

of

[17]

ro

[16]

-p

[15]

re

[14]

lP

[13]

M.C. Gravielle, Regulation of GABAA receptors by prolonged exposure to endogenous and exogenous ligands, Neurochem Int 118 (2018) 96-104. M.C. Gravielle, R. Faris, S.J. Russek, D.H. Farb, GABA Induces Activity Dependent Delayed-onset Uncoupling of GABA/Benzodiazepine Site Interactions in Neocortical Neurons, J. Biol. Chem. 280 (2005) 20954-20960. C. Heninger, N. Saito, J.F. Tallman, K.M. Garrett, M.P. Vitek, R.S. Duman, D.W. Gallager, Effects of continuous diazepam administration on GABAA subunit mRNA in rat brain, Journal of molecular neuroscience : MN 2 (1990) 101-107. T.D. Hernandez, C. Heninger, M.A. Wilson, D.W. Gallager, Relationship of agonist efficacy to changes in GABA sensitivity and anticonvulsant tolerance following chronic benzodiazepine ligand exposure, Eur J Pharmacol 170 (1989) 145-155. R.A. Holt, A.N. Bateson, I.L. Martin, Chronic treatment with diazepam or abecarnil differently affects the expression of GABAA receptor subunit mRNAs in the rat cortex, Neuropharmacology 35 (1996) 1457-1463. R.A. Holt, A.N. Bateson, I.L. Martin, Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam, J Neurochem 72 (1999) 2219-2222. X.J. Hu, M.K. Ticku, Chronic benzodiazepine agonist treatment produces functional uncoupling of the gamma-aminobutyric acid-benzodiazepine receptor ionophore complex in cortical neurons, Mol Pharmacol 45 (1994) 618-625. F. Impagnatiello, C. Pesold, P. Longone, H. Caruncho, J.M. Fritschy, E. Costa, A. Guidotti, Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam, Mol Pharmacol 49 (1996) 822-831. T.C. Jacob, G. Michels, L. Silayeva, J. Haydon, F. Succol, S.J. Moss, Benzodiazepine treatment induces subtype-specific changes in GABAA receptor trafficking and decreases synaptic inhibition, Proc Natl Acad Sci U S A (2012). M. Katsura, M. Shibasaki, K. Kurokawa, A. Tsujimura, S. Ohkuma, Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4and 1,5-benzodiazepines in cerebrocortical neurons, J Neurochem 103 (2007) 25182528. R.L. Klein, P.J. Whiting, R.A. Harris, Benzodiazepine treatment causes uncoupling of recombinant GABAA receptors expressed in stably transfected cells, J Neurochem 63 (1994) 2349-2352. H. Kubová, Z. Bendová, S. Moravcová, D. Pačesová, L.L. Rocha, P. Mareš, Neonatal Clonazepam Administration Induces Long-Lasting Changes in Glutamate Receptors, Frontiers in Molecular Neuroscience 11 (2018). D. Laurie, W. Wisden, P. Seeburg, The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development, J. Neurosci. 12 (1992) 4151-4172. R.G. Lister, D.J. Nutt, Mice and rats are sensitized to the proconvulsant action of a benzodiazepine-receptor inverse agonist (FG 7142) following a single dose of lorazepam, Brain Res 379 (1986) 364-366. P. Longone, F. Impagnatiello, A. Guidotti, E. Costa, Reversible modification of GABAA receptor subunit mRNA expression during tolerance to diazepam-induced cognition dysfunction, Neuropharmacology 35 (1996) 1465-1473.

ur na

[12]

[25]

[26]

[32]

[33]

[34]

[35]

[36]

[37]

of

Jo

[38]

ro

[31]

-p

[30]

re

[29]

lP

[28]

J.M. Lorenz-Guertin, M.J. Bambino, S. Das, S.T. Weintraub, T.C. Jacob, Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking, Front Cell Neurosci 13 (2019) 163. H.R. Lyons, M.B. Land, T.T. Gibbs, D.H. Farb, Distinct signal transduction pathways for GABA-induced GABA(A) receptor down-regulation and uncoupling in neuronal culture: a role for voltage-gated calcium channels, J Neurochem 78 (2001) 1114-1126. R.J. Marley, D.W. Gallager, Chronic diazepam treatment produces regionally specific changes in GABA-stimulated chloride influx, Eur J Pharmacol 159 (1989) 217-223. J. Muir, J.T. Kittler, Plasticity of GABAA receptor diffusion dynamics at the axon initial segment, Front Cell Neurosci 8 (2014) 151. M.W. Nicholson, A. Sweeney, E. Pekle, S. Alam, A.B. Ali, M. Duchen, J.N. Jovanovic, Diazepam-induced loss of inhibitory synapses mediated by PLCdelta/ Ca(2+)/calcineurin signalling downstream of GABAA receptors, Molecular psychiatry 23 (2018) 1851-1867. C. Pesold, H.J. Caruncho, F. Impagnatiello, M.J. Berg, J.M. Fritschy, A. Guidotti, E. Costa, Tolerance to diazepam and changes in GABA(A) receptor subunit expression in rat neocortical areas, Neuroscience 79 (1997) 477-487. A. Prasad, J.N. Reynolds, Uncoupling of GABA-benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites, Brain Res 591 (1992) 327-331. J.A. Pratt, R.R. Brett, D.J. Laurie, Benzodiazepine Dependence: From Neural Circuits to Gene Expression, Pharmacology Biochemistry and Behavior 59 (1998) 925-934. T.C. Roberts, J.R. Hart, M.U. Kaikkonen, M.S. Weinberg, P.K. Vogt, K.V. Morris, Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR, Nat Protoc 10 (2015) 1198-1211. D. Roca, G. Schiller, L. Friedman, I. Rozenberg, T. Gibbs, D. Farb, gammaAminobutyric acid A receptor regulation in culture: altered allosteric interactions following prolonged exposure to benzodiazepines, barbiturates, and methylxanthines, Mol Pharmacol 37 (1990) 710-719. H.C. Rosenberg, T.H. Chiu, Tolerance during chronic benzodiazepine treatment associated with decreased receptor binding, Eur J Pharmacol 70 (1981) 453-460. R.S. Saliba, Z. Gu, Z. Yan, S.J. Moss, Blocking L-type voltage-gated Ca2+ channels with dihydropyridines reduces gamma-aminobutyric acid type A receptor expression and synaptic inhibition, J Biol Chem 284 (2009) 32544-32550. R.S. Saliba, K. Kretschmannova, S.J. Moss, Activity-dependent phosphorylation of GABA(A) receptors regulates receptor insertion and tonic current, Embo J 31 (2012) 2937-2951. R.S. Saliba, G. Michels, T.C. Jacob, M.N. Pangalos, S.J. Moss, Activity-dependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites, J Neurosci 27 (2007) 13341-13351. E.I. Tietz, T.H. Chiu, H.C. Rosenberg, Regional GABA/benzodiazepine receptor/chloride channel coupling after acute and chronic benzodiazepine treatment, Eur J Pharmacol 167 (1989) 57-65.

ur na

[27]

[39]

[40]

[41]

[48]

[49]

[50]

[51]

Jo

[52]

of

[47]

ro

[46]

-p

[45]

re

[44]

lP

[43]

E.I. Tietz, X.J. Zeng, S. Chen, S.M. Lilly, H.C. Rosenberg, P. Kometiani, Antagonist-induced reversal of functional and structural measures of hippocampal benzodiazepine tolerance, J Pharmacol Exp Ther 291 (1999) 932-942. M. Uusi-Oukari, E.R. Korpi, Regulation of GABAA receptor subunit expression by pharmacological agents, Pharmacol Rev 62 (2010) 97-135. C. van Rijnsoever, M. Tauber, M.K. Choulli, R. Keist, U. Rudolph, H. Mohler, J.M. Fritschy, F. Crestani, Requirement of alpha5-GABAA receptors for the development of tolerance to the sedative action of diazepam in mice, J Neurosci 24 (2004) 6785-6790. C.H. Vinkers, B. Olivier, Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABA(A) Receptor Modulators?, Advances in pharmacological sciences 2012 (2012) 416864. J. Vlainic, D. Pericic, Effects of acute and repeated zolpidem treatment on pentylenetetrazole-induced seizure threshold and on locomotor activity: comparison with diazepam, Neuropharmacology 56 (2009) 1124-1130. A.E. West, W.G. Chen, M.B. Dalva, R.E. Dolmetsch, J.M. Kornhauser, A.J. Shaywitz, M.A. Takasu, X. Tao, M.E. Greenberg, Calcium regulation of neuronal gene expression, Proc Natl Acad Sci U S A 98 (2001) 11024-11031. G. Wong, T. Lyon, P. Skolnick, Chronic exposure to benzodiazepine receptor ligands uncouples the gamma-aminobutyric acid type A receptor in WSS-1 cells, Mol Pharmacol 46 (1994) 1056-1062. P.T. Wong, Y.L. Yoong, M.C. Gwee, Acute tolerance to diazepam induced by benzodiazepines, Clinical and experimental pharmacology & physiology 13 (1986) 1-8. Y. Wu, H.C. Rosenberg, T.H. Chiu, T.J. Zhao, Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam, Journal of molecular neuroscience : MN 5 (1994) 105-120. K. Xiang, D.E. Earl, K.M. Davis, D.R. Giovannucci, L.J. Greenfield, Jr., E.I. Tietz, Chronic benzodiazepine administration potentiates high voltage-activated calcium currents in hippocampal CA1 neurons, J Pharmacol Exp Ther 327 (2008) 872-883. K. Xiang, E.I. Tietz, Chronic benzodiazepine-induced reduction in GABA(A) receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection, Neuroscience 157 (2008) 153-163.

ur na

[42]