SEARCH

SEARCH BY CITATION

Keywords:

  • benzodiazepine;
  • drug dependence;
  • KCC2;
  • morphine;
  • nucleus accumbens;
  • zolpidem

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K+-Cl co-transporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ-aminobutyric acid A-type receptor (GABAAR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP-1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ-PP-1-KCC2 pathway by chronic treatment with zolpidem.

Abbreviations used
GABAAR

γ-aminobutyric acid A-type receptor

HVA

homovanillic acid

KCC2

K+-Cl co-transporter 2

LFB

limbic forebrain

LMB

lower midbrain

MPE

maximum possible effect

PBS

phosphate buffer saline

PP-1

protein phosphatase-1

pPKCγ

phosphorylated protein kinase C γ

VTA

ventral tegmental area

Benzodiazepines are used as sedatives, sleeping aids, and anti-anxiety drugs. However, the long-term use of benzodiazepines induces psychological and physiological dependence. Psychological dependence is considered to accompany the neuroplastic changes in the dopaminergic system which projects from the ventral tegmental area to the nucleus accumbens, and activation of the mesolimbic dopaminergic system induces the rewarding effects and sensitization to locomotor activity induced by opioids and psychostimulants (Funada et al. 1993; Narita et al. 2003, 2004, 2006; Fukakusa et al. 2008). On the other hand, even though benzodiazepines induce psychological dependence without increasing the release of dopamine (Invernizzi et al. 1991), the mechanisms by which benzodiazepines induce psychological dependence are not fully understood.

γ-aminobutyric acid A-type receptor (GABAAR) consists of a five-subunit complex (2 α, 2 β, and 1 γ subunit), containing Cl channels, and the activation of GABAAR by GABA results in an increase in Cl influx. Benzodiazepines bind to the benzodiazepine receptor that is located on GABAAR between α and γ subunits. The activation of GABAAR containing α1 subunit is associated with sleeping, sedation, abuse potential, and anti-epilepsia. The binding of benzodiazepines to GABAAR induces anti-anxiety effects, which are mediated by the α2 and α3 subunits (Rudolph and Mohler 2006). The self-administration of midazolam could be suppressed by mutation of the GABAAR α1 subunit (Tan et al. 2010). Furthermore, the GABAAR α1 subunit agonist zolpidem induces physical and psychological dependence in humans (Pitchot and Ansseau 2009). It is also reported that subunit selective benzodiazepines sparing α1 induce the reduction in addiction liability (Tan et al. 2010). Moreover, stimulation of GABAAR α1 receptors is sufficient, but not necessary, for mediation on addiction of these drugs (Rowlett et al. 2005). Thus, these reports indicate that GABAAR α1 plays a key role in the dependence on benzodiazepines.

Neural response is regulated by neurotransmitters such as noradrenaline, serotonin, dopamine, glutamate, and GABA. As noted above, GABA has inhibitory effects by modulating Cl. Transmembrane Cl gradient is maintained primarily by cation-chloride co-transporters such as Na+-K+-2Cl co-transporter 1 (NKCC1) and K+-Cl co-transporter 2 (KCC2) (Price et al. 2009). Cl homeostasis in neurons is modulated by KCC2, which can regulate Cl efflux to equilibrate the membrane potential in mature neurons, and NKCC1, which can regulate Cl influx (Ben-Ari 2002). Since changes in the expression and/or function of these co-transporters are an important for pathophysiological mechanism response, chronic treatment with benzodiazepines causes the influence on Cl homeostasis by NKCC1 and KCC2. Thus, it is possible that increase in Cl influx via GABAAR by benzodiazepine affects the cascade of Cl efflux by KCC2 as Cl homeostasis. Especially, The membrane stability and activity of KCC2 are believed to be regulated by the phosphorylation of serine 940 within the C-terminal of KCC2, which is modulated by PKC (a serine/threonine kinase) and protein phosphatase-1 (PP-1) (a serine/threonine phosphatase) (Lee et al. 2007, 2011). Furthermore, it has been reported that PP-1 is downstream of PKC transduction (Eto et al. 2004). Therefore, neural activity is regulated by PP-1 as well as PKC.

The reinforcing/rewarding effects of opioids are mediated by activation of the mesolimbic dopaminergic system by the inhibition of GABAnergic neurons in the ventral tegmental area (Johnson and North 1992). It is reported that GABAA receptors might be involved in the acquisition of morphine-induced sensitization (Zarrindast et al. 2007). Moreover, our previous study has indicated that GABAergic receptors may be involved in morphine-induced locomotor sensitization (Narita et al. 2003). The GABAAR-dependent reward mechanism is sensitive to drug dependence through dopamine-independent system in opiate-naïve animals and a dopamine-dependent system in morphine-dependent or morphine-withdrawn animals (Laviolette et al. 2004). Furthermore, opioids and benzodiazepines have different effects on behavior (e.g., hyperlocomotion vs. hypolocomotion), and are co-abuse in humans (Browne et al. 1998; Rooney et al. 1999). However, it is not yet clear how benzodiazepines influence the behavioral effects of opioids. Therefore, this study was designed to investigate the interaction between zolpidem and opioid by measuring the effects of chronic treatment with zolpidem on morphine-induced hyperlocomotion. In this study, we confirmed that GABAAR α1 subunit agonist zolpidem enhanced morphine-induced hyperlocomotion mediated by changes in KCC2 in the nucleus accumbens.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals

This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University. Experiments were performed using male ICR mice (Tokyo Animal Science Laboratories, Tokyo, Japan), weighing 20–22 g at the beginning of experiments, which were housed in groups of six in a temperature (23 ± 1°C)- and humidity (55%)-controlled room. They were maintained on a 12-h light–dark cycle with laboratory mouse food and water available ad libitum.

Locomotor activity

The locomotor activity of mice was measured by an ambulometer (ANB-M20, O'Hara, Co., Ltd, Tokyo, Japan) as described previously (Narita et al. 1993). Briefly, a male ICR mouse was placed in a tilting-type round activity cage of 20 cm in diameter and 19 cm in height. Any slight tilt of the activity cage caused by horizontal movement of the animal was detected by microswitches. Total activity counts in each 10-min segment were automatically recorded for 30 min prior injection and for 180 min after morphine administration. Zolpidem (30 mg/kg, i.p.) or diazepam (10 mg/kg, i.p.) was administered once a day for 7 days. To confirm the acute effects of zolpidem, it was administered 1 h or 24 h before treatment with morphine treatment. Some animals were pre-treated with furosemide 10 min before chronic treatment with zolpidem for 7 days.

In vivo microdialysis study and quantification of dopamine and its metabolites

Before implantation of a cannula, all of the mice were anesthetized with 3% isoflurane for surgery. Briefly, the anesthetized animal was placed in a stereotaxic apparatus, the skull was exposed, and a small hole was made using a dental drill. A guide cannula (AG-6; Eicom, Kyoto, Japan) was implanted into the NAc (from bregma: anterior, +1.5 mm; lateral, −0.9 mm; ventral, −4.9 mm) according to the atlas of Paxinos and Watson and fixed to the skull with cranioplastic cement. At 24 h after surgery, microdialysis probes (A-I-6–02; 1 mm membrane length; Eicom) were slowly inserted into the NAc through guide cannulas under anesthesia with diethyl ether, and the mice were placed in experimental cages (30 cm wide × 30 cm deep × 30 cm high). The probes were perfused continuously (2 μL/min) with artificial cerebrospinal fluid: 0.9 mM MgCl2, 147.0 mM NaCl, 4.0 mM KCl, and 1.2 mM CaCl2. Outflow fractions were collected every 20 min. After three baseline fractions were collected from the NAc, mice were given morphine (10 mg/kg, s.c.). Dialysis samples were collected for 180 min after treatment and analyzed by high-performance liquid chromatography (HPLC; Eicom) with electrochemical detection (ECD; Eicom). Dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were separated by column chromatography and identified and quantified by the use of standards.

Neuropathic pain model

Mice were anesthetized with 3% isoflurane. We proposed a partial sciatic nerve injury by tying a tight ligature with 8-0 silk suture around approximately one third to half the diameter of the sciatic nerve located on the right side under light scope (SD30; Olympus, Tokyo, Japan) as described previously (Seltzer et al. 1990; Malmberg and Basbaum 1998). In sham-operated mice, the nerve was exposed without ligation.

Assessment of antinociception

The morphine-induced antinociceptive response was evaluated by recording the latency to paw licking or tapping in the hot-plate test (55 ± 0.5°C, Muromachi Kikai Co., Ltd., Tokyo, Japan). The hot-plate latencies were measured 30, 60, 90 min after saline or morphine (10 mg/kg, s.c.) injection. Zolpidem (30 mg/kg, i.p.) was administered once a day for 7 days before the hot-plate test. Antinociception was calculated as percentage of the maximum possible effect (% MPE) according to the following formula: % MPE = (test latency − pre-drug latency)/(cutoff time − pre-drug latency) × 100. The cut-off time that was set at 30 s for the hot-plate test or 10 s for the tail-flick test to prevent tissue damage. Antinociceptive response is expressed by as the mean with SEM of % MPE.

Measurement of thermal hyperalgesia

To assess the sensitivity to thermal stimulation, the right plantar surface of mice was tested individually using a well-focused, radiant heat light source (model 33 Analgesia Meter; IITC Inc./Life Science Instruments, Woodland Hills, CA, USA). The thermal hyperalgesia was measured 30 min after morphine (5 mg/kg, s.c.) injection after 7 days from neuropathic pain model was operated. Zolpidem (30 mg/kg, i.p.) was administered once a day for 7 days before the thermal hyperalgesia test. The intensity of the thermal stimulus was adjusted to achieve an average baseline paw withdrawal latency of approximately 8–10 s in naive mice. The paw withdrawal latency was determined as the average of three measurements per paw. Only quick hind paw movements (with or without licking of hind paws) away from the stimulus were considered to be a withdrawal response. Paw movements associated with locomotion or weight shifting were not counted as a response. Before testing of the behavioral responses to the thermal stimulus, mice were habituated for at least 30 min in a clear acrylic cylinder (15 cm high and 8 cm in diameter). Under these conditions, the latency of paw withdrawal in response to the thermal stimulus was tested. The data represent the average value for the paw withdrawal latency of the right hind paws.

Immunohistochemistry

Male ICR mice were deeply anesthetized by pentobarbital (70 mg/kg i.p.) and intracardially perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). After perfusion, the brains were quickly removed and thick coronal sections of the nucleus accumbens were rapidly dissected and stored in paraformaldehyde at 4°C overnight. They were then washed with 0.1 M PB. Transverse sections 50 μm thick were cut using a microtome (Microslicer DTK-1000; Dousaka EM, Kyoto, Japan). The sections were blocked in 5% normal donkey serum in 0.01 M phosphate buffer saline (PBS) for 1 h at 20°C. The primary antibodies were diluted in 0.01 M PBS containing 5% normal donkey serum [1 : 750 KCC2 (Millipore, Milano, Italy) and 1 : 60 dopamine and cAMP-regulated neuronal phosphoprotein 32 (DARPP32) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)]. The samples were then rinsed and incubated with the appropriate secondary antibody conjugated with Alexa 488 and Alexa 546 (Molecular Probes, Eugene, OR, USA) for 2 h at 20°C. The slides were then coverslipped with ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (Molecular Probes). The fluorescence of immunolabeling was detected and photographed with a microscopic system (BZ-9000; Keyence, Osaka, Japan).

Co-immunoprecipitation

Tissues from the limbic forebrain were homogenized on ice with lysis buffer (10 mM Tris-HCl pH-7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 0.5% Triton X-100) containing a phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolic, IN, USA). The homogenates were centrifuged at 1000 g for 10 min and the supernatant was used. The homogenates were subjected to immunoprecipitation for 1.5 h [1 : 75 KCC2 (Millipore) and 1 : 60 PP-1 (Santa Cruz Biotechnology, Inc.)], followed by rotation at 4°C for 1 h with 15 μL dynabeads protein A (Molecular Probes). The beads were washed three times with lysis buffer. Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting.

Western blotting

Tissues from the limbic forebrain including N.Ac., frontal cortex including cingulate cortex, and lower midbrain including ventral tegmental area (VTA) and substantia nigra (SN) were retrieved from animals, and fractions containing membranes and cytosol including microsomes were prepared by centrifugation with lysis buffer (10 mM Tris-HCl pH-7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 0.5% Triton X-100) containing phosphatase inhibitor cocktail (Roche Diagnostics). The homogenate was centrifuged at 1000 g for 10 min, and the supernatant was used. The supernatant was centrifuged at 20 000 g for 35 min and membranes or cytosol containing microsome allowed to stand. Protein concentration was quantified by the Lowry protein assay (Bio-Rad Laboratories, GmbH, Munich, Germany). Briefly, equal amounts of protein concentration (12 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) and then transferred to polyvinylidene difluoride membranes (ATTO, Tokyo, Japan). After samples were blocked with Tris-buffered saline (TBS) containing 5% milk 0.1% Tween for 1 h at 20°C, the primary antibodies were diluted in TBS containing 5% milk 0.1% Tween [1 : 10 000 KCC2 (Millipore), 1 : 1000 NKCC1 (Millipore), 1 : 3000 PP-1 (Santa Cruz Biotechnology, Inc.) and 1 : 5000 phosphorylated protein kinase C γ (pPKCγ) (Cell Signaling Technology, Beverly, MA, USA)] at 4°C overnight. The membrane was washed three times in TBS containing 0.05% Tween. The secondary antibodies were diluted in TBS containing 5% milk 0.1% Tween horseradish peroxidase (HRP) conjugate [1 : 10 000 Goat anti-Mouse IgG (Southern Biotechnology Associates, Birmingham, AL, USA) or 1 : 10 000 Goat anti-Rabbit IgG (Southern Biotechnology Associates)] and washed for three times. Proteins were visualized by an enhanced chemiluminescence detection system (PIERCE, Thermo Fisher Scientific Inc., Rockford, IL, USA). Equal loading was confirmed by the use internal control [1 : 500 β-tubulin (Santa Cruz Biotechnology, Inc.)]. Western blot results were analyzed by a FluorChem3 system (Laboratory & Medical Supplies, Tokyo, Japan). All results were normalized by β-tubulin.

Drugs

The drugs used in this study were zolpidem (Myslee; Astellas Pharma Inc., Tokyo, Japan), morphine hydrochloride (Daiichi-Sankyo, Co. Tokyo, Japan), furosemide (Sigma-Aldrich, Co., St. Louis, MO, USA), and diazepam (Wako Pure Chemical Industries, Ltd, Tokyo, Japan).

Statistical analysis

Comparisons of data were analyzed by one-way or two-way anova with Bonferroni's multiple comparison test, except for those in Fig. 1d and Figure S2 which were analyzed by Wilcoxon matched-pairs signed rank test. Values are expressed as the mean ± SEM. Statistical significance was set at p < 0.05 and p < 0.01. All statistical analyses were performed with Prism version 5.0a (GraphPad Software, Inc., La Jolla, CA, USA).

image

Figure 1. Effects of zolpidem (30 mg/kg, i.p.) and diazepam (10 mg/kg, i.p.) on morphine-induced hyperlocomotion. Zolpidem was administered as pre-treatment 1 h (a) or 24 h (b) or chronically for 7 days (c) before morphine. Effects of chronic treatment with diazepam on morphine-induced hyperlocomotion (d) *p < 0.05 versus saline-treated mouse.

Download figure to PowerPoint

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chronic treatment with zolpidem enhances morphine-induced hyperlocomotion

To confirm the effects of chronic treatment with zolpidem in the mesolimbic system, we examined morphine-induced hyperlocomotion after chronic treatment with diazepam (10 mg/kg, i.p.) or zolpidem (30 mg/kg, i.p.) for 7 days. Chronic treatment with zolpidem significantly enhanced morphine-induced hyperlocomotion in a dose-dependent manner (F(1,56) = 5.100, p < 0.05, Fig. 1c). In contrast, a single treatment with zolpidem (1 h or 24 h pre-treatment) had no effect on morphine-induced hyperlocomotion (Fig. 1a and b). Furthermore, diazepam (a GABAAR α subunit agonist) also significantly enhanced morphine-induced hyperlocomotion (p < 0.05, Fig. 1d).

However, administration of morphine after chronic treatment with zolpidem did not produce any change in increase in dopamine or its metabolites in the nucleus accumbens (NAc) (Fig. 2).

image

Figure 2. Effects of chronic treatment with zolpidem (30 mg/kg, i.p.) (Zol: open) or saline (Sal: closed) on the levels of extracellular dopamine (DA) (a) and its metabolites, such as 3,4-dihydroxyphenylacetic acid (DOPAC) (b) and homovanillic acid (HVA) (c), in the nucleus accumbens. Animals received morphine (10 mg/kg, s.c.) (Mor: square) or saline (Sal: circle) at 0 min. Levels of DA and its metabolites are given as the mean with SEM of the percent changes from the basal levels.

Download figure to PowerPoint

Interaction of KCC2 with PP-1 through pPKCγ

As shown in Fig. 3, we investigated whether pPKCγ or PP-1 is associated with KCC2 by a co-immunoprecipitation experiment. In this study, PP-1, but not pPKCγ, could be co-immunoprecipitated with KCC2 from samples of the limbic forebrain (LFB) (Fig. 3a, lower, middle). Furthermore, pPKCγ and KCC2 are co-immunoprecipitated with PP-1 (Fig. 3b, upper, middle). In this study, we did not examine the reversed co-immunoprecipitated with pPKCγ, since the pPKCγ antibody used in this study did not work well for co-immunoprecipitation.

image

Figure 3. Protein–protein interactions between K+-Cl co-transporter 2 (KCC2) and KCC2 modulators. Limbic forebrain lysate from mice was immunoprecipitated with KCC2 (a) or protein phosphatase-1 (PP-1) (b) antibodies followed by western blot analysis using KCC2, phosphorylated protein kinase C γ (pPKCγ) or PP-1 antibodies.

Download figure to PowerPoint

Influence of chronic treatment with zolpidem on the levels of pPKCγ and PP-1 in the mouse limbic forebrain

We next investigated the effects of treatment with zolpidem (1 h and 24 h pre-treated and for 7 days) on the changes in the protein levels of pPKCγ and its downstream PP-1 in the LFB including the NAc. pPKCγ levels were significantly increased whereas PP-1 levels were decreased by treatment with zolpidem for 7 days. However, pre-treatment with zolpidem (1 h and 24 h) had no effect on the levels of pPKCγ (F(3,20) = 3.999, p < 0.05, Fig. 4) and PP-1 (F(3,12) = 5.167, p < 0.05, Fig. 4) levels.

image

Figure 4. Western blot analysis of phosphorylated protein kinase C γ (pPKCγ) and protein phosphatase-1 (PP-1) in limbic forebrain. Zolpidem (30 mg/kg, i.p.) was administered as pre-treatment 1 h or 24 h or chronically for 7 days before sample preparation. All data were normalized by β-tubulin. *p < 0.05 versus saline-treated mice.

Download figure to PowerPoint

Expression and changes in KCC2 in DARPP32-positive neurons

To determine the localization of KCC2 and GABAAR α1 subunit, we examined the immuno-staining of KCC2 or GABAAR α1 subunit with DARPP32 as a maker of medium spiny neurons (MSN) in the NAc. Our results confirmed that KCC2 was localized at MSN in the NAc and frontal cortex, which is projected of dopaminergic neuron (Fig. 5 and Figure S2). Furthermore, GABAAR α1 subunit was also localized at MSN in the NAc (Fig. 5). KCC2 was detected in the VTA and SN, whereas DARPP32 positive cells were not detected. Moreover, the protein level of KCC2 in the LFB (F(3,24) = 6.268, p < 0.01, Figure S2) and frontal cortex (FC; p < 0.05, Fig. 5i, Figure S2) was clearly increased after treatment with zolpidem for 7 days, whereas NKCC1 level was not changed in the LFB (Figure S1). On the other hand, KCC2 protein level was not changed in lower midbrain (LMB) including VTA and SN (Figure S2) by zolpidem.

image

Figure 5. γ-aminobutyric acid A-type receptor (GABAAR) α1 subunit (a) and K+-Cl co-transporter 2 (KCC2) (e) expression on DARPP32 (b and f)-positive neuron in the NAc. GABAA α1 subunit and KCC2 exists in DARPP32-positive neuron (c and g). Scale bar 50 μm. Western blot analysis of KCC2 in the limbic forebrain (i). Zolpidem (30 mg/kg, i.p.) was administered as pre-treatment 1 h or 24 h or chronically for 7 days before sample preparation. All data were normalized by β-tubulin. **p < 0.01 versus saline-treated mice.

Download figure to PowerPoint

Effects of furosemide, a KCC2 inhibitor, on zolpidem-induced sensitization to morphine-induced hyperlocomotion

To determine the direct involvement of KCC2 in the zolpidem-induced enhancement of the hyperlocomotion induced by morphine, we finally examined the effects of the KCC2 inhibitor furosemide on morphine-induced hyperlocomotion after chronic treatment with zolpidem. The enhancement of morphine-induced hyperlocomotion by chronic treatment with zolpidem is significantly suppressed by pre-treatment with furosemide (30 nmol/mice i.c.v.) for 7 days (F(3,87) = 5.287, p < 0.05, Fig. 6).

image

Figure 6. Effect of furosemide (30 nmol/mouse, i.c.v.) the enhancement of morphine (10 mg/kg, s.c.)-induced hyperlocomotion by zolpidem (30 mg/kg, i.p.). *p < 0.05 versus vehicle-saline-treated mouse, #< 0.05 versus vehicle-zolpidem-treated mice.

Download figure to PowerPoint

Effects of chronic treatment with zolpidem on morphine-induced antinociception

We examined the effect of chronic treatment with zolpidem on morphine-induced antinociception using the hot-plate test. Morphine-induced antinociception was not affected by chronic treatment with zolpidem and diazepam (Fig. 7a). Furthermore, we investigated the effect of chronic treatment with zolpidem on antinociception induced by morphine under partial nerve ligation of the sciatic nerve. Partial nerve ligation of the sciatic nerve caused a significant decrease in the latency of paw withdrawal after the thermal stimulus only on the ipsilateral side of mice (vs. sham group, F(7,32) = 16.32, p < 0.001). Whereas morphine reversed these decrease in the latency of paw withdrawal, chronic treatment with zolpidem were not affected these effect of morphine (Fig. 7b).

image

Figure 7. Effects of chronic treatment with zolpidem (30 mg/kg i.p.) on morphine-induced antinociception. (a) Antinociceptive effect of morphine (10 mg/kg, s.c.) by hot-plate test. Antinociception was expressed as% of maximum possible effect (MPE). Each data represent the mean ± SEM. (b) Effect of antinociception by morphine (5 mg/kg, s.c.) following chronic treatment with zolpidem on the thermal hyperalgesia in sham-operated or nerve-ligated mice. Each column represents the mean ± SEM. *p < 0.005 versus ligation–saline group **p < 0.001 versus sham–saline group.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study demonstrated that chronic treatment with zolpidem enhanced mesolimbic dopaminergic activity through the up-regulation of post-synaptic KCC2 in the NAc. It has been reported that the mesolimbic dopaminergic system is important for morphine-induced hyperlocomotion (Mori et al. 2004). And that systemic administration of alprazolam promotes the heroin-induced place preference (Walker and Ettenberg 2005). Furthermore, the co-abuse of opioids and benzodiazepines is widespread in several countries (Iguchi et al. 1993; Darke et al. 1995; Segura et al. 2001). However, diazepam can attenuate the release of dopamine and induce sedation (Invernizzi et al. 1991; Giorgetti et al. 1998), whereas morphine and amphetamine induce hyperlocomotion and a place preference (Leri and Franklin 2000; Panhelainen et al. 2011). In contrast, it has been reported that stimulation of the GABAAR α1 subunit by zolpidem has the discriminative effects, which are different from those induced by stimulation of the GABAAR α2, α3 subunits (Vinkers et al. 2011). Furthermore, the GABAAR α1 subunit exists on MSN in the NAc (Fig. 5). Thus, it is likely that the modulation of dopamine sensitivity is enhanced by stimulation of the GABAAR α1 subunit on MSN in the NAc by chronic treatment with zolpidem.

Previous reports suggested that withdrawal after chronic treatment with diazepam increased [3H]MK801 binding and NR1 and NR2B NMDA receptor subunit protein in rat cerebrocortical tissue (Tsuda et al. 1998a, b). Moreover, the GABAAR α1 subunit-preferring benzodiazepine-site ligand zolpidem also produced an increase in the AMPA/NMDA ratio in VTA dopamine neurons. These findings suggest a role of mesolimbic dopamine system in the initial actions of and on neuronal adaptation to benzodiazepines (Heikkinen et al. 2009). In previous study, morphine-induced hyperlocomotion and place preference were accompanied by up-regulation of PKC (Narita et al. 2001, 2002). Furthermore, benzodiazepines can increase Ca2+ currents (Xiang et al. 2008), and Ca2+ signaling is well known to regulate PKC activity (Morgan et al. 1998). These previous studies may support our present finding that chronic treatment with zolpidem induced the up-regulation of pPKCγ. In addition, it has been reported that PP-1 is a downstream of PKC (Eto et al. 2004), and that the regulation of PP-1 modulates membrane stability of KCC2 (Lee et al. 2007, 2011). Furthermore, co-immunoprecipitation studies suggested that PP-1 directly associated with pPKCγ and KCC2, but did not associate proteins between pPKCγ and KCC2 (Fig. 3). These reports raise the possibility that chronic treatment with zolpidem-induced Ca2+ signaling through the NMDA may up-regulate the KCC2 levels in the membrane to down-regulate PP-1 via the up-regulation of pPKCγ.

Our results implicated that changes in PKC/PP-1 response by chronic treatment with zolpidem caused the reduction in down-regulation of KCC2. On the other hand, it is reported that alteration of intracellular concentration of chloride ion affects the transcriptional factor and causes neuronal plasticity (Yang et al. 2012). Decrease in intracellular chloride concentration promotes the inflammation of endothelial cells by activation of nuclear factor-κB pathway. Especially, it is implicated that with-no-lysine kinase 3 (WNK3) is a putative chloride-sensing kinase and WNK3 regulates the neuronal splicing factor Fox-1 (Lee et al. 2012).

Fox-1 splices mRNAs encoding proteins are important in the synaptic transmission and membrane excitation. Thus, alteration of intracellular concentration of chloride ion by chronic treatment with zolpidem may cause the up-regulation of KCC2 and sensitization to morphine-induced hyperlocomotion. Furthermore, previous studies have indicated that the modulation of KCC2 may regulate a neuroplastic changes (Rivera et al. 2004; Liu et al. 2009) and suppress of excitatory synapses mediated by the activation of AMPA receptor (Gauvain et al. 2011). In addition, KCC2 can regulate Cl efflux to modulate the Cl equilibrium of the membrane potential in mature neurons (Ben-Ari 2002). Therefore, our present results suggest that chronic treatment with zolpidem can promote morphine-induced hyperlocomotion mediated by neuroplastic changes in KCC2 on MSN in the NAc. Furthermore, we also demonstrated that furosemide, a KCC2 inhibitor, suppressed the enhancing effects of chronic treatment with zolpidem on morphine-induced hyperlocomotion. Our findings suggest that chronic treatment with zolpidem may modulate KCC2, and these changes may enhance the response of mesolimbic dopaminergic system.

Morphine is a prototypical μ-opioid receptor agonist that serves as the standard analgesic for severe pain. In clinical fields, it is reported that the patients with a history of alcohol dependence were easy to be addicted to opioids even under pain control (Ballantyne and LaForge 2007; Hojsted and Sjogren 2007). Moreover, GABAA α1 subunit is related to ethanol-potentiating effects (Rudolph et al. 1999). Our recent study suggested that chronic treatment with ethanol enhanced the rewarding effects of morphine (Shibasaki et al. 2013). Thus, these changes by chronic treatment with zolpidem may influence the vulnerability to opioid abuse in chronic pain control.

In conclusion, this study suggested that chronic treatment with zolpidem enhanced the activation of the mesolimbic dopaminergic system accompanied by changes in KCC2 alteration via a pPKCγ-PP-1-KCC2 pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

All authors declare that they have no conflicts of interest in this study.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Ballantyne J. C. and LaForge K. S. (2007) Opioid dependence and addiction during opioid treatment of chronic pain. Pain 129, 235255.
  • Ben-Ari Y. (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat. Rev. Neurosci. 3, 728739.
  • Browne R., Sloan D., Fahy S., Keating S., Moran C. and O'Connor J. (1998) Detection of benzodiazepine abuse in opiate addicts. Ir. Med. J. 91, 1819.
  • Darke S. G., Ross J. E. and Hall W. D. (1995) Benzodiazepine use among injecting heroin users. Med. J. Aust. 162, 645647.
  • Eto M., Kitazawa T. and Brautigan D. L. (2004) Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Proc. Natl Acad. Sci. USA 101, 88888893.
  • Fukakusa A., Nagai T., Mizoguchi H. et al. (2008) Role of tissue plasminogen activator in the sensitization of methamphetamine-induced dopamine release in the nucleus accumbens. J. Neurochem. 105, 436444.
  • Funada M., Suzuki T., Narita M., Misawa M. and Nagase H. (1993) Modification of morphine-induced locomotor activity by pertussis toxin: biochemical and behavioral studies in mice. Brain Res. 619, 163172.
  • Gauvain G., Chamma I., Chevy Q., Cabezas C., Irinopoulou T., Bodrug N., Carnaud M., Levi S. and Poncer J. C. (2011) The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines. Proc. Natl Acad. Sci. USA 108, 1547415479.
  • Giorgetti M., Javaid J. I., Davis J. M., Costa E., Guidotti A., Appel S. B. and Brodie M. S. (1998) Imidazenil, a positive allosteric GABAA receptor modulator, inhibits the effects of cocaine on locomotor activity and extracellular dopamine in the nucleus accumbens shell without tolerance liability. J. Pharmacol. Exp. Ther. 287, 5866.
  • Heikkinen A. E., Moykkynen T. P. and Korpi E. R. (2009) Long-lasting modulation of glutamatergic transmission in VTA dopamine neurons after a single dose of benzodiazepine agonists. Neuropsychopharmacology 34, 290298.
  • Hojsted J. and Sjogren P. (2007) Addiction to opioids in chronic pain patients: a literature review. Eur. J. Pain 11, 490518.
  • Iguchi M. Y., Handelsman L., Bickel W. K. and Griffiths R. R. (1993) Benzodiazepine and sedative use/abuse by methadone maintenance clients. Drug Alcohol Depend. 32, 257266.
  • Invernizzi R., Pozzi L. and Samanin R. (1991) Release of dopamine is reduced by diazepam more in the nucleus accumbens than in the caudate nucleus of conscious rats. Neuropharmacology 30, 575578.
  • Johnson S. W. and North R. A. (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J. Neurosci. 12, 483488.
  • Laviolette S. R., Gallegos R. A., Henriksen S. J. and van der Kooy D. (2004) Opiate state controls bi-directional reward signaling via GABAA receptors in the ventral tegmental area. Nat. Neurosci. 7, 160169.
  • Lee H. H., Walker J. A., Williams J. R., Goodier R. J., Payne J. A. and Moss S. J. (2007) Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2. J. Biol. Chem. 282, 2977729784.
  • Lee H. H., Deeb T. Z., Walker J. A., Davies P. A. and Moss S. J. (2011) NMDA receptor activity downregulates KCC2 resulting in depolarizing GABAA receptor-mediated currents. Nat. Neurosci. 14, 736743.
  • Lee A. Y., Chen W., Stippec S., Self J., Yang F., Ding X., Chen S., Juang Y. C. and Cobb M. H. (2012) Protein kinase WNK3 regulates the neuronal splicing factor Fox-1. Proc. Natl Acad. Sci. USA 109, 1684116846.
  • Leri F. and Franklin K. B. (2000) Diazepam in the ventral striatum dissociates dopamine-dependent and dopamine-independent place conditioning. NeuroReport 11, 25532557.
  • Liu Y., Chen J., Song T., Hu C., Tang Y., Zhang X. and Zhao J. (2009) Contribution of K+-Cl- cotransporter 2 in MK-801-induced impairment of long term potentiation. Behav. Brain Res. 201, 300304.
  • Malmberg A. B. and Basbaum A. I. (1998) Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates. Pain 76, 215222.
  • Morgan E. T., Sewer M. B., Iber H. et al. (1998) Physiological and pathophysiological regulation of cytochrome P450. Drug Metab. Dispos. 26, 12321240.
  • Mori T., Ito S., Narita M., Suzuki T. and Sawaguchi T. (2004) Combined effects of psychostimulants and morphine on locomotor activity in mice. J. Pharmacol. Sci. 96, 450458.
  • Narita M., Takahashi Y., Takamori K., Funada M., Suzuki T., Misawa M. and Nagase H. (1993) Effects of kappa-agonist on the antinociception and locomotor enhancing action induced by morphine in mice. Jpn. J. Pharmacol. 62, 1524.
  • Narita M., Aoki T., Ozaki S., Yajima Y. and Suzuki T. (2001) Involvement of protein kinase Cgamma isoform in morphine-induced reinforcing effects. Neuroscience 103, 309314.
  • Narita M., Mizuo K., Shibasaki M. and Suzuki T. (2002) Up-regulation of the G(q/11alpha) protein and protein kinase C during the development of sensitization to morphine-induced hyperlocomotion. Neuroscience 111, 127132.
  • Narita M., Shibasaki M., Mizuo K. and Suzuki T. (2003) Changes in G-protein activity mediated through the stimulation of dopamine and GABA(B) receptors in the mesolimbic dopaminergic system of morphine-sensitized mice. Addict. Biol. 8, 319325.
  • Narita M., Akai H., Nagumo Y., Sunagawa N., Hasebe K., Nagase H., Kita T., Hara C. and Suzuki T. (2004) Implications of protein kinase C in the nucleus accumbens in the development of sensitization to methamphetamine in rats. Neuroscience 127, 941948.
  • Narita M., Nagumo Y., Hashimoto S. et al. (2006) Direct involvement of orexinergic systems in the activation of the mesolimbic dopamine pathway and related behaviors induced by morphine. J. Neurosci. 26, 398405.
  • Panhelainen A. E., Vekovischeva O. Y., Aitta-Aho T., Rasanen I., Ojanpera I. and Korpi E. R. (2011) Diazepam-induced neuronal plasticity attenuates locomotor responses to morphine and amphetamine challenges in mice. Neuroscience 192, 312321.
  • Pitchot W. and Ansseau M. (2009) Zolpidem dependence and withdrawal seizure. Rev. Med. Liege 64, 407408.
  • Price T. J., Cervero F., Gold M. S., Hammond D. L. and Prescott S. A. (2009) Chloride regulation in the pain pathway. Brain Res. Rev. 60, 149170.
  • Rivera C., Voipio J., Thomas-Crusells J. et al. (2004) Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J. Neurosci. 24, 46834691.
  • Rooney S., Kelly G., Bamford L., Sloan D. and O'Connor J. J. (1999) Co-abuse of opiates and benzodiazepines. Ir. J. Med. Sci. 168, 3641.
  • Rowlett J. K., Platt D. M., Lelas S., Atack J. R. and Dawson G. R. (2005) Different GABAA receptor subtypes mediate the anxiolytic, abuse-related, and motor effects of benzodiazepine-like drugs in primates. Proc. Natl Acad. Sci. USA 102, 915920.
  • Rudolph U. and Mohler H. (2006) GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 1823.
  • Rudolph U., Crestani F., Benke D., Brunig I., Benson J. A., Fritschy J. M., Martin J. R., Bluethmann H. and Mohler H. (1999) Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 401, 796800.
  • Segura M., Barbosa J., Torrens M., Farre M., Castillo C., Segura J. and de la Torre R. (2001) Analytical methodology for the detection of benzodiazepine consumption in opioid-dependent subjects. J. Anal. Toxicol. 25, 130136.
  • Seltzer Z., Dubner R. and Shir Y. (1990) A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43, 205218.
  • Shibasaki M., Watanabe K., Takeda K., Itoh T., Tsuyuki T., Narita M., Mori T. and Suzuki T. (2013) Effect of chronic ethanol treatment on mu-opioid receptor function, interacting proteins and morphine-induced place preference. Psychopharmacology (in press).
  • Tan K. R., Brown M., Labouebe G., Yvon C., Creton C., Fritschy J. M., Rudolph U. and Luscher C. (2010) Neural bases for addictive properties of benzodiazepines. Nature 463, 769774.
  • Tsuda M., Chiba Y., Suzuki T. and Misawa M. (1998a) Upregulation of NMDA receptor subunit proteins in the cerebral cortex during diazepam withdrawal. Eur. J. Pharmacol. 341, R1R2.
  • Tsuda M., Shimizu N., Yajima Y., Suzuki T. and Misawa M. (1998b) Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: evidence for upregulation of NMDA receptors. Naunyn Schmiedebergs Arch. Pharmacol. 357, 309315.
  • Vinkers C. H., Olivier B., Hanania T., Min W., Schreiber R., Hopkins S. C., Campbell U. and Paterson N. (2011) Discriminative stimulus properties of GABAA receptor positive allosteric modulators TPA023, ocinaplon and NG2-73 in rats trained to discriminate chlordiazepoxide or zolpidem. Eur. J. Pharmacol. 668, 190193.
  • Walker B. M. and Ettenberg A. (2005) Intra-ventral tegmental area heroin-induced place preferences in rats are potentiated by peripherally administered alprazolam. Pharmacol. Biochem. Behav. 82, 470477.
  • Xiang K., Earl D. E., Davis K. M., Giovannucci D. R., Greenfield L. J. Jr and Tietz E. I. (2008) Chronic benzodiazepine administration potentiates high voltage-activated calcium currents in hippocampal CA1 neurons. J. Pharmacol. Exp. Ther. 327, 872883.
  • Yang H., Huang L. Y., Zeng D. Y. et al. (2012) Decrease of intracellular chloride concentration promotes endothelial cell inflammation by activating nuclear factor-kappaB pathway. Hypertension 60, 12871293.
  • Zarrindast M. R., Heidari-Darvishani A., Rezayof A., Fathi-Azarbaijani F., Jafari-Sabet M. and Hajizadeh-Moghaddam A. (2007) Morphine-induced sensitization in mice: changes in locomotor activity by prior scheduled exposure to GABAA receptor agents. Behav. Pharmacol. 18, 303310.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12258-sup-0001-FigureS1-S2.pdfapplication/PDF3201K

Figure S1. Western blot analysis of NKCC1 in the limbic forebrain.

Figure S2. KCC2 expression on DARPP32-positive neuron in the ventral tegmental area (VTA: a–d), substantial nigra (SN: e–h), and frontal cortex (FC: i–l).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.