Physiology & Behavior 139 (2015) 393–396
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Effects of morphine on stress induced anxiety in rats: Role of nitric oxide and Hsp70 Jagdish C. Joshi, Arunabha Ray, Kavita Gulati ⁎ Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India
H I G H L I G H T S • • • • •
Acute restraint stress induced anxiogenesis, reduced brain NOx and enhanced Hsp70 level. Morphine and L-arginine reversed anxiety of acute RS and brain NOx but augmented Hsp70. Chronic stress induced adaptation in behavior and brain NOx but accentuated brain Hsp70. Morphine and L-arginine attenuated the chronic stress effects on brain Hsp70. NO and Hsp70 may be involved in morphine induced anxiety modulation during stress.
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 15 November 2014 Accepted 17 November 2014 Available online 21 November 2014 Keywords: Stress Morphine L-Arginine Nitric oxide Hsp70
a b s t r a c t The present study evaluated the effects of morphine on acute and chronic restraint stress (RS) induced anxiety modulation and the possible involvement of nitric oxide (NO) and heat shock proteins (Hsp70) during such effects. Acute RS (× 1) induced anxiogenesis in the elevated plus maze (EPM) test which was associated with lowered brain NO metabolites (NOx) and elevated Hsp70 levels. Pretreatment with morphine (1 and 5 mg/kg) and L-arginine (500 mg/kg) attenuated the RS effects on EPM activity and brain NOx, whereas, Hsp70 levels were further augmented. Co-administration of both agents showed synergistic effects. By contrast, repeated RS (×15) did not induce any significant changes in EPM activity or brain NOx, but brain Hsp70 levels stayed elevated. Administration of morphine or L-arginine prior to chronic RS did not influence such chronic stress induced changes in behavioral and biochemical markers, but appreciably attenuated chronic RS induced elevation in Hsp70 levels. These results suggest that acute and chronic RS induced anxiety modulations were differentially influenced by morphine and L-arginine and that complex interactions involving brain NO and unregulated Hsp70 could regulate such effects. © 2014 Published by Elsevier Inc.
1. Introduction In response to external or internal stressors, the brain activates the autonomic nervous system and the neuroendocrine axis which attempt to restore the disrupted physiological homeostasis by inducing adaptive responses or allostasis. The ability or lack of it to cope with such stressful situations is a key determinant of health and disease [1]. Emotional stressors like restraint stress are known to induce neurobehavioral responses like anxiogenesis and both typical and atypical neural pathways have been proposed for such effects. Endogenous opioids subserve a key role during stress reactions and, both endogenous and exogenous opiates are known to induce variable effects. For example, both peripheral and central administrations of morphine and naloxone/naltrexone have been shown to modulate restraint stress induced gastrointestinal and ⁎ Corresponding author. Tel.: +91 9818033085. E-mail address: [email protected] (K. Gulati).
http://dx.doi.org/10.1016/j.physbeh.2014.11.056 0031-9384/© 2014 Published by Elsevier Inc.
immunological responses and the involvement of limbic areas like the amygdaloid complex has been proposed in such opioid effects [2]. However, inconsistencies have been reported in the nature of opioid effects during stress and the possibility of alternative signaling pathways cannot be totally discounted. Nitric oxide (NO) is an atypical, ubiquitous gasotransmitter, formed from L-arginine by enzymatic reaction involving NO synthases, and has multidimensional functions [3]. Studies from our laboratory have indicated that NO acts as an important modulator during stress susceptibility and adaptation [4,5]. It has also been suggested that opioid receptors and NO synthase enzymes are co-localized in the hypothalamus [6]. Cellular proteins such as heat shock proteins (Hsps) are molecular chaperones which confer cytoprotection against various stressors by maintaining homeostasis of protein by regulating their proliferation, differentiation and apoptotic pathways [7]. A recent study suggested that Hsp70 may play a crucial role in neuroprotective effects of morphine in vivo and in vitro neuronal systems [8]. Studies also suggested that mu receptor activation releases
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NO which is involved in the modulation of opioid effects by decreasing development of tolerance and dependence to morphine [9]. However, the mechanisms involved during various stress effects on behavior and their modulation by opioids and NO are still not clearly defined. In view of the above, the present study was designed to investigate the possible cellular/molecular mechanisms of morphine and L-arginine during acute and chronic stress in rats.
2. Materials and methods 2.1. Animals Male Wistar rats (180–220 g) were used and were fed on standard chow diet and water ad libitum. They were acclimatized in experimental laboratory conditions at an ambient temperature (22 ± 2 °C) and animal care was as per guidelines for Care and Use of Animals in Scientific Research prepared by the Indian National Sciences Academy (INSA), New Delhi. The experimental protocol was approved by the Institutional Animal Ethical Committee.
2.2. Drugs and chemicals Morphine sulfate was purchased from Verve Health Care Ltd., Delhi (a govt. authorized dealer) while L-arginine hydrochloride was purchased from Sigma-Aldrich. The drugs were dissolved in distilled water and injected intraperitoneally (i.p.) in a volume of 1 ml/kg, and pretreatment times were 30 min for morphine and 60 min for L-arginine. All other chemicals required for biochemical estimation were procured from SRL Labs, Delhi and were of analytical grade.
2.3. Stress procedure Restraint stress (RS) was used as the experimental stressor and rats were immobilized in adjustable Plexiglas restrainers (INCO, Ambala) for 1 h at room temperature. Immobilization is a well validated and widely used method for inducing emotional stress and all classical stress responses are consistently expressed [10]. After completion of the immobilization stress procedure the animals were observed for neurobehavioral parameters in the elevated plus maze (EPM) test. In the acute stress paradigm, rats were exposed to RS for 1 h only once, whereas, for the chronic stress experiments, 15 daily RS (1 h) sessions were given. Immediately after the last session of RS, rats were exposed to the EPM test.
2.5. Biochemical assays 2.5.1. Preparation of brain homogenate Immediately after the behavioral test, rats were sacrificed by using an overdose of ether anesthesia, the skulls were opened and brains taken out. The brains were washed in ice cold isotonic saline and weighed. They were then minced, and the homogenates (10% w/v) were prepared in chilled phosphate buffer. These homogenates were used for estimating brain Hsp70 and NOx levels. 2.5.2. Brain NOx level Stable NO metabolite (NOx) was determined according to the method of Tracey et al. [12], in which Aspergillus nitrate reductase was coupled with NADPH and FAD to convert all nitrates present in the sample into nitrites. Homogenized brain samples were centrifuged at 10,000 g for 15 min at 4 °C. Assay mixture contained 50 μl of test sample/supernatant, 10 μl of 0.86 mM/l NADPH, 10 μl of 0.11 mM/l FAD, 10 μl of nitrate reductase (2 U/ml) and 20 μl of 310 mM/l potassium phosphate buffer in a total assay volume of 100 μl. Samples were allowed to incubate at 37 °C for 1 h in the dark, followed by an addition of 5 μl of 1 M/l zinc sulfate to precipitate proteins. After centrifugation of microtubes, 50 μl of supernatant from each microtube was transferred into individual wells of 96 well microplate followed by an addition of 100 μl Griess reagent, for color development. The absorbance was measured at 540 nm after 10 min in 96 well assay plate using Spectra Max M3 photometric microplate reader (Molecular Devices). Standard curve was generated using known concentration of sodium nitrate and converted to NOx content by using a nitrate standard curve. Brain protein was estimated by [13] and NOx data was expressed as nmol/mg protein. 2.5.3. Heat shock protein level estimation Quantitative determination of Hsp70 (pg/ml of 10% brain homogenate) was carried out by using commercially available ELISA kit (USCN Life Science and Technology Co., Ltd.) with an experimental protocol designed according to manufacturer's instruction. The amount of Hsp in each sample was determined by interpolating OD values of Hsp concentration using the standard curve and expressed as pg/ml of brain homogenate. 3. Statistical analysis The data were expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison test. The p value b 0.05 was considered to be statistically significant.
2.4. Elevated plus maze test
4. Results
The EPM consists of two open opposite arms (40 × 10 cm) and two enclosed arms, of the same measurement, with 40 cm high walls. The arms are connected in such a way that the maze has a plus sign (+) look. The entire maze is elevated 50 cm above ground and placed in a quiet dimly lit room. Experimental rats were placed individually in the center of the maze facing the closed arms and were observed for 5 min by using any maze software and video tracking system (Stoelting, USA). The following parameters were measured: number of open and closed arm entries and time spent on open and closed arms. Crossing of 75% of the body into any arm is defined as an entry by the software. Subsequently, the percentage of open arm entries and the percentage of time spent on open arm with respect to total number of entries and total time i.e. 5 min were calculated. The EPM test is a widely used method for testing anxiety like behavior, in which rats exposed to a conflict situation have a natural tendency to move and stay in the closed arms. An increase in the percent entries and/or percent time in open arms is suggestive of an anxiolytic state [11].
4.1. Studies during acute restraint stress Acute exposure to restraint stress for 1 h (RS × 1) significantly suppressed the % open arm entries by 64% as well as % time spent in open arm by 88% in the EPM test, as compared to that in control rats (p b 0.05). Pretreatment with morphine (1 and 5 mg/kg), 30 min prior to RS, reversed the stress induced behavioral suppression i.e. both % open arm entries as well as % time spent in open arm in a dose related manner. Similar reversals in RS induced neurobehavioral suppression in the EPM were also seen after administration of the NO donor, L -arginine (500 mg/kg) when given 1 h prior to RS. Further, coadministration of L-arginine (500 mg/kg) and morphine (1 mg/kg) before acute RS showed greater attenuations in RS induced changes in EPM parameters as compared to either drug alone (p b 0.05). Assay of rat brain homogenates for biochemical parameters showed that acute RS decreased levels of stable NO metabolites (NOx) in comparison to controls (no RS), whereas, there was a marked increase (by about
J.C. Joshi et al. / Physiology & Behavior 139 (2015) 393–396
400%) in levels of Hsp70 in these samples as compared to controls. Administration of morphine (1 and 5 mg/kg) prior to RS attenuated RS induced suppression of NOx levels which were returned to near normal values (p N 0.05 vs controls). Pretreatment with L-arginine (500 mg/kg) also induced similar reversals in RS induced suppressions in brain NOx levels and these values were restored to near control values. On the other hand, stress induced elevations in Hsp70 levels were further augmented after morphine administration (1 and 5 mg/kg), by 420% and 510%, respectively, and the effects with the latter dose being statistically significant (p b 0.05) as compared to controls. Similar enhanced expression of Hsp70 was also seen in brain homogenates in L-arginine treated rats. Co-administration of morphine (1 mg/kg) and L-arginine clearly potentiated the expression of Hsp70 in brain homogenates of RS exposed rats and the effects of combined treatment were greater than those seen with either drug alone. These results are summarized in Table 1.
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Table 2 Effects of morphine and L-arginine on elevated plus maze activity, brain NOx and Hsp70 levels in chronic stressed (RS × 15) rats. Treatment (mg/kg)
Elevated plus maze % OA entries
% OA time
Controls RS Mor (1) + RS Mor (5) + RS
35.1 41.6 35.8 36.6 39.8
11.6 6.4 6.6 8.8 7.2
L-Arg
(500) + RS
L-Arg
+ Mor (1) + RS
± ± ± ± ±
7.6 2.6 7.7 7.6 7.7
60.0 ± 9.9
± ± ± ± ±
3.5 1.6 2.3 3.8 2.3
14.6 ± 4.0
Brain NOx (nmol/mg prot.)
Brain Hsp70 (pg/ml)
4.3 3.8 4.3 4.8 4.4
149.5 802.8 670.5 659.3 577.2
± ± ± ± ±
0.4 0.2 0.6 0.5 0.7
5.4 ± 0.8
± ± ± ± ±
4.5 28.1a 24.6b 27.3b 48.3b
503.2 ± 35.2b
All data are mean ± SE; OA — open arm; Mor — morphine; L-Arg — L-arginine. a p b 0.05 (vs controls). b p b 0.05 (vs RS).
5. Discussion 4.2. Studies during chronic restraint stress Chronic exposure to RS (1 h daily × 15 such) did not induce any appreciable alterations in the behavioral parameters in the EPM when compared to controls. It was apparent that the reductions in open arm entries and open arm time seen after acute RS in the earlier experiment were not seen. Pretreatment with morphine (1 or 5 mg/kg prior to RS) as well as L-arginine (500 mg/kg) was not able to influence the behavioral effects of chronic RS by any significant extent (p N 0.05). This was again in contrast to the findings seen after acute RS, wherein both morphine and L-arginine showed attenuating effects on stress induced anxiogenesis. Co-administration of morphine and L-arginine prior to RS showed enhancements in the behavioral parameters in the EPM, but these changes were not statistically significant (p N 0.05). Assay of rat brain homogenates for biochemical parameters showed that in chronic RS exposed rats there were no significant alterations in levels of stable NO metabolites (NOx) in comparison to controls (no RS), but interestingly, there was a marked increase in levels of Hsp70 (by N500%). Administration of morphine (1 and 5 mg/kg) or L-arginine prior to RS was unable to influence NOx levels in both the control and the RS group of rats (p N 0.05 in each case). However, chronic RS induced elevations in Hsp70 levels in the brain were significantly attenuated after either morphine (1 or 5 mg/kg) or L-arginine (500 mg/kg) when compared to corresponding controls (p b 0.05). The most marked reductions of Hsp70 levels in brain homogenates were seen in the morphine + L-arginine treatment group when compared to the chronic RS or when either drug was given alone prior to RS. This was in contrast to what was seen during acute RS, wherein augmentations in Hsp70 levels were seen after all treatments. These results are summarized in Table 2.
Table 1 Effects of morphine and L-arginine on elevated plus maze activity, brain NOx and Hsp70 levels in acute stressed (RS × 1) rats. Treatment (mg/kg)
Elevated plus maze % OA entries
% OA time
Controls RS Mor (1) + RS Mor (5) + RS
30.9 9.9 18.2 27.3 21.7
11.9 1.4 4.8 12.1 8.8
L-Arg L-Arg
(500) + RS
+ Mor (1) + RS
± ± ± ± ±
3.0 3.1a 2.6 1.8b 2.2b
27.9 ± 1.9b
± ± ± ± ±
1.7 0.4a 1.6 2.3b 1.3b
13.6 ± 2.7b
Brain NOx (nmol/mg prot.)
Brain Hsp70 (pg/ml)
4.8 1.3 2.7 4.2 4.9
70 .1 355.2 364.0 427.0 431.0
± ± ± ± ±
0.4 0.2a 0.2b 0.2b 0.2b
5.3 ± 0.5b
± ± ± ± ±
2.5 4.6a 18.6 14.2b 18.6b
569.0 ± 14.1b
All data are mean ± SE; OA — open arm; Mor — morphine; L-Arg — L-arginine. a p b 0.05 (vs controls). b p b 0.05 (vs RS).
Stressful experiences can influence the neurobehavioral profile of the organism and precipitate neuropsychiatric states like anxiety and depression [14]. Experimental data has shown that endogenous opioids regulate stress responses elicited by physical and psychological stimuli, viz. immobilization, cold-water immersion, and electric foot shock [15] probably by increasing the synthesis of pro-opiomelanocortin (POMC), as well as release of β-endorphin from the arcuate nucleus of the hypothalamus [16]. Restraint stress (RS) is a widely used experimental model for emotional/psychological stress and the EPM test is one of the validated methods to evaluate the effects of anxiogenic/anxiolytic agents in rodents [11,17]. Our study evaluated the effects of morphine and its cellular mechanisms during acute and chronic RS induced anxiety modulation in rats by using the EPM test. Our earlier studies have shown that NO plays a crucial role in stress regulation and that morphine induced reversal of stress effects may be mediated through NO [4,5,18]. The present study further evaluated the cellular mechanisms in the anxiolytic effects of morphine and its interactions with NO during both acute and chronic stress in rats. To correlate the behavioral response with changes in brain NO, the levels of stable NO metabolites (NOx), which are considered a reliable marker of NO activity in vivo [19], were estimated in brain homogenates. The present results showed that acute RS resulted in anxiogenesis which was manifested as reduced % open arm entries and % open arm time and this was attenuated by prior administration of morphine (1 or 5 mg/kg) in a dose related manner. Measurement and interpretation of anxiety in the EPM could be influenced by changes in locomotor activity. In our study, the total number of closed arm entries, which is a clear index of general motor activity, was always recorded, and the differences between the number of total arm entries (open arm + closed arm) in controls, RS exposed and drug treated rats were marginal (data not shown) — indicating that locomotor activity was possibly not influencing our interpretation of anxiety modulation in the EPM. Such anxiolytic effects of morphine have also been reported earlier in other experimental models [18,20,21]. Further, biochemical analysis of brain homogenates revealed lowered levels of NOx (stable metabolites of nitric oxide) and elevated Hsp70 expression after RS exposure in comparison to controls. Hsp70 is a heat shock protein present in the cytosol, nucleus and endoplasmic reticulum, which is not usually detectable under normal conditions and is specifically induced in response to a variety of stressors, and is often regarded as a diagnostic marker for stress [22]. The stress proteins of the Hsp70 family function as chaperones, interacting transiently with many proteins and preventing its denaturation in an ATP-dependent manner to protect cells from cell death [7]. Interestingly, morphine treatment attenuated the acute RS effects on brain NOx, but further augmented the enhancements in Hsp70 levels. Thus it can be speculated that stimulation of Hsp70 by morphine may be involved in its anxiolytic effect in stressed rats as there are reports that Hsp70 acts to restore protein structure and functions [23].
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L-Arginine (a NO precursor) pretreatment also produced similar effects on neurobehavioral and biochemical parameters in RS exposed rats. A similar anxiolytic effect of NO mimetics has also been reported by Spiacci et al. [24]. In addition some of the earlier studies reported that NO or its donor induces Hsp70 levels in various experimental models [25]. Further, our present study also showed that co-administration of morphine and L-arginine showed greater magnitude of behavioral and biochemical effects than that seen with either drug alone — suggesting synergistic effects of both agents during acute stress. In contrast to acute stress, repeated exposure to daily RS for 15 days resulted in restoration of the % open arm entries and open arm time in the EPM test and brain NOx levels which were also not much different from the respective controls. After exposure to acute stress, homeostatic mechanisms are activated and may be associated with reduced activity of NO synthase enzyme and hence low NO levels. However, repeated RS exposure induced reversal in neurobehavioral suppression and brain NOx levels which could have been due to development of adaptation/ tolerance to the stressor. Adaptation to stress is an autoregulatory mechanism which helps to reduce the impact of stressors on the biological system via induction of adaptive responses [26]. Such adaptation after repeated stress exposure is widely reported [27,28]. The fact that daily prior administration of morphine (before RS exposure) did not further augment EPM parameters or brain NOx values indicates that either tolerance could have developed to the anti-anxiety effect of morphine. Alternatively, a ceiling effect in these responses could also have occurred and that NO could possibly be contributing to this phenomenon. Interestingly, as in acute RS, the Hsp70 levels were also enhanced after chronic RS which suggests a differential role of these biomarkers in the adaptation process. There may also be a different temporal relationship between these two biomarkers during chronic stress. But in contrast to that seen after acute RS, chronic RS induced increase in Hsp70 was attenuated after morphine or L-arginine treatment. In addition, co-administration of morphine and L-arginine showed synergistic effects on neurobehavioral (EPM data) and biochemical (brain NOx and Hsp70) parameters — suggesting interactions of NO and morphine during chronic RS. This reduction in Hsp70 may be due to the fact that its levels were already raised beyond an optimum level after chronic RS and morphine could have induced beneficial effects by lowering this cellular marker. A report has suggested that long term use of morphine decreased Bax and Hsp70 in cultured rat primary neurons while providing protection against cytotoxicity [29]. In addition, another study has indicated the involvement of oxidative stress in such morphine effects on Hsp70 [30]. Interactions between reactive oxygen and reactive nitrogen species are known and an antioxidant role for NO has been proposed during stress [26]. Similarly, pretreatment with L-arginine decreased the Hsp70 and increased the adaptive response after repeated stress. Further, administration of L-arginine with morphine prior to the restraint stress enhanced brain NOx and decreased Hsp70 levels as well as appreciably potentiated the anxiolytic and adaptive effects of morphine. It thus appears that NO may be involved in the effects of morphine during acute and chronic stress induced anxiety modulation and their interactions with Hsp70 may be contributing to these effects. Taken together, it is inferred that morphine induces differential anxiety modulation during acute and chronic stress. Further, interactions involving NO and Hsp70 exert complex regulatory influences on these effects. These results are of translational value as Hsp70 levels can be used as an effective marker/target for stress induced anxiety and anxiolytic agents.
Acknowledgments The authors thank the Council of Scientific and Industrial Research (CSIR) 37(1454)/10/EMR-II, New Delhi, for providing the financial support for the study.
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Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Effects of morphine on stress induced anxiety in rats: Role of nitric oxide and Hsp70 Jagdish C. Joshi, Arunabha Ray, Kavita Gulati ⁎ Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India
H I G H L I G H T S • • • • •
Acute restraint stress induced anxiogenesis, reduced brain NOx and enhanced Hsp70 level. Morphine and L-arginine reversed anxiety of acute RS and brain NOx but augmented Hsp70. Chronic stress induced adaptation in behavior and brain NOx but accentuated brain Hsp70. Morphine and L-arginine attenuated the chronic stress effects on brain Hsp70. NO and Hsp70 may be involved in morphine induced anxiety modulation during stress.
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 15 November 2014 Accepted 17 November 2014 Available online 21 November 2014 Keywords: Stress Morphine L-Arginine Nitric oxide Hsp70
a b s t r a c t The present study evaluated the effects of morphine on acute and chronic restraint stress (RS) induced anxiety modulation and the possible involvement of nitric oxide (NO) and heat shock proteins (Hsp70) during such effects. Acute RS (× 1) induced anxiogenesis in the elevated plus maze (EPM) test which was associated with lowered brain NO metabolites (NOx) and elevated Hsp70 levels. Pretreatment with morphine (1 and 5 mg/kg) and L-arginine (500 mg/kg) attenuated the RS effects on EPM activity and brain NOx, whereas, Hsp70 levels were further augmented. Co-administration of both agents showed synergistic effects. By contrast, repeated RS (×15) did not induce any significant changes in EPM activity or brain NOx, but brain Hsp70 levels stayed elevated. Administration of morphine or L-arginine prior to chronic RS did not influence such chronic stress induced changes in behavioral and biochemical markers, but appreciably attenuated chronic RS induced elevation in Hsp70 levels. These results suggest that acute and chronic RS induced anxiety modulations were differentially influenced by morphine and L-arginine and that complex interactions involving brain NO and unregulated Hsp70 could regulate such effects. © 2014 Published by Elsevier Inc.
1. Introduction In response to external or internal stressors, the brain activates the autonomic nervous system and the neuroendocrine axis which attempt to restore the disrupted physiological homeostasis by inducing adaptive responses or allostasis. The ability or lack of it to cope with such stressful situations is a key determinant of health and disease [1]. Emotional stressors like restraint stress are known to induce neurobehavioral responses like anxiogenesis and both typical and atypical neural pathways have been proposed for such effects. Endogenous opioids subserve a key role during stress reactions and, both endogenous and exogenous opiates are known to induce variable effects. For example, both peripheral and central administrations of morphine and naloxone/naltrexone have been shown to modulate restraint stress induced gastrointestinal and ⁎ Corresponding author. Tel.: +91 9818033085. E-mail address: [email protected] (K. Gulati).
http://dx.doi.org/10.1016/j.physbeh.2014.11.056 0031-9384/© 2014 Published by Elsevier Inc.
immunological responses and the involvement of limbic areas like the amygdaloid complex has been proposed in such opioid effects [2]. However, inconsistencies have been reported in the nature of opioid effects during stress and the possibility of alternative signaling pathways cannot be totally discounted. Nitric oxide (NO) is an atypical, ubiquitous gasotransmitter, formed from L-arginine by enzymatic reaction involving NO synthases, and has multidimensional functions [3]. Studies from our laboratory have indicated that NO acts as an important modulator during stress susceptibility and adaptation [4,5]. It has also been suggested that opioid receptors and NO synthase enzymes are co-localized in the hypothalamus [6]. Cellular proteins such as heat shock proteins (Hsps) are molecular chaperones which confer cytoprotection against various stressors by maintaining homeostasis of protein by regulating their proliferation, differentiation and apoptotic pathways [7]. A recent study suggested that Hsp70 may play a crucial role in neuroprotective effects of morphine in vivo and in vitro neuronal systems [8]. Studies also suggested that mu receptor activation releases
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J.C. Joshi et al. / Physiology & Behavior 139 (2015) 393–396
NO which is involved in the modulation of opioid effects by decreasing development of tolerance and dependence to morphine [9]. However, the mechanisms involved during various stress effects on behavior and their modulation by opioids and NO are still not clearly defined. In view of the above, the present study was designed to investigate the possible cellular/molecular mechanisms of morphine and L-arginine during acute and chronic stress in rats.
2. Materials and methods 2.1. Animals Male Wistar rats (180–220 g) were used and were fed on standard chow diet and water ad libitum. They were acclimatized in experimental laboratory conditions at an ambient temperature (22 ± 2 °C) and animal care was as per guidelines for Care and Use of Animals in Scientific Research prepared by the Indian National Sciences Academy (INSA), New Delhi. The experimental protocol was approved by the Institutional Animal Ethical Committee.
2.2. Drugs and chemicals Morphine sulfate was purchased from Verve Health Care Ltd., Delhi (a govt. authorized dealer) while L-arginine hydrochloride was purchased from Sigma-Aldrich. The drugs were dissolved in distilled water and injected intraperitoneally (i.p.) in a volume of 1 ml/kg, and pretreatment times were 30 min for morphine and 60 min for L-arginine. All other chemicals required for biochemical estimation were procured from SRL Labs, Delhi and were of analytical grade.
2.3. Stress procedure Restraint stress (RS) was used as the experimental stressor and rats were immobilized in adjustable Plexiglas restrainers (INCO, Ambala) for 1 h at room temperature. Immobilization is a well validated and widely used method for inducing emotional stress and all classical stress responses are consistently expressed [10]. After completion of the immobilization stress procedure the animals were observed for neurobehavioral parameters in the elevated plus maze (EPM) test. In the acute stress paradigm, rats were exposed to RS for 1 h only once, whereas, for the chronic stress experiments, 15 daily RS (1 h) sessions were given. Immediately after the last session of RS, rats were exposed to the EPM test.
2.5. Biochemical assays 2.5.1. Preparation of brain homogenate Immediately after the behavioral test, rats were sacrificed by using an overdose of ether anesthesia, the skulls were opened and brains taken out. The brains were washed in ice cold isotonic saline and weighed. They were then minced, and the homogenates (10% w/v) were prepared in chilled phosphate buffer. These homogenates were used for estimating brain Hsp70 and NOx levels. 2.5.2. Brain NOx level Stable NO metabolite (NOx) was determined according to the method of Tracey et al. [12], in which Aspergillus nitrate reductase was coupled with NADPH and FAD to convert all nitrates present in the sample into nitrites. Homogenized brain samples were centrifuged at 10,000 g for 15 min at 4 °C. Assay mixture contained 50 μl of test sample/supernatant, 10 μl of 0.86 mM/l NADPH, 10 μl of 0.11 mM/l FAD, 10 μl of nitrate reductase (2 U/ml) and 20 μl of 310 mM/l potassium phosphate buffer in a total assay volume of 100 μl. Samples were allowed to incubate at 37 °C for 1 h in the dark, followed by an addition of 5 μl of 1 M/l zinc sulfate to precipitate proteins. After centrifugation of microtubes, 50 μl of supernatant from each microtube was transferred into individual wells of 96 well microplate followed by an addition of 100 μl Griess reagent, for color development. The absorbance was measured at 540 nm after 10 min in 96 well assay plate using Spectra Max M3 photometric microplate reader (Molecular Devices). Standard curve was generated using known concentration of sodium nitrate and converted to NOx content by using a nitrate standard curve. Brain protein was estimated by [13] and NOx data was expressed as nmol/mg protein. 2.5.3. Heat shock protein level estimation Quantitative determination of Hsp70 (pg/ml of 10% brain homogenate) was carried out by using commercially available ELISA kit (USCN Life Science and Technology Co., Ltd.) with an experimental protocol designed according to manufacturer's instruction. The amount of Hsp in each sample was determined by interpolating OD values of Hsp concentration using the standard curve and expressed as pg/ml of brain homogenate. 3. Statistical analysis The data were expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison test. The p value b 0.05 was considered to be statistically significant.
2.4. Elevated plus maze test
4. Results
The EPM consists of two open opposite arms (40 × 10 cm) and two enclosed arms, of the same measurement, with 40 cm high walls. The arms are connected in such a way that the maze has a plus sign (+) look. The entire maze is elevated 50 cm above ground and placed in a quiet dimly lit room. Experimental rats were placed individually in the center of the maze facing the closed arms and were observed for 5 min by using any maze software and video tracking system (Stoelting, USA). The following parameters were measured: number of open and closed arm entries and time spent on open and closed arms. Crossing of 75% of the body into any arm is defined as an entry by the software. Subsequently, the percentage of open arm entries and the percentage of time spent on open arm with respect to total number of entries and total time i.e. 5 min were calculated. The EPM test is a widely used method for testing anxiety like behavior, in which rats exposed to a conflict situation have a natural tendency to move and stay in the closed arms. An increase in the percent entries and/or percent time in open arms is suggestive of an anxiolytic state [11].
4.1. Studies during acute restraint stress Acute exposure to restraint stress for 1 h (RS × 1) significantly suppressed the % open arm entries by 64% as well as % time spent in open arm by 88% in the EPM test, as compared to that in control rats (p b 0.05). Pretreatment with morphine (1 and 5 mg/kg), 30 min prior to RS, reversed the stress induced behavioral suppression i.e. both % open arm entries as well as % time spent in open arm in a dose related manner. Similar reversals in RS induced neurobehavioral suppression in the EPM were also seen after administration of the NO donor, L -arginine (500 mg/kg) when given 1 h prior to RS. Further, coadministration of L-arginine (500 mg/kg) and morphine (1 mg/kg) before acute RS showed greater attenuations in RS induced changes in EPM parameters as compared to either drug alone (p b 0.05). Assay of rat brain homogenates for biochemical parameters showed that acute RS decreased levels of stable NO metabolites (NOx) in comparison to controls (no RS), whereas, there was a marked increase (by about
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400%) in levels of Hsp70 in these samples as compared to controls. Administration of morphine (1 and 5 mg/kg) prior to RS attenuated RS induced suppression of NOx levels which were returned to near normal values (p N 0.05 vs controls). Pretreatment with L-arginine (500 mg/kg) also induced similar reversals in RS induced suppressions in brain NOx levels and these values were restored to near control values. On the other hand, stress induced elevations in Hsp70 levels were further augmented after morphine administration (1 and 5 mg/kg), by 420% and 510%, respectively, and the effects with the latter dose being statistically significant (p b 0.05) as compared to controls. Similar enhanced expression of Hsp70 was also seen in brain homogenates in L-arginine treated rats. Co-administration of morphine (1 mg/kg) and L-arginine clearly potentiated the expression of Hsp70 in brain homogenates of RS exposed rats and the effects of combined treatment were greater than those seen with either drug alone. These results are summarized in Table 1.
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Table 2 Effects of morphine and L-arginine on elevated plus maze activity, brain NOx and Hsp70 levels in chronic stressed (RS × 15) rats. Treatment (mg/kg)
Elevated plus maze % OA entries
% OA time
Controls RS Mor (1) + RS Mor (5) + RS
35.1 41.6 35.8 36.6 39.8
11.6 6.4 6.6 8.8 7.2
L-Arg
(500) + RS
L-Arg
+ Mor (1) + RS
± ± ± ± ±
7.6 2.6 7.7 7.6 7.7
60.0 ± 9.9
± ± ± ± ±
3.5 1.6 2.3 3.8 2.3
14.6 ± 4.0
Brain NOx (nmol/mg prot.)
Brain Hsp70 (pg/ml)
4.3 3.8 4.3 4.8 4.4
149.5 802.8 670.5 659.3 577.2
± ± ± ± ±
0.4 0.2 0.6 0.5 0.7
5.4 ± 0.8
± ± ± ± ±
4.5 28.1a 24.6b 27.3b 48.3b
503.2 ± 35.2b
All data are mean ± SE; OA — open arm; Mor — morphine; L-Arg — L-arginine. a p b 0.05 (vs controls). b p b 0.05 (vs RS).
5. Discussion 4.2. Studies during chronic restraint stress Chronic exposure to RS (1 h daily × 15 such) did not induce any appreciable alterations in the behavioral parameters in the EPM when compared to controls. It was apparent that the reductions in open arm entries and open arm time seen after acute RS in the earlier experiment were not seen. Pretreatment with morphine (1 or 5 mg/kg prior to RS) as well as L-arginine (500 mg/kg) was not able to influence the behavioral effects of chronic RS by any significant extent (p N 0.05). This was again in contrast to the findings seen after acute RS, wherein both morphine and L-arginine showed attenuating effects on stress induced anxiogenesis. Co-administration of morphine and L-arginine prior to RS showed enhancements in the behavioral parameters in the EPM, but these changes were not statistically significant (p N 0.05). Assay of rat brain homogenates for biochemical parameters showed that in chronic RS exposed rats there were no significant alterations in levels of stable NO metabolites (NOx) in comparison to controls (no RS), but interestingly, there was a marked increase in levels of Hsp70 (by N500%). Administration of morphine (1 and 5 mg/kg) or L-arginine prior to RS was unable to influence NOx levels in both the control and the RS group of rats (p N 0.05 in each case). However, chronic RS induced elevations in Hsp70 levels in the brain were significantly attenuated after either morphine (1 or 5 mg/kg) or L-arginine (500 mg/kg) when compared to corresponding controls (p b 0.05). The most marked reductions of Hsp70 levels in brain homogenates were seen in the morphine + L-arginine treatment group when compared to the chronic RS or when either drug was given alone prior to RS. This was in contrast to what was seen during acute RS, wherein augmentations in Hsp70 levels were seen after all treatments. These results are summarized in Table 2.
Table 1 Effects of morphine and L-arginine on elevated plus maze activity, brain NOx and Hsp70 levels in acute stressed (RS × 1) rats. Treatment (mg/kg)
Elevated plus maze % OA entries
% OA time
Controls RS Mor (1) + RS Mor (5) + RS
30.9 9.9 18.2 27.3 21.7
11.9 1.4 4.8 12.1 8.8
L-Arg L-Arg
(500) + RS
+ Mor (1) + RS
± ± ± ± ±
3.0 3.1a 2.6 1.8b 2.2b
27.9 ± 1.9b
± ± ± ± ±
1.7 0.4a 1.6 2.3b 1.3b
13.6 ± 2.7b
Brain NOx (nmol/mg prot.)
Brain Hsp70 (pg/ml)
4.8 1.3 2.7 4.2 4.9
70 .1 355.2 364.0 427.0 431.0
± ± ± ± ±
0.4 0.2a 0.2b 0.2b 0.2b
5.3 ± 0.5b
± ± ± ± ±
2.5 4.6a 18.6 14.2b 18.6b
569.0 ± 14.1b
All data are mean ± SE; OA — open arm; Mor — morphine; L-Arg — L-arginine. a p b 0.05 (vs controls). b p b 0.05 (vs RS).
Stressful experiences can influence the neurobehavioral profile of the organism and precipitate neuropsychiatric states like anxiety and depression [14]. Experimental data has shown that endogenous opioids regulate stress responses elicited by physical and psychological stimuli, viz. immobilization, cold-water immersion, and electric foot shock [15] probably by increasing the synthesis of pro-opiomelanocortin (POMC), as well as release of β-endorphin from the arcuate nucleus of the hypothalamus [16]. Restraint stress (RS) is a widely used experimental model for emotional/psychological stress and the EPM test is one of the validated methods to evaluate the effects of anxiogenic/anxiolytic agents in rodents [11,17]. Our study evaluated the effects of morphine and its cellular mechanisms during acute and chronic RS induced anxiety modulation in rats by using the EPM test. Our earlier studies have shown that NO plays a crucial role in stress regulation and that morphine induced reversal of stress effects may be mediated through NO [4,5,18]. The present study further evaluated the cellular mechanisms in the anxiolytic effects of morphine and its interactions with NO during both acute and chronic stress in rats. To correlate the behavioral response with changes in brain NO, the levels of stable NO metabolites (NOx), which are considered a reliable marker of NO activity in vivo [19], were estimated in brain homogenates. The present results showed that acute RS resulted in anxiogenesis which was manifested as reduced % open arm entries and % open arm time and this was attenuated by prior administration of morphine (1 or 5 mg/kg) in a dose related manner. Measurement and interpretation of anxiety in the EPM could be influenced by changes in locomotor activity. In our study, the total number of closed arm entries, which is a clear index of general motor activity, was always recorded, and the differences between the number of total arm entries (open arm + closed arm) in controls, RS exposed and drug treated rats were marginal (data not shown) — indicating that locomotor activity was possibly not influencing our interpretation of anxiety modulation in the EPM. Such anxiolytic effects of morphine have also been reported earlier in other experimental models [18,20,21]. Further, biochemical analysis of brain homogenates revealed lowered levels of NOx (stable metabolites of nitric oxide) and elevated Hsp70 expression after RS exposure in comparison to controls. Hsp70 is a heat shock protein present in the cytosol, nucleus and endoplasmic reticulum, which is not usually detectable under normal conditions and is specifically induced in response to a variety of stressors, and is often regarded as a diagnostic marker for stress [22]. The stress proteins of the Hsp70 family function as chaperones, interacting transiently with many proteins and preventing its denaturation in an ATP-dependent manner to protect cells from cell death [7]. Interestingly, morphine treatment attenuated the acute RS effects on brain NOx, but further augmented the enhancements in Hsp70 levels. Thus it can be speculated that stimulation of Hsp70 by morphine may be involved in its anxiolytic effect in stressed rats as there are reports that Hsp70 acts to restore protein structure and functions [23].
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L-Arginine (a NO precursor) pretreatment also produced similar effects on neurobehavioral and biochemical parameters in RS exposed rats. A similar anxiolytic effect of NO mimetics has also been reported by Spiacci et al. [24]. In addition some of the earlier studies reported that NO or its donor induces Hsp70 levels in various experimental models [25]. Further, our present study also showed that co-administration of morphine and L-arginine showed greater magnitude of behavioral and biochemical effects than that seen with either drug alone — suggesting synergistic effects of both agents during acute stress. In contrast to acute stress, repeated exposure to daily RS for 15 days resulted in restoration of the % open arm entries and open arm time in the EPM test and brain NOx levels which were also not much different from the respective controls. After exposure to acute stress, homeostatic mechanisms are activated and may be associated with reduced activity of NO synthase enzyme and hence low NO levels. However, repeated RS exposure induced reversal in neurobehavioral suppression and brain NOx levels which could have been due to development of adaptation/ tolerance to the stressor. Adaptation to stress is an autoregulatory mechanism which helps to reduce the impact of stressors on the biological system via induction of adaptive responses [26]. Such adaptation after repeated stress exposure is widely reported [27,28]. The fact that daily prior administration of morphine (before RS exposure) did not further augment EPM parameters or brain NOx values indicates that either tolerance could have developed to the anti-anxiety effect of morphine. Alternatively, a ceiling effect in these responses could also have occurred and that NO could possibly be contributing to this phenomenon. Interestingly, as in acute RS, the Hsp70 levels were also enhanced after chronic RS which suggests a differential role of these biomarkers in the adaptation process. There may also be a different temporal relationship between these two biomarkers during chronic stress. But in contrast to that seen after acute RS, chronic RS induced increase in Hsp70 was attenuated after morphine or L-arginine treatment. In addition, co-administration of morphine and L-arginine showed synergistic effects on neurobehavioral (EPM data) and biochemical (brain NOx and Hsp70) parameters — suggesting interactions of NO and morphine during chronic RS. This reduction in Hsp70 may be due to the fact that its levels were already raised beyond an optimum level after chronic RS and morphine could have induced beneficial effects by lowering this cellular marker. A report has suggested that long term use of morphine decreased Bax and Hsp70 in cultured rat primary neurons while providing protection against cytotoxicity [29]. In addition, another study has indicated the involvement of oxidative stress in such morphine effects on Hsp70 [30]. Interactions between reactive oxygen and reactive nitrogen species are known and an antioxidant role for NO has been proposed during stress [26]. Similarly, pretreatment with L-arginine decreased the Hsp70 and increased the adaptive response after repeated stress. Further, administration of L-arginine with morphine prior to the restraint stress enhanced brain NOx and decreased Hsp70 levels as well as appreciably potentiated the anxiolytic and adaptive effects of morphine. It thus appears that NO may be involved in the effects of morphine during acute and chronic stress induced anxiety modulation and their interactions with Hsp70 may be contributing to these effects. Taken together, it is inferred that morphine induces differential anxiety modulation during acute and chronic stress. Further, interactions involving NO and Hsp70 exert complex regulatory influences on these effects. These results are of translational value as Hsp70 levels can be used as an effective marker/target for stress induced anxiety and anxiolytic agents.
Acknowledgments The authors thank the Council of Scientific and Industrial Research (CSIR) 37(1454)/10/EMR-II, New Delhi, for providing the financial support for the study.
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