Chronic variable stress induces supersensitivity of central α2-adrenoceptors which modulate the jaw-opening reflex in the rat

Brain Research 801 Ž1998. 72–77

Research report

Chronic variable stress induces supersensitivity of central a 2-adrenoceptors which modulate the jaw-opening reflex in the rat Pascual Garcıa-Vallejo ´ a b

a, )

a , Francisco M. Gomez , Carlos Infante a , Eduardo Ginestal a , ´ M. Teresa Giralt b

Department of Stomatology, Faculty of Medicine and Odontology, UniÕersity of the Basque Country, Leioa, Apdo. 699-48080 Bilbao, Vizcaya, Spain Department Pharmacology, Faculty of Medicine and Odontology, UniÕersity of the Basque Country, Leioa, Apdo. 699-48080 Bilbao, Vizcaya, Spain Accepted 19 May 1998

Abstract In a previous study, we found that the sensitivity of central postsynaptic a 2-adrenoceptors which modulate, in an inhibitory way, the activity of the jaw-opening reflex ŽJOR. is reduced after chronic repeated stress Žtail pinch. in the rat. The aim of this study was to assess the effects of exposure to a chronic variable stress regime on these adrenoceptors. To do this, the digastric electromyographic responses elicited by orofacial electrical stimulation after the intravenous administration of cumulative doses Ž=3.3. of the a 2-adrenoceptor agonist, clonidine Ž0.1–10 000 mgrkg., were recorded. As expected, in unmanipulated control rats, clonidine inhibited the reflex, in a dose-dependent manner, until abolition ŽED50 s 17.3 " 2.2 mgrkg.. Single tail pinch did not significantly alter the ability of clonidine to abolish the reflex. However, chronic variable stress led to an enhancement of the inhibitory effect of clonidine on the amplitude of JOR, resulting in a shift to the left of the dose-response curve in comparison with that of the control group ŽED50 was reduced by 37%, P s 0.032., without affecting either the estimated maximum effect for the agonist or the slope of the inhibitory function. This in vivo result indicates that chronic variable stress leads to an increased sensitivity of central a 2-adrenoceptors which modulate JOR, in contrast to the desensitization of these adrenoceptors found after repeated exposure to the same stressor. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Jaw-opening reflex; Central a 2 -adrenoceptor sensitivity; Chronic variable stress; Rat

1. Introduction Central noradrenergic ŽNAergic. systems play a relevant role in mediating an organism’s response to stress w4,31x. Acute exposure to many stressful stimuli elevates the activity in central NAergic neurons, provoking noradrenaline ŽNE . release in their terminal fields w25,30,34,38x. Numerous studies have shown that chronic stress also alters the functioning of the NAergic systems and their receptors w1,15,21,24,27x. More specifically, in relation to the effects of either acute or chronic stress on the density of central a 2-adrenoceptors in several brain regions, disparate results have been obtained w9,32,39x. Functional studies have provided more information about how stress modifies the sensitivity of these adrenoceptors w7,24,27x. In this context, the evalu) C orresponding [email protected]

author.

Fax:

q 34-4-4801899;

E-m ail:

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 4 7 - 2

ation of clonidine-induced inhibition of the jaw-opening reflex ŽJOR. in the rat, is considered a useful in vivo method for studying the regulatory mechanisms of central a 2-adrenoceptors w13x. JOR, which is mediated at the brainstem level, is regarded as the main relevant orofacial protective reflex w19x, and can be evoked by orofacial electrical stimulation w17x. Although other brain neurotransmitters are also involved, NE, via central a 2-adrenoceptors, plays a relevant role in the control of JOR in rodents w10,17x. The activation of postsynaptic central a 2adrenoceptors in the rat after the administration of cumulative doses of clonidine, an a 2-adrenoceptor agonist, induces a dose-dependent inhibition, until abolition, of JOR evoked by orofacial electrical stimulation ŽOF–JOR. w13x. Recently, we have found that chronic repeated exposure to tail pinch provokes a reduction in the ability of clonidine to inhibit OF–JOR, which indicates a desensitization of the a 2-adrenoceptors which regulate the reflex w14x. It has been suggested that an organism adapts more slowly to

P. Garcıa-Vallejo et al.r Brain Research 801 (1998) 72–77 ´

chronic diverse stressors than when it is repeatedly exposed to the same stressor w4x. In relation to the effects of chronic variable stress on central adrenoceptors, the few studies which have been carried out have mainly been focused on b-adrenoceptor density and have not yielded consistent results w6,21,35x. In this study, we tested the in vivo effects of exposure to a chronic variable stress regime on the sensitivity of a 2-adrenoceptors which modulate OF–JOR in the rat, in order to further our understanding of the adaptive mechanisms which central adrenoceptors develop in response to chronic stress.

2. Materials and methods 2.1. Animals Male Sprague–Dawley rats with an initial weight of 310–325 g were housed in groups of four per cage in a temperature-controlled room Ž22–248C. and maintained under a 12-h lightrdark cycle Žlight cycle was from 0800 to 2000 h.. Food in pellet form and water were available ad libitum. Rats were allowed at least one week to acclimatize before being subjected to any treatment, and were then randomly divided into the different experimental groups. Animals were treated in accordance with institutional guidelines and the appropriate EC directives ŽEuropean Economic Community Council Directive 86r609rEEC, November 24, 1986.. The experimental procedures to which the rats were subjected were reviewed and approved by the Ethics Committee of the Faculty of Medicine and Odontology of the University of the Basque Country. 2.2. Experimental stress Rats were either subjected to a single tail pinch for 5 min Ž n s 11., which was carried out by placing a clothes pin 8 cm from the tip of the tail, or exposed to a chronic stress regime using different stressors over 14 days Ž n s 15., following a paradigm similar to that described by Murua ´ and Molina w23x. Tail pinch was chosen as it is regarded as the stressor which most clearly induces gnawingrbiting in rodents w5x, and this oral response may trigger changes in the activity of the jaw reflexes. In the chronically stressed group, one stressor was applied per day during the light cycle, in a laboratory adjacent to the room where the rats were housed. The schedule of the stress regime is presented in Table 1. A group of rats Ž n s 21. was unmanipulated and used as a control. The rats from this group were daily transported to the same laboratory and remained there for 2 h, at a time which did not coincide with the stress procedure. As useful indicators of the severity of the perceived stress w8,35x, the postmortem adrenal gland weight in milligrams and the gain in body weight expressed as a

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Table 1 Chronic variable stress regime Day Procedure 1 2 3 4 5 6 7

Day Procedure

Tail pinched for 5 mina 8 Cold exposure at 48C for 120 minb 9 Motion stress for 60 minc 10 Cold swim at 48C for 5 min 11 Immobilization for 120 mind 12 Cold exposure Žas on day 2. 13 Motion stress Žas on day 3. 14

cold swim Žas on day 4. immobilization Žas on day 5. cold exposure motion stress cold swim immobilization tail pinch Žas on day 1.

a

Tail pinch was carried out using a plastic clothes pin whose elliptical opening was 3 mm in diameter and placed 8 cm from the tip of the tail. b Cold exposure consisted of placing rats individually into plastic cages and putting them in a cold room. c Motion stress consisted of horizontal shakes Ž38 mm-excursions at 60 times per min.. Rats were placed individually into a plastic cage that was put into an Unitronic 320-OR shaker ŽSelectaw , Spain.. d Rats were immobilized in plastic disposable rodent restrainers ŽBraintree Scientific, Braintree, MA, USA..

percentage of the initial weight of each animal Žmean " S.D.., were assessed in the group subjected to chronic variable stress and compared with those values obtained in the control group. 2.3. Preparation, stimulation and recording of the jawopening reflex Rats were anaesthetized with sodium pentobarbitone Ž50 mgrkg, i.p.. after which, the left digastric muscle was located and the right femoral vein was cannulated for drug administration. Animals were placed in a supine position over a heating pad to maintain body temperature, which was recorded by a rectal thermistor, at 37 " 18C. A supplementary dose of pentobarbitone Ž15 mgrkg, i.v.. was routinely administered 35 min after the first intraperitoneal anaesthetic injection, which was given 15 min before beginning the electromyographic ŽEMG. recordings of the reflex. As an expression of JOR, the EMG responses from the anterior belly of the left digastric muscle were recorded using a concentric bipolar needle electrode ŽRef. DMC 25, Medelec, Surrey, UK.. The methodology described by Garcıa-Vallejo et al. w13x was followed for both stimulation ´ and recordings. All recordings of OF–JOR were carried out 24 h after exposure to the last stressor. Two initial recordings were obtained in the absence of the drug to assess the basal value of OF–JOR, then, digastric EMG responses were measured after cumulative intravenous administration of increasing doses Ž=3.3. of clonidine Ž0.1– 10 000 mgrkg., at 4-min intervals, until the complete abolition of the reflex. The volume of each i.v. administration was held constant at 0.1 ml. Cumulative dose-response curves for the agonist were determined by evaluating the fractional variations of the sum of amplitudes Ž S amplitude. of OF–JOR in relation to the previously obtained basal value. The S amplitude was defined as the

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arithmetical addition of each peak-to-peak value of every wave that formed the configuration of the reflex. Cumulative dose-response curves for clonidine were assessed in stressed rats and then compared with those obtained in control rats. 2.4. Drugs Clonidine HCl ŽSigma, St. Louis, MO, USA. was dissolved in saline solution ŽNaCl 0.9%.. 2.5. Dose-effect curÕe analysis The cumulative dose-response curves were analyzed using the non-linear least-squares fitting programme GraFit Žversion 2.0; Erithacus Software, Staines, UK, 1990.. Experimental data from each group was pooled and analyzed for the best, simple, non-linear fit to the three-parameter n logistic equation: E s Emax w A x nrŽED50 q w A x n ., where w A x is the i.v. dose of agonist, ED50 the effective dose of agonist which produces 50% of the maximum effect Ž Emax . and n is the slope factor of the inhibitory function w11,26x.

proach is statistically optimal, as experimental errors are not systematically distorted w11,22x. As previously described by Pineda et al. w28x firstly, dose-effect curves from two different experimental data sets were analyzed simultaneously, but independently Žwithout constraints., to determine the overall value for the sum of squares ŽSS1 . and the number of degrees of freedom Ž df 1 .. Secondly, the dose-effect curves were simultaneously analyzed, successively constraining them to share common ED50 , Emax andror n and further values for the sum of squares ŽSS 2 . and the number of degrees of freedom Ž df 2 . were determined. Following this, the F ratio value was calculated by the equation F s ŽSS 2 y SS1 . df 1rŽ df 2 y df 1 .SS1. A significant impairment in the goodness of fit Ž P - 0.05. when ED50 , Emax or n were shared indicated that the parameters of the two dose-effect curves were different; that implies that the analysis that permitted one of the parameters to be shared without a significant increase in the residual variance was taken to be the best fit. The level of significance was set at P - 0.05.

3. Results 2.6. Statistical analysis Differences in the percentage of body weight gain and in adrenal gland weight between the control group and the chronically stressed group were analyzed by the Mann– Whitney U-test. The basal amplitude of OF–JOR Ž S amplitude. is expressed as the mean " S.E.M., and the non-parametric Kruskal–Wallis analysis of variance of ranks was used to evaluate differences among groups. Dose-effect curves obtained under different experimental conditions were compared in pairs by determining the goodness of fit of a model, with and without sharing the same parameters, by means of an F-test w22x. This ap-

3.1. Effect of the administration of cumulatiÕe doses of clonidine on the actiÕity of OF–JOR in control rats Orofacial electrical stimulation by trains of 10 rectangular pulses Ž0.1 ms duration. of 50 V Žsupramaximal voltage., presented at 1 Hz, elicited polyphasic EMG responses from the digastric muscle ipsilateral to stimulation. Under basal conditions, the elicited EMG showed a mean latency to onset of 4.9 " 0.8 ms, a mean duration of 6.0 " 1.9 ms, a mean maximum amplitude of 10.1 " 1.2 mV and a mean S amplitude of 20.6 " 2.3 mV in control rats ŽTable 2.. As expected, the administration of increas-

Table 2 Effects of single tail pinch and chronic variable stress on the clonidine-induced inhibition of the jaw-opening reflex evoked by orofacial electrical stimulation ŽOF–JOR. in the rat Groups

Controla Single tail pinchb Chronic variable stress c a

Basal valuese S amplitude ŽmV.

Clonidine-induced inhibition of the OF–JORd f Ž ED50 mgrkg. Emax Ž% inhibition.

ng

Ž n.

20.6 " 2.3 18.3 " 2.6 19.2 " 3.3

17.3 " 2.2 17.9 " 4.3 10.9 " 1.9 h

0.89 " 0.09 0.70 " 0.10 0.74 " 0.08

21 11 15

97.0 " 2.0 96.9 " 4.1 101.7 " 2.8

Rats were unmanipulated. Rats were exposed to a single tail pinch for 5 min, 24 h before the beginning of registration of the EMG OF–JOR. c Rats were chronically exposed to variable stressors for 14 days Žsee Table 1 for details.. Twenty-four hours after exposure to the last stressor, EMG recordings of OF–JOR responses to clonidine were carried out. d Cumulative dose-response curves for clonidine Ž0.1–10 000 mgrkg, i.v.. were determined to estimate, for each dose of agonist, the reduction in the basal values in the sum of amplitudes Ž S amplitude. of OF–JOR. Dose-response parameters for clonidine were determined directly by simultaneous analysis of Ž n. experiments and are expressed as the best fit values" S.E. calculated by non-linear regression analysis. e Basal values of the sum Ž S . of amplitudes represent the mean " S.E.M. of Ž n. determinations. f ED50 represents the dose of clonidine that provokes 50% of the maximum effect Ž Emax .. g n is the slope factor of the inhibitory function. h P s 0.032 vs. control Ž F-test.. b

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in control rats Ž Fw3,349x s 1.0, P s 0.41; F-test.. The basal value of S amplitude of OF–JOR, 24 h after exposure to tail pinch was not significantly different from those of control or chronic variable stress groups Ž P s 0.73; Kruskal–Wallis test. ŽTable 2.. 3.3. Effect of chronic Õariable stress on the clonidine-induced inhibition of OF–JOR

Fig. 1. Cumulative dose-response curves for clonidine-induced inhibition of the jaw-opening reflex Ž S of amplitudes. elicited by orofacial electrical stimulation ŽOF–JOR. in rats subjected to chronic variable stress for 14 days Ž ns15; v ., and unmanipulated control rats Ž ns 21; `.. For each dose of clonidine, the digital average of ten digastric electromyographic signals evoked by a train of 10 rectangular pulses Ž0.1 ms duration each pulse., presented at 1 Hz, was obtained, then S of amplitudes calculated. In the group subjected to chronic variable stress, the experiments were carried out 24 h after exposure to the last stressor. Data shown is the mean"S.E.M. of Ž n. experiments per group. Curves represent the computer fitted hyperbolic functions described by equation: n Es Ema x w A x n rŽED50 qw A x n ..

ing doses of the a 2-adrenoceptor agonist clonidine Ž0.1– 10 000 mgrkg, i.v.. resulted in a dose-dependent reduction of the basal values of S amplitude of the digastric EMG responses elicited by orofacial electrical stimulation ŽFig. 1.. The dose-dependence was assessed by analyzing untransformed experimental data according to the generic hyperbolic function proposed by Parker and Waud w26x. This model described the relation between the clonidine doses and the changes in S amplitude well, as revealed by the existence of a significant correlation Ž r 2 s 0.845; P 0.001. and non-systematic deviation from the model Žruns test P ) 0.05.. The ED50 value for clonidine which reduced S amplitude of OF–JOR ŽED50 s 17.3 " 2.2 mgrkg. indicated a high sensitivity of central a 2-adrenoceptors which regulate the activity of the reflex. In the present model, clonidine acted as a full agonist, as the maximum effect Ž Emax . was not different from 100 Ž Fw1,237x s 2.0, P s 0.16; F-test. and the obtained slope factor Ž n s 0.89 " 0.09. was not different from unity Ž Fw1,237x s 1.3, P s 0.25; F-test. ŽTable 2 and Fig. 1.. 3.2. Effect of a single tail pinch on the clonidine-induced inhibition of OF–JOR Exposure to a single tail pinch Žfor 5 min., which is the last stressor in the chronic variable stress regime ŽTable 1., did not significantly modify the inhibitory dose-response curve for clonidine of OF–JOR in relation to that obtained

Exposure to the chronic variable stress regime increased the ability of clonidine to inhibit OF–JOR, with the doseresponse curve for the clonidine-induced inhibition of OF– JOR being significantly different in relation to that of the control rats Ž Fw3,392x s 5.0, P s 0.002; F-test.. ED50 for clonidine in rats subjected to chronic variable stress decreased by 37% Ž Fw1,392x s 4.6, P s 0.032; F-test. when compared with the ED50 of the control group, without affecting the slope of the inhibitory function n Ž Fw1,392x s 1.3, P s 0.26; F-test. and the estimated Emax for the agonist Ž Fw1,392x s 2.0, P s 0.16; F-test. ŽTable 2 and Fig. 1.. Chronic stress did not alter the basal value of S amplitude of OF–JOR Ž P s 0.73; Kruskal–Wallis test. ŽTable 2.. A significant increase in the adrenal gland weight Ž38.5 " 6.0 mg. was found in the rats exposed to chronic variable stress when compared to corresponding values from unmanipulated rats Ž31.5 " 3.0 mg. Ž P - 0.001; Mann–Whitney U-test.. Stressed rats also gained less body weight Ž13 " 6%. than control rats Ž18 " 3%. Ž P s 0.015; Mann–Whitney U-test..

4. Discussion The results confirm previous findings which indicated that clonidine has a great ability to inhibit the OF–JOR through the activation of postsynaptic a 2-adrenoceptors w13x, and show how the sensitivity of these adrenoceptors is modulated by chronic variable stress. The changes caused by stress in central NAergic transmission have been widely documented. Stress, in general, provokes increases in NE release in several NAergic brain regions, which depend on the type, severity and duration of the stressor applied. An immediate elevation of extracellular NE levels in hippocampus, amygdala, prefrontal cortex and hypothalamic paraventricular nucleus has been reported after acute tail pinch, returning to their basal values within a few hours w12,16,30,34x. Confirming previous results w14x, 24 h after a single exposure to tail pinch, we did not find significant alterations in the sensitivity of a 2-adrenoceptors which modulate OF–JOR, which is in agreement with the short-lived neurochemical changes reported after acute tail pinch. In contrast, exposure to chronic variable stress induced a clear shift to the left of the inhibitory dose-effect curve for clonidine, indicative of a supersensitivity of post-

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synaptic a 2-adrenoceptors which regulate OF–JOR. To our knowledge, however, there have been no in vivo studies assessing the effect of chronic exposure to different stressors on the sensitivity of central a 2-adrenoceptors. Nevertheless, in an autoradiographic study w35x, using a variable stress regime which lasted for 21 days, changes were not found in a 2-adrenoceptor binding in numerous brain regions of Sprague–Dawley rats 48 h after the last exposure to stress. The discrepancy between the findings obtained from our in vivo study and that previously referred to, may be due not only to the possible varying degrees of the severity of the diverse stressors used, which could lead to a different adrenoceptor modulation, but also to a functional approach being a more suitable way of detecting slight changes in central adrenoceptors provoked by stress. On the other hand, the area in which the postsynaptic a 2-adrenoceptors regulating JOR are probably located was not analyzed in the aforementioned study. Recently, after repeated stress Žtail pinched for 5 min, every 12 h, for 14 days., a desensitization of a 2-adrenoceptors which modulate OF–JOR has been found w14x. Diverse effects on central NAergic neurotransmission induced by the two different chronic stress regimes applied could be responsible for the opposite modifications observed in the sensitivity of these receptors, and it is widely accepted that the sensitivity of adrenoceptors is modulated by the concentration of NE available. Thus, the desensitization of a 2-adrenoceptors modulating OF–JOR after repeated tail pinch is compatible with the fact that NE content in brain is rarely depleted 24 h after chronic exposure to the same stressor w1,15,29x. However, a slower neuroendocrine adaptation seems to occur if the chronic stress regime involves different types of stressors w8x. In this study, the rats subjected to the variable stress paradigm for 14 days showed both a lesser body weight gain and adrenal hypertrophy, thus suggesting that this regime effectively produced the stress response sought. Acute exposure to unpredictable or inescapable stress causes greater NE reductions in many brain regions than when animals are able to predict or control the stress w3,4,36,40x. In contrast to the down-regulation in central b-adrenoceptors commonly reported after repeated stress wsee Ref. w31xx, an up-regulation in cortical b-adrenergic binding, which was prevented by imipramine, was found in a study w21x which used a similar chronic variable stress regime to the one followed in this work. Under certain stressful conditions which lead to changes in endogenous NAergic neurotransmitter levels, NE concentration has a greater correlation with the modulation of central a 2-adrenoceptors than with that of b-adrenoceptors w33x. As the trigeminal motor nucleus, which contains the digastric motoneurons, receives a relevant NAergic innervation from the lateral tegmental area w18x, the supersensitivity of postsynaptic a 2-adrenoceptors which modulate the reflex induced by chronic variable stress, could be the final result of a reduction in the availability of endogenous NE in trigemi-

nal target cells. In line with this hypothesis, a functional up-regulation of postsynaptic a 2-adrenoceptors has been reported after NE depletion w20,37x, and acute administration of reserpine also led to a leftward shift of the inhibitory dose-response curve for clonidine of the OF–JOR w13x. Furthermore, inescapable stress-induced NE depletion was observed 48 h post-stress in both locus coeruleus and hypothalamus w40x. However, increased NE levels in the brainstem were reported 2 h after acute exposure to unpredictable stress w3x, and reductions in NE content in diverse brain regions were not found in rats 24 h after chronic exposure to a mild unpredictable stress regime unable to provoke adrenal hypertrophy w2x. Moreover, as has been previously mentioned, the neurochemical NAergic changes elicited by tail pinch, which is the last stressor applied in our chronic variable stress paradigm, are short-lasting, although tail pinch-induced NE release in terminal NAergic fields was greater and more prolonged in rats chronically stressed than in naive rats w12x. In summary, the above mentioned evidence highlights the complexity of the response to stress in central NAergic systems and the difficulty of associating chronic stress-induced adaptive changes to central adrenoceptor sensitivity and the neurochemical alterations provoked by stressors. Nevertheless, we cannot rule out that the supersensitivity of central postsynaptic a 2-adrenoceptors induced by chronic variable stress may, in fact, reflect the organism’s failure to cope with stress, in contrast with the possible adaptation Ždesensitization. developed in response to repeated stress w14x.

Acknowledgements The authors wish to thank Adele ` Hopley for correcting the English version of the manuscript. This study was supported by the Basque Government ŽGrant No. PI 96r44..

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