Effect And Possible Mechanism Of Habenular Nucleus In The Regulation Of Adenosine On Behavior Of Depressed Rats Induced By CUMS

Stressful life events are considered an important factor in the development of centralnervous system disorders such as depression, which is one of the most commonpsychological problems, affecting nearly everyone through either personal experience orthrough depression in a family member. Recent studies have indicated that chronic stressand depression have been linked in a variety of ways by altering the activity of HPA andHPT axis. To our knowledge the habenula regulates the activity of many systems that mayalso play a major role during depression. The habenula have the connection of neuronfibers and function with the raphe nuclei and the hippocampus. Thus, the habenula appearsto be necessary for the induction of learned helplessness/behavioral depression.Accordingly, we investigated the role and mechanism exhibited by the habenula after acuteand chronic administration of adenosine and the change in the behavioral responsed elicitduring chronic stress.1 Methodological considerations of the study:1.1 The animal model1.1.1 Chronic Unpredictable Mild Stress (CUMS)Principally, we used this animal model of depression in these studies inorder toprovide a good model for studying the effect and mechanism of the habenular nucleus inthe regulation of adenosine on behavior of depressed rats during chronic stress. In ourstudies we exposed rats to chronic unpredictable mild stress every day to make adepression model. These data are in accordance with previous studies, which have shownthat chronic stress is able to evoke behavioral changes resembling clinical depression, suchas locomotor activity deficit. So the chronic unpredictable mild model of stress applied inthis work was developed to mimic depressive disorders. Moreover, rats that undergo thistreatment show increase level of cortisol, T3 and T4, which are indicatives of depression.1.2 Choice of Habenula and Dorsal Raphe for the chronic treatmentIn previous studies suggest that there is a functional relationship between the habenulaand the dorsal raphe during chronic stress. The habenula can influence the activity ofserotonergic neurons in the raphe and that in turn modulate the release of cerebral serotonin.Electrophysiological studies indicate that LHb converge to inhibit single neurons in theDR.1.3 Choice of behavioral testsThe open field test and forced swimming test were used first, primarily as apreliminary screening test in order to detect possible deficits occurred in locomotorfunctions. Thus, the main aim of the present work was the determination of the ratbehavioral structure during OFT and FST.1.4 Choice of adenosine for treatmentsIt had been reported that adenosine and its metabolites play an important role in theregulation of many physiological processes, one of which is adrenocorticotophic hormones(ACTH) corticosterone signaling. During chronic stress a high level of cortisol wasobserved due to the release of ACTH, and this prolonged increase in cortisol levels canfinally lead to changes in mood. Accordingly, we would like to investigate the link betweenthe hypothalamo-pituitary-adrenal (HPA) axis and adenosine. As in vitro and in vivo theeffects of adenosine on the hypothalamo-pituitary-adrenal (HPA) axis are well documented.Clinical depression is a complex condition likely resulting from a combination of inherited,biochemical, and environmental factors. Elevation of cortisol through a prolonged stressresponse is only one factor that may be related to the mood changes associated withdepression, and a causal relationship between elevated cortisol levels and depression hasnot been firmly established. Also, there was an explanatory hypothesis for the mechanismof T4 increase in depression is based on the increase of cortisol (hypercortisolism ofdepression), believed to lead to an activation of the hypothalamic neurons which producethe thyrotrophin releasing hormone (TRH) and, consequently, of the thyroid function. Thus,we need to investigate the link between the hypothalamo-pituitary-adrenal (HPA) axis andthe hypothalamo-pituitary –thyroid (HPT) axis on the basis of adenosine administration. Aswell as, the role of the habenula-raphe-hippocampus pathway during this endocrinologicalprocess.2 Experiment.12.1 The Efficiency of CUMS Procedure2.1.1 Effects of CUMS on Open Field Test (OFT) Session:Statistical analyses showed a significant reduction (P <0.001) in thenumber of crossing and rearing in the control group versus rats that underwentthis treatment (49±15.53 vs. 13.13±11.67) and (37±2.27 vs. 6.13±3.83)respectively. However, in the model group, immobility time significantly (P<0.001) increased from (73.75±17.59 to 208.38±12.84), when compared to thecontrol group. Also, rats exposed to CUMS showed fewer tendencies ingrooming behavior time when compared to the control group. Whereas, theincidence of defecation slightly increased with time compared to the controlgroup.2.1.2 Effects of CUMS on Forced Swimming Test (FST) Session:The duration of climbing and swimming behavior within 5-min FST weresignificantly decreased (127.75±19.92 sec vs. 66±29.61 sec and 122±2.56 secvs. 99.38±4.78 sec respectively, P <0.001), in the model group when comparedto the control, whereas, the immobility behavior time was significantlyincreased (49.5±4.72 sec vs. 136.25±44.1 sec, P <0.001), in the treated modelgroup as compared with the control group. These results indicated that chronicvariable stress could lead to depressive behavior.2.2 Effects of CUMS on C-fos Expression in the HippocampusAfter CUMS procedure, T-test showed a significant increase in the c-fospercentage expression in the hippocampus (18.57±079 vs.60.71±3.45), whencompared to the control group.2.3 Effect of CUMS on body weight changeOn the 22nd of stress, in the model group (221.38±8.94) was less than that in thecontrol group (262.38±11.29), and there was a notable significance between them (P< .0001).2.4 Effect of CUMS on cortisol, T3 and T4 profilesThe stress model group showed a high plasma cortisol concentration(16.65±6.03) compared to the control group (10.43±3.22) and there was anotable significance between them (P <0.05). Likewise, a significant increase(P <0.01) in T3 and T4 levels (from 0.47±0.14 to 0.84±0.09 and from40.96±4.31 to 65.81±11.27) were observed in the model rats compared to thecontrol group respectively.2.5 Effect of CUMS on Systolic Blood Pressure and Heart RateOur model presented a significant ( P < .001) high systolic blood pressure and heartrate during chronic stress procedure in the model group (from 92.5±5.98 to 121.13±4.94and from 381.25±23.11 to 465.63±47.17) when compared to the control group respectively.These above findings ensured that our model showed a high efficiency forexperiencing depression disorders and selectively appear to affect the search strategies inthe animal behavioral model test.3 Experiment.23.1 The Role and mechanisms of habenula lesion on animal behavioralresponses3.1.1 Histological analysisWe applied CUMS to investigate the influence of the bilateral electrolytic lesions ofthe lateral habenula on animal behavioral responses.3.1.2 Behavioral findingsThe effect of the lateral habenula lesion on behavioral responses during open field testand forced swimming test showed an active behavior when compared to the model group.During OFT the crossing and rearing behavior significantly increased (P <0.001) from(13.13±11.67 to 37.5±6.21 and from 6.13±3.83 to 24±2.93) in the lesion group whencompared to the model respectively. Whereas, the immobility behavior significantlydecreased (P <0.001) in the lesion group when compared to the model group(208.38±12.84 to 97.88±4.40). Results showed that the immobility and swimming behaviortime during FST decreased from (136.25±44.1 to 121.75±4.65 and from 99.38±4.78 to54.25±2.43) after the damage of the lateral habenula (P <0.001) when compared to themodel group respectively. Whereas, the climbing behavior increased (66±26.61 vs.124±4.31) in the lesion group when compared to the model group (P <0.001).According to these results, damage to the lateral habenula proves that habenula sharedirectly on the amelioration of the depressive behaviors that exhibited either by the chronicstress or after the chronic and acute administration of adenosine. Previous studies indicatedthat the habenula can influence the activity of serotonergic neurons in the raphe, and in turnmodulate the release of cerebral serotonin. So the decrease of serotonin in the raphe nucleiis the main cause of depression. This ensures that depression on the raphe is relieved afterhabenula lesion in which habenula influence the synthesis and release of serotonin uptakein the raphe nuclei.4 Experiment 3:4.1 The role and mechanisms of LHb after R-PIA administration and the changes inthe behavioral responses produced by CUMS.4.1.1 Histological analysisHistological analysis of injection sites showed that all animals originally included inthe study received microinjections of the drugs.4.1.2 Behavioral findings(1) Changes in Behavior during open field test (Pretreatment of DPCPX)There was a significant effect of adenosine agonist R-PIA treatment on the crossing,rearing and immobility behavior time in the open field test. Animal behavior wassignificantly reduced by a direct application of adenosine agonist R-PIA in dose of 0.1μg,0.5μg, 1μg and 5μg into the LHb during OFT and FST. Pretreatment of DPCPX 1μg intothe LHb alter the effects of R-PIA.(2) Changes in Behavior during forced swimming test (Pretreatment of DPCPX)Adenosine agonist showed the same patterns as in OFT, where there was a significanteffect of adenosine agonist R-PIA treatment on the immobility, climbing and swimmingbehavior time in the forced swim test. Animal behavior was significantly reduced by adirect application of adenosine agonist R-PIA in dose of 0.1μg, 0.5μg, 1μg and 5μg into theLHb during and FST. Pretreatment DPCPX 1μg into the LHb alters the effect of R-PIA.(3) Changes in Behavior during open field test (Pretreatment of 4-AP)Adenosine agonist R-PIA treatment showed a significant effect on the crossing,rearing and immobility behavior time in the open field test. Animal behavior wassignificantly reduced by a direct application of adenosine agonist R-PIA in dose of 1μg and5μg into the LHb during OFT. Pretreatment of 4-AP 5μg into the LHb alter the effects ofR-PIA.(4) Changes in Behavior during forced swimming test (Pretreatment of 4-AP)Administration of adenosine agonist R-PIA represented the same manner as in OFTwhere, there was a significant effect of adenosine agonist R-PIA treatment on theimmobility, climbing and swimming behavior time in the forced swim test. Animalbehavior was significantly reduced by a direct application of adenosine agonist R-PIA indose of 1μg and 5μg into the LHb during and FST. Pretreatment of 4-AP 5μg into the LHbalters the effect of R-PIA.5 Experiment 45.1 The effect of microinjection of R-PIA agonist into the LHb on C-fos expression inHippocampus induced by CUMS.Rats exposed to CUMS and R-PIA administration showed a significant increase in thepercentage of C-fos expression in the hippocampus when compared to the control group.To our knowledge that there is relation of functions and structures between habenula, raphenuclei and hippocampus. During depression the activity of habenula increased due to theglutamate receptor activation and this lead to the increase in the c-fos expression. So weanalyzed the changes of c-fos expression in the hippocampus during chronic stress afteradenosine treatment. Our result showed that c-fos expression in the hippocampus obviouslyincreased when compared to the control and there were a notable significance betweenthem (P <0.001). We observed that R-PIA increased the c-fos expression in thehippocampus and that DPCPX and 4-AP pretreatment reduce the c-fos expression. Thesefindings give us a good insight to investigate more about the role and mechanism of thelateral habenular nucleus in the reglulation of adenosine on animal behavior of chronicallydepressed rats.6 Experiment 5:6.1 Effect of microinjection of R-PIA into the LHb on the single unit discharges ofDorsal Raphe neuronsIn our present work a total of 47 single unit discharges of serotonergic efferentneurons (slow discharge frequency, S cell) were recorded from the DR. Poststimulusinhibitions of 31/47 5-HT neurons (66%), (regular, slow firing and broad spike width) wererecorded after microinjection of R-PIA (1ug and 5ug) into the LHb. The effects due to thechanges in firing rate last for 6.091±3.548 and 6.481±5.12 min respectively. The drugduration of R-PIA 1ug last about 15.876±14.008 and for R-PIA 5ug about 26.777±22.103min. The remaining single unit discharges in DR 6 of them were increased (21%) and 10unit discharges showed no changes or tend to decrease (13%). R-PIA (1ug and 5ug)showed a significant (P <0.05) reduction in the firing rate before and after drugadministration. These results suggest that the habenula is necessary for the differentialactivation of the dorsal raphe and the state of behavioral despair produced by CUMS andadenosine.According to our study Adenosine enhanced depression by prolonging the behavioractivity deficit during open field test and forced swimming test as well as, increasing thecortisol levels and suppressing T3 and T4 levels during CUMS procedure. These key pointsensure that the Habenula site play an important role for modulating Adenosine action. So,we concluded that:(1) Our model (CUMS) procedure able to evoke behavioral changes resemblingclinical depression by lowering the animal locomotary activity during open field test andforced swimming test.(2) Chronic and acute application of Adenosine produce depressant like effects in doserelated manner (prolonged locomotor activity deficit) by decreasing the animal behavioractivity (OFT and FST) via the activation of A1 receptor subtype and its mechanisms wereinvolved in K+ channel activation (Avoltage-dependent 4-AP).(3) Adenosine induces a state of 'learned helplessness' similar to that observed in ananimal model of depression by regulating the release of cortisol, T3 and T4. As well as,chronic Adenosine administration can regulates blood pressure and heart rate.(4) The habenula is activated by CUMS procedure, which is necessary for stressinduced alteration in the firing rate in the dorsal raphe. Thus, the habenula appears to benecessary for the induction of learned helplessness/behavioral depression and may be oneof the main targets that indoors for depression treatment and prevention.(5) Adenosine enhances the locomotary activity deficit after habenula lesion duringopen field test and forced swimming test, whereas, habenula lesion induces antidepressanteffects during CUMS procedure.(6) Microinjection of Adenosine into the Habenula enhance depression during openfield and forced swimming tests and that may probably due to the effect of adenosine insuppressing the GABAergic neurons in the habenula which in turn lowering the firing ratein the Raphe.(7) Adenosine plays a key role in modulating the depression episode process via theHabehula by decreasing the firing rate in the dorsal raphe neurons and increasing thepercentage of C-fos expression in the Hippocampus.The innovation of the studyThis new insight suggests a novel approach of the Habenula in regulating thebehavioral deficits exerted during depression through the involvement of adenosine A1receptor subtype. Habenula appear to be 'hot spot' in a broader circuit underlyingdepression and, as such, may be particularly one of the effective targets that collaboratesfor the prevention and treatment of depression. Accordingly, considerable furtherinvestigation will be necessary to understand the role of Habenula in the prevention andtreatment of depressive disorders.