| 1. Methodological considerations of the study1.1. The animal modelWe used the intermittent access to20%ethanol using a two-bottle-choice paradigm drinking procedure in order to provide a good model for studying the role and mechanisms of lateral habenula after chronic ethanol intake. The combination of voluntary oral ethanol consumption followed by abstinence that is utilized in the intermittent-access20%ethanol drinking paradigm provides a unique opportunity to investigate neurochemical and ehavioral changes induced by high ethanol consumption overtime. In our study, we exposed the rats to this paradigm for35days to make the ethanol model. The data in this study are in accordance with previous studies, which have shown a rapid and robust escalation of ethanol consumption in Wistar rats until they reach a stable consumption (Sims et al.,2008). 1.2. Choice of Habenula and striatum for the studyThe importance of the habenula is based mainly on the prevalence of its connectivity (Ramon y Cajal,1995). The habenular nucleus is a point of convergence for neural information from the basal ganglia and limbic forebrain. From here, the information is passed on to regulatory monoaminergic mid-and hindbrain areas. Several studies have shown that neurons of LHb and MHb possess different morphological and electrical properties and are connected with different brain areas (Kim,2009).The importance of striatum in this study is related to the fact that all drug of abuse have in common that they increase striatal dopamine release, which is believed to convey a rewarding feeling (Baldwin et al.,2012). Previous studies have shown that nicotine increase dopamine release in the striatum, which is the reason why nicotine is addictive. In addition, during the withdrawal the dopamine concentration is decreased (Zhang et al.,2011). Therefore the activity of dopaminergic cells in the VTA/SNC is of importance to addictive behaviors. Niotine, cocaine and ethanol are all drugs of abuse, the lateral habenula also be involved in the regulation of ethanol effect. The level of activity in the lateral habenula are a major modulator of the activity of the VTA/SNC.1.3. Choice of behavioral testsThe open field and forced swimming test are behavioral tests that are predicative of antidepressant activity in rodents. When the rats are exposed to those paradigms, they typically display an immobile posture which said to reflect a state of behavioral despair (Porsolt et al.,1978). Due to the connectivity of habenula, we decide to investigate the role of habenula in learning and memory and the underlying possible mechanisms. We used different tests to explore the effect of ethanol consumption and LHb lesion on learning and memory. Animal behavioral studies largely confirm that hippocampus-dependent memory tasks are more affected by alcohol than are hippocampus-independent tasks (Matthews et al.,1995; Melchior et al.,1993; Bammer et al.,1982). Importantly, hippocampal long term potentiation, an electrophysiological correlate of memory and learning, is highly sensitive to alcohol (Sinclair and Lo,1986). Morris watermaze, which is one of the most widely used paradigm to assess learning and memory in rodents. Also, we used another test for declarative memory:novel object recognition task. In this task the rodents are trained to be familiar with objects and then a new object replaced an old one. The animal supposed to increase the exploratory time of the new object compared to the familiar one.To assess the effect of ethanol intake and the role of lateral habenula in circadian rhythm and sleep, we chose respectively two test:Wheel running test and EEG recording.1.4. Choice of FosB/△FosBRepeated exposure to abused drugs can cause several neuroadaptations in the mesocorticolimbic dopaminergic system. One such adaptation is the altered expression of transcription factors, which give rise to changes in gene expression and may lead to alterations in sensitivity to drugs of abuse (Ketz et al.,1999; McClung et al.,2004; Nestler et al.,2001).AFosB represents one mechanism by which drugs of abuse produce relatively stable changes in the brain that contribute to the addiction phenotype.△FosB, a member of the Fos family of transcriptions factors accumulate within a subset of neurons of nucleus accumbens and dorsal striatum (brain regions important for addiction) after repeated exposure to drugs of abuse. We choose to investigate the level of induction of△FosB instead of c-Fos because of after repeated exposure to stress the c-Fos response is desensitized, but△FosB like immunoreactivity remains high and persists after chronic stimulation. 2. Experiment1:Role of lateral habenula on depression, learning and memory after chronic ethanol consumption2.1. Effect of LHb lesion on ethanol consumptionThe intermittent-access20%ethanol2bottle-choice drinking paradigm showed an overall increase in ethanol intake (g/kg/24h) over time (Fig.2). We observed an overall main effect on ethanol consumption in each group. Repeated measures ANOVA determined that mean ethanol consumption during sessions significantly differed between groups F (2,15)=18.461P<0.0007. Posthoc Tukey analysis revealed that lesioned rats increased their ethanol intake compared to the model group and sham group. This significant increase of ethanol intake occured in sessions6,8,9and13. Lesioned rats consumed the highest level of ethanol in session9(7.15±1g/kg/24h) compared to model group (5.22±1.44g/kg/24h, P=0.02) and sham group5.08±0.67g/kg/24h, P=0.013). However, we did not observed a significant difference in ethanol intake between sham rats and model rats P=0.099.2.2. Effect of LHb lesion on depression after chronic ethanol intake2.2.1. Forced-swimming testStatistical analysis using one way ANOVA showed a significant effect on immobility [F(3,20)=4.24, P=0.018] and climbing [F(3,20)=6.41, P=0.003] behaviors among different groups. Post-hoc Tukey test showed a significant increase of immobility time in model group (93.5±15.29s) compared to the control group (63.5±11.14), P=0.030. The immobility time of lesioned rats decreased significantly (63.50±15.16s) compared to the model rats, P=0.030. Model rats showed a significant decrease of climbing time with (61.5±11.14s) compared to the control group (105.5±17.31s), P=0.002 2.2.2. Open field testStatistical analysis showed a significant difference on time spent in the center of open field between groups F(3,20)=123.88P<0.001. Lesioned group (43.91±6.03s) shows a significant difference on time spent in the center of arena compared respectively to the control group (3.57±0.39s) P<0.001, model group (21.57±4.64s) P<0.001and sham group (7.68±2.33) P《0.001. Also, we can observe a significant increase of time spent in the center of arena in model compared to control (P<0.001) and model compared to the sham (P=0.001).Also, we observed a significant difference on mobility time on open field test between groups F(3,20)=6.798P=0.002(one way ANOVA followed by Tukey’s test). Post hoc analysis revealed a significant increase of mobility duration of lesioned group (34.08±6.61s) compared to the control group (20.74±3.15s) P=0.004. Also we observed a significant increase of mobility duration in model group (33.28±8.71s) compared to the control group, P=0.0072.3. Effect of LHb lesion on cognition after chronic ethanol intake2.3.1. Morris watermaze test2.3.1.1. Effect of LHb lesion on escape latency after a chronic ethanol intakeWe observed an overall decrease of escape latency time over days. For the first day of training, we did not observe any significant difference in escape latency between groups. However, from the second day until the reversal day, the mean escape latency time was significantly different, F(3,20)=6.54P=0.003and F(3,20)=60.62P<0.001, respectively.One way ANOVA followed by Tukey’s post hoc test revealed that there is a significant difference between groups for the escape latency time during reversal F(3,20)=60.6, P=0.000Post hoc analysis revealed that mean escape latency time significantly increased for lesioned group (52.69±3.51s) compared with control group (23.9±4.12s), model group (31.43±3.7s) and sham group (30.56±4.32) P<0.001.2.3.1.2. Effect of LHb lesion on distance traveled after a chronic ethanol intakeStatistical analysis showed a significant intergroup difference on distance traveled in Morris watermaze. We observe an overall decrease of the distance traveled to find the platform overdays. One way ANOVA with repeated measures revealed that, except for the day2of the training, there was a significant difference between groups.During the day test day (reversal), we observed a significant difference between groups (one way way ANOVA followed by Tukey’s test) F (3,20)=17.696P<0.001. The lesioned group showed an increase of the distance traveled (1133.94±256.85cm) compared to control group (442±117.52cm) P<0.001, model (674±97cm) P<0.01and sham group (740.81±150.68cm) P<0.05, respectively2.3.2. Novel Object Recognition TaskANOVA one way followed by Tukey’s test revealed that there is a significant difference of distance moved in NORT between groups F3,20=28.06P<0.001.Bonferroni’s multiple comparison test revealed that there is a significant increase of exploratory time of novel object compared to the familiar object in control group (familiar:2.15±0.46s; novel:4.7±1.21s) P=0.039<0.05, model group (familiar:3.75±0.63s; novel:9.45±1.78s) P=0.005<0.001and sham group (familiar:1.55±0.46s; novel:4.86±1.05s) P=0.011<0.05, respectively. Meanwhile, there was no significant difference in exploratory time of novel object compared to the familiar one in lesioned group. However we observed a significant difference in exploratory time of novel object in lesioned group (3.43±0.82s) compared to the model group (9.45±1.78s) P<0.001 3. Experiment2:Effect of LHb lesion on circadian rhythm and sleep after chronic ethanol intake3.1. Effect of LHb lesion on circadian rhythm after chronic ethanol intake3.1.1. Wheel running testWe observed a significant increase of count bin/min in the daytime from8.13±1.95in control group to22.13±4.994model group,[F(3,28)=3.41, P=0.031].Also, there was a significant increase of mean count/min in night time from213.13±33.712in control group to379±44.58model group,[F (3,28)=3.864, P=0.02.In the daytime, we observed an increased of mean count/min in lesioned rats compared to the control and the sham. However, this difference was not significantly different.3.1.2. Effect of LHb lesion on sleep after chronic ethanol intake3.1.2.1. EEG recordingWe observed a significant increase of wake time from309.52±10.02min to373.7±7.59min [F(3,26)=8.073, P=0.001] when comparing control group to the lesion group, respectively. Lesion group showed a significant increase of wake time compared to the model (337.22±6.51min). Meanwhile, the NREMS time significantly decreased from275.15±9.59min to217±7.98min [F(3,26)=6.395, P=0.002. Also, we observed a significant difference in REMS between groups [F(3,26)=11.49, P<0.001]. REMS sleep increased from15.28±2.48min to23.46±1.86min between control group and model group, respectively.Statistical analysis showed a significant difference of total sleep time between different group [F(3,26)=8.073, P=0.001. The total sleep time decrease from290.47±10.02min to226.3±7.59min, when comparing control to lesion group respectively. Also, the lesion group showed a significant decrease of sleep time compared to the model group (262.77±18.43min). 4. Experiment3:Induction of△FosB/FosB in habenula, striatum and hippocampus after chronic ethanol intake4.1. Induction of AFosB in striatal regionsChronic voluntary ethanol consumption significantly increased FosB/△FosB positive cells in the striatum. The most dramatic induction of△FosB was seen in the nucleus accumbens (t=-3.986, df=6, P=0.007) and caudate putamen (CPu)(t=-3.452, df=6, P=0.014), where we observed a high amount of△FosB. In NAc, the number of FosB/△FosB positive cells in control rats was382±22and523±27.94in model group.△FosB induction was seen in both the core and shell subregions of the nucleus accumbens, with more induction seen in the core. Robust induction of△FosB was also observed in the CPu, with219±22.213FosB/AFosB positive cells in control group and317.5±17.68in model group. Interestingly, ethanol produced the greatest induction of△FosB in the nucleus accumbens core.4.2. Induction of AFosB in hippocampusBeyond the striatal complex, chronic self-administration of ethanol induced△FosB in several other brain areas such as hippocampus. Ethanol induced high levels of△FosB in the CA3-CA1subfields compared to the dentate gyrus. Statistical analysis showed a significant increase of FosB/AFosB in the hippocampus (t=-6.522, df=6, P=0.001). The number of FosB/AFosB positive cells in control rats was258.25±18.56and476.25±27.79in model group.4.3. Induction of AFosB in habenulaWe observed a significant increase of FosB/AFosB in the lateral habenula (t=-6.319, df=6, P=0.001) and also the medial habenula (t=-4.760, df=6, P=0.003). In LHb, the number of FosB/△FosB positive cells in model group significantly increase from78.75±4.53in control rats to143±9.1in model rats. In MHb, we also observed a significant increase of FosB/△FosB positive cells in model rats compared to the control rats. The number of FosB/AFosB positive cells in control rats was59.5±3.06and88.25±5.20in model groupAccording to our study, these key points ensure that habenula play an important role in regulation of depression, cognition and sleep after chronic ethanol consumption:(1) LHb lesions induced increase of ethanol intake in rats, the high consumption of alcohol in habenula lesioned rat could be the result of the lack of inhibition of LHb on the VTA(2) Lesioned habenula rats showed a decrease of the immobility time in forced-wimming test, which might result with the improvement of behavioral response in depressed rats by increasing the level of serotonin level in dorsal raphe nucleus(3) Lesioned of lateral habenula showed marked spatial memory impairment, impairment in spatial memory was marked in ethanol drinking groups. This could suggest that the ethanol drinking might exacerbate cognition impairments(4) Lesioned of LHB significantly decrease of exploratory time of the novel object in rats. This suggests that LHb lesions lead to object recognition memory impairments(5) Chronic voluntary ethanol intake robustly induces FosB/△FosB positive cells in the striatum and hippocampus providing further support that the induction of FosB/△FosB in related brain regions is a common chronic adaptation to drugs of abuse (6) Habenula (LHb and MHb) shows an increase of FosB/△FosB immunoreactive cells, this confirms the involvement of habenula in mechanisms underlying ethanol addiction, thus the important role that habenula plays in addiction(7) LHb lesions altered the amount of daily activity. This effect might be due to the destruction of habenula efferents to serotoninergic neurons in the raphe nuclei, which have been implicated in both photic and non-photic regulation of the circadian system(8) Our observation strongly argues that the time spent in night-time sleep is also decreased, consistent with other studies which showed rat’s decreased duration of sleep bouts/REM sleep and altered sleep patterns after LHb lesion. This supports the habenula as a candidate site that regulates circadian rhythm and sleep... |
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