| Objective:Substance addiction is a state of compulsive drug use even if one has the intention to control their use.Morphine has a long history as an addictive drug,and the treatment of morphine addiction and relapse prevention is a worldwide challenge.Previous research has shown that addiction is associated with alterations in the structure and function of the limbic reward system,and that susceptibility to addiction and withdrawal difficulties may be related to an individual’s early life experiences.Animal models of maternal separation（MS）are widely used to study the neurobiological and behavioral changes of mood disorders,and early life experiences not only play an important role in psychological development but also influence the development of the animal brain.Oxytocin（OT）is a peptide hormone synthesized by large cell neurons in the paraventricular nucleus of the hypothalamus（PVN）and supraoptic nucleus of the hypothalamus（SON）and secreted by the pituitary gland.OT neurons in the brain project to brain regions associated with social-behavioral regulation and play an important role in attachment formation,social behavior,and stress regulation.In order to investigate the relationship between maternal separation and addiction and the effects of OT during this process,this experiment used C57BL/6 mice to establish a maternal separation model to investigate the effects of intranasal OT administration on addiction susceptibility and neuronal responsiveness in specific brain regions.Methods:1 Establishment of maternal separation model.C57BL/6 mice were randomly divided into separation group（MS）and control group（C）after birth,The mice of MS group was separated 6h per day after birth until 21 days（P21）,C group was kept together with their mothers.8 groups were randomly divided according to sex and separation after P30.The 8 groups were divided into male control normal saline group（C+NS）,male maternal separate normal saline group（MS+NS）,male control OT group（C+OT）,male maternal separate OT group（MS+OT）,female control normal saline group（C+NS）,female maternal separate normal saline group（MS+NS）,female control OT group（C+OT）,Female maternal separate OT group（MS+OT）.2 Behavioral test.Conditional position preference（CPP）was used to evaluate the effect of different interventions on morphine reward.In the training stage,initial preference was recorded and mice with obvious preference were excluded;The mice were given morphine hydrochloride solution（5 mg/kg,i.p.）and NS（0.2 ml,i.p.）solution alternately in specific compartments during the conditioning phase,and the small door between the compartments was opened during the testing phase to record the time spent in the different compartments.The open-field test was performed 72 h after the end of CPP,and the three-chamber social test was performed 72 h after the open-field test.3 Tissue preparation and immunostaining of sections.72 h after the end of the three-chamber social test,mice in each group were given morphine hydrochloride intraperitoneally,followed by intranasal administration of OT or NS,After anesthesia,blood was collected from orbit,and serum was isolated to detect serum OT and glucocorticoid（GC）levels.After blood collection,the mice were perfused,brains were coronally sectioned with a cryostat,c-fos and OTR expression levels in the corresponding brain regions were measured by immunofluorescence histochemistry combined with semi-quantitative techniques.Results:1 Male mice1.1 CPP test.The mice preference for morphine was higher in the MS+NS group than in the C+NS group.In mice given OT,preference was significantly lower in the MS+OT group than in the C+OT group.Also in MS mice,preference for morphine was higher in the MS+NS group than in the MS+OT group,but in control mice,preference for morphine was higher in the C+OT group than in the C+NS group.1.2 Behavioral test.In the open field test,the activity of mice increased after morphine administration,compared with the C+OT group mice,the C+NS group mice spent more time in the center,and the mice total moving distance in the center was higher in the MS+NS group than in the C+NS group.In the three-chamber social test,mice social tendency disappeared after morphine administration,and the MS+OT group spent more time in the nonsocial area.Mice social novelty preference was higher in the MS+NS group than in the C+NS and MS+OT groups after morphine administration.1.3 Serological detection.Mice serum GC level in the MS+NS group was higher than that in the C+NS group after morphine administration.In mice without social separation,GC level in the C+OT group was higher than in the C+NS group.After OT was given,the GC level in the MS+OT group was lower than in the C+OT group.Mice overall serum OT level did not change significantly,and the total serum OT level did not change significantly after nasal OT administration.1.4 Brain sectioning for c-Fos staining and immunohistochemistry.After morphine administration,c-fos staining of consecutive coronal sections of the whole mouse brain revealed that the number of c-fos positive cells in the nucleus accumbens septi（NAc）region was higher in the C+NS group than in the MS+NS group,and this difference was completely reversed in the ventral tegmental area（VTA）of the midbrain,where the number of c-fos positive cells in the C+NS group was lower than in the MS+NS group.C+NS group had fewer positive cells in this area than the MS+NS group.The c-fos positive cells in the VTA area,medial geniculate bodies（MGB）and periaqueductal grey region（PAG）of the midbrain were increased in both groups after OT administration.Immunofluorescence staining for c-fos and oxytocin receptor（OTR）in the paraventricular nucleus（PVN）of the hypothalamus showed that the number of c-fos positive cells was higher in the C+NS group than in the MS+NS and C+OT groups,and the number of c-fos positive cells in the PVN area decreased after OT administration.The number of OTR-positive cells changed in the same trend as c-fos positive cells in all groups,and there were obvious co-labeling of OTR and c-fos in the PVN region.2.Female mice2.1 CPP test.The mice of C+OT group scored highest in morphine-induced conditioned positional preference,and after nasal administration of OT,the MS+OT group had essentially the same level of preference as the C+NS group..2.2 Behavioral test.In the open field test,the activity of mice increased after morphine administration,and the MS+NS group mice spent more time in the field center than the C+NS group,and the MS+OT group mice spent less time in the field center after OT administration compared to the C+OT group.In the three-chamber social test,the social tendency of mice disappeared after morphine administration,and there was no significant difference between the groups of social novelty preference.2.3 Serological detection.The alteration of serum GC levels in female mice after morphine administration was different from that in males;OT administration led to an increase in serum GC levels,and this change was most pronounced in the C+NS and MS+NS groups.The mice serum OT levels were higher in the MS+NS group than in the C+NS group,and intranasal administration increased the serum OT levels,while there was no such change in males,and the change was most obvious in the C+NS group compared with the C+OT group.2.4 Brain sectioning for c-Fos staining and immunohistochemistry.After morphine administration,c-fos staining of whole brain coronal sections of female mice revealed that c-fos positive cells in the NAc region were higher in the MS+OT group than in the MS+NS group.In the VTA,c-fos positive cells in the MS+NS group were higher than those in the C+NS group.After nasal administration of OT,the number of c-fos positive cells in the VTA increased in both groups of mice,and the rise in the MS+OT group was higher than that in the C+OT group.In the MGB area,c-fos positive cells were higher in the C+NS group than in the C+OT group.c-fos positive cells were lower in the MS+NS group than in the C+NS group in the PAG region.c-fos and OTR fluorescent labeling staining in the PVN region showed that the number of c-fos positive cells was higher in the C+OT group than in the C+NS and MS+OT groups in mice,while there was no significant difference in the change of OTR positive cells.There was significant co-labeling of OTR and c-fos in the PVN region.3.Comparison of experimental results of mice of different genders3.1 The above experimental results showed no statistically significance in morphine-induced positional preference between female and male mice.Morphineinduced positional preference was diminished in the MS+OT group compared with the C+OT group,and was enhanced in the C+OT group compared with the C+NS group.3.2 In the open field test,the mice were more active after morphine injection,and there was no significant difference in freezing time or moving distance between male and female mice.In the three-chamber social test,mice lost their social tendency after morphine administration,males in the MS+OT group had lower social novelty preference compared to females,and males in the MS+NS group had higher social novelty preference than females.3.3 There were significant gender differences in blood serum GC levels between the C+NS and MS+OT groups.In addition,OT administration was found to increase serum GC levels in both females and males.The fluctuation level of serum OT in males after OT administration was less obvious than that in females,and serum OT levels were higher in females in the MS+NS,C+OT and MS+OT groups.3.4 C-fos staining of brain sections showed differences in the number of positive cells in different brain regions of male and female mice.After morphine administration,the number of c-fos positive cells in the NAc region of female mice was more than that of males.In the MS+NS and MS+OT groups,maternal separation resulted in a decrease in the number of c-fos positive cells in the NAC region of male mice,but there was no significant change in the number of c-fos positive cells in the NAC region of female mice in all groups.In addition,maternal separation resulted in an increase in the number of c-fos positive cells in the VTA region in males and a decrease in females.The number of males c-fos-positive cells was higher in the C+NS group than in females,but higher in the MS+NS and C+OT groups in females than in males.In the MGB region,more c-fos positive cells were found in males than females after OT administration;the number of c-fos positive cells in the PAG region increased more significantly in females in the C+NS group.In the PVN region,the number of c-fos positive cells in male mice in C+NS group was more than that in female mice,and the number of c-fos positive cells decreased in this group of mice after OT administration.Female mice in the C+OT and MS+NS groups had more OTR-positive cells in the PVN region than males.Conclusion:Maternal separation had a synergistic effect on morphine-induced rewarding behaviors,and also affects behaviors such as anxiety and social tendencies.Among them,OT administered intranasally to maternal separation mice attenuated morphineinduced rewarding behavior,a process that involves multiple brain regions related to the regulation of rewarding and social behavior,and showed gender differences,suggesting that OT may have an ameliorative effect on addicts who have experienced early attachment impairment. |
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