The Role Of The Dopamine System In Paternal Behavioral Sensitization In ICR Mice

In some mammal species,both mother and fathers play important roles for survival and development of offspring.For examples,experiencing early maternal separation will induce serious mental disorders in their adulthood.Besides,early paternal deprivation could impair the social cognition,increase level of anxiety,inhibit the formation of pair bonding and reduce levels of sociability and paternal care for offspring in monogamous rodents.However,although the roles of paternal care have been investigated profoundly,how the paternal care is initiated and maintained,and its underlying neural mechanisms remain unclear.Unlike mothers,neurobiological and hormonal reorganization via experiencing copulation,pregnancy and delivery were essential to the onset of maternal behaviors,males had not experienced the gestation process before becoming fathers.The onset of paternal behavior may be induced by behavioral experience.For examples,males,after mating,showed significantly lower infanticide behavior and transiently started nursing unfamiliar pups earlier compared with virginal male mice.Fathers experiencing paternal care showed higher level of licking,retrieval,and nest building compared with new father.Thus,it is suggested that pup sensitization via repeated exposure may be one of factors in the onset of paternal care.But less is known about whether pup exposure has an effect on initiation of male parental care and what neurotransmitter system is involved in it.One possible neurobiological mechanism may be the involvement of the dopamine(DA)system.In particular,DA neuron in ventral tegmental area(VTA)projecting to nucleus accumbens(NAc)was also a key component of motivation system.NAc lesion can induce a significant defect in retrieving behavior in the male mice.Two weeks interaction with female can promote paternal behavior,the DA release and the Dopamine 1 receptor expression in NAc.Therefore,the DA system may act on the initiation phase of paternal behavior.In addition,the DA release in the NAc in virgin male prairie voles had a 38.3%increase immediately for the first time exposure to pups.In brief,the mPOA-VTA-NAc-ventral pallidum(VP)neural circuit plays an important role in regulating parental behavior.However,it is not clear whether long-term pup exposure could alter the DA system of males.The mainly limitation of the study of paternal behavior is mainly due to the selection of pup sensitization models.In about 95%mammal species,male do not take care of their offspring spontaneously in nature.It is generally believed that laboratory mice Mus musculus have no spontaneous paternal behavior.However,male ICR mice,could be induced to produce retrieval behavior by cohabitation with their partner and offspring,and the process was similar to sensitization.Hence,we want to further explore whether pup exposure could facilitate paternal behavior in virgin male ICR mice,and how the dopamine system is involved in this process.Here,we exposed virgin Slc:ICR males to alien pups 6 days with 20 min everyday.Their paternal behaviors were observed two days after repeated pup exposure.The content of dopamine and the numbers of Fos-ir neurons in the medial prefrontal cortex(mPFc),NAc,mPOA,bed nucleus of the stria terminalis(BNST),paraventricular nucleus(PVN)and VTA were detected by high performance liquid chromatography(HPLC)and immunofluorescence respectively.Finally,Designer Receptors Exclusively Activated Designer Drugs(DREADDS),a chemogenetic tool,was used to inhibit the DA neurons projection from VTA to the NAc in several male mice with repeated pups exposure,and then their paternal behaviors were observed.In this study,we demonstrated that male paternal level had a significant increase in ICR virgin male mice after a 6-day period of contact with foster pups.Males in SEN group had shorter latency to paternal behavior,longer paternal care duration and shorter sniffing time than CON group.SEM male retrieved more pups,and had shorter latency to retrieve the first pup,the second pup and the third pup,compared controls.Besides,no significant difference was founded on the latency to approach the pups.Repeated measures ANOVA indicated that pup exposure altered the paternal behavior in SEN group.In detail,the total sniffing time of males with repeated exposure to pups,differed on day 2-8 compared to day 1.The total time of paternal behavior responses to pups significantly differed between day 8 and day 1.A significant increase in the number of retrieved pups was found on day 8.In addition,the latencies to retrieve the first,second and third pup were shortened on day 8 compared to day 1.The effect of prior pup exposure on the c-fos protein expression on day 8 was detected with immunofluorescence analysis.T-test showed that the numbers of Fos-ir neurons in the NAc and mPOA were significantly higher in the SEN group compared to CON group.The Fos-ir neurons decreased in PrL of SEN group.Numbers of Fos-ir neurons in the SEN and CON groups did not show significant difference in the Cg IL,BNST,PVN.In addition,HPLC assay showed that the levels of DA were significantly higher in the NAc and VTA in SEN groups compared with CON groups,while levels of DA had no significant difference in the mPFc,mPOA,BNST and PVN between two groups.In addition,the number of TH-ir neuron in the VTA of the SEN group was significantly higher compared to the CON group.Inhibition of the activity of the DA nerve terminal in the NAc reduced the level of paternal behavior induced by repeated pup exposure.Result indicated that the duration of paternal behavior and the latencies of paternal behavior and retrieval of the first and second pup were significantly different between hM4Di group and mCherry group on day 9 but without significant difference on day 1 and day 8.Nonetheless,the sniffing time and the latency of retrieving the third pup on day 9 had no difference between the two groups.Moreover,there were significant differences with intra-group showed on the duration of sniffing and paternal behavior and the latencies of paternal behavior and retrieving the first,second and the third pup.Inhibition of activation of projection from VTA to NAC of mice in hM4Di group reduced duration of paternal behavior and increased the latencies to paternal care and retrieval of the first,second and the third pup while CNO was injected via comparison with the behavior tested after saline infusion on day 8,but it had no effects in the mCherry group.In addition,one way ANOVA showed that CNO infusion significantly changed the number of Fos-ir neurons in the hM4Di group.Number of Fos-ir neurons in hM4Di group,treated with CNO,was decreased significantly compared to hM4Di-saline,mCherry-CNO or mCherry-saline group.In summary,as our finding,pup exposure at least 20 minutes a day for six days can effectively promote a variety of paternal behaviors such as licking,huddling and retrieving behavior in virgin ICR male mice,which can be used to investigate the neuroendocrine mechanism underlying pup-induced paternal behavior.Repeated pup exposure increased the neural activity of mPOA and NAc during interaction with pups.MPOA are the core areas that dominate parental care and could be activated when received a pup-signal.The circuit,dopamine neuron in VTA projected to the NAc,was activated subsequently.The PrL activity was reduced after experiencing pup-sensitization,and it plays a major role in emotion and cognition.Thus,repeated pup exposure could possibly reduce levels of anxiety and decreased the activity of PrL while they interacted with pups.Present study finds that dopamine levels in the VTA and NAc were increased significantly,which is consistent with the immunofluorescence result that the number of TH-ir neuron was increased after repeated exposure to pups.Tyrosine hydroxylase is a speed-limiting enzyme that works in the process of dopamine synthesis,and its levels could indirectly reflect the amount of the DA.Nucleus accumbens and the DAergic projection from the VTA of the midbrain are considered to be significant anatomical substrates for goal-induced behavior.Dopamine plays an important role in such cognitive processes as gradual forming of reward-stimulus reinforcement learning.Another interesting finding is that inhibition of DA projection from VTA to NAc using DREADDS method inhibited levels of paternal care induced by repeated pup exposure.It further indicated that inhibition of DA neurons in NAc may lead to a temporary decrease in DA release,and reduced the activity of DA neuron in NAc,so that the onset of paternal behavior was hindered.In general,present study showed that the VTA-NAc neural circuit may be indispensable in the onset of the pup-induced paternal care.Thus,present study found that dopamine system play an important role in regulation of paternal behavior induced by repeated pup exposure.This finding sheds light on the underlying mechanism of paternal care.