Involvement Of Orexins In The Regulation Of Neuronal Activity And Emotional Behaviors In Central Amygdala In Rats

The central amygdala?CeA?is an important output nucleus of the amygdala.As an integrated nerve center for anxiety and a complex nucleus regulating emotional behavior,the central amygdala plays an important role in emotional regulation.Orexin is a hypothalamic neuropeptide.Central amygdala receives orexinergic fibers originating from the lateral hypothalamus and expresses orexin-1?OX1?receptors in rats.Orexins are involved in emotional regulation.We speculated that orexin-A could regulate emotional behaviors by regulating the spontaneous firing of central amygdala neurons.Objective:In the present study,we aimed to investigate the modulation of exogenous and endogenous orexins on the spontaneous firing rate and firing patterns of the central amygdala neurons in rats by using orexin-A,orexin-B,OX1 receptor antagonist SB-334867 and OX2 receptor antagonist TCS-OX₂-29.Furthermore,we observed the effects of orexins in the central amygdala on anxiety-like behavior.Methods:In vivo extracellular single unit recordings were used to detect the effects of orexins on the spontaneous firing rate and firing patterns.Open field test?OFT?and elevated plus maze test?EPM?were performed to observe the effects of orexins on emotional activity.Results:1.In normal rats,the basal spontaneous firing rate of central amygdala neurons ranged from 0.31 Hz to 8.79 Hz.The average firing rate was 2.95±0.16 Hz?n=159?.2.Micro-pressure ejection of 0.01mmol/L orexin-A significantly increased the firing rate from 2.38±0.45 Hz to 4.40±0.67 Hz in 18 out of the 31 central amygdala neurons?t=-7.619,df=17,P<0.001?.The average increase was 109.76±15.12%,which was significantly different from that of vehicle group?Z=-4.180,P=0.000?.There was a negative correlation between orexin-A-induced excitation and basal firing rate in central amygdala neurons?r=-0.508,P=0.031?,which indicated that the low-frequency neurons displayed a strong excitation of orexin-A.In other 13 out of the 31 neurons,the increase in firing rate induced by orexin-A was less than 2 SDs?a criterion to define significant effects?,which was considered non-responsive to orexin-A.In the 18 neurons responsive to orexin-A,there were 8 neurons with irregular firing patterns and 10 neurons with bursting firing patterns.In other 13 non-responsive neurons,there were 6 neurons with irregular firing patterns and 7 neurons with bursting firing patterns.We also analyzed the correlation between the orexin-A-induced effects on firing activity of the central amygdala neurons and the distinct firing patterns.The results showed that the excitatory effects induced by orexin-A was independent of the different firing patterns?X²=0.009,P=0.606?.3.Micro-pressure ejection of SB-334867 was performed to observe the modulation of endogenous orexins via OX1 receptors.In 14 out of the total 33 central amygdala neurons recorded,micro-pressure ejection of 0.01mmol/L SB-334867significantly decreased the firing rate from 3.23±0.47 Hz to 1.14±0.28 Hz?t=5.78,df=13,P<0.001?.The average decrease was 67.37±5.68%,which was significantly different from that of vehicle group?Z=-3.970,P=0.000?.In other 19 central amygdala neurons,SB-334867 did not change the firing rate significantly?less than mean±2SD?.4.In 15central amygdala neurons,application of TCS-OX₂-29 did not change the firing rate significantly?basal:3.27±0.61 Hz;TCS-OX2-29:3.26±0.61 Hz;t=0.175,df=14,P=0.864?.The average change was 2.23±4.97%,which was not significantly different from that of vehicle group?Z=-0.439,P=0.660?.5.Furthermore,both of orexin-A and receptor antagonists were co-applicated to observe the receptor mechanisms of orexin-A-induced electrophysiological effects.In 9 orexin-A responsive neurons,micro-pressure ejection of orexin-A alone significantly increased the firing rate from 2.11±0.55Hz to 5.14±0.97 Hz.The average increase was 128.32±17.13%.After completely recovery,co-application of SB-334867 and orexin-A increased the firing rate from2.45±0.59 Hz to 2.76±0.61 Hz.The average increase was 17.68±3.68%,which was significantly decreased compared to that of orexin-A alone?Z=-3.576,P=0.000?.6.In another 7 orexin-A responsive neurons,micro-pressure ejection of orexin-A alone significantly increased the firing rate from 1.93±0.59 Hz to 4.14±1.32 Hz.The average increase was 122.65±30.38%.After completely recovery,co-application of TCS-OX₂-29and orexin-A increased the firing rate from 2.02±0.64 Hz to 4.97±1.59 Hz with the average of 133.80±26.38%.There was no significant difference between orexin-A alone and TCS-OX2-29 with orexin-A?Z=-0.575,P=0.565?.7.In 23 central amygdala neurons,orexin-B changed the firing rate from 3.05±0.54 Hz to 3.33±0.57 Hz?less than mean±2SD?.The average change was 14.19±4.78%,which was not significantly different from that of vehicle group?Z=-0.274,P=0.784?.8.In the open field test,compared with the normal saline group,bilateral microinjection of orexin-A into the central amygdala increased the time spent in the center zones significantly?P<0.001?.Co-application of SB-334867 with orexin-A significantly blocked orexin-A-induced effects on the time spend in the center zone?P=0.015 compared with orexin-A alone?.Orexin-A did not change the total distance moved in the open field test?P=0.172compared with vehicle?.9.In the elevated maze test,micro-injection of orexin-A into the central amygdala of rats significantly increased the time spent in the open arms?P=0.007compared with control?.Co-application of SB-334867 with orexin-A could significantly block orexin-A-induced effects on the time spent in the open arms?P=0.006 compared with orexin-A alone?.Orexin-A also increased the total number of entries in the open arms?P=0.036 compared with control?.Besides,orexin-A did not change the number of entries in closed arms?P=0.116 compared with control?.Conclusion:Our in vivo electrophysiological studies indicated that exogenous application of orexin-A increased the spontaneous firing rate of central amygdala neurons via OX1 receptors.Endogenous orexins also modulated the spontaneous firing of the central amygdala neurons through OX1 receptors.Further behavioral studies using open field test and elevated plus-maze test revealed that central amygdala orexin-A could improve emotional behavior in rats.Orexinergic system in the central amygdala may regulate emotional behaviors through its modulation of the spontaneous firing of central amygdala neurons.The present studies may provide a theoretical evidence for the involvement of orexinergic system in emotional regulation.