Galaninergic Neurons In The Ventrolateral Preoptic Nucleus Inactivate Histaminergic Neurons In The Tuberomammillary Nucleus Through GalR1 To Promote Slow Wave Sleep

A"flip-flop"switch model of sleep-wake transitions hypothesizes that the ventrolateral preoptic nucleus（VLPO）and tuberomammillary nucleus（TMN）mutually inhibit each other to realize the conversion between sleep and wakefulness.Approximately 80%of the neurons in VLPO contain the inhibitory neurotransmitter gamma-aminobutyric acid（GABA）and galanin（GAL）.The inhibitory effect of the VLPO GABAergic neurons on the TMN histaminergic neurons to promote sleep has been confirmed by several studies,while few evidences reveal whether the VLPO galaninergic neurons are involved in the inactivation of TMN histaminergic neurons to promote sleep also.Moreover,the neural pathway of the VLPO galaninergic neurons projecting to TMN histaminergic neurons,and exact subtype of GAL receptors（GalRs）involved in the inactivation of TMN histaminergic neurons remain to be elucidated.Electroencephalogram（EEG）and electromyography（EMG）were used to define sleep-wake states including wakefulness（W）,slow wave sleep（SWS）and paradoxical sleep（PS）in the present study.The EEG power spectrum at the different frequency ranges（delta:0.5-4 Hz,theta:4.5-8.5 Hz,alpha:9-14 Hz,beta:14.5-30 Hz）were analyzed by the fast Fourier transform.Firstly,the effects of GAL（0.1,0.25 and 0.5 nmol）on the amount of each state,architecture of sleep-wake states and EEG power spectrum were investigated after central administration in freely moving rats during active phase（20:00-08:00 h）.The immediate early gene c-Fos as the marker of neuronal activation was performed with immunohistochemistry to identify the distributions of GAL-induced activated and inactivated neurons.Dual-immunofluorescence was employed to identify the subtype of GalRs in the neurons that play a key role in sleep-wake transitions.Secondly,based on the results above,the distributional characteristics of galaninergic neurons in the VLPO were labeled with immunohistochemistry for identifying the injecting site of virus tracer,and then the methods of adeno-associated virus（AAV）anterograde and retrograde tracing combined with dual-immunoflurescences were used to determine the pathway of VLPO galaninergic neurons projecting to TMN histaminergic neurons.Finally,TMN microinjection of GAL,GalR1 agonist,combined with non-selective GalRs antagonist or selective GalR2 antagonist were respectively microinjected into the bilateral TMN to clarify which subtype of GalRs was involved in the regulation of sleep-wake states.The bilateral TMN administration of GalR1agonist was performed to investigate whether it attenuates the wakefulness elicited by histamine H3 receptor（H3R）antagonist ciproxifan（CIP）which disinhibits TMN histaminergic neurons to induce a histamine（HA）release.Results:GAL（0.1-0.5 nmol）central action,as compared with saline（Sal）,dose-dependently shortened sleep latency,and increased SWS accompanied with an enhancement of EEG delta（0.5-4 Hz）activities and decreased W accompanied with a reduction of EEG beta（14.5-30 Hz）activities.The amount of SWS after GAL 0.25 and 0.5 nmol administration for 6 h respectively increased by 9.4%（P<0.01）and 20.5%（P<0.001）.The increased SWS by GAL at dose of0.25 nmol was due to prolonged episode mean duration（2.63±0.23 min vs.2.34±0.19 min,P<0.05）,and at dose of 0.5 nmol was due to increasing both episode number（79.13±7.92 vs.71.36±4.83,P<0.01）and mean duration（2.62±0.28 min vs.2.34±0.19 min,P<0.05）.Meanwhile GAL 0.25 and 0.5 nmol respectively decreased W by 12.4%（P<0.01）and 22.3%（P<0.001）through decreasing episode mean duration.The increased SWS induced by GAL was completely blocked by M40（2 nmol）,a non-selective GalRs antagonist.Central administration of GAL induced an increase of the number of Fos immunoreactive（-ir）neurons by 166.4%（57.8±3.6 vs.21.3±2.3,P<0.01）in the VLPO and a decrease of that by 65.1%（20.2±3.0 vs.56.8±4.5,P<0.01）in the TMN.Dual-immunoflurescences demonstrated that the percentage of Fos-ir neurons in the TMN histaminergic neurons was decreased by 77.2%（P<0.01）.The 72.8±3.0%of TMN histaminergic neurons expressed GalR1.The 82.8±2.3%of neurons in the VLPO were GAL-ir neurons.The density fibers labeled with AAV9-CAG-GFP were found in the ipsilateral TMN histaminergic neurons following VLPO injected with the anterograde tracer.The neurons tagged with rAAV_（retro）-hSyn-mCherry co-expressing in GAL-ir neuron in the VLPO were observed following the TMN injected with the retrograde tracer.M617（0.25 nmol/side）,a selective GalR1 agonist,bilaterally injected into the TMN decreased the SWS latency,and induced an increase in SWS for 12 h and a reduction in W.The M617-induced SWS was blocked by M40.Bilateral administration of GAL（0.25 nmol/side）into the TMN enhanced SWS also,the GAL-induced SWS was blocked by M40,but not by M871,a selective GalR2 antagonist.CIP（1 mg/kg,i.p.）promoted W accompanied with an enhancement of EEG beta activities,meanwhile,reduced SWS.The CIP-induced W and EEG beta activities were attenuated by M671 bilaterally injected into the TMN.Conclusion:GAL central action promotes SWS,meanwhile W is reduced.The number of activated neuron following GAL central administration significantly increases in the VLPO and decreases in the TMN histaminergic neurons.A great number of the TMN histaminergic neurons express GalR1.The VLPO galaninergic neurons project to the TMN histaminergic neurons.GAL action on the TMN through GalR1 attenuates the wakefulness induced by HA release.These results suggest that the VLPO galaninergic neurons project to the TMN and inactivate histaminergic neurons through GalR1 to promote SWS.