小鼠下丘脑外侧区食欲素神经元向中脑腹侧被盖区的投射

Orexinergic projection from the lateral hypothalamic area to the ventral tegmental area in mice

目的:研究小鼠下丘脑外侧区(LHA)内食欲素(orexin)神经元向腹侧被盖区(VTA)的投射及VTA靶神经元的类型及其所占比例。方法:利用脑立体定向技术将逆向示踪病毒AAVretro-DIO-mCherry注射至Hcrt-Cre小鼠的VTA,待病毒表达之后制备包含LHA的间脑切片,用免疫荧光染色法进行orexin阳性神经元染色,计算病毒表达阳性神经元总数占orexin染色阳性神经元数量的比例。将顺向示踪病毒AAV2/1-Ef1α-DIO-EGFP注射至Hcrt-Cre小鼠的LHA,同时在VTA注射含神经元特异性启动子(hSyn)的表达Cre酶的重组腺相关病毒AAV2/9-hSyn-Cre感染VTA的神经元。病毒表达后切取含VTA的中脑切片,用免疫荧光染色法分别进行多巴胺、谷氨酸和γ-氨基丁酸(GABA)阳性神经元染色,计算各类免疫荧光染色阳性同时病毒表达阳性的神经元的数量占病毒表达阳性神经元总数的比例。结果:LHA内有(52.41±15.22)%的orexin阳性神经元向VTA发出投射。在接受orexin传入终末投射的VTA的靶神经元中,多巴胺能神经元占(62.04±12.87)%,谷氨酸能和GABA能神经元分别占(25.22±13.09)%和(17.16±4.77)%。结论:LHA中半数以上的orexin神经元向VTA发出投射;VTA中谷氨酸、GABA、多巴胺阳性神经元均接受来自LHA的orexin投射纤维的支配,其中尤以多巴胺能神经元为主。

Objective:The projections from orexin neurons in the lateral hypothalamic area(LHA)to the ventral tegmental area(VTA)and the types and percentage of target neurons in the VTA were studied in mice.Methods:The retrograde tracing virus AAVretro-DIO-mCherry was injected into the VTA in Hcrt-Cre mice by stereoscopic positioning technique.After the virus was expressed,the diencephalon sections containing the LHA were prepared,and the orexin-positive neurons were stained by immunofluorescence staining.The proportion of the total virus-expressed neurons to the orexin-positive neurons was calculated.The anterograde tracing virus AAV2/1-Ef1α-DIO-EGFP was injected into the LHA in Hcrt-Cre mice,and the recombinant adeno-associated virus expressing Cre enzyme with neuron specific promoter(hSyn)(AAV2/9-hSyn-Cre)was simultaneously injected into VTA to infect neurons in the VTA.After the virus was expressed,the midbrain sections containing VTA were prepared,then dopamine,glutamate,andγ-aminobutyric acid(GABA)-positive neurons were stained by immunofluorescence staining,respectively.The proportions of virus-expressed neurons with immunofluorescent staining for each antibody to total virus-expressed neurons were calculated.Results:There were(52.41±15.22)%of total orexin-positive neurons in the LHA projecting to the VTA.The proportions of dopaminergic,glutamatergic and GABAergic neurons in VTA receiving orexin afferents were(62.04±12.87)%,(25.22±13.09)%and(17.16±4.77)%,respectively.Conclusion:More than half of the orexin neurons in the LHA project to the VTA.Dopaminergic,glutamatergic,and GABAergic neurons in the VTA are all innervated by orexinergic afferents and among them the innervation to dopaminergic neurons is predominant.