Whole-brain Three-dimensional Mapping Of Histaminergic Circuit In The Mouse Brain

Histamine(HA),an important neuro-transmitter and moderator in the mammalian brain,is generally believed to regulate a series of physiological functions and is associated with a multitude of pathological process.Histaminergic neurons are the exclusive resource of neuronal histamine,located only in the tuberomammillary nucleus(TMN)of the posterior hypothalamus and sending neuronal fibers to nearly all major brain regions,and they also receive neural inputs from several brain regions.However,the precise 3D output and input of histaminergic neurons in the brain have not been fully analyzed yet.Deconstruction of its precise neural circuit basis has great significance for the subsequent development of precise drug targets.In this study,we constructed trans genetic HDC-Cre ERT2 mice,combined with Ai47 reporter mice,virus tracing,molecular biology technology,and fluorescence micro-optical sectioning tomography(f MOST),to three-dimensional reconstruct and analyze the histaminergic circuits in the whole mouse brain.And further verified the function of key histaminergic neural circuits by using in vivo fluorescent histamine senser and optogenetic tools.Firstly,we made reconstruction and analysis of the histaminergic neurons of HDCCre CERT2:Ai47 mouse brains by f MOST system,finding that the number of histaminergic neuronal somata in the whole mouse brain is about 5000,and precisely reconstructing their distribution in the TMN.To reconstructed the output map of mouse central histaminergic neurons,we labeled the somata and fibers of mouse histaminergic neurons with anterograde tracing virus tools,and then quantified the distribution density of histaminergic nerve fibers in 588 nuclei of the whole mouse brain by calculating fluorescent pixels for the first time.We found that the histaminergic fiber distribution density in different brain regions varies,and then classified them into three groups by fiber density grade: “dense”,“moderate”,and “few”.Among them,the projection density of several sub regions of hypothalamus,and globus pallidus is high.Then we used the combination of in vivo fluorescent histamine sensor and optogenetic tools to verify the correlation between the histaminergic nerve fiber density and the amount of histamine release.We dynamic detected the histamine concentration change in the nuclei belong to “dense”(MS),“moderate”(Central amygdalar nucleus,Ce A),and “few”(CA3),finding that there was a positive correlation between the distribution density of histaminergic nerve fibers and the amount of histamine release in the downstream brain regions.To analyze the spatial fiber distribution characteristics of histaminergic neuron in the single cell level,we used the virus tool that can sparsely label neurons to track the individual complete structure of 60 histaminergic neurons.It was found that most histaminergic neurons do not have extensive and diffuse fiber projection,and the projection characteristics of histaminergic neuron groups significantly differ.However,taking MS and SC as typical downstream backtracking analysis,it shows that the structure of histaminergic neural output network is complicated,and there is no obvious spatial preference in the distribution of histaminergic neurons in different subgroups.Through the pharmacological manipulation and optogenetic technology,we found that histaminergic neural circuit directly from TMN to MS regulating the feeding behavior of mice through histamine release,and this function depends not on classic H1 Rs but H2 Rs.Finally,we reconstruct the input map of central histaminergic neurons in mice by using retrograde monosynaptic tracing virus,and then obtained the distribution and quantity information of histaminergic upstream neurons in the whole brain.We further used immunohistochemical staining to explore the types of neurons in the main upstream brain regions,finding that except the anterior hypothalamic region,the main input nuclei of histaminergic neurons were mainly excitatory.Cortical neurons usually have high-level function and complex structure,we thus finely analyzed the characteristics of histaminergic upstream neurons located in the Isocortex,finding that majority of them are located in the layer V and VIa.After tracing and reconstruction of the complete structure of 150 cortical neurons,we analyzed the fiber structure and terminal distribution of different cortical regions,and summarized the similarities and differences between them.Above all,using anterograde and retrograde labeling virus tools,combined with transgenic mice and f MOST technology,we obtained a comprehensive and single cell accuracy 3D map containing the output and input information of the whole histaminergic nervous system,and verified the correlation between histamine projection density and function by combining histamine probe,optogenetics and pharmacology.What’s more,the histaminergic neural circuit projected to MS closely regulates feeding behavior through H2 Rs.These findings provide a solid foundation for the further exploration of the histaminergic neural circuit functions,and also provide potential breakthroughs and basis for the further mechanism research and precise drug target development with histaminergic nervous system as the core.