OBJECTIVE Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions.Understanding the precise structure of histaminergic network is the cor-nerstone in elucidating its function.METHODS Herein,using novel HDC-CreERT2 mice and genetic labeling strategies,we reconstructed a whole brain 3D structure of histaminergic neurons and their outputs at 0.32×0.32×2 μm3 pixel resolution with a cutting-edge fluorescence micro-optical sectioning tomography system(fMOST).And we dissect an appetite control circuit originating from the TMN to medial septal nucleus(MS)using fiber photometry,optogenetics,and chemogenetics interfer-ence.RESULTS We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions.The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stim-ulation or physiological aversive stimulation.Moreover,we reconstructed fine morphological structure of 60 hista-minergic neurons via sparse labeling,and uncovered the largely heterogeneous projection pattern of individual his-taminergic neuron.Lastly,we found that MS-projecting histaminergic circuit is functionally inhibited during food consumption,and bidirectionally modulates feeding behavior via downstream H2,but not H1,receptors on MS glutamatergic neurons.CONCLUSION Collectively,this study reveals an unprecedented whole-brain quanti-tative analysis of histaminergic projections at the meso-scopic level,providing a foundation for future functional histaminergic study.And we also demonstrate that this MS-projecting histaminergic circuit is critically involved in feeding,and H2Rs in MS glutamatergic neurons is a promising target for treating body weight problems.

Objective To establish an in vitro model of cultured mouse testis using rotary aerobic culture. Methods Rotary aerobic incubation with optimized culture conditions was used for in vitro culture of mouse testis, and the morphology of the cultured testicular tissues was compared with that cultured in Transwell chambers. The changes in the testicular tissue structure were examined using HE staining, and the cell proliferation was assessed with BrdU staining. Testosterone concentrations in the culture medium were tested with radioimmunoassay and the expression of the functionally related proteins in the testis was detected using immunohistochemistry. Results The testicular tissue cultured by optimized rotary aerobic culture presented with more intact histological structure with the size of the testis ranged from 0.3 to 0.8 mm3. In the two culture systems, the prolifeation index of the spermatogonia increased and that of Sertoli cells decreased with time, and such changes in spermatogonia and Sertoli cell proliferation indices became statistically significant at 3 days (P<0.05) and 5 days (P<0.05) of culture, respectively, as compared with those at 1 day. The concentration of testoerone in the culture media decreased significantly with incubation time (P<0.05). At 3 days of culture, the protein expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase and cholesterol side-chain cleavage enzyme was detected in Leydig cell cytoplasm and vimentin expression in Sertoli cell cytoplasm. Conclusion An in vitro model of cultured mouse testis has been successfully established using rotary aerobic incubation.

Objective To establish an in vitro model of cultured mouse testis using rotary aerobic culture. Methods Rotary aerobic incubation with optimized culture conditions was used for in vitro culture of mouse testis, and the morphology of the cultured testicular tissues was compared with that cultured in Transwell chambers. The changes in the testicular tissue structure were examined using HE staining, and the cell proliferation was assessed with BrdU staining. Testosterone concentrations in the culture medium were tested with radioimmunoassay and the expression of the functionally related proteins in the testis was detected using immunohistochemistry. Results The testicular tissue cultured by optimized rotary aerobic culture presented with more intact histological structure with the size of the testis ranged from 0.3 to 0.8 mm3. In the two culture systems, the prolifeation index of the spermatogonia increased and that of Sertoli cells decreased with time, and such changes in spermatogonia and Sertoli cell proliferation indices became statistically significant at 3 days (P<0.05) and 5 days (P<0.05) of culture, respectively, as compared with those at 1 day. The concentration of testoerone in the culture media decreased significantly with incubation time (P<0.05). At 3 days of culture, the protein expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase and cholesterol side-chain cleavage enzyme was detected in Leydig cell cytoplasm and vimentin expression in Sertoli cell cytoplasm. Conclusion An in vitro model of cultured mouse testis has been successfully established using rotary aerobic incubation.

ObjectiveTo establish a method for seahorse identification based on graphene oxide fluorescence sensing technology， and to provide a new research idea for identification of traditional Chinese medicine. MethodThe fluorophore FAM was labeled at the 5' end of the specificity upstream primer Ja-F of Hippocampus japonicus as the nucleic acid probe FAM-ssDNA （single strand DNA）. The recognition site of RNA polymerase Ⅱ was added to its specific downstream primer Ja-R as Ja-R1. The seahorse samples were amplified with Ja-F/Ja-R1 primers， and the ssDNA of H. japonicus was obtained by reverse transcription of the amplification products using vitro transcription method. The 20 μL nucleic acid probe FAM-ssDNA （500 nmol·L^-1） was incubated at 90 ℃ for 5 min， and was gradually cooled to room temperature. Different volume of graphene oxide solution （100 mg·L^-1） and Tris hydroxymethyl amino methane HCl （Tris-HCl） buffer （50 mmol·L^-1） were added into each probe solution to make a final reaction volume of 1 mL. The fluorescence intensity of each sample was measured after mixing and placing different times at room temperature away from the light. So that the most appropriate graphene oxide concentration and reaction time were screened for constructing the best nucleic acid probe-graphene oxide biosensor. Adding probe complementary sequence FAM-ssDNA-match solution into the nucleic acid probe-graphene oxide solution， the fluorescence intensity of the reaction mixture was measured after being placed different times at room temperature. Therefore， the optimal reaction time of fluorescence recovery was screened and the feasibility of the sensor was tested. The sensitivity was detected via adding ddH₂O as the blank control and different concentration H. japonicus ssDNA into each nucleic acid probe-graphene oxide solution， respectively. Finally， the commercial hippocampal were identified using the optimal experimental condition， and the feasibility of this method for the identification of Chinese medicinal materials was verified. ResultThe fluorescence of 1 mL reaction mixture including 10 nmol·L^-1 nucleic acid probe FAM-ssDNA and 12 mg·L^-1 go solution for 20 min at room temperature away from the light could be completely quenched. Feasibility test of the biosensor showed that when probe complementary sequence FAM-ssDNA-match solution （final concentration 90 nmol·L^-1） was added to the biosensor solution and reacted 1 h reaction at room temperature， the fluorescence signal was significantly enhanced. Sensitivity test showed that the minimum concentration of ssDNA detected by this method was about 10 mg·L^-1. This method was used to detect commercial seahorses， and only H. japonicus samples had obvious fluorescence signal. ConclusionThe graphene oxide-based fluorescent sensing technology could be used for zoological origin survey of commercial hippocampus.