Spatial Distribution And Morphological Characteristics Of Main Neuron Types In Hypothalamus Orexin-distributed Areas During Adulthood And Development In Mice

The hypothalamus?HP?is a large and heterogeneous area with several distinct nuclear groups,as a major component of the diencephalon.It can receive a vast array of interoceptive and exteroceptive information and modulate cognitive,sleep-awake,and feeding behaviors.The function depends on the coordination and output of the cell network structure;however,because of the heterogeneity of the network and the complexity of cell structure,it leads to functional differences.The diversity of cell types is the basic element of the network.The main neurons that constitute the cell network structure of hypothalamus include orexin/hypocretin,melanin-concentrating hormone?MCH?,?-aminobutyric acid?GABA?and glutamate?Glut?neurons,which are distributed in lateral hypothalamic area?LHA?,perifornical area?PeF?,zona incerta?ZI?and dorsomedial hypothalamus?DMH?with close intercommunications,and maintain the function of hypothalamus together.Previous studies evaluated the distribution pattern of orexin and MCH neurons from a two-dimensional view.Orexin and MCH neurons are intermingled in the hypothalamus but distinguished from each other.Although the tridimensional?3D?spatial distribution pattern of MCH neurons has been investigated,the 3D distribution characteristics of orexin neurons and its spatial relationship with MCH neurons have not been well studied.Moreover,the co-distribution pattern of GABA neurons in orexin-distributed area is still not clear.In the study on the developmental expression of orexin and MCH neurons,it was found that the mRNA and protein expression of rat orexin could be detected at embryonic 12?E12?,while the mRNA and protein expression of MCH was detected at E16.The embryonic development of mouse orexin neurons was consistent with that of rats,while the distribution and development of MCH neurons in rats and mice were different.In rats,the peak of MCH neurons occurred slightly later than that of orexin neurons.In mice,the peak of MCH and orexin neurons coincided.However,in mice,whether the development of orexin and MCH neurons in postnatal period is synchronized,whether neuron subgroup migration happens and the quantity of neurons changes during their development to adulthood,and when the quantity,size and distribution distance of adult neurons reach consistency obtain no effective conclusion.In this study,we want to clarify the 3D structure of orexin and MCH neurons and their relationship with GABA neurons,the subgroup characteristics of distribution in each group and morphological changes in adulthood and developmental stages.We used immunofluorescence and 3D reconstruction method to explore the 3D structures of orexin and MCH neurons,quantified the number of the two type neurons,the size of single neuron and the length of distribution distance.We aim to present a morphological evidence of orexin,MCH and GABA neurons.The major results are as follows:1.Adult orexin and MCH neurons have different distribution patterns in different sub-brain regionsTo analyze distribution patterns of the two types of neurons in the HP-orexin region,a comparison was carried out in accordance with the Allen Brain Atlas of the mouse brain,and orexin and MCH neurons were restored into the brain region,followed by 3D reconstruction of neurons in different brain regions.Furthermore,the number,neuron size and distribution distance were statistically analyzed.The results showed that adult orexin neurons were mainly distributed in LHA,forming a dimensional central region.MCH neurons distributed around orexin neurons in a wrapped mode,and only a small number of MCH neurons were mixed with orexin neurons.The number of orexin neurons in unilateral HP of adult mice was2212±144,and the number of MCH neurons was 3032±82,the number of MCH neurons was about 1.3 times more than that of orexin neurons in the unilateral brain region.In addition,the distribution of orexin neurons on dorsal–ventral?D-V?axis was longer than that of MCH neurons,but shorter on rostra–caudal?R-C?axis and medial–lateral?M-L?axis than that of MCH neurons.Orexin and MCH neurons were mainly distributed in six brain sub-regions:LHA,ZI,DMH,posterior hypothalamus?PH?,preparasubthalamus?PST?and parasubthalamus?PSTN?.However,there were significant differences regarding distribution patterns and numbers in different sub-regions of the brain.Moreover,the number of two neurons in LHA was far more than that in other brain regions,and the number of orexin neurons was even higher.Meanwhile,the number of the two neurons in PSTN were relatively evenly distributed,while ZI,DMH,and PH was dominated by MCH neurons.2.Adult orexin,MCH,and GABA neurons exhibit different distribution patterns along the R-C axisTotal numbers of the two neurons were significantly different on R-C axis in each brain region.To obtain the quantitative distribution of neurons in different levels of R-C axis with higher accuracy,the R-C axis was segmented from the rostral side to the caudal side,with each of the unit strip?S?of about 120?m.The whole R-C axis was divided into 9 strips,starting from the rostral side?S1?.The general trend of the quantitative distribution of the two neurons was increased in the beginning and then decreased from S1 to S9 in different sub-regions.From the rostral side to the caudal side,the proportion of orexin neurons showed an increasing trend and the distribution was relatively concentrated,and the proportion of MCH and GABA neurons showed a decreasing trend mainly featured by the decrease of GABA neurons before the level with the biggest proportion of orexin neurons(the 5^th level).After the level with the biggest proportion of orexin neurons(the 5^th level),the proportion of orexin neurons decreased and the distribution was scattered,and the proportion of MCH and GABA neurons showed an increasing trend mainly featured by the increase of MCH neurons.At the rostral side(from the 2^th level to the 5^th level),MCH neurons were mainly distributed in the dorsal and medial sides of orexin neurons,with wide presence,where the relative cell density was relatively low.At the caudal side(from the 6^th level to the 8^th level),MCH neurons were mainly distributed in the lateral side of orexin neurons,with concentrated cell bodies,where the relative cell density was relatively high.No significant change was found in the density and distribution of GABA neurons.3.The orexin neurons projecting to the paraventricular thalamus?PVT?appear to increase first and then decrease along the R-C axis.In order to clarify the single output from orexin neurons to PVT neurons,we injected AAV-DIO-TVA-EGFP and AAV-DIO-RVG into PVT in Vglut-2-cre mice.Three weeks later,the retrograde tracing virus-rabies virus?RV?was injected into the same region,and single synaptic retrograde tracing after the formation of the starting cells.The distribution of orexin neurons targeting PVT could be directly observed.The results show,RV co-labeled with orexin neurons are located in LHA,and the distribution of first increase and decrease from the rostral side to the caudal side.In the presence of co-labeled neurons,the most common neurons were at the central level?bregma-1.70mm?of orexin neurons,with about 12 neurons,while the number of unilateral co-labeled orexin neurons occupies about 12%of total unilateral orexin neurons population?n=3?.4.The changes of neuron size in different developmental stages of orexin and MCH neuronsThere is no standard comparison of mouse brain atlases in different developmental stages.Therefore,we using Clifford method to divide orexin neurons in hypothalamus into DMH₂,PeF₂ and LHA₂ regions?different from brain areas in the second part?,and analyzed some indicators during different developmental stages.First,we analyzed the size of neurons by double-labeling immunofluorescence of orexin and MCH neurons.Under the same magnification,the changes in neuron size could be clearly observed.With transection major axis?TMaA?and transection minor axis?TMiA?as measures of neuron size,the results show:compared with postnatal 1?P1?,TMaA and TMiA of orexin and MCH neurons enlarged significantly in P7?n=3,P<0.05?,while TMaA and TMiA showed no obvious difference between P14 and P7?n=3,P>0.05?.Interestingly,TMaA and TMiA of the two neurons reached the size of adult neurons at P21.However,there was no agreement between P14 and P21 in the section length of neurons.Therefore,we cannot fully believe that in P21,the size of neurons is at the adult level.5.The changes of distribution distance in different developmental stages of orexin and MCH neuronsThen,further analysis was performed based on three distribution distances.The length of M-L axis and D-V axis of orexin neurons gradually increased to P14,while MCH neurons showed increased length of R-C axis only at P7 and P21.However,the R-C axis of the two neurons did not reach the stable adult state at P21.In addition,the growth rate of orexin neurons on the D-V axis was faster than that on the M-L axis and R-C axis,while the growth rate of MCH neurons on the M-L axis was faster than that on the other two axes.The comparison between the two neurons showed more regularity.From P1 to P56,obvious differences were detected in the length of D-V axis between the two neurons.The adult orexin and MCH neurons showed significant differences in the three distribution distances,which might be caused by a bias in the range of neuronal expansion during their development from P21 to P56.6.The changes of quantity and 3D distribution patterns at different developmental stages of orexin and MCH neuronsThe total number of orexin and MCH neurons reached adult level at P14,and did not changed at P21.The number in various brain regions during development showed some differences.The number of orexin neurons in PeF₂ from P7 to P14,and in LHA₂ from P1 to P7 increased significantly?n=3,P<0.05?.The number of MCH neurons in DMH₂ from P7 to P14,and in LHA₂ from P1 to P7 increased significantly?n=3,P<0.05?.In the whole development period,the number of orexin neurons in DMH₂ and MCH neurons in PeF₂slightly increased,without statistical differences?n=3,P>0.05?.These results indicated that although the number of orexin and MCH neurons reached the adult level at P14,it was caused by the increased number in different brain subregions.In conclusion,our study made a clear presentation and description of 3D distribution patterns of orexin and MCH neurons in adult and developmental mice,location relationship with GABA neurons in orexin-distributed areas.In addition,we described and counted the number of orexin and MCH neurons,the size of a single neuron and distribution distance during development in unilateral hypothalamus.We expected that this kind of basic morphological research will provide some new insights for future research regarding the functional properties and connectivity of these cells.