The Subregional Difference Of Reciprocal Projections Between Lateral Hypothalamic Orexin Neurons And Medial Prefrontal Cortex

The medial prefrontal cortex(mPFC)is essential for higher cognitive functions,such as awareness,attention,decision making,learning and memory.Anatomically,the mPFC can be classified as anterior cingulate cortex(AC),prelimbic cortex(PL)and infralimbic cortex(IL)at dorso-ventral axis.Also,it is divided into rostral,middle and caudal part according to different rostro-caudal levels.Functional difference exists in the m PFC subregions.The AC is associated with cognitive accuracy;the PL plays an important role in emotion-related cognitive processes;the IL is essential for controlling impulsivity.From rostro-caudal aspect,the activation of m PFC exhibits apparent rostro-caudal hierarchy in goal-directed behaviors.Reward-related neural activities are highest in the rostral part of the m PFC and decrease progressively towards the caudal m PFC.The accomplishment of m PFC-related functions depends on efficient arousal state,which is the result of excitation of mPFC by specific arousal-promoting systems in the brain.At present,the relationship between arousal-promoting systems and subregional difference of the m PFC constructions and functions is still unclear.Orexin neurons in the lateral hypothalamus(LH)have been recognized as key components in generating and maintaining of arousal.Previous studies demonstrated that LH neurons project to the mPFC with distinguishable subregional diversity.Retrograde tracing results showed a higher density of LH-originated fibers in the AC region than in the PL and IL region.However,the distribution patterns of fibers from the LH in rostro-caudal axis of the mPFC and the distribution characteristics of mPFC-projecting neurons in the LH remain unknown.It was reported that LH neurons also receive projections from the mPFC neurons.But the distribution of LH-targeting neurons in different m PFC subregions needs to be further clarified.As one of the major neuronal types in the LH,orexin neurons are widely projected to the whole central nervous system.Earlier studies have identified the distribution of orexin receptors(including type 1 and 2,OX1 R and OX2R)and orexinergic fibers in the mPFC.Results from our lab showed that orexin-A increases the excitability of m FPC neurons by acting the membrane ionic channels.Therefore,the arousal-promoting orexin system plays an essential role in regulating the mPFC activities.Nevertheless,little is known about the subregioal difference of mPFC in receiving the orexinergic innervation and the projections from mPFC to orexin neurons.In this study,we used neuronal tracing(anterograde and retrograde)methods and inmmnohistochemistry to explore the reciprocal projections between LH orexin neurons and the mPFC subregions.The main results are as follows:1.The LH neurons project to the m PFC with an increased fiber density from rostral to caudal part.In order to clarify the subregional difference of LH-originated fiber density in the m PFC,we injected the anterograde tracer biotin dextran amine(BDA)in the LH.Immunohistochemical results showed that BDA-positive fibers were identified in the mPFC with an apparent subregional difference.Firstly,the BDA-positive fibers were distributed in the PL and IL subregion with an increased density from rostral to caudal part,indicating that LH neurons project to the m PFC with a rostro-caudal hierarchy.Secondly,the density of positive fibers was highest in AC region,and decreased consecutively in PL and IL subregion,which was consistent with previous results.2.The number of mPFC-targeting neuronal soma decreased from rostral to caudal part of the LH.(1)Whole brain mapping of monosynaptic input to the m PFC subregionsRabies viruses(RV)were injected into the m PFC subregions to selectively label the monosynaptic input of infected neurons.As for PL subregion,the highest monosynaptic input was observed in hippocampus,and decreased successively in cingulate cortex,thalamic nucleus,motor cortex,diagonal band nucleus,orbital cortex,preoptic area,claustrum,lateral hypothalamic area,pallidum and amygdaloid nucleus.The highest amount of monosynaptic input to IL subregion was detected in hippocampus,and decreased consecutively in thalamic nucleus,cingulate cortex,lateral hypothalamic area,motor cortex,preoptic area,orbital cortex,amygdaloid nucleus,diagonal band nucleus,and perirhinal cortex.(2)The amount of mPFC-targeting neuronal soma decreased from rostral to caudal part of LH.To illustrate the distribution pattern of neurons that projecting to the m PFC,we calculated the number of RV-positive neuronal soma in rostral,middle,and caudal part of LH.After injection of the RV in PL or IL subregion,we found that the amount of neurons that expressing RV decreased from the rostral to caudal part of LH.3.The LH receives projections mainly from PL subregion in mPFCIn order to demonstrate the projections from different mPFC subregions,we firstly injected the anterograde tracer BDA into the mPFC,and the BDA-positive fibers were detected in the LH according to the inmmunohistochemical results,which indicated that LH receives projections from m PFC.Then,the retrograde tracer CTB was injected into the LH and CTB-positive neurons were counted to further clarify the subregional difference of distribution of mPFC-projecting neurons in the LH.Interestingly,the amount of CTB-positive neurons was significantly higher in the PL than in the AC and IL subregion.Thus,the neurons that project to the LH are mainly located in the PL subregion.4.The distribution of orexinergic fibers and OX1 Rs exhibits rostro-caudal increasing hierarchy in the m PFCThe BDA-mediated anterograde tracing is unable to distinguish specific neuronal types.We incubated the aforementioned slices with BDA tracing experiments in the orexin-A antibodies to specify the distribution of orexinergic fibers in the mPFC subregions.Results showed that the density of orexinergic fibers was higher in caudal AC than in the middle AC subregion.In terms of the PL and IL region,a rostro-caudal increasing hierarchy was observed concerning the orexinergic fiber density.In the next step,we investigated the expression of OX1 Rs in different mPFC subregions by incubation of slices with OX1 R antibodies.Interestingly,compared with IL region,the level of OX1 Rs was expressed significantly higher in AC and PL region.In consistent with the distribution of orexinergic fibers,the amount of OX1 R expression was increased from rostral to caudal part in these three subregions.In terms of different layers,abundant OX1 Rs was detected in layer II/III and V in the mPFC,and the amount of OX1 Rs was increased along the rostro-caudal axis.5.PL and IL receive monosynaptic input from orexin neuronsThe aforementioned PL and IL slices with RV injection in the LH were incubated in the orexin-A antibodies.We found that RV positive neurons were co-labeled with orexin-A positive neurons,indicating that orexin neurons monosynaptically project to the PL and IL subregion.6.Orexin neurons receive projections from m PFC neuronsThe LH slices with experiments of BDA injection in the mPFC were incubated with orexin-A antibodies.Immunohistochemical results demonstrated that orexin neurons are surrounded by abundant BDA-positive fibers,indicating that orexin neurons receive innervation from the m PFC neurons.In the present study,we found that,on one hand,LH orexin neurons project to the three m PFC subregions with an increased fiber density from rostral to caudal part,while the number of m PFC-targeted LH neurons is distributed in a rostro-caudal decreasing pattern.Also,the distribution of OX1 Rs is increased from rostral to caudal mPFC,regardless of AC,PL,IL subregion or different layers.On the other hand,the mPFC neurons,which are mainly located in the PL subregion,send their fibers to the LH,and connect with orexin neurons.These results provide anatomical and morphological evidences for further clarifying the neural modulatory role of arousal-promoting orexin system in regulating the m PFC-related higher cognitive functions.