The Neural Mechanism Of Feeding Behavior And Attention Dysfunction In Extreme Temperature Environment

The appropriate ambient temperature is the necessary condition for life to survive.The extreme ambient temperature has an important influence on the nervous system.Both the instinctive behavior and advanced cognitive behavior are affected by the ambient temperature The nervous system performs an adaptive adjustment of the body behavior through the sensation of the temperature change,in order to better adapt to the extreme environment According to the professional background and research direction,this paper analyzed the adaptive adjustment ability of the nervous system at ambient temperature from the instinctive feeding behavior and advanced attention function of mouse and humanPart 1 The neural circuits for temperature-dependent feeding behavior mediated by ventromedial preoptic areaFrom rodents to humans,there is a well-developed system of temperature regulation.The stability of core body temperature is a necessary condition for the physiological activities of the body's cells,tissues and organs.The thermogenesis and dissipation that the body brings through food intake and energy consumption are the decisive factors of constant body temperature.When the ambient temperature changes,the body's thermal demand and heat load change,correspondingly,the body will adapt to adjust food intake and energy consumption,but the the neural mechanism of the adaptive adjustment is not completely clear.As the center of temperature sensation and regulation,the preoptic area(POA)receives the internal temperature signals of peripheral spinal cord,lateral parabrachial nuclei,and reacts differently to the dorsomedial hypothalamus(DMH)to regulate the heat production and energy metabolism rate through brown adipose tissue.On the other hand,after facing different ambient temperature,the body will make temperature-dependent adjustment to food intake according to energy demand and thermal load,for example,the amount of intake in high and low temperature environment will increase and decrease accordingly,but the underlying neural mechanism is not clear.Due to the unclear mechanism,we still still can not fully understand the neural mechanism of temperature control,but also lack of in-depth understanding and coping strategies for clinical febrile feeding abnormalities and other diseases,such as inflammatory anorexia,cancer and late-stage AIDS fever anorexiaThe ventromedial preoptic area(VMPO)which located at the edge of the third ventricle,with weak blood-brain barrier,has a large number of receptors to feel cerebrospinal fluid osmosis pressure receptors,sex hormones,glycopeptides,leptin and so on,and plays a vital role in the regulation of thermal balance,water salt balance,mating behavior,maternal behavior and other aspects.After structural damage to the perventricle circumstites of the third ventricle in VMPO,the animals showed a balance disorder in the intake of food and water.Injecting growth hormone release factors or glycerides in the VMPO increases animal intake,and activating or blocking the appetite hormone Orexin-A receptor at the VMPO site can also affect feeding behavior.However,it is not clear whether VMPO does have the effect of regulating feeding behavior and the specific neural circuit remains unknownBased on the backgrounds above,the purpose of this paper is to analyze the neural circuit mechanism of feeding behavior mediated by VMPO sensing ambient temperature information,and to reveal the temperature sensation characteristics and functional significance of temperature-dependent feeding behavior mediated by VMPO.We conducted the following experiments:1.First of all,we use morphological dyeing technology,among the multiple sub-regions in the POA sensitive to the temperature we found the expression of hunger-related C-FOS in the VMPO.And then we identified the specific neuron types in the VMPO which may be involved in feeding behavior through immunofluorescent co-localizing,fiber photometry and optogenetic technology;2.Then,using anteograded tracing technology,we identified the brain regions which the VMPO projects to.Combined with optogenetic,chemicogenetics and neuro-induced apoptosis technology,we screened out the regions one by one which VMPO orchestrates to find the possible regions which regulate feed behavior;3.After that,using morphological retrograded tracing technology,we identified the morphological basis of VMPO projection pathway.Combined with C-FOS dyeing and optogenetic technology,we revealed the temperature perception characteristics and functional significances of VMPO projection pathway;4.Finally,using in vivo fiber photometry and retrograded tracing technology,we further uncovered the temperature perception characteristics of the VMPO projection pathways,which were functional related with the feeding behaviorThe main findings were:1.Under different temperature environment(hyperthermia:33.5±2?,normothermia:25±2?,hypothermia:10±2?)for a short period(2h)and long time(12h)temperature exposure,the mice showed a feeding behavior dependent on the temperature.Compared with normothermia,the amount of food intake during hypothermia increased significantly,and the food intake in the hyperthermia was significantly reduced.Further,we measured the levels of the main appetite-related hormones in the blood of mice exposed at different temperatures within 12h,including ghrelin,leptin,and neuropeptide Y.We found that blood hormone levels did not show significant temperature dependence.Then we investigated the expression of C-FOS,a marker of neuronal activity in POA after 12 hour-fasting,and found that only VMPO in the multiple temperature-sensitive subregions of POA expressed a large number of C-FOS after fasting.Subsequently,on Ai9::Vglut2-cre and Ai9::Gad2-cre double-positive transgenetic mice,we found that the fasting-induced C-FOS at VMPO site had a high co-locolization with excitatory Vglut2-positive neurons.The co-locolization with Gad2-positive neurons was lower.To this end,we further used fiber photometry technology to record the calcium signal activity in the VMPO after injection calcium indicator Gcamp6s in Vglut2-cre and Gad2-cre mice.The result showed that the calcium signal in the Vglut2-cre mice gradually decreased during feeding after 12 hour-fasting.But in the Gad2-cre mice,there was no significant change during feeding after 12h fasting.These results indicated that Vglut2-positive neurons in VMPO site presented excited state in hunger,and their activity is gradually reduced when the hunger state was relieved during feeding,which meaned that Vglut2-positive neuron in VMPO was related to the hunger state of mice and may be involved in the regulation of feeding behavior.Finally,the optical sensitive virus CHR2 was injected into the VMPO of the Vglut2-cre and Gad2-cre mice,and the neurons of Vglut2 and Gad2 in the VMPO were activated by optogenetic techniques,and the positive neurons of Vglut2 in the VMPO were found to decrease the rectal temperature of the mice.At the same time,the feeding behavior also decreased.But the activation of Gad2-positive neuron did not alter the body temperature or feeding behavior significantly,suggesting that the Gad2 positive neurons in the VMPO may not be involved into the regulation of feeding behavior;2.We further perform anteograded tracing and functional analysis on the projection pathway of excitory glutamatergic neurons in VMPO,aiming to find the projection pathway of the VMPO which could specifically regulate feeding behavior without causing other nonspecific behavior indicators to change.We injected the anteograded tracing virus AAV-DIO-CHR2-EYFP into the VMPO of Vglut2-cre mouse,and we found that the VMPO glutamate neurons project from the rostral to caudate of the hypothalamus and thalamus,with multiple nuclei included,specifically the nuclei of thermal sensation and regulation,for example,the median preoptic nucleus(MnPO),the dorsomedial hypothalamic nucleus(DMH),the nuclei of feeding behavior,such as the lateral hypothalamic area(LH),the paraventricular nucleus of hypothalamus(PVH),the arcuate nucleus(ARC),the tuberal nucleus(TN)and so on,and the motivation and reward brain nuclei,such as the periaqueductal gray(PAG),the ventral tegmental area(VTA),and stress and emotion regulation-related brain nuclei such as the bed nucleus of the stria terminalis(BNST),the anterior paraventricular nucleus of thalamus(PVA),the paraventricular thalamic nucleus(PVT),amygdalohippoal area(AHiPM).Using optogenetic techniques to activate CHR2 on the fiber terminals of the VMPO downstream pathway,and to record the amount of food intake in mice within 2 hours after 12h fasting,we found that activating the VMPO? ARC pathway significantly increased the food intake,while activating the VMPO?PVH pathway significantly inhibited food intake.The mice that activated the two pathways,VMPO? ARC and VMPO?PVH,showed specific changes in feeding,without core body temperature,and other behavioral abnormalities.The VMPO?DMH pathway showed inhibition on the feeding behavior,but at the same time the mice also showed significant cold defense(nesting behavior).Furthermore,we used the technique of transsynaptic virus and apoptosis,with injection of the recombinant enzyme AAV1-cre of the transsynaptic in the VMPO site,and the AAV-flex-tacasp3 virus in the ARC,PVH and DMH sites,inducing the apoptosis of post-synatic neurons in the ARC,PVH and DMH.The result further confirmed that VMPO?ARC,VMPO?PVH can specifically regulate feeding behavior,without causing changes in core body temperature and other indicators and behaviors,while the VMPO?DMH pathway had no specific effect on the feeding behavior,but rather caused a partial loss of body temperature regulation during high and low temperature conditions.Finally,we used chemicogenetic techniques to inject the recombinant enzyme AAV 1-cre,which was transsynaptic,and the AAV-DIO-hM4Di-mcherry virus in the ARC and PVH sites,to inhibit the VMPO?ARC,VMPO?ARC.After the inhibition of the VMPO-ARC pathway,the mice showed reduced in the amount of food consumed in different temperature environments,while after the inhibition of the VMPO?PVH pathway,the food intake of mice during different temperature environments were increased,and the mice have significant normal weight and body temperature regulation.Thus we further clarified that VMPO regulated the feeding behavior mainly through VMPO?ARC,VMPO?PVH.In addition,according to the food intake after inhibition,the VMPO?ARC,VMPO?PVH pathways did not cause the mouse serious anorexia and excessive feeding,Therefore,these two pathways seemed to regulate the food consumption within an appropriate amount;3.Using retrograded morphology tracing technology and optogenetic regulation technology we further uncovered the thermal sensation characteristics of the VMPO?ARC and VMPO?PVH pathways.Using the combination of AAVretro-cre recombinant enzyme and Ai9 reporterline,we found that a large number of retrograded neurons in the VMPO sites were projected to the ARC and PVH sites,and were co-localized with fasting-induced C-FOS.Further through morphological staining,we found that the cells of VMPO projected to the ARC were co-localized only with C-FOS induced by cold stimulation(10±2?,2h),but no co-localization with C-FOS induced by hot stimulation(33.5±2?,2h).There are co-localization for the neurons of VMPO projected to the PVH with the C-FOS induced both by heat and cold.We further used optogenetic technology combined with retrograded tracing technology to inject the AAVretro-DIO-flp at ARC and PVH sites,and inject AAV-fDIO-CHR2 virus at VMPO site to stimulate the neurons retrograded from ARC and PVH retrograde to VMPO site.We found that after the activation of the projected neurons of the two pathways in the VMPO site,the mice showed a consistent thermal autonomic regulation,with a significant decrease in core body temperature,faster heat dissipation,and a decrease in brown adipose tissue(BAT)heat production.However,after the activation of the two pathways,the mice showed inconsistent behavioral thermoregulation patterns.In the two-box place preference test(cold box 12?,hot box 38?),the mice preferred to stay at thehot box before the light stimulation of the two pathways,and the stay time accounted for about 80%.After using optogenetic activation on the VMPO?ARC,there was no significant change in mouse position preference,still preferred to stay at the hot box,while VMPO?PVH was activated,the stay time in the cold box increased significantly,the hot box stay time decreased significantly,each accounted for about 50%.The above results show that the two paths of VMPO?ARC and VMPO?PVH have completely different thermal sensation characteristics.VMPO?ARC may reflect a kind of sensing cold information for the VMPO,promoting feeding behavior.And VMPO?PVH has a more complex temperature characteristics,may reflect a kind of bidirectional sensation to the hot and cold information to the balance of feeding behavior.4.Combined with the retrograded tracing technique,we further used the calcium activity indicator Gcamp6m to record the calcium signal response of VMPO?ARC and VMPO?PVH pathway of free-moving mice under different temperature conditions.We injected retroactive recombinant enzyme AAVretro-cre at ARC and PVH sites,cre-dependent AAV-DIO-Gcamp6m at VMPO site,and recorded the signal response of Gcamp6m on the retrograded neurons at VMPO site via optical fiber.We recorded the activity of calcium signals in mice facing local and systemic temperature stimulation using a temperature-controlled metal plate(hyperthermia:40?,normothermia:25?,hypothermia:10?)and in the thermostatic control chamber(hyperthermia:35?,hypothermia:10?)respectively.The results showed that VMPO?ARC has two completely different thermal sensation properties than VMPO?PVH.VMPO?ARC showed significant calcium signal increase in the face of systemic cold stimulation,while VMPO?PVH has significant bidirectional temperature sensation characteristics,with significant calcium signal increase in the face of systemic hot stimulation,but a significant decrease in calcium signal in the face of systemic and local cold stimulation.Both pathways showed a weak or no significant calcium signal response in the face of local stimulation(except for local cold stimulation by the VMPO?PVH pathway),and only significant changes in calcium signal when systemic ambient temperature stimulation was presented.The different thermal sensation characteristics of the VMPO?ARC and VMPO?PVH pathways may be the functional basis for their regulation of temperature-dependent feeding behavior.Summarizing the above results,the glutamatergic neurons in the VMPO site and their projection pathways were closely related to temperature-dependent feeding behavior.As an important area of thermal sensation and regulation,VMPO regulated the feeding behavior by the glutamatergic VMPO?ARC and VMPO?PVH projection pathways after sensing the thermal stimulation of the whole body.The role of VMPO in temperature-dependent feeding behavior and the analysis of the mechanism of neural circuits will not only help us to further deepen our understanding of the relationship between feeding and thermal regulation,but also help to provide a new entry point and theoretical basis for the treatment of clinical febrile anorexia.Part 2 The resting-state neural mechanism of selective attention deficits during hyperthermiaAttention is an important cognitive ability.Better attention enables the brain rapidly respond to objective information and subjective consciousness.The brain receives the relevant audio-visual signals through the sensory system,and makes a quick selection of the external complex and redundant information,and selectively retains and processes the information closely related to its own concern.Attention is always responsible for being highly alert,monitoring,and processing external environmental events,and making choices,but in reality,the external environment also reshapes the attentional activity of our brains Previous studies have shown that attention activity can change over time,tasks,and cognitive state.However,it is not clear how the attention varies with the parameters of our living environment,such as the temperature of the environmentHyperthermia is an important risk factor in many professional situations.During hyperthermia,the brain becomes less alert to surrounding targets.Hyperthermia increases the depletion rate of attention resources,which affects human behavior.Our previous studies also showed that the central fatigue increased significantly in the sustained attention tasks during hyperthermia,with compensating local blood flow perfusion in the prefrontal cortex significantly inhibited.The attention deficits will undoubtedly greatly increase the occupational accident rate.However,the underlying neural mechanism of attention dysfunction during hyperthermia is not well explained.In the previous study,we found that in the visual attention network test,the human brain showed selective attention deficit,mainly decreased executive control,but preserved alert and orient ability.However,the decline in executive control may not be entirely due to abnormal brain activation during the task.In previous reports,even when no cognitive tasks were performed,the baseline state of the brain had changed,such as an increase in central fatigue.The change in the baseline activity of the brain may have contributed to a decrease in behavior during subsequent task execution However,it is not clear whether the attention to resting-state baseline activity during hyperthermia is related to selective attention disordersBased on the above background,we performed the following experiments in this part:1.Using group independent component analysis(ICA)method,we investigated the resting-state functional connectivity(FC)changes during hyperthermia.Using 3D arterial spin labelling technology(ASL),we collected images of cerebral blood flow(CBF)during hyperthermia,and coupled with the FC images to calculate CBF-FC correlation,CBF/FC ratio and other indicators to further explore the correlation between neural activity and vascular reaction.At the same time,we performed multiple regression analysis of imaging metrics and behavioral performances to further reveal the predictive effect of resting neural activity on attention behavior.2,We further extracted the default mode network(DMN)and dorsal attention network(DAN)components during hyperthermia.The change of FC within the DMN and DAN networks was analyzed.Then,using the functional network connectivity(FNC)technology,we calculated the changes of FNC within-network and between-network of the DMN and DAN networks,thus revealing the change of DMN-DAN network anti-correlation under the same modal data.And again,using the multi-linear regression analysis method,the correlation between DMN-DAN anticorrelation coefficient and attention performance was explored.The main results and conclusions are as follows:1.Using conjoint analysis of multiple neuroimaging indicators(FC,CBF,CBF-FC correlation and CBF/FC ratio),we confirmed that the resting-state attention network was reconstructed in response to the hyperthermia,with significant decreased DAN activity,but enhanced ventral attention network(VAN)activity.The main performance is as follows:(1)CBF:the CBF in the DAN network reduced mainly in the bilateral intraparietal sulcus(IPS)and front eye al field(FEF)area.In contrast,CBF in the VAN showed an elevated trend,especially in the right ventral frontal cortex(VFC)region;(2)CBF-FC correlation:The CBF-FC correlation in the bilateral FEF region within the DAN was found to be significantly reduced,while the CBF-FC correlation in the IPS region remained unchanged.Within the VAN network,the CBF-FC correlation was significantly increased,while the correlation at the temporal-parietal junction(TPJ)area remained the same;In the VAN network,the hyperthermia did not cause a significant change in the CBF/FC ratio2.We found that the decreased FC in the medial prefrontal cortex/anterior cingulate cortex in the anterior DMN was correlated with executive control performance.The FNC analysis revealed that the decreased FC between IC20 components(mainly MPFC/ACC region)and IC14 components(mainly IPS regions)in the DAN was related to the executive control,indicating that the inherent anti-correlation between DMN and DAN during hyperthermia became weakening.These results meant that the execution control was related to the decreased FC within the DMN and the weakening anti-correlation with the DAN.The alert was obviously affected during hyperthermia may be due to the compensation within the DAN,especially increased FC between FEF and IPS area,resulting in no obvious behavioral deficit.Summarizing the above results,this paper revealed the resting-state brain activity characterized by decreased activity in DAN and increased activity in the VAN.The changes of CBF,FC and CBF-FC in the DAN and VAN were associated with selective attention deficits,suggesting that the altered attention behavior may be associated with abnormal baseline activity of the brain during hyperthermia.The changes in the FC between DMN and DAN indicated that the brain reconstructs attention resources,giving priority to performing tasks that require less cognitive resources,such as alertness,resulting in selective deficits,such as the ability to control conflict processing that require higher cognitive resources.This paper provided further evidence of neuroimaging for brain dysfunction during hyperthermia,and novel strategic basis for the scientific planning of operational tasks in high temperature occupational environment.