{"paper_id":"15e82e2a-42b9-497c-8b01-0bf17d3abde6","body_text":"5-hydroxytryptamine Distribution Alterations in both Neurons and Synapses: A Potential Pathogenesis of Neuron Death in Tg(SOD1*G93A)1gur Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 5-hydroxytryptamine Distribution Alterations in both Neurons and Synapses: A Potential Pathogenesis of Neuron Death in Tg(SOD1*G93A)1gur Mice Shishi Jiang, Menghua Li, Qi Dai, Xiwang Liu, Cheng Li, Huifeng Jiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3939628/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, the accurate pathogenesis of ALS hasn’t been found up to now. The previous studied results revealed that the abnormal alterations of some non-motor neurons (MN) were one of potential pathogenesis of MN death in ALS. Therefore, we studied the altered features of 5-hydroxytryptamine (5-HT) distribution and expression in the spinal cord and brainstem of both Tg(SOD1*G93A)1Gur (TG) and wild-type (WT) mice through the fluorescent immunohistochemistry and Western blot methods using the biomarkers of 5-HT neuron and synapse (both 5-HT and Tryptophan hydroxylase 2). Our results revealed that 5-HT synapses mainly distributed in the funiculus lateralis, the anterior horn, the posterior horn, the central lateral column and the around central canal in the cervical, thoracic and lumbar segments of spinal cord, as well as both the raphe nucleus and the lateral paragigantocellular nucleus of brainstem, and gradually reduced following by the age increase in WT mice. However, both 5-HT synapses and 5-hydroxytryptamine receptor 1A (5-HTR1A), but not 5-HTR2A, in spinal cord and 5-HT neurons in brainstem gradually increased following by the progression of disease and presented the significantly negative correlation between the increased distribution of both 5-HT synapses and neurons and neural cell death at the onset and/or progression stages of TG mice. Therefore, it is speculated that the distribution changes of 5-HT synapses in spinal cord and 5-HT neurons in brainstem are closely associated with neuron death, is a potential pathogenesis of ALS. Amyotrophic lateral sclerosis 5-hydroxytryptamine neuron 5-hydroxytryptamine receptor spinal cord brainstem pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease mainly damaged motor neuron (MN) [ 1 , 2 ]. Approximate 10% of ALS patients are familial ALS (fALS), the rest of 90% patients are sporadic ALS (sALS) [ 3 ]. The average onset age of ALS is 60 ± 5 years [ 4 ]. The average disease course of ALS is about 4.4 years from the begin of diagnosis [ 5 ]. ALS patients usually die from the respiratory failure [ 4 – 6 ]. ALS mainly is featured by the selection and progression death of both superior and inferior MN involving cerebrum, brainstem as well as spinal cord, which results in the muscle atrophy of laryngopharyngeal, limbs and even whole body [ 3 ]. To date, the pathogenesis about ALS has been incompletely understood. Based on the current studied results, it is suggested that the following potential pathogenesis might be closely related to the degeneration of MN in ALS, which are involved in the toxic of mutative ribonucleic acid, the excitatory toxicity, the disorder of protein balance, the defection of axon transportation, the excessive production of reactive oxygen species, the lesion of mitochondria function and the abnormal alterations of non-MN [ 6 – 11 ]. In addition, it is hypothesized that ALS should be not only the sole neurodegeneration and death of MN, but also might affects other neural cells besides MN based on the currently studied reports about the pathogenesis of ALS [ 6 , 8 – 11 ]. The currently researched evidences demonstrate that the traditional viewpoint which ALS only lonely damages MN isn’t completely accurate, and more and more evidences have proved that other nervous systems besides motor systems also are involved in the pathogenesis of ALS [ 6 – 12 ]. The staging pattern of ALS based on the pathological alterations of phosphorylated TAR deoxyribonucleic acid-binding protein (pTDP-43) is consisted of stages 1–4. The staging pattern of ALS based on the spread of pTDP-43 further fully proves that the ALS pathological lesion in nervous systems is far beyond the motor areas in the cerebral cortex, brainstem and the anterior horn of spinal cord [ 13 – 19 ]. Raphe nuclei (RN) is major anatomical regions of 5-hydroxytryptamine (serotonin, 5-HT) neuron distribution, approximate 80% of 5-HT neurons in central nervous system (CNS) distribute in RN. The related molecules of 5-HT neuron functions such as 5-HT1/2A, 5-HT2B/C receptor agonist or 5-HT precursor 5-hydroxytryptophan (5-HTP) may partially protect MN functions, even significantly reverse the progression of ALS [ 20 , 21 ]. In general, the potential effects about 5-HT in the pathogenesis of ALS theoretically have obtained new understanding, especially because several related papers recently were published [ 21 – 23 ]. It is well known that 5-HT is an important monoamine neurotransmitter to transfer happy feel in CNS [ 24 ]. Approximate 90% of 5-HT primarily distribute in the enterochromaffin cell of gastro-intestine tracts besides CNS [ 25 ]. The residual 5-HT are synthesized in the 5-HT neurons of CNS and the majority of 5-HT distribute in the RN of brainstem, exerts some important physiological functions such as regulating mood, appetite, sleep and memory and learn cognition. Therefore, 5-HT neurons in RN are the major sources synthesizing and releasing 5-HT neurotransmitter in CNS [ 26 , 27 ]. Axons projecting from RN neurons to other neural structures form 5-HT-nergic neuro-transmitter systems to reach nearly all parts of CNS. 5-HT-nergic-neuron axons in the inferior RNs terminate to cerebellum and spinal cord, but the 5-HT-nergic axons from the superior RNs almost project to entire brain. Although the current investigation evidences showed that 5-HT neurotransmitter might participate and play some roles in the pathogenesis of ALS, the accurate mechanisms and effects about 5-HT neurotransmitter on ALS haven't been very clarified and exist some debate at present yet. To this end, we observed and analyzed the altered features of 5-HT distribution in neurons and synapses in both spinal cord and brainstem of mainly damaged regions in ALS, and the relationship between 5-HT alterations and neural cell death applying Tg(SOD1*G93A)1Gur (TG) and wild-type (WT) mice. Our results revealed that 5-HT neurotransmitter significantly decreased or increased in both spinal cord and brainstem of TG mice compared with WT mice. Our data showed a closed relationship between 5-HT neurotransmitter alterations and neural cell death in TG mice. This study suggested that the 5-HT neurotransmitter alterations in the neurons and synapses of spinal cord and brainstem might be the potential pathogenesis of neuronal death in the pathogenesis of ALS. Results The Alterations of 5-HT Distributed Features among the Cervical, Thoracic and Lumbar Segments of Adult Spinal Cord at the Pre-Onset, Onset and Progression Stages between WT and TG Mice 5-HT mainly expressed in the grey matter of adult spinal cord, including the cervical, thoracic and lumbar anterior horn (AH), posterior horn (PH), central lateral column (CLC) and around central canal (CC). In the spinal cervical segment, the 5-HT distribution in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage showed a significant decrease, but that in AH, PH and CC at the progression stage significantly increased while compared WT with TG mice (Fig. 1 A, B). In the thoracic segment, the distribution of 5-HT in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage significantly decreased, but that in AH at the onset stage and that in AH, PH and CC at the progression stage significantly increased while compared WT with TG mice (Fig. 2 A, B). In the lumbar segment, the distribution of 5-HT in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage exhibited a significant decrease, but that in AH at the onset stage and that in AH and PH at the progression stage significantly increased while compared WT with TG mice (Fig. 3 A, B). The comparison of 5-HT distribution in the cervical, thoracic and lumbar AH, PH, CLC, CC and the entire spinal cord at the stages of pre-onset, onset and progression (Fig. 4 ), results demonstrated that 5-HT distribution in the cervical, thoracic and lumbar AH, PH, CC and the entire spinal cord of WT mice at the stages of onset and progression displayed a significant decrease (Fig. 4 A, C, G, I), and the 5-HT distribution in the cervical, thoracic and lumbar CLC of WT mice at the progression stage (Fig. 4 E) significantly decreased compared with the pre-onset stage. Meanwhile, the 5-HT distribution in AH and the entire cervical, thoracic and lumbar segments of TG mice at the stages of onset and progression (Fig, 4B, J), as well as that in PH at the progression stage (Fig. 4 D) showed a significant increase, and the 5-HT distribution in the cervical, thoracic and lumbar PH of TG mice at the onset stage significantly decreased compared with the pre-onset stage. The comparison of 5-HT distribution in the cervical, thoracic and lumbar CLC and CC of TG mice at the stages of pre-onset, onset and progression didn't reveal any significant changes (Fig. 4 F, H). To further confirm the alterations of 5-HT expression in spinal cord with the disease progression, Western blot analysis was used to examine TPH2 levels in the cervical, thoracic and lumbar segments of spinal cord at the different stages of TG mice and the age-matched same periods of WT mice. In the cervical segment, TPH2 expression decreased at the onset and progression stages of WT mice, while increased at the progression stage of TG mice (Fig. 5 A and D). In the thoracic segment, TPH2 expression gradually decreased at the onset and progression stages of WT mice, while significantly increased at the onset stage of TG mice (Fig. 5 B and E). In the lumbar segment, TPH2 expression gradually decreased at the onset and progression stages of WT mice (Fig. 5 C and F). These results confirmed that the entire spinal cord of WT mice displayed a significant decrease of TPH2 at the onset and progression stages, while both the cervical segment of spinal cord in TG mice at the progression stage and the thoracic segment of spinal cord in TG mice at the onset stage showed the significant up-regulation of TPH2. Next, we further examined whether the specific 5-HT receptor subtypes increased in TG mice. 5-HTR1A is a highly characterized spinal 5-HTR, is reported to increase the spinal MN excitability [ 28 ]. 5-HTR1A expression in the thoracic segment of spinal cord was evaluated at three stages of both WT and TG mice. Western blot analysis showed that 5-HTR1A levels decreased at the onset stage of WT mice, which was rescued in TG mice (Fig. 6 A and C). 5-HTR2A is another 5-HT receptor subtype which is reported to be involved in mediating 5-HT effects on MN [ 29 ]. However, 5-HTR2A expression was similar at all three stages of WT mice, while decreased at the onset stage of TG mice (Fig. 6 B and D). These results suggested that 5-HTR1A, but not 5-HTR2A, might be involved in ALS. However, the roles of other 5-HT receptor subtypes in ALS can’t be excluded yet. The Alterations of TPH2 Distributed Features in the Cervical, Thoracic and Lumbar Segments of Adult Spinal Cord at the Pre-Onset, Onset and Progression Stages between WT and TG Mice TPH2 mainly expressed in the adult spinal white matter, especially among funiculus lateralis (FL) (Fig. 7 A). While compared between WT and TG mice, TPH2 in FL significantly decreased at the pre-onset stage, but TPH2 among the cervical segment showed a significant increase at the progression stage (Fig. 7 B). TPH2 distribution in the cervical segment was more than that among both thoracic and lumbar segments at the progression stage, TPH2 at both pre-onset and onset stages didn’t show the significant difference between the cervical, thoracic and lumbar segments (Fig. 7 B). In the cervical segment, TPH2 distribution at the progression stage significantly increased compared with that at both pre-onset and onset stages, and was the highest distribution (Fig. 7 B). The Overlapped Distribution of TPH2, 5-HT, DAPI, GFAP, Vimentin and NeuN in the Adult Spinal Cord The representative images of TPH2 and DAPI double staining in the lateral sagittal and transverse spinal cord (Fig. 8 A, B). The representative images of TPH2 and GFAP double staining in the lateral transverse spinal cord (Fig. 8 C), the representative images of 5-HT and Vimentin (Fig. 8 D) and 5-HT and NeuN double staining (Fig. 9 ) in the AH, PH, CLC and CC of lateral transverse spinal cord showed no double staining among them. Results indirectly indicated that both TPH2 and 5-HT didn’t express in neuronal bodies, astrocytes, neural precursor cells (NPCs) and neurons, and indirectly implied that both TPH2 and 5-HT expressed in the neuronal synapses based on the anatomic distribution features of neuronal synapses in the adult spinal cord. The Alterations of TPH2 Distributed Features in the Adult Brainstem at the Pre-Onset, Onset and Progression Stages between WT and TG Mice TPH2 mainly expressed in brainstem nuclei, including dorsal RN (DRN), median RN (MRN), raphe magnus nucleus (RMG), raphe obscurus nucleus (ROB), raphe pallidus nucleus (RPA), raphe pontine nucleus (RPN) and lateral paragigantocellular nucleus (LPGI). In WT mice, TPH2 distribution in DRN was the most abundant, the secondary abundance was in MNR and RMG, the third was in ROB, RPA, RPN and LPGI. While compared between WT and TG mice, TPH2 in ROB at the pre-onset stage significantly decreased, TPH2 in RPN at both onset and progression stages significantly increased (Fig. 10 B). The Overlapped Distribution of TPH2, Nestin, NeuN and Vimentin in the Adult Brainstem The representative images of TPH2 double staining with Nestin, NeuN and Vimentin showed that all TPH2 doubly stained with NeuN (Fig. 11 A), didn’t detect any double staining with Nestin (Fig. 11 B) or Vimentin (No data showed because of similar negative results with Nestin results). The results showed that TPH2 expressed in neurons, but not in the NPCs of brainstem. Correlation between Neural Cell Number and both 5-HT and TPH2 Distribution in Spinal Cord and TPH2 Distribution in Brainstem The reduction of neural cell number showed a negative correlation with 5-HT and TPH2 distribution increase in the spinal cervical (Fig. 12 A, B), thoracic (Fig. 12 C, D) and lumbar (Fig. 12 E, F) segments and the TPH2 distribution increase in the RNP nuclei (Fig. 12 G) of brainstem in TG mice, which indirectly implied that the alterations of 5-HT and TPH2 distribution in both spinal cord and brainstem exhibited a negative correlation with neural cell death in both spinal cord and brainstem 5-HT nuclei (Fig. 12 H-J). Results reminded us that the increase of both 5-HT and TPH2 distribution in both spinal cord and brainstem might be one of potential candidate factors in neurons death in the pathogenesis of ALS. Discussion In our study, we observed and analyzed the distributed features of 5-HT neurotransmitter in the different anatomical regions and segments of spinal cord, and the different brainstem nuclei in both WT and TG mice applying the fluorescence immunohistochemistry staining of 5-HT and TPH2 biomarkers, performed the comparison of their distribution at the pre-onset, onset and progression stages between WT and TG mice, and observed the relationship between 5-HT distribution and neurons, glial cells and NPCs. Our results showed that 5-HT biomarker mainly distributed in the spinal AH, PH, CLC and CC of grey matter with stripe shape (Fig. 1 - 3 A), the distribution in both AH and PH was the most abundant and the secondary was in the cervical, thoracic and lumbar CLC and CC (Fig. 1 - 3 B). The 5-HT distribution in AH, PH, CLC and/or CC significantly decreased at the pre-onset stages (Fig. 1 – 7 ) compared TG with WT mice, however, they significantly increased at the progression stages of TG mice. Although what happen about the mechanism of abnormal 5-HT distribution in the spinal cord of TG mice wasn’t clear in our study yet, it implied that the abnormal alterations of 5-HT distribution occurred in AH, PH and/or CC, especially in AH. The abnormal distribution of 5-HT in the spinal cord of TG mice was in agreement with the damaged regions of spinal cord in ALS, such as AH. Therefore, we speculated that the abnormal 5-HT distribution in the spinal cord of TG mice participated in the pathogenesis of G93A-SOD1 TG mice, might be related to neuron death in AH in the pathogenesis of ALS. TPH2 biomarker mainly distributed in the FL of spinal white matter by linear shape, the distribution in the cervical, thoracic and lumbar FL significantly decreased at the pre-onset stage, and that among the cervical segment significantly increased at the progression stage (Fig. 7 ), the distribution alterations of 5-HT were responsible to the spinal FL of ALS majorly damaged region. Therefore, we speculated that the abnormal 5-HT distribution in spinal cord was closely associated with FL damage in ALS. Both 5-HT and TPH2 biomarkers in spinal cord didn't overlap stain with neurons, glial cells and NPCs biomarkers (Fig. 7 , 8 ). We hypothesized that the stripe or linear distribution of 5-HT and TPH2 biomarkers in spinal cord were 5-HT synapses projected into spinal cord based on the anatomic features of 5-HT synapse distribution because the synapses of 5-HT neurons comprehensively projected into the FL of white matter and the AH, PH, CLC and CC of grey matter in spinal cord [ 30 , 31 ]. Meanwhile, we conducted the comparison of 5-HT synapses in spinal cord between WT and TG mice, results showed 5-HT synapses in the spinal cervical, thoracic and lumbar AH, PH, CC and/or FL significantly reduced at both pre-onset and/or onset stages, however, 5-HT synapses in the cervical, thoracic and lumbar AH, PH, CLC, CC, and/or FL significantly increased at the progression and/or stages (Fig. 1 – 7 ). Our data revealed that 5-HT synapses in AH reduced at the pre-onset stage, but that at the stages of both onset and progression increased in comparison of WT mice, which further identified that the abnormal 5-HT distribution closely was associated with the damage of neurons in AH in the pathogenesis of ALS. In brainstem, 5-HT neurons mainly distributed in DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei, the brain region of the most abundant 5-HT distribution was in DRN, the secondary was in both MNR and RMG, the third was in ROB, RPA, RPN and LPGI nuclei (Fig. 10 A, B). TPH2 biomarker in brainstem only overlapped with neuron biomarker, which indicated that TPH2 biomarker expressed cells were neuronal cells (Fig. 11 ). Our results further identified that 5-HT neurons existed in the DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei of brainstem [ 30 ]. Our results showed that 5-HT neurons in the ROB of brainstem significantly reduced at the pre-onset stage, however, 5-HT neurons in the RPN of brainstem demonstrated a significant increase at the stages of both TG onset and progression in TG mice. Therefore, our data further revealed that the abnormal alterations of 5-HT neuron distribution exhibited a close relationship to neural cell death among brainstem during the pathogenesis of ALS, especially in distribution in RPN nuclei. TPHs are the enzymes synthesized 5-HT neurotransmitter. Human and other mammal animals have 2 unique TPH genes, distribute in the eleventh and twelfth chromosome, encode 2 distinct homogenous enzymes of TPH1 and TPH2 [ 32 ]. TPH1 mostly expresses in the periphery tissues such as skin, gut and pineal gland, and also expresses among CNS. TPH2 solely expresses among neurons in CNS, is an initial limiting-rate enzyme during synthesizing 5-HT neurotransmitter, mainly expresses among brain 5-HT neurons, largely distributes among midbrain RN [ 33 , 34 ]. Therefore, we labeled 5-HT neurons in both spinal cord and brainstem applying TPH2 fluorescent staining. Our results revealed that 5-HT synapses comprehensively projected into FL, AH, PH, CLC and CC as well as cortex and sub-cortex. 5-HT neurons mainly distributed in the RN nuclei of brainstem including DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei. Moreover, the abnormal alterations in the 5-HT neuron distribution of both ROB and RPN nuclei were found at the onset and/or progression stages of TG mice, and a significant decrease in ROB nuclei at the onset stage and a significant increase in RPN nuclei at both onset and progression stages were showed. Our data further indicated that 5-HT neurotransmitter participated in the pathogenesis of G93A-SOD1 TG mice because 5-HT neurons in ROB extensively project into both spinal cord and medulla nuclei including ambiguous nucleus [ 31 ], and 5-HT neurons in RPN mainly project into various constructs of cortex and sub-cortex [ 30 ], and might exist a potential relationship to the neuronal damage in the commonest damaged region in ALS, such as cortex, sub-cortex, spinal cord and brainstem nuclei, especially in the MN of AH, cortex and sub-cortex as well as the ambiguous nucleus of medulla. 5-HT nuclei usually are classified into 2 major clusters, caudal and rostral clusters, which contain three and four nuclei respectively. The caudal cluster consists of ROB (B2 nucleus), RMG (also known as B3 nucleus), RPA (B1 nucleus) and lateral medullary reticular nucleus (LMR), which project into brainstem and spinal cord including FL, AH, PH, CLC and CC as well as brainstem related regions, which are involve in the motor activity of both spinal cord and brainstem [ 30 , 31 ]. Rostral cluster composes of caudal linear nucleus (CLN) (B8 nucleus), DRN (B6 and B7 nucleus), MNR (B5, B8 and B9 nucleus) and RPN, project to various constructs of cortex and sub-cortex [ 30 ]. RMG, ROB, RPA and LMR project into brainstem and spinal cord, and CLN, DRN, MNR and RPN project into cortex and sub-cortex regions, which is involved in motor functions through 5-HT pathways [ 30 , 31 ]. RN is a moderate-size cluster of nuclei in brainstem. Based on the sequence from caudal to rostral, RN is divided into ROB, RPA, RMG, RPN, MNR, DRN and caudal linear nucleus. Overall, all caudal RN including RMG, RPA and ROB project to spinal cord and brainstem [ 31 ]. Almost all rostral nuclei included RPN, MNR and DRN project towards the brain areas of higher functions [ 31 ]. In our study, we observed that the number increase of 5-HT neurons in RPN presented a significantly negative correlation with neural cell death at both onset and progression stages of TG mice (Fig. 12 G, J), which indicated that the increased alterations of 5-HT neurons projected into the brain areas of higher functions participated in the damage of motor and/or non-MN among cerebrum, was one of possible factors that non-MN involved in the pathogenesis of ALS. Moreover, our results showed that the 5-HT ROB projected into spinal cord and brainstem as well as the RPN nuclei projected into various constructs of cortex and sub-cortex significantly increased following by ALS progression respectively, which further indicated that the increase of both 5-HT neurons and synapses among the 5-HT nucleus of brainstem was closely associated with the pathogenesis of MN and/or non-MN death in spinal cord and brainstem as well as cortex and sub-cortex regions in the pathogenesis of ALS. Although our data illustrated that the abnormal alterations of 5-HT synapses in spinal cord and 5-HT neurons in brainstem were closely associated with the pathogenesis of ALS, it isn’t clearly known that the 5-HT abnormal alterations in both spinal cord and brainstem is whether damage or protect neural cells in the pathogenesis of ALS. Based on the current clinical and experimental evidences as well as our data [ 35 – 38 ], we hypothesized that 5-HT neurotransmitter might play a potential neuronal protective role in ALS probably via 5-HTR1A, non-MN such as 5-HT neurons might play some roles in the pathogenesis of ALS through moderating the 5-HT neurotransmitter releasing into the projecting neurons, and 5-HT neurons participated in the damage course of MN in cortex and sub-cortex regions, brain stem nuclei and AH, was the potential pathogenesis of neuron death in the pathogenesis of ALS. Materials and Methods G93A-SOD1 Mice The G93A-SOD1 TG mice from Jackson laboratory (cat#002726, Jackson laboratory, Bar Harbour, ME, USA) was bred by mating male TG mice with female WT mice (cat#000664, Jackson laboratory, Bar Harbour, ME, USA) in The First Affiliated Hospital of Nanchang University. The experimental mice were isolated genome deoxyribonucleic acid (DNA) from mice tail by Rapid Animal Genomic DNA Isolation Kit (cat#B518221, Sangon Biotech, Shanghai, China). G93A-SOD1 TG mice were identified whether or not G93A-SOD1 positive TG mice by the polymerase chain reaction (PCR) of genome DNA. The used primers (Sangon Biotech, Shanghai, China) in PCR were following, the forward primer of IL-2 (PCR internal reference) was 5'-CTA GGC CAC AGA ATT GAA AGA TCT-3', the reverse primer of IL-2 was 5'-GTA GGT GGA AAT TCT AGC ATC ATC C-3', the forward primer of hmSOD1 G93A was 5'-CAT CAG CCC TAA TCC ATC TGA-3', and the reverse primer of hmSOD1 G93A was 5'-CGC GAC TAA CAA TCA AAG TGA-3'. The condition of PCR was: degenerating at 94°C three secs, annealing at 60°C one min, extending at 72°C one min, conducting thirty-five cycles. Animals were sacrificed after anesthetized with CO 2 at three time points of pre-onset (60–70 days), onset (90–100 days) and progression (120–130 days). The severity of muscle atrophy in hind limbs was determined by the hematoxylin eosin staining of gastrocnemius muscles at different stages, and histologically evaluated different stages by observing changes in muscle structures under light microscopy [ 39 – 42 ]. All experimental mice were housed under 20–27°C, 40–50% humidity, 12 hrs light/dark cycle and free access to food or water. ALS Therapy Development Institute (ALSTDI) score was used in monitoring the health and behavioral disorders of mice in order to further determine the disease courses and stages [ 43 , 44 ]. Experiments were designed according to the way minimized total number and suffering of used animals. All used mice in this study were manipulated by Chinese Guide for the Care and Use of Laboratory Animals and reviewed by Animal Care and Use Ethics Committee of The First Affiliated Hospital of Nanchang University. The Fluorescence Immunohistochemistry Staining of both Spinal Cord and Brainstem Both spinal cord and brain were stripped after adequately perfused by 20ml 0.9% saline and 40ml 4% 1xPBS pH 7.5 paraformaldehyde solution (cat#P7059, Sigma-Aldrich, St. Louis, MO, USA) via right ventricle at room temperature (RT) after anesthetized with CO 2 . The detailed protocols were similar with the experimental methods of Zhou et al. [ 42 ]. Both spinal cord and brain were immediately put into 4% 1xPBS pH 7.5 paraformaldehyde solution (cat#F8775, Sigma-Aldrich, St. Louis, MO, USA) overnight after taken out, then incubated in 20% 1xPBS pH 7.5 sucrose, embedded using optimal cutting temperature compound (OCT, cat#4583, Sakura Finetek, Chuo-ku, Tokyo, Japan). Both spinal cord and brainstem tissues were successively cut 12µm coronal section by a freezing microtome and put slices on Superfrost Plus slide. The fluorescence immunohistochemistry staining of spinal cord and brainstem was conducted by following processes, sections were permeabilized with 0.2% TritonX-100 (cat#T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked with 10% 1xPBS bovine serum albumin (cat#SRE0098, Sigma-Aldrich, St. Louis, MO, USA) after rehydrated by pH 7.4 1xPBS. The following primary antibodies were added according to the requirement of experimental designs: 1:100 rabbit anti-Vimentin (cat#EPR3776 RRID:AB_92547, Abcam, Cambridge, MA, USA), 1:100 rabbit anti-Nestin (cat#SP103 RRID:AB_105389, Abcam, Cambridge, MA, USA), 1:200 rabbit anti-NeuN (cat#EPR12763 RRID:AB_ab177487, Abcam, Cambridge, MA, USA), 1:1000 rabbit anti-GFAP (cat#EPR19996 RRID:AB_ab7260, Abcam, Cambridge, MA, USA), 1:400 rat anti-serotonin (5-HT) (cat#YC5/45 RRID:AB_ab6336, Abcam, Cambridge, MA, USA) or 1:600 mouse anti-TPH2 (cat#CL2990 RRID:AB_ ab211528, Abcam, Cambridge, MA, USA) monoclonal antibodies. All primary antibodies were incubated overnight at 4°C, then lightly washed by 0.2% 1X PBS Triton X-100 buffer for 6 times, each times 5 minutes. The following secondary antibodies conjugating to green or red rhodamine fluorescence were incubated for 2 hrs at RT: 1:250 donkey anti-rabbit (RRID:AB_ab150073, Abcam, Cambridge, MA, USA), 1:250 Donkey Anti-Rat (RRID:AB_ab150153, Abcam, Cambridge, MA, USA) or 1:200 donkey anti mouse (RRID:AB_ab150105, Abcam, Cambridge, MA, USA) IgG (H + L) Alexa Fluor 488 cross-adsorbed second antibody, followed staining by DAPI (blue) and treating anti-fluorescence fade after lightly washing 6 times, each times 5 minutes. All slices were observed and taken pictures by a fluorescence microscope equipped with digital camera (Nikon E800, Sumida-ku, Tokyo, Japan) and Photoshop software (Adobe Systems, San Francisco, CA, USA). The staining of doubly labeled fluorescence was performed by simultaneously using Vimentin, Nestin, NeuN, GFAP, 5-HT and TPH2 antibodies and DAPI to doubly stain to identify the proliferation and the distributed cellular types of 5-HT neurons. The Analysis of 5-HT and TPH2 Distribution and Positive Cells The analysis of 5-HT and TPH2 distribution and positive cells was measured the fluorescence intensity of 5-HT and TPH2 stripe or linear structures in the positive cells of ten spinal cord and brainstem sections under magnified 200 times, calculated the sum of ten sections. Total fluorescence intensity were divided by total section number, 5 mice each group, ten slices from each animal. The mean fluorescence intensity was applied to conduct a quantitation analysis. We identified and selected the interest nervous regions of cervical, thoracic and lumbar segments of spinal cord and brainstem based on the mouse spinal cord and brain anatomic atlas according to our previous study protocols [ 42 , 45 , 46 ]. Image J software (1.8.0 version) was used to quantify 5-HT and TPH2 positive cells in images. The Analysis of TPH2, 5HTR1A and 5HTR2A Levels in Spinal Cord by Western Blot The temporal changes of protein levels were quantified by Western blot analysis. Spinal cords were homogenized in lysis buffer containing phenylmethylsulphonyl fluoride and protease inhibitor after quickly removed from deeply anesthetized male WT and TG mice. The protein concentrations were then assessed by a BCA assay. After separated by 8% SDS-PAGEDs with 3 mg proteins in each lane, total proteins were transferred to a PVDF membrane. After blocked with 10% nonfat milk at 4°C for 2 hrs, PVDF membranes were incubated by following primary antibodies: 1:200 mouse anti-TPH2 (ab211528, Abcam Ltd., Cambridge, MA, USA), 1:200 rabbit anti-5HTR1A (AF5453, Affinity Biosciences Ltd., Jiangshu, China), 1:200 rabbit anti-5HTR2A (bs-12049R, Beijing Biosynthesis Biotechnology co., Ltd., Bejing, China) or 1:200 rabbit anti-GAPDH (ab245355, Abcam Ltd., Cambridge, MA, USA) antibodies at 4°C overnight. After incubated by 1:5,000 horseradish peroxidase-labeled goat anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 2 hrs, protein bands were visualized with enhanced chemiluminescence reagents (ECL, Pierce, Rockford, IL, USA). The protein levels were quantified and normalized to GAPDH internal controls. 3 mice per group were used. For the quantification analysis of Western blots, gray values were extracted using Image J software, and the gray values of objective proteins were divided by the gray values of internal reference proteins to obtain ratio for each group. The statistical analysis of these ratios was performed using GraphPad Prism (v. 7, GraphPad Software, San Diego, CA, USA). Ten samples per mouse and 5 mice per group were used, and Western blot was repeated three times per sample, and average values were compared and analyzed. Data Analysis Experimental data were indicated by mean ± standard deviation. Comparisons between two groups were applied the analysis of variance and Student's t test. The comparisons of multiple groups were performed using one-way analysis of variance with a Turkey post hoc test. Spearman correlation analysis was used to examine the correlation between positive cells and neural cells. p < 0.05 indicated a significantly statistical difference. Abbreviations ALS, Amyotrophic lateral sclerosis; MN, motor neuron; 5-HT, 5-hydroxytryptamine; TPH2, Tryptophan hydroxylase 2; 5-HTR1A, Serotonin receptor 1A; 5-HTR2A, Serotonin receptor 2A; pTDP-43, phosphorylated TAR deoxyribonucleic acid-binding protein; RN, raphe nuclei; 5-HTP, 5-hydroxytryptophan; CNS, central nervous system; AH, anterior horn; PH, posterior horn; CLC, central lateral column; CC, around central canal; FL, funiculus lateralis; NPCs, neural precursor cells; DRN, dorsal RN; MRN, median RN; RMG, raphe magnus nucleus; ROB, raphe obscurus nucleus; RPA, raphe pallidus nucleus; RPN, raphe pontine nucleus; LPGI, lateral paragigantocellular nucleus. Declarations Ethics approval All animal studies and experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the ethics committee for animal care and use of The First Affiliated Hospital of Nanchang University, China (Ethic approval number: 2015-3-12). Consent to participate No applicable. Consent for publication No applicable. Availability of data and materials All data and materials are included in this article. Competing interests The authors declare that they have no conflict of interest. Funding The authors received no financial support for the research, authorship, and/or publication of this article. This study was supported by the research grants to Renshi Xu from National Natural Science Foundation of China (30560042, 81160161, 81360198, 82160255), Education Department of Jiangxi Province (GJJ13198, GJJ170021), Jiangxi provincial department of science and technology ([2014]-47, 20142BBG70062, 20171BAB215022, 20192BAB205043), Health and Family Planning Commission of Jiangxi province (20181019 and 202210002), and Jiangxi Provincial Department of Science and Technology Gan Po Elite 555 (Jiangxi Finance Elite Education Refers to [2015] 108). Authors' contributions S. Jiang, M. Li, Q. Dai, H. Nie, H. Pan, R. Xu performed experiments, analyzed the data and wrote the manuscript; S. Jiang, M. Li, Q. Dai, X. Liu, C. Li, H. Jiao, H. Nie, H. Pan, R. Xuconducted statistical analyses; S. Jiang, M. Li, Q. Dai were the common jointed authors and equally contributed to this study. R. Xu conceived the project and wrote the manuscript. R. Xu, H. Pan, H. Nie revised the manuscript. All authors read the approved the final manuscript. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (30560042, 81160161, 81360198, 82160255), Education Department of Jiangxi Province (GJJ13198, GJJ170021), Jiangxi provincial department of science and technology ([2014]-47, 20142BBG70062, 20171BAB215022, 20192BAB205043), Health and Family Planning Commission of Jiangxi province (20181019 and 202210002), and Jiangxi Provincial Department of Science and Technology Gan Po Elite 555 (Jiangxi Finance Elite Education Refers to [2015] 108) for Renshi Xu. The partial schematics of spinal cord in Figure 1 and 7 were download from the public websites, these schematics don’t explicitly show any authors and published information, and the schematic of brain stem in Figure 10 C was cited from the second edition of mouse brain stereotaxic coordinates edited by professor George Paxinos and professor Keith B. J. Franklin. Here, specially stated and sincerely thank them. We are sincerely grateful of Dr. Dongyuan Yao for carefully revising our manuscript. References Chiò A, Logroscino G, Traynor BJ et al (2013) Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature. Neuroepidemiology 41:118–130. https://doi.org/10.1159/000351153 Mehta P, Kaye W, Bryan L et al (2016) Prevalence of Amyotrophic Lateral Sclerosis - United States, 2012–2013. MMWR Surveill Summ 65:1–12. https://doi.org/10.15585/mmwr.ss6508a1 Ludolph AC, Brettschneider J, Weishaupt JH (2012) Amyotrophic lateral sclerosis. Curr Opin Neurol 25:530–535. https://doi.org/10.1097/WCO.0b013e328356d328 Orsini M, Oliveira AB, Nascimento OJM et al (2015) Amyotrophic Lateral Sclerosis: New Perpectives and Update. Neurol Int 7:5885. https://doi.org/10.4081/ni.2015.5885 Kiernan MC, Vucic S, Cheah BC et al (2011) Amyotrophic lateral sclerosis. Lancet 377:942–955. https://doi.org/10.1016/S0140-6736(10)61156-7 Oskarsson B, Gendron TF, Staff NP (2018) Amyotrophic Lateral Sclerosis: An Update for 2018. Mayo Clin Proc 93:1617–1628. https://doi.org/10.1016/j.mayocp.2018.04.007 Ferraiuolo L, Kirby J, Grierson AJ et al (2011) Molecular pathways of motor neuron injury in amyotrophic lateral sclerosis. Nat Rev Neurol 7:616–630. https://doi.org/10.1038/nrneurol.2011.152 Liang H, Wu C, Deng Y et al (2017) Aldehyde Dehydrogenases 1A2 Expression and Distribution are Potentially Associated with Neuron Death in Spinal Cord of Tg(SOD1*G93A)1Gur Mice. Int J Biol Sci 13:574–587. https://doi.org/10.7150/ijbs.19150 Li J, Lu Y, Liang H et al (2016) Changes in the Expression of FUS/TLS in Spinal Cords of SOD1 G93A Transgenic Mice and Correlation with Motor-Neuron Degeneration. Int J Biol Sci 12:1181–1190. https://doi.org/10.7150/ijbs.16158 Lu Y, Tang C, Zhu L et al (2016) The Overexpression of TDP-43 Protein in the Neuron and Oligodendrocyte Cells Causes the Progressive Motor Neuron Degeneration in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis. Int J Biol Sci 12:1140–1149. https://doi.org/10.7150/ijbs.15938 Zhang J, Huang P, Wu C et al (2018) Preliminary Observation about Alteration of Proteins and Their Potential Functions in Spinal Cord of SOD1 G93A Transgenic Mice. Int J Biol Sci 14:1306–1320. https://doi.org/10.7150/ijbs.26829 Zhang J, Liang H, Zhu L et al (2018) Expression and Distribution of Arylsulfatase B are Closely Associated with Neuron Death in SOD1 G93A Transgenic Mice. Mol Neurobiol 55:1323–1337. https://doi.org/10.1007/s12035-017-0406-9 Li F, Zhou F, Huang M et al (2017) Frequency-Specific Abnormalities of Intrinsic Functional Connectivity Strength among Patients with Amyotrophic Lateral Sclerosis: A Resting-State fMRI Study. Front Aging Neurosci 9:351. https://doi.org/10.3389/fnagi.2017.00351 Silani V, Ludolph A, Fornai F (2017) The emerging picture of ALS: a multisystem, not only a motor neuron disease. Arch Ital Biol 155:99–109. https://doi.org/10.12871/00039829201741 Verde F, Del Tredici K, Braak H, Ludolph A (2017) The multisystem degeneration amyotrophic lateral sclerosis - neuropathological staging and clinical translation. Arch Ital Biol 155:118–130. https://doi.org/10.12871/00039829201746 Zhou F, Gong H, Li F et al (2013) Altered motor network functional connectivity in amyotrophic lateral sclerosis: a resting-state functional magnetic resonance imaging study. NeuroReport 24:657–662. https://doi.org/10.1097/WNR.0b013e328363148c Zhou F, Xu R, Dowd E et al (2014) Alterations in regional functional coherence within the sensory-motor network in amyotrophic lateral sclerosis. Neurosci Lett 558:192–196. https://doi.org/10.1016/j.neulet.2013.11.022 Brettschneider J, Del Tredici K, Toledo JB et al (2013) Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol 74:20–38. https://doi.org/10.1002/ana.23937 Sofic E, Riederer P, Gsell W et al (1991) Biogenic amines and metabolites in spinal cord of patients with Parkinson’s disease and amyotrophic lateral sclerosis. J Neural Transm Park Dis Dement Sect 3:133–142. https://doi.org/10.1007/BF02260888 Turner BJ, Lopes EC, Cheema SS (2003) The serotonin precursor 5-hydroxytryptophan delays neuromuscular disease in murine familial amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 4:171–176. https://doi.org/10.1080/14660820310009389 Dentel C, Palamiuc L, Henriques A et al (2013) Degeneration of serotonergic neurons in amyotrophic lateral sclerosis: a link to spasticity. Brain 136:483–493. https://doi.org/10.1093/brain/aws274 El Oussini H, Bayer H, Scekic-Zahirovic J et al (2016) Serotonin 2B receptor slows disease progression and prevents degeneration of spinal cord mononuclear phagocytes in amyotrophic lateral sclerosis. Acta Neuropathol 131:465–480. https://doi.org/10.1007/s00401-016-1534-4 El Oussini H, Scekic-Zahirovic J, Vercruysse P et al (2017) Degeneration of serotonin neurons triggers spasticity in amyotrophic lateral sclerosis. Ann Neurol 82:444–456. https://doi.org/10.1002/ana.25030 Young SN (2007) How to increase serotonin in the human brain without drugs. J Psychiatry Neurosci 32:394–399 Yano JM, Yu K, Donaldson GP et al (2015) Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161:264–276. https://doi.org/10.1016/j.cell.2015.02.047 Siegel GJ (1999) Basic neurochemistry: molecular, cellular, and medical aspects, 6th edn. Lippincott Williams & Wilkins, Philadelphia Binder MD, Hirokawa N, Windhorst U (2009) Encyclopedia of neuroscience. Springer, Berlin Wu X, Kushwaha N, Albert PR, Penington NJ (2002) A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels. J Physiol 538:41–51. https://doi.org/10.1113/jphysiol.2001.012668 Gilmore J, Fedirchuk B (2004) The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres. J Physiol 558:213–224. https://doi.org/10.1113/jphysiol.2004.064717 Jacobs BL, Fornal CA (2010) CHAPTER 2.1 - Activity of Brain Serotonergic Neurons in Relation to Physiology and Behavior. In: Müller CP, Jacobs BL (eds) Handbook of Behavioral Neuroscience. Elsevier, pp 153–162 Törk I (1990) Anatomy of the serotonergic system. Ann N Y Acad Sci 600:9–34 discussion 34–35. https://doi.org/10.1111/j.1749-6632.1990.tb16870.x Walther DJ, Bader M (2003) A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 66:1673–1680. https://doi.org/10.1016/s0006-2952(03)00556-2 Walther DJ, Peter J-U, Bashammakh S et al (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76. https://doi.org/10.1126/science.1078197 Zill P, Büttner A, Eisenmenger W et al (2007) Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study. J Psychiatr Res 41:168–173. https://doi.org/10.1016/j.jpsychires.2005.05.004 Fomina T, Weichwald S, Synofzik M et al (2017) Absence of EEG correlates of self-referential processing depth in ALS. PLoS ONE 12:e0180136. https://doi.org/10.1371/journal.pone.0180136 Dupuis L, Spreux-Varoquaux O, Bensimon G et al (2010) Platelet serotonin level predicts survival in amyotrophic lateral sclerosis. PLoS ONE 5:e13346. https://doi.org/10.1371/journal.pone.0013346 Holecek V, Rokyta R (2018) Possible etiology and treatment of amyotrophic lateral sclerosis. Neuro Endocrinol Lett 38:528–531 Koschnitzky JE, Quinlan KA, Lukas TJ et al (2014) Effect of fluoxetine on disease progression in a mouse model of ALS. J Neurophysiol 111:2164–2176. https://doi.org/10.1152/jn.00425.2013 Gurney ME, Pu H, Chiu AY et al (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264:1772–1775. https://doi.org/10.1126/science.8209258 Henriques A, Pitzer C, Schneider A (2010) Characterization of a novel SOD-1(G93A) transgenic mouse line with very decelerated disease development. PLoS ONE 5:e15445. https://doi.org/10.1371/journal.pone.0015445 Rosen DR, Siddique T, Patterson D et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59–62. https://doi.org/10.1038/362059a0 Zhou Y, Lu Y, Fang X et al (2015) An astrocyte regenerative response from vimentin-containing cells in the spinal cord of amyotrophic lateral sclerosis’s disease-like transgenic (G93A SOD1) mice. Neurodegener Dis 15:1–12. https://doi.org/10.1159/000369466 Knippenberg S, Thau N, Dengler R, Petri S (2010) Significance of behavioural tests in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Behav Brain Res 213:82–87. https://doi.org/10.1016/j.bbr.2010.04.042 Scott S, Kranz JE, Cole J et al (2008) Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler 9:4–15. https://doi.org/10.1080/17482960701856300 Xu R, Wu C, Tao Y et al (2008) Nestin-positive cells in the spinal cord: a potential source of neural stem cells. Int J Dev Neurosci 26:813–820. https://doi.org/10.1016/j.ijdevneu.2008.06.002 Xu R, Wu C, Tao Y et al (2010) Description of distributed features of the nestin-containing cells in brains of adult mice: a potential source of neural precursor cells. J Neurosci Res 88:945–956. https://doi.org/10.1002/jnr.22263 Additional Declarations No competing interests reported. Supplementary Files H1AWesternblotRawdata.tif H2AWesternblotRawData.tif TPH2Westernblotrawdata.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3939628\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":273442868,\"identity\":\"f336fabe-dd5b-4466-98de-88a591b96a5f\",\"order_by\":0,\"name\":\"Shishi Jiang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangxi Provincial People’s Hospital, Clinical College of Nanchang Medical College, First Affiliated Hospital of Nanchang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shishi\",\"middleName\":\"\",\"lastName\":\"Jiang\",\"suffix\":\"\"},{\"id\":273442869,\"identity\":\"fd2fc613-4be5-47c6-a3ab-1c9c3ebf31d2\",\"order_by\":1,\"name\":\"Menghua Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"First Affiliated Hospital of Nanchang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Menghua\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":273442870,\"identity\":\"21911f30-af92-4ee1-b3ef-40c7bebfaaa3\",\"order_by\":2,\"name\":\"Qi Dai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanchang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qi\",\"middleName\":\"\",\"lastName\":\"Dai\",\"suffix\":\"\"},{\"id\":273442871,\"identity\":\"8243f959-bf87-4d1e-8f0a-c60c15cf9e38\",\"order_by\":3,\"name\":\"Xiwang Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanchang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiwang\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":273442872,\"identity\":\"c4ec0682-c7eb-49d0-a98b-09f9efc273ea\",\"order_by\":4,\"name\":\"Cheng Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangxi Provincial People’s Hospital, Clinical College of Nanchang Medical College, First Affiliated Hospital of Nanchang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Cheng\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":273442873,\"identity\":\"ceaffd25-84d7-4b56-b4b4-538481a4451a\",\"order_by\":5,\"name\":\"Huifeng Jiao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanchang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Huifeng\",\"middleName\":\"\",\"lastName\":\"Jiao\",\"suffix\":\"\"},{\"id\":273442874,\"identity\":\"ca6489b9-33d8-445d-9766-a7085f51db66\",\"order_by\":6,\"name\":\"Hongbing Nie\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangxi Provincial People’s Hospital, Clinical College of Nanchang Medical College, First Affiliated Hospital of Nanchang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hongbing\",\"middleName\":\"\",\"lastName\":\"Nie\",\"suffix\":\"\"},{\"id\":273442875,\"identity\":\"4409111c-9549-4f65-9bc4-f129b83f797d\",\"order_by\":7,\"name\":\"Haili Pan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiangxi Provincial People’s Hospital, Clinical College of Nanchang Medical College, First Affiliated Hospital of Nanchang Medical College\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Haili\",\"middleName\":\"\",\"lastName\":\"Pan\",\"suffix\":\"\"},{\"id\":273442876,\"identity\":\"8c250719-bd11-4312-9a1e-ec88f3ef53ef\",\"order_by\":8,\"name\":\"Renshi Xu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3PsYrCQBCA4VkWtlrMlROQ+AorgXCdr7KLkM5DsNlCzgiyKS6HrY9haZkgpFp7u4v4BHYWFqb3cGNnsX89HzMD4PO9YSxYX843gYvR32nZSD13kx6WseD6U0I53ovG1m4SgUw+uNUtSdPwtKIdDmtHITT4RTKbaJUxCPIf+ZzQrG6GBmeUFMlR7fqA9rB1bKlyoQwSQ3lLLAOBExcZM6xaUjCeTJWhXUjKwsyi2nCWQjeClsagMRZI9yhtzZ2/DNYFOYP4jgSS5eWq51GQ/z4nD/HXxn0+n8/3b3fYbUaPKz4QOAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Jiangxi Provincial People’s Hospital, Clinical College of Nanchang Medical College, First Affiliated Hospital of Nanchang Medical College\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Renshi\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-02-08 10:34:45\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3939628/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3939628/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":51335059,\"identity\":\"d4020d1e-2db0-4f5b-bbf0-7aa7eafe9388\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1137204,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e5-HT distribution in the cervical AH, PH, CLC and CC of adult spinal cord. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of 5-HT distribution in the cervical segment of both WT and TG mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The comparison of 5-HT distribution in the adult cervical AH, PH, CLC and CC between WT and TG mice at the pre-onset, onset and progression stages. 5 mice per group. *p\\u0026lt;0.05. 5-HT distribution in the cervical AH, PH and CC at the pre-onset stage as well as in the cervical PH at the onset stage significantly decreased, but significantly increased in the cervical AH, PH and CC at the progression stage in TG mice. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) The schematic diagram of spinal cord anatomic regions.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/bc41e6a2886798995c82b6c7.png\"},{\"id\":51335060,\"identity\":\"42cae917-f169-4958-a0b3-0b54fb3a1c95\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":873795,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e5-HT distribution in the thoracic AH, PH, CLC and CC of adult spinal cord. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of 5-HT distribution in the thoracic segment of both WT and TG mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The comparison of 5-HT distribution in the adult thoracic AH, PH, CLC and CC between WT and TG mice at the pre-onset, onset and progression stages. 5 mice per group, *p\\u0026lt;0.05. The 5-HT distribution in the thoracic AH, PH and CC at the pre-onset stage as well as in the thoracic AH and PH at the onset stage significantly decreased, but significantly increased in the thoracic AH, PH, CLC and CC at the progression stage in TG mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/9d56541a6562abbdb883fb77.png\"},{\"id\":51335061,\"identity\":\"11375652-d401-4abc-b27b-601920c387b5\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":869955,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e5-HT distribution in the lumbar AH, PH, CLC and CC of adult spinal cord.\\u003cstrong\\u003e \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of 5-HT distribution in the lumbar segment of both WT and TG mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The comparison of 5-HT distribution in the adult lumbar AH, PH, CLC and CC between WT and TG mice at the pre-onset, onset and progression stages. 5 mice per group, *p\\u0026lt;0.05. 5-HT distribution in the lumbar AH, PH and CC at the pre-onset stage as well as in the lumbar PH at the onset stage significantly decreased, but significantly increased in the lumbar AH and PH at the progression stage in TG mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/d33178f1a6c8f4b77b691059.png\"},{\"id\":51335066,\"identity\":\"9c7df040-b12e-42d2-8ec3-67738a71f78a\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":982890,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe comparison of 5-HT distribution in AH, PH, CLC, CC and total 5-HT distribution in spinal cord between WT and TG mice.\\u003cstrong\\u003e \\u003c/strong\\u003e(\\u003cstrong\\u003eA-B\\u003c/strong\\u003e) The comparison of 5-HT distribution in the AH of different spinal cord segments of WT (\\u003cstrong\\u003eA\\u003c/strong\\u003e) and TG (\\u003cstrong\\u003eB\\u003c/strong\\u003e) mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eC-D\\u003c/strong\\u003e)\\u003cstrong\\u003e \\u003c/strong\\u003eThe comparison of 5-HT distribution in the PH of different spinal cord segments of WT (\\u003cstrong\\u003eC\\u003c/strong\\u003e) and TG (\\u003cstrong\\u003eD\\u003c/strong\\u003e) mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eE-F\\u003c/strong\\u003e)\\u003cstrong\\u003e \\u003c/strong\\u003eThe comparison of 5-HT distribution in the CLC of different spinal cord segments of WT (\\u003cstrong\\u003eE\\u003c/strong\\u003e) and TG (\\u003cstrong\\u003eF\\u003c/strong\\u003e) mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eG-F\\u003c/strong\\u003e)\\u003cstrong\\u003e \\u003c/strong\\u003eThe comparison of 5-HT distribution in the CC of different spinal cord segments of WT (\\u003cstrong\\u003eG\\u003c/strong\\u003e) and TG (\\u003cstrong\\u003eH\\u003c/strong\\u003e) mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eI-J\\u003c/strong\\u003e)\\u003cstrong\\u003e \\u003c/strong\\u003eThe comparison of total 5-HT distribution in the different spinal cord segments of WT (\\u003cstrong\\u003eI\\u003c/strong\\u003e) and TG (\\u003cstrong\\u003eJ\\u003c/strong\\u003e) mice at the pre-onset, onset and progression stages. 5 mice per group, *p\\u0026lt;0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/ece604cc748b6cc8494a4dbf.png\"},{\"id\":51335062,\"identity\":\"c71bbead-da5a-467a-88d1-f6302b3be99e\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2038247,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe alterations of TPH2 levels in the adult spinal cords at the different stages of TG mice and the age-matched same periodsof WT mice. (\\u003cstrong\\u003eA-C\\u003c/strong\\u003e) The representative Western blot bands of TPH2 in the cervical, thoracic and lumbar segments of spinal cord. (\\u003cstrong\\u003eD-F\\u003c/strong\\u003e) Western blot analysis showed the protein levels of TPH2 in the cervical, thoracic and lumbar segments of spinal cord at the pre-onset, onset and progression stages of TG mice and the age-matched same periodsof WT mice. 9 mice per group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/a9979737780db1e83591ab5c.png\"},{\"id\":51335070,\"identity\":\"95ffc4df-d77f-4dc7-b493-693ec5cd451e\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:21\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1587085,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe alterations of both 5-HTR1A and 5-HTR2A levels in adult spinal cords at the different stages of TG mice and the age-matched same periodsof WT mice. (\\u003cstrong\\u003eA\\u003c/strong\\u003e and \\u003cstrong\\u003eB\\u003c/strong\\u003e) The representative Western blot bands of 5-HTR1A and 5-HTR2A in the thoracic spinal cord. (\\u003cstrong\\u003eC\\u003c/strong\\u003e and \\u003cstrong\\u003eD\\u003c/strong\\u003e) Western blot analysis showed the protein levels of both 5-HTR1A and 5-HTR2A in the thoracic spinal cord at the pre-onset, onset and progression stages of TG mice and the age-matched same periods of WT mice. 9 mice per group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/a1cb1097f47b3f629ce45ec3.png\"},{\"id\":51335063,\"identity\":\"a0262e0c-4964-4a0c-b455-2c8ac7089d00\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":793917,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTPH2 distribution in the funiculus lateralis (FL) of adult spinal cord. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of TPH2 distribution in the cervical, thoracic and lumbar FL at the pre-onset, onset and progression stages of both WT and TG mice. 5 mice per group; *p\\u0026lt;0.05. FL was the most TPH2 distribution in adult spinal cord. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The comparison of TPH2 distribution in the cervical, thoracic and lumbar FL at the pre-onset, onset and progression stages of both WT and TG mice. 5 mice per group, *p\\u0026lt;0.05. TPH2 distribution in the cervical, thoracic and lumbar FL significantly decreased at the pre-onset stage, but that in the cervical segment significantly increased at the progression stage in TG mice. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) The schematic diagram of spinal cord anatomic regions.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/141dfa8b80e7f0c7f18e1ca5.png\"},{\"id\":51335069,\"identity\":\"63239c93-fedc-4cf7-9b6b-f726dcc63ad2\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:21\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1812818,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe double staining of TPH2/DAPI/GFAP and 5-HT/Vimentin. (\\u003cstrong\\u003eA-B\\u003c/strong\\u003e)\\u003cstrong\\u003e \\u003c/strong\\u003eThe representative images of TPH2 and DAPI double stain in the sagittal (\\u003cstrong\\u003eA\\u003c/strong\\u003e) and transverse (\\u003cstrong\\u003eB\\u003c/strong\\u003e) spinal cord. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) The representative images of TPH2 and GFAP double staining in the transverse spinal cord. (\\u003cstrong\\u003eD\\u003c/strong\\u003e) The representative images of 5-HT and Vimentin double staining in the spinal AH, CLC and CC. The distribution of TPH2 wasn't doubly stained with DAPI, GFAP and Vimentin.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/fc8bde8c93b8908a2ae1e3bb.png\"},{\"id\":51335064,\"identity\":\"84afb8a9-a72f-49b3-ba4e-32dacc59e9ec\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:20\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2007117,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe double staining of 5-HT and NeuN in the AH, PH, CLC and CC of adult spinal cord. The distribution of 5-HT wasn't doubly stained with NeuN in the spinal AH, PH, CLC and CC.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/a779750d9313d99c35b623e5.png\"},{\"id\":51335067,\"identity\":\"87d1d6da-9551-4675-aa67-cb23bab5c93a\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:21\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1668128,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTPH2 distribution in the nuclei of adult brainstem. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of TPH2 distribution in the different regions of adult brainstem of both WT and TG mice at the pre-onset, onset and progression stages. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The comparison of TPH2 distribution in the different nucleus of adult brainstem between both WT and TG mice at the pre-onset, onset and progression stages. 5 mice per group, *p\\u0026lt;0.05. TPH2 distribution in the ROB nucleus of adult brainstem significantly decreased at the onset stage, but that in RPN significantly increased at both onset and progression stages in TG mice. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) The schematic diagram of brainstem anatomic regions. The image in Fig. 10 C was partially reproduced from the published book of the second edition of mouse brain stereotaxic coordinates edited by professor George Paxinos and professor Keith B. J. Franklin.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/1304845e4788fc819ffbc474.png\"},{\"id\":51335369,\"identity\":\"58940606-06b5-47e3-94a6-cf014417d0b7\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 19:06:20\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":949534,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe double staining of TPH2/NeuN/Nestin in adult brainstem. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) The representative images of TPH2 and NeuN double staining in adult brainstem. The distribution of all TPH2 doubly stained with NeuN in adult brainstem. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The representative images of TPH2 and Nestin double stainingin adult brainstem. The distribution of TPH2 wasn't doubly stained with Nestin in brainstem.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/149ce35d250d7209294e61a7.png\"},{\"id\":51335071,\"identity\":\"6101ad0f-7f30-4388-9b4d-9f5e500d4af8\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:21\",\"extension\":\"png\",\"order_by\":12,\"title\":\"Figure 12\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":531400,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCorrelation between 5-HT and TPH2 distribution and neural cell death in both spinal cord and brainstem at the pre-onset, onset and progression stages of TG mice. (\\u003cstrong\\u003eA-F\\u003c/strong\\u003e) The correlation of both 5-HT (\\u003cstrong\\u003eA\\u003c/strong\\u003e, \\u003cstrong\\u003eC\\u003c/strong\\u003e, \\u003cstrong\\u003eE\\u003c/strong\\u003e) and TPH2 (\\u003cstrong\\u003eB\\u003c/strong\\u003e, \\u003cstrong\\u003eD\\u003c/strong\\u003e, \\u003cstrong\\u003eF\\u003c/strong\\u003e) distribution with neural cell death in the cervical, thoracic and lumbar segments at the different stages. 9 mice per group. (\\u003cstrong\\u003eG\\u003c/strong\\u003e) Correlation between TPH2 distribution and neural cell death in the RPN nuclei of brainstem at the pre-onset, onset and progression stages. 5-HT distribution in both thoracic and lumbar segments, TPH2 distribution in the spinal cervical, thoracic and lumbar segments as well as TPH2 positive cells in the RPN nuclei of brainstem presented a significantly negative correlation with neural cell death at the progression stage of TG mice. (\\u003cstrong\\u003eH-J\\u003c/strong\\u003e) Alteration tendency between 5-HT (\\u003cstrong\\u003eH\\u003c/strong\\u003e) and TPH2 (\\u003cstrong\\u003eI\\u003c/strong\\u003e) distribution in spinal cord and TPH2 positive cells (\\u003cstrong\\u003eJ\\u003c/strong\\u003e) in brainstem as well as neural cell death at the pre-onset, onset and progression stages of both WT and TG mice. The increase of both 5-HT and TPH2 distribution was accompanying with neural cell death increase in the spinal cervical, thoracic and lumbar segments in TG mice. The increase of TPH2 distribution was accompanying by neural cell death increase in the RPN nuclei of brainstem in TG mice. 5 mice per group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"RevisedFigure12.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/65bbbcd2edbc46575285118a.png\"},{\"id\":56344714,\"identity\":\"9b3a9939-5f1f-4dde-bbe7-b1891148ca37\",\"added_by\":\"auto\",\"created_at\":\"2024-05-13 01:54:09\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":11537691,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/0be8e6ad-49b4-4f32-b3a3-e52f8af8c018.pdf\"},{\"id\":51335074,\"identity\":\"fc21ae9a-152b-415e-9bb2-0852dfebd832\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:27\",\"extension\":\"tif\",\"order_by\":14,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":108131040,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"H1AWesternblotRawdata.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/d9c0e4393410389da10a36b8.tif\"},{\"id\":51335073,\"identity\":\"c550b863-9a9c-42c7-9334-4c1f447597a8\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:27\",\"extension\":\"tif\",\"order_by\":15,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":96875488,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"H2AWesternblotRawData.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/083f238f74dbcc2f96ad0202.tif\"},{\"id\":51335072,\"identity\":\"25726aa5-1c09-434e-b2c4-a2b4b4276e50\",\"added_by\":\"auto\",\"created_at\":\"2024-02-19 18:58:23\",\"extension\":\"tif\",\"order_by\":16,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":30729288,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TPH2Westernblotrawdata.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3939628/v1/762e748b510d4107e3237f2c.tif\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"5-hydroxytryptamine Distribution Alterations in both Neurons and Synapses: A Potential Pathogenesis of Neuron Death in Tg(SOD1*G93A)1gur Mice\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease mainly damaged motor neuron (MN) [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Approximate 10% of ALS patients are familial ALS (fALS), the rest of 90% patients are sporadic ALS (sALS) [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. The average onset age of ALS is 60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5 years [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. The average disease course of ALS is about 4.4 years from the begin of diagnosis [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. ALS patients usually die from the respiratory failure [\\u003cspan additionalcitationids=\\\"CR5\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. ALS mainly is featured by the selection and progression death of both superior and inferior MN involving cerebrum, brainstem as well as spinal cord, which results in the muscle atrophy of laryngopharyngeal, limbs and even whole body [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTo date, the pathogenesis about ALS has been incompletely understood. Based on the current studied results, it is suggested that the following potential pathogenesis might be closely related to the degeneration of MN in ALS, which are involved in the toxic of mutative ribonucleic acid, the excitatory toxicity, the disorder of protein balance, the defection of axon transportation, the excessive production of reactive oxygen species, the lesion of mitochondria function and the abnormal alterations of non-MN [\\u003cspan additionalcitationids=\\\"CR7 CR8 CR9 CR10\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. In addition, it is hypothesized that ALS should be not only the sole neurodegeneration and death of MN, but also might affects other neural cells besides MN based on the currently studied reports about the pathogenesis of ALS [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR9 CR10\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. The currently researched evidences demonstrate that the traditional viewpoint which ALS only lonely damages MN isn\\u0026rsquo;t completely accurate, and more and more evidences have proved that other nervous systems besides motor systems also are involved in the pathogenesis of ALS [\\u003cspan additionalcitationids=\\\"CR7 CR8 CR9 CR10 CR11\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. The staging pattern of ALS based on the pathological alterations of phosphorylated TAR deoxyribonucleic acid-binding protein (pTDP-43) is consisted of stages 1\\u0026ndash;4. The staging pattern of ALS based on the spread of pTDP-43 further fully proves that the ALS pathological lesion in nervous systems is far beyond the motor areas in the cerebral cortex, brainstem and the anterior horn of spinal cord [\\u003cspan additionalcitationids=\\\"CR14 CR15 CR16 CR17 CR18\\\" citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eRaphe nuclei (RN) is major anatomical regions of 5-hydroxytryptamine (serotonin, 5-HT) neuron distribution, approximate 80% of 5-HT neurons in central nervous system (CNS) distribute in RN. The related molecules of 5-HT neuron functions such as 5-HT1/2A, 5-HT2B/C receptor agonist or 5-HT precursor 5-hydroxytryptophan (5-HTP) may partially protect MN functions, even significantly reverse the progression of ALS [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. In general, the potential effects about 5-HT in the pathogenesis of ALS theoretically have obtained new understanding, especially because several related papers recently were published [\\u003cspan additionalcitationids=\\\"CR22\\\" citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. It is well known that 5-HT is an important monoamine neurotransmitter to transfer happy feel in CNS [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Approximate 90% of 5-HT primarily distribute in the enterochromaffin cell of gastro-intestine tracts besides CNS [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The residual 5-HT are synthesized in the 5-HT neurons of CNS and the majority of 5-HT distribute in the RN of brainstem, exerts some important physiological functions such as regulating mood, appetite, sleep and memory and learn cognition. Therefore, 5-HT neurons in RN are the major sources synthesizing and releasing 5-HT neurotransmitter in CNS [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Axons projecting from RN neurons to other neural structures form 5-HT-nergic neuro-transmitter systems to reach nearly all parts of CNS. 5-HT-nergic-neuron axons in the inferior RNs terminate to cerebellum and spinal cord, but the 5-HT-nergic axons from the superior RNs almost project to entire brain.\\u003c/p\\u003e \\u003cp\\u003eAlthough the current investigation evidences showed that 5-HT neurotransmitter might participate and play some roles in the pathogenesis of ALS, the accurate mechanisms and effects about 5-HT neurotransmitter on ALS haven't been very clarified and exist some debate at present yet. To this end, we observed and analyzed the altered features of 5-HT distribution in neurons and synapses in both spinal cord and brainstem of mainly damaged regions in ALS, and the relationship between 5-HT alterations and neural cell death applying Tg(SOD1*G93A)1Gur (TG) and wild-type (WT) mice. Our results revealed that 5-HT neurotransmitter significantly decreased or increased in both spinal cord and brainstem of TG mice compared with WT mice. Our data showed a closed relationship between 5-HT neurotransmitter alterations and neural cell death in TG mice. This study suggested that the 5-HT neurotransmitter alterations in the neurons and synapses of spinal cord and brainstem might be the potential pathogenesis of neuronal death in the pathogenesis of ALS.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eThe Alterations of 5-HT Distributed Features among the Cervical, Thoracic and Lumbar Segments of Adult Spinal Cord at the Pre-Onset, Onset and Progression Stages between WT and TG Mice\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003e5-HT mainly expressed in the grey matter of adult spinal cord, including the cervical, thoracic and lumbar anterior horn (AH), posterior horn (PH), central lateral column (CLC) and around central canal (CC). In the spinal cervical segment, the 5-HT distribution in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage showed a significant decrease, but that in AH, PH and CC at the progression stage significantly increased while compared WT with TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, B). In the thoracic segment, the distribution of 5-HT in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage significantly decreased, but that in AH at the onset stage and that in AH, PH and CC at the progression stage significantly increased while compared WT with TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, B). In the lumbar segment, the distribution of 5-HT in AH, PH and CC at the pre-onset stage as well as that in PH at the onset stage exhibited a significant decrease, but that in AH at the onset stage and that in AH and PH at the progression stage significantly increased while compared WT with TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA, B).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe comparison of 5-HT distribution in the cervical, thoracic and lumbar AH, PH, CLC, CC and the entire spinal cord at the stages of pre-onset, onset and progression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), results demonstrated that 5-HT distribution in the cervical, thoracic and lumbar AH, PH, CC and the entire spinal cord of WT mice at the stages of onset and progression displayed a significant decrease (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, C, G, I), and the 5-HT distribution in the cervical, thoracic and lumbar CLC of WT mice at the progression stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE) significantly decreased compared with the pre-onset stage. Meanwhile, the 5-HT distribution in AH and the entire cervical, thoracic and lumbar segments of TG mice at the stages of onset and progression (Fig, 4B, J), as well as that in PH at the progression stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD) showed a significant increase, and the 5-HT distribution in the cervical, thoracic and lumbar PH of TG mice at the onset stage significantly decreased compared with the pre-onset stage. The comparison of 5-HT distribution in the cervical, thoracic and lumbar CLC and CC of TG mice at the stages of pre-onset, onset and progression didn't reveal any significant changes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF, H).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo further confirm the alterations of 5-HT expression in spinal cord with the disease progression, Western blot analysis was used to examine TPH2 levels in the cervical, thoracic and lumbar segments of spinal cord at the different stages of TG mice and the age-matched same periods of WT mice. In the cervical segment, TPH2 expression decreased at the onset and progression stages of WT mice, while increased at the progression stage of TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and D). In the thoracic segment, TPH2 expression gradually decreased at the onset and progression stages of WT mice, while significantly increased at the onset stage of TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB and E). In the lumbar segment, TPH2 expression gradually decreased at the onset and progression stages of WT mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC and F). These results confirmed that the entire spinal cord of WT mice displayed a significant decrease of TPH2 at the onset and progression stages, while both the cervical segment of spinal cord in TG mice at the progression stage and the thoracic segment of spinal cord in TG mice at the onset stage showed the significant up-regulation of TPH2.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eNext, we further examined whether the specific 5-HT receptor subtypes increased in TG mice. 5-HTR1A is a highly characterized spinal 5-HTR, is reported to increase the spinal MN excitability [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. 5-HTR1A expression in the thoracic segment of spinal cord was evaluated at three stages of both WT and TG mice. Western blot analysis showed that 5-HTR1A levels decreased at the onset stage of WT mice, which was rescued in TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA and C). 5-HTR2A is another 5-HT receptor subtype which is reported to be involved in mediating 5-HT effects on MN [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. However, 5-HTR2A expression was similar at all three stages of WT mice, while decreased at the onset stage of TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB and D). These results suggested that 5-HTR1A, but not 5-HTR2A, might be involved in ALS. However, the roles of other 5-HT receptor subtypes in ALS can\\u0026rsquo;t be excluded yet.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eThe Alterations of TPH2 Distributed Features in the Cervical, Thoracic and Lumbar Segments of Adult Spinal Cord at the Pre-Onset, Onset and Progression Stages between WT and TG Mice\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eTPH2 mainly expressed in the adult spinal white matter, especially among funiculus lateralis (FL) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA). While compared between WT and TG mice, TPH2 in FL significantly decreased at the pre-onset stage, but TPH2 among the cervical segment showed a significant increase at the progression stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). TPH2 distribution in the cervical segment was more than that among both thoracic and lumbar segments at the progression stage, TPH2 at both pre-onset and onset stages didn\\u0026rsquo;t show the significant difference between the cervical, thoracic and lumbar segments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). In the cervical segment, TPH2 distribution at the progression stage significantly increased compared with that at both pre-onset and onset stages, and was the highest distribution (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eThe Overlapped Distribution of TPH2, 5-HT, DAPI, GFAP, Vimentin and NeuN in the Adult Spinal Cord\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe representative images of TPH2 and DAPI double staining in the lateral sagittal and transverse spinal cord (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA, B). The representative images of TPH2 and GFAP double staining in the lateral transverse spinal cord (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC), the representative images of 5-HT and Vimentin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eD) and 5-HT and NeuN double staining (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e) in the AH, PH, CLC and CC of lateral transverse spinal cord showed no double staining among them. Results indirectly indicated that both TPH2 and 5-HT didn\\u0026rsquo;t express in neuronal bodies, astrocytes, neural precursor cells (NPCs) and neurons, and indirectly implied that both TPH2 and 5-HT expressed in the neuronal synapses based on the anatomic distribution features of neuronal synapses in the adult spinal cord.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eThe Alterations of TPH2 Distributed Features in the Adult Brainstem at the Pre-Onset, Onset and Progression Stages between WT and TG Mice\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eTPH2 mainly expressed in brainstem nuclei, including dorsal RN (DRN), median RN (MRN), raphe magnus nucleus (RMG), raphe obscurus nucleus (ROB), raphe pallidus nucleus (RPA), raphe pontine nucleus (RPN) and lateral paragigantocellular nucleus (LPGI). In WT mice, TPH2 distribution in DRN was the most abundant, the secondary abundance was in MNR and RMG, the third was in ROB, RPA, RPN and LPGI. While compared between WT and TG mice, TPH2 in ROB at the pre-onset stage significantly decreased, TPH2 in RPN at both onset and progression stages significantly increased (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThe Overlapped Distribution of TPH2, Nestin, NeuN and Vimentin in the Adult Brainstem\\u003c/h2\\u003e \\u003cp\\u003eThe representative images of TPH2 double staining with Nestin, NeuN and Vimentin showed that all TPH2 doubly stained with NeuN (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eA), didn\\u0026rsquo;t detect any double staining with Nestin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eB) or Vimentin (No data showed because of similar negative results with Nestin results). The results showed that TPH2 expressed in neurons, but not in the NPCs of brainstem.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eCorrelation between Neural Cell Number and both 5-HT and TPH2 Distribution in Spinal Cord and TPH2 Distribution in Brainstem\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe reduction of neural cell number showed a negative correlation with 5-HT and TPH2 distribution increase in the spinal cervical (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eA, B), thoracic (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eC, D) and lumbar (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eE, F) segments and the TPH2 distribution increase in the RNP nuclei (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eG) of brainstem in TG mice, which indirectly implied that the alterations of 5-HT and TPH2 distribution in both spinal cord and brainstem exhibited a negative correlation with neural cell death in both spinal cord and brainstem 5-HT nuclei (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eH-J). Results reminded us that the increase of both 5-HT and TPH2 distribution in both spinal cord and brainstem might be one of potential candidate factors in neurons death in the pathogenesis of ALS.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn our study, we observed and analyzed the distributed features of 5-HT neurotransmitter in the different anatomical regions and segments of spinal cord, and the different brainstem nuclei in both WT and TG mice applying the fluorescence immunohistochemistry staining of 5-HT and TPH2 biomarkers, performed the comparison of their distribution at the pre-onset, onset and progression stages between WT and TG mice, and observed the relationship between 5-HT distribution and neurons, glial cells and NPCs. Our results showed that 5-HT biomarker mainly distributed in the spinal AH, PH, CLC and CC of grey matter with stripe shape (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e-\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), the distribution in both AH and PH was the most abundant and the secondary was in the cervical, thoracic and lumbar CLC and CC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e-\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). The 5-HT distribution in AH, PH, CLC and/or CC significantly decreased at the pre-onset stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e) compared TG with WT mice, however, they significantly increased at the progression stages of TG mice. Although what happen about the mechanism of abnormal 5-HT distribution in the spinal cord of TG mice wasn\\u0026rsquo;t clear in our study yet, it implied that the abnormal alterations of 5-HT distribution occurred in AH, PH and/or CC, especially in AH. The abnormal distribution of 5-HT in the spinal cord of TG mice was in agreement with the damaged regions of spinal cord in ALS, such as AH. Therefore, we speculated that the abnormal 5-HT distribution in the spinal cord of TG mice participated in the pathogenesis of G93A-SOD1 TG mice, might be related to neuron death in AH in the pathogenesis of ALS.\\u003c/p\\u003e \\u003cp\\u003eTPH2 biomarker mainly distributed in the FL of spinal white matter by linear shape, the distribution in the cervical, thoracic and lumbar FL significantly decreased at the pre-onset stage, and that among the cervical segment significantly increased at the progression stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e), the distribution alterations of 5-HT were responsible to the spinal FL of ALS majorly damaged region. Therefore, we speculated that the abnormal 5-HT distribution in spinal cord was closely associated with FL damage in ALS. Both 5-HT and TPH2 biomarkers in spinal cord didn't overlap stain with neurons, glial cells and NPCs biomarkers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). We hypothesized that the stripe or linear distribution of 5-HT and TPH2 biomarkers in spinal cord were 5-HT synapses projected into spinal cord based on the anatomic features of 5-HT synapse distribution because the synapses of 5-HT neurons comprehensively projected into the FL of white matter and the AH, PH, CLC and CC of grey matter in spinal cord [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Meanwhile, we conducted the comparison of 5-HT synapses in spinal cord between WT and TG mice, results showed 5-HT synapses in the spinal cervical, thoracic and lumbar AH, PH, CC and/or FL significantly reduced at both pre-onset and/or onset stages, however, 5-HT synapses in the cervical, thoracic and lumbar AH, PH, CLC, CC, and/or FL significantly increased at the progression and/or stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Our data revealed that 5-HT synapses in AH reduced at the pre-onset stage, but that at the stages of both onset and progression increased in comparison of WT mice, which further identified that the abnormal 5-HT distribution closely was associated with the damage of neurons in AH in the pathogenesis of ALS.\\u003c/p\\u003e \\u003cp\\u003eIn brainstem, 5-HT neurons mainly distributed in DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei, the brain region of the most abundant 5-HT distribution was in DRN, the secondary was in both MNR and RMG, the third was in ROB, RPA, RPN and LPGI nuclei (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eA, B). TPH2 biomarker in brainstem only overlapped with neuron biomarker, which indicated that TPH2 biomarker expressed cells were neuronal cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003e). Our results further identified that 5-HT neurons existed in the DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei of brainstem [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Our results showed that 5-HT neurons in the ROB of brainstem significantly reduced at the pre-onset stage, however, 5-HT neurons in the RPN of brainstem demonstrated a significant increase at the stages of both TG onset and progression in TG mice. Therefore, our data further revealed that the abnormal alterations of 5-HT neuron distribution exhibited a close relationship to neural cell death among brainstem during the pathogenesis of ALS, especially in distribution in RPN nuclei.\\u003c/p\\u003e \\u003cp\\u003eTPHs are the enzymes synthesized 5-HT neurotransmitter. Human and other mammal animals have 2 unique TPH genes, distribute in the eleventh and twelfth chromosome, encode 2 distinct homogenous enzymes of TPH1 and TPH2 [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. TPH1 mostly expresses in the periphery tissues such as skin, gut and pineal gland, and also expresses among CNS. TPH2 solely expresses among neurons in CNS, is an initial limiting-rate enzyme during synthesizing 5-HT neurotransmitter, mainly expresses among brain 5-HT neurons, largely distributes among midbrain RN [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Therefore, we labeled 5-HT neurons in both spinal cord and brainstem applying TPH2 fluorescent staining. Our results revealed that 5-HT synapses comprehensively projected into FL, AH, PH, CLC and CC as well as cortex and sub-cortex. 5-HT neurons mainly distributed in the RN nuclei of brainstem including DRN, MNR, RMG, ROB, RPA, RPN and LPGI nuclei. Moreover, the abnormal alterations in the 5-HT neuron distribution of both ROB and RPN nuclei were found at the onset and/or progression stages of TG mice, and a significant decrease in ROB nuclei at the onset stage and a significant increase in RPN nuclei at both onset and progression stages were showed. Our data further indicated that 5-HT neurotransmitter participated in the pathogenesis of G93A-SOD1 TG mice because 5-HT neurons in ROB extensively project into both spinal cord and medulla nuclei including ambiguous nucleus [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e], and 5-HT neurons in RPN mainly project into various constructs of cortex and sub-cortex [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], and might exist a potential relationship to the neuronal damage in the commonest damaged region in ALS, such as cortex, sub-cortex, spinal cord and brainstem nuclei, especially in the MN of AH, cortex and sub-cortex as well as the ambiguous nucleus of medulla.\\u003c/p\\u003e \\u003cp\\u003e5-HT nuclei usually are classified into 2 major clusters, caudal and rostral clusters, which contain three and four nuclei respectively. The caudal cluster consists of ROB (B2 nucleus), RMG (also known as B3 nucleus), RPA (B1 nucleus) and lateral medullary reticular nucleus (LMR), which project into brainstem and spinal cord including FL, AH, PH, CLC and CC as well as brainstem related regions, which are involve in the motor activity of both spinal cord and brainstem [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Rostral cluster composes of caudal linear nucleus (CLN) (B8 nucleus), DRN (B6 and B7 nucleus), MNR (B5, B8 and B9 nucleus) and RPN, project to various constructs of cortex and sub-cortex [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. RMG, ROB, RPA and LMR project into brainstem and spinal cord, and CLN, DRN, MNR and RPN project into cortex and sub-cortex regions, which is involved in motor functions through 5-HT pathways [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. RN is a moderate-size cluster of nuclei in brainstem. Based on the sequence from caudal to rostral, RN is divided into ROB, RPA, RMG, RPN, MNR, DRN and caudal linear nucleus. Overall, all caudal RN including RMG, RPA and ROB project to spinal cord and brainstem [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Almost all rostral nuclei included RPN, MNR and DRN project towards the brain areas of higher functions [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. In our study, we observed that the number increase of 5-HT neurons in RPN presented a significantly negative correlation with neural cell death at both onset and progression stages of TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eG, J), which indicated that the increased alterations of 5-HT neurons projected into the brain areas of higher functions participated in the damage of motor and/or non-MN among cerebrum, was one of possible factors that non-MN involved in the pathogenesis of ALS. Moreover, our results showed that the 5-HT ROB projected into spinal cord and brainstem as well as the RPN nuclei projected into various constructs of cortex and sub-cortex significantly increased following by ALS progression respectively, which further indicated that the increase of both 5-HT neurons and synapses among the 5-HT nucleus of brainstem was closely associated with the pathogenesis of MN and/or non-MN death in spinal cord and brainstem as well as cortex and sub-cortex regions in the pathogenesis of ALS.\\u003c/p\\u003e \\u003cp\\u003eAlthough our data illustrated that the abnormal alterations of 5-HT synapses in spinal cord and 5-HT neurons in brainstem were closely associated with the pathogenesis of ALS, it isn\\u0026rsquo;t clearly known that the 5-HT abnormal alterations in both spinal cord and brainstem is whether damage or protect neural cells in the pathogenesis of ALS. Based on the current clinical and experimental evidences as well as our data [\\u003cspan additionalcitationids=\\\"CR36 CR37\\\" citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e], we hypothesized that 5-HT neurotransmitter might play a potential neuronal protective role in ALS probably via 5-HTR1A, non-MN such as 5-HT neurons might play some roles in the pathogenesis of ALS through moderating the 5-HT neurotransmitter releasing into the projecting neurons, and 5-HT neurons participated in the damage course of MN in cortex and sub-cortex regions, brain stem nuclei and AH, was the potential pathogenesis of neuron death in the pathogenesis of ALS.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eG93A-SOD1 Mice\\u003c/h2\\u003e \\u003cp\\u003eThe G93A-SOD1 TG mice from Jackson laboratory (cat#002726, Jackson laboratory, Bar Harbour, ME, USA) was bred by mating male TG mice with female WT mice (cat#000664, Jackson laboratory, Bar Harbour, ME, USA) in The First Affiliated Hospital of Nanchang University. The experimental mice were isolated genome deoxyribonucleic acid (DNA) from mice tail by Rapid Animal Genomic DNA Isolation Kit (cat#B518221, Sangon Biotech, Shanghai, China). G93A-SOD1 TG mice were identified whether or not G93A-SOD1 positive TG mice by the polymerase chain reaction (PCR) of genome DNA. The used primers (Sangon Biotech, Shanghai, China) in PCR were following, the forward primer of IL-2 (PCR internal reference) was 5'-CTA GGC CAC AGA ATT GAA AGA TCT-3', the reverse primer of IL-2 was 5'-GTA GGT GGA AAT TCT AGC ATC ATC C-3', the forward primer of hmSOD1 G93A was 5'-CAT CAG CCC TAA TCC ATC TGA-3', and the reverse primer of hmSOD1 G93A was 5'-CGC GAC TAA CAA TCA AAG TGA-3'. The condition of PCR was: degenerating at 94\\u0026deg;C three secs, annealing at 60\\u0026deg;C one min, extending at 72\\u0026deg;C one min, conducting thirty-five cycles. Animals were sacrificed after anesthetized with CO\\u003csub\\u003e2\\u003c/sub\\u003e at three time points of pre-onset (60\\u0026ndash;70 days), onset (90\\u0026ndash;100 days) and progression (120\\u0026ndash;130 days). The severity of muscle atrophy in hind limbs was determined by the hematoxylin eosin staining of gastrocnemius muscles at different stages, and histologically evaluated different stages by observing changes in muscle structures under light microscopy [\\u003cspan additionalcitationids=\\\"CR40 CR41\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAll experimental mice were housed under 20\\u0026ndash;27\\u0026deg;C, 40\\u0026ndash;50% humidity, 12 hrs light/dark cycle and free access to food or water. ALS Therapy Development Institute (ALSTDI) score was used in monitoring the health and behavioral disorders of mice in order to further determine the disease courses and stages [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Experiments were designed according to the way minimized total number and suffering of used animals. All used mice in this study were manipulated by Chinese Guide for the Care and Use of Laboratory Animals and reviewed by Animal Care and Use Ethics Committee of The First Affiliated Hospital of Nanchang University.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eThe Fluorescence Immunohistochemistry Staining of both Spinal Cord and Brainstem\\u003c/h3\\u003e\\n\\u003cp\\u003eBoth spinal cord and brain were stripped after adequately perfused by 20ml 0.9% saline and 40ml 4% 1xPBS pH 7.5 paraformaldehyde solution (cat#P7059, Sigma-Aldrich, St. Louis, MO, USA) via right ventricle at room temperature (RT) after anesthetized with CO\\u003csub\\u003e2\\u003c/sub\\u003e. The detailed protocols were similar with the experimental methods of Zhou et al. [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. Both spinal cord and brain were immediately put into 4% 1xPBS pH 7.5 paraformaldehyde solution (cat#F8775, Sigma-Aldrich, St. Louis, MO, USA) overnight after taken out, then incubated in 20% 1xPBS pH 7.5 sucrose, embedded using optimal cutting temperature compound (OCT, cat#4583, Sakura Finetek, Chuo-ku, Tokyo, Japan). Both spinal cord and brainstem tissues were successively cut 12\\u0026micro;m coronal section by a freezing microtome and put slices on Superfrost Plus slide. The fluorescence immunohistochemistry staining of spinal cord and brainstem was conducted by following processes, sections were permeabilized with 0.2% TritonX-100 (cat#T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked with 10% 1xPBS bovine serum albumin (cat#SRE0098, Sigma-Aldrich, St. Louis, MO, USA) after rehydrated by pH 7.4 1xPBS. The following primary antibodies were added according to the requirement of experimental designs: 1:100 rabbit anti-Vimentin (cat#EPR3776 RRID:AB_92547, Abcam, Cambridge, MA, USA), 1:100 rabbit anti-Nestin (cat#SP103 RRID:AB_105389, Abcam, Cambridge, MA, USA), 1:200 rabbit anti-NeuN (cat#EPR12763 RRID:AB_ab177487, Abcam, Cambridge, MA, USA), 1:1000 rabbit anti-GFAP (cat#EPR19996 RRID:AB_ab7260, Abcam, Cambridge, MA, USA), 1:400 rat anti-serotonin (5-HT) (cat#YC5/45 RRID:AB_ab6336, Abcam, Cambridge, MA, USA) or 1:600 mouse anti-TPH2 (cat#CL2990 RRID:AB_ ab211528, Abcam, Cambridge, MA, USA) monoclonal antibodies. All primary antibodies were incubated overnight at 4\\u0026deg;C, then lightly washed by 0.2% 1X PBS Triton X-100 buffer for 6 times, each times 5 minutes. The following secondary antibodies conjugating to green or red rhodamine fluorescence were incubated for 2 hrs at RT: 1:250 donkey anti-rabbit (RRID:AB_ab150073, Abcam, Cambridge, MA, USA), 1:250 Donkey Anti-Rat (RRID:AB_ab150153, Abcam, Cambridge, MA, USA) or 1:200 donkey anti mouse (RRID:AB_ab150105, Abcam, Cambridge, MA, USA) IgG (H\\u0026thinsp;+\\u0026thinsp;L) Alexa Fluor 488 cross-adsorbed second antibody, followed staining by DAPI (blue) and treating anti-fluorescence fade after lightly washing 6 times, each times 5 minutes. All slices were observed and taken pictures by a fluorescence microscope equipped with digital camera (Nikon E800, Sumida-ku, Tokyo, Japan) and Photoshop software (Adobe Systems, San Francisco, CA, USA). The staining of doubly labeled fluorescence was performed by simultaneously using Vimentin, Nestin, NeuN, GFAP, 5-HT and TPH2 antibodies and DAPI to doubly stain to identify the proliferation and the distributed cellular types of 5-HT neurons.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThe Analysis of 5-HT and TPH2 Distribution and Positive Cells\\u003c/h2\\u003e \\u003cp\\u003eThe analysis of 5-HT and TPH2 distribution and positive cells was measured the fluorescence intensity of 5-HT and TPH2 stripe or linear structures in the positive cells of ten spinal cord and brainstem sections under magnified 200 times, calculated the sum of ten sections. Total fluorescence intensity were divided by total section number, 5 mice each group, ten slices from each animal. The mean fluorescence intensity was applied to conduct a quantitation analysis. We identified and selected the interest nervous regions of cervical, thoracic and lumbar segments of spinal cord and brainstem based on the mouse spinal cord and brain anatomic atlas according to our previous study protocols [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Image J software (1.8.0 version) was used to quantify 5-HT and TPH2 positive cells in images.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThe Analysis of TPH2, 5HTR1A and 5HTR2A Levels in Spinal Cord by Western Blot\\u003c/h2\\u003e \\u003cp\\u003eThe temporal changes of protein levels were quantified by Western blot analysis. Spinal cords were homogenized in lysis buffer containing phenylmethylsulphonyl fluoride and protease inhibitor after quickly removed from deeply anesthetized male WT and TG mice. The protein concentrations were then assessed by a BCA assay. After separated by 8% SDS-PAGEDs with 3 mg proteins in each lane, total proteins were transferred to a PVDF membrane. After blocked with 10% nonfat milk at 4\\u0026deg;C for 2 hrs, PVDF membranes were incubated by following primary antibodies: 1:200 mouse anti-TPH2 (ab211528, Abcam Ltd., Cambridge, MA, USA), 1:200 rabbit anti-5HTR1A (AF5453, Affinity Biosciences Ltd., Jiangshu, China), 1:200 rabbit anti-5HTR2A (bs-12049R, Beijing Biosynthesis Biotechnology co., Ltd., Bejing, China) or 1:200 rabbit anti-GAPDH (ab245355, Abcam Ltd., Cambridge, MA, USA) antibodies at 4\\u0026deg;C overnight. After incubated by 1:5,000 horseradish peroxidase-labeled goat anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4\\u0026deg;C for 2 hrs, protein bands were visualized with enhanced chemiluminescence reagents (ECL, Pierce, Rockford, IL, USA). The protein levels were quantified and normalized to GAPDH internal controls. 3 mice per group were used.\\u003c/p\\u003e \\u003cp\\u003eFor the quantification analysis of Western blots, gray values were extracted using Image J software, and the gray values of objective proteins were divided by the gray values of internal reference proteins to obtain ratio for each group. The statistical analysis of these ratios was performed using GraphPad Prism (v. 7, GraphPad Software, San Diego, CA, USA). Ten samples per mouse and 5 mice per group were used, and Western blot was repeated three times per sample, and average values were compared and analyzed.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eData Analysis\\u003c/h2\\u003e \\u003cp\\u003eExperimental data were indicated by mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. Comparisons between two groups were applied the analysis of variance and Student's t test. The comparisons of multiple groups were performed using one-way analysis of variance with a Turkey post hoc test. Spearman correlation analysis was used to examine the correlation between positive cells and neural cells. p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 indicated a significantly statistical difference.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eALS, Amyotrophic lateral sclerosis; MN, motor neuron; 5-HT, 5-hydroxytryptamine; TPH2, Tryptophan hydroxylase 2; 5-HTR1A, Serotonin receptor 1A; 5-HTR2A, Serotonin receptor 2A; pTDP-43, phosphorylated TAR deoxyribonucleic acid-binding protein; RN, raphe nuclei; 5-HTP, 5-hydroxytryptophan; CNS, central nervous system; AH, anterior horn; PH, posterior horn; CLC, central lateral column; CC, around central canal; FL, funiculus lateralis; NPCs, neural precursor cells; DRN, dorsal RN; MRN, median RN; RMG, raphe magnus nucleus; ROB, raphe obscurus nucleus; RPA, raphe pallidus nucleus; RPN, raphe pontine nucleus; LPGI, lateral paragigantocellular nucleus.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u0026nbsp;\\u003c/strong\\u003eAll animal studies and experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the ethics committee for animal care and use of\\u0026nbsp;The First Affiliated Hospital of Nanchang University, China (Ethic approval number: 2015-3-12).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to participate\\u0026nbsp;\\u003c/strong\\u003eNo applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003eNo applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u0026nbsp;\\u003c/strong\\u003eAll data and materials are included in this article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003eThe authors declare that they have no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003eThe authors received no financial support for the research, authorship, and/or publication of this article. This study was supported by the research grants to Renshi Xu from\\u0026nbsp;National Natural Science Foundation of China (30560042, 81160161, 81360198,\\u0026nbsp;82160255), Education Department of Jiangxi Province (GJJ13198, GJJ170021), Jiangxi provincial department of science and technology ([2014]-47, 20142BBG70062, 20171BAB215022, 20192BAB205043), Health and Family Planning Commission of Jiangxi province (20181019 and 202210002),\\u0026nbsp;and Jiangxi Provincial Department of Science and Technology Gan Po Elite 555 (Jiangxi Finance Elite Education Refers to [2015] 108).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u0026nbsp;\\u003c/strong\\u003eS. Jiang, M. Li, Q. Dai, H. Nie, H. Pan, R. Xu\\u0026nbsp;performed experiments, analyzed the data and wrote the manuscript;\\u0026nbsp;S. Jiang, M. Li, Q. Dai, X. Liu, C. Li, H. Jiao, H. Nie, H. Pan, R. Xuconducted statistical analyses;\\u0026nbsp;S. Jiang, M. Li, Q. Dai were the common jointed authors and equally contributed to this study.\\u0026nbsp;R. Xu\\u0026nbsp;conceived the project and wrote the manuscript.\\u0026nbsp;R. Xu, H. Pan, H. Nie\\u0026nbsp;revised the manuscript. All authors read the approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003eThis work was supported by grants from National Natural Science Foundation of China (30560042, 81160161, 81360198, 82160255), Education Department of Jiangxi Province (GJJ13198, GJJ170021), Jiangxi provincial department of science and technology ([2014]-47, 20142BBG70062, 20171BAB215022, 20192BAB205043), Health and Family Planning Commission of Jiangxi province (20181019 and 202210002), and Jiangxi Provincial Department of Science and Technology Gan Po Elite 555 (Jiangxi Finance Elite Education Refers to [2015] 108) for Renshi Xu. The partial schematics of spinal cord in Figure 1 and 7 were download from the public websites, these schematics don\\u0026rsquo;t explicitly show any authors and published information, and the schematic of brain stem in Figure 10 C was cited from the second edition of mouse brain stereotaxic coordinates edited by professor George Paxinos and professor Keith B. J. Franklin. Here, specially stated and sincerely thank them. We are sincerely grateful of Dr. Dongyuan Yao for carefully revising our manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eChi\\u0026ograve; A, Logroscino G, Traynor BJ et al (2013) Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature. Neuroepidemiology 41:118\\u0026ndash;130. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1159/000351153\\u003c/span\\u003e\\u003cspan address=\\\"10.1159/000351153\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMehta P, Kaye W, Bryan L et al (2016) Prevalence of Amyotrophic Lateral Sclerosis - United States, 2012\\u0026ndash;2013. MMWR Surveill Summ 65:1\\u0026ndash;12. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.15585/mmwr.ss6508a1\\u003c/span\\u003e\\u003cspan address=\\\"10.15585/mmwr.ss6508a1\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLudolph AC, Brettschneider J, Weishaupt JH (2012) Amyotrophic lateral sclerosis. Curr Opin Neurol 25:530\\u0026ndash;535. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1097/WCO.0b013e328356d328\\u003c/span\\u003e\\u003cspan address=\\\"10.1097/WCO.0b013e328356d328\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOrsini M, Oliveira AB, Nascimento OJM et al (2015) Amyotrophic Lateral Sclerosis: New Perpectives and Update. Neurol Int 7:5885. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.4081/ni.2015.5885\\u003c/span\\u003e\\u003cspan address=\\\"10.4081/ni.2015.5885\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKiernan MC, Vucic S, Cheah BC et al (2011) Amyotrophic lateral sclerosis. Lancet 377:942\\u0026ndash;955. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/S0140-6736(10)61156-7\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/S0140-6736(10)61156-7\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOskarsson B, Gendron TF, Staff NP (2018) Amyotrophic Lateral Sclerosis: An Update for 2018. Mayo Clin Proc 93:1617\\u0026ndash;1628. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.mayocp.2018.04.007\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.mayocp.2018.04.007\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFerraiuolo L, Kirby J, Grierson AJ et al (2011) Molecular pathways of motor neuron injury in amyotrophic lateral sclerosis. Nat Rev Neurol 7:616\\u0026ndash;630. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/nrneurol.2011.152\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/nrneurol.2011.152\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiang H, Wu C, Deng Y et al (2017) Aldehyde Dehydrogenases 1A2 Expression and Distribution are Potentially Associated with Neuron Death in Spinal Cord of Tg(SOD1*G93A)1Gur Mice. Int J Biol Sci 13:574\\u0026ndash;587. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.7150/ijbs.19150\\u003c/span\\u003e\\u003cspan address=\\\"10.7150/ijbs.19150\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi J, Lu Y, Liang H et al (2016) Changes in the Expression of FUS/TLS in Spinal Cords of SOD1 G93A Transgenic Mice and Correlation with Motor-Neuron Degeneration. Int J Biol Sci 12:1181\\u0026ndash;1190. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.7150/ijbs.16158\\u003c/span\\u003e\\u003cspan address=\\\"10.7150/ijbs.16158\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLu Y, Tang C, Zhu L et al (2016) The Overexpression of TDP-43 Protein in the Neuron and Oligodendrocyte Cells Causes the Progressive Motor Neuron Degeneration in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis. Int J Biol Sci 12:1140\\u0026ndash;1149. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.7150/ijbs.15938\\u003c/span\\u003e\\u003cspan address=\\\"10.7150/ijbs.15938\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang J, Huang P, Wu C et al (2018) Preliminary Observation about Alteration of Proteins and Their Potential Functions in Spinal Cord of SOD1 G93A Transgenic Mice. Int J Biol Sci 14:1306\\u0026ndash;1320. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.7150/ijbs.26829\\u003c/span\\u003e\\u003cspan address=\\\"10.7150/ijbs.26829\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang J, Liang H, Zhu L et al (2018) Expression and Distribution of Arylsulfatase B are Closely Associated with Neuron Death in SOD1 G93A Transgenic Mice. Mol Neurobiol 55:1323\\u0026ndash;1337. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s12035-017-0406-9\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s12035-017-0406-9\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi F, Zhou F, Huang M et al (2017) Frequency-Specific Abnormalities of Intrinsic Functional Connectivity Strength among Patients with Amyotrophic Lateral Sclerosis: A Resting-State fMRI Study. Front Aging Neurosci 9:351. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3389/fnagi.2017.00351\\u003c/span\\u003e\\u003cspan address=\\\"10.3389/fnagi.2017.00351\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSilani V, Ludolph A, Fornai F (2017) The emerging picture of ALS: a multisystem, not only a motor neuron disease. Arch Ital Biol 155:99\\u0026ndash;109. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.12871/00039829201741\\u003c/span\\u003e\\u003cspan address=\\\"10.12871/00039829201741\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVerde F, Del Tredici K, Braak H, Ludolph A (2017) The multisystem degeneration amyotrophic lateral sclerosis - neuropathological staging and clinical translation. Arch Ital Biol 155:118\\u0026ndash;130. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.12871/00039829201746\\u003c/span\\u003e\\u003cspan address=\\\"10.12871/00039829201746\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhou F, Gong H, Li F et al (2013) Altered motor network functional connectivity in amyotrophic lateral sclerosis: a resting-state functional magnetic resonance imaging study. NeuroReport 24:657\\u0026ndash;662. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1097/WNR.0b013e328363148c\\u003c/span\\u003e\\u003cspan address=\\\"10.1097/WNR.0b013e328363148c\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhou F, Xu R, Dowd E et al (2014) Alterations in regional functional coherence within the sensory-motor network in amyotrophic lateral sclerosis. Neurosci Lett 558:192\\u0026ndash;196. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.neulet.2013.11.022\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.neulet.2013.11.022\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBrettschneider J, Del Tredici K, Toledo JB et al (2013) Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol 74:20\\u0026ndash;38. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1002/ana.23937\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/ana.23937\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSofic E, Riederer P, Gsell W et al (1991) Biogenic amines and metabolites in spinal cord of patients with Parkinson\\u0026rsquo;s disease and amyotrophic lateral sclerosis. J Neural Transm Park Dis Dement Sect 3:133\\u0026ndash;142. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/BF02260888\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/BF02260888\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTurner BJ, Lopes EC, Cheema SS (2003) The serotonin precursor 5-hydroxytryptophan delays neuromuscular disease in murine familial amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 4:171\\u0026ndash;176. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1080/14660820310009389\\u003c/span\\u003e\\u003cspan address=\\\"10.1080/14660820310009389\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDentel C, Palamiuc L, Henriques A et al (2013) Degeneration of serotonergic neurons in amyotrophic lateral sclerosis: a link to spasticity. Brain 136:483\\u0026ndash;493. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1093/brain/aws274\\u003c/span\\u003e\\u003cspan address=\\\"10.1093/brain/aws274\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEl Oussini H, Bayer H, Scekic-Zahirovic J et al (2016) Serotonin 2B receptor slows disease progression and prevents degeneration of spinal cord mononuclear phagocytes in amyotrophic lateral sclerosis. Acta Neuropathol 131:465\\u0026ndash;480. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s00401-016-1534-4\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s00401-016-1534-4\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEl Oussini H, Scekic-Zahirovic J, Vercruysse P et al (2017) Degeneration of serotonin neurons triggers spasticity in amyotrophic lateral sclerosis. Ann Neurol 82:444\\u0026ndash;456. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1002/ana.25030\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/ana.25030\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYoung SN (2007) How to increase serotonin in the human brain without drugs. J Psychiatry Neurosci 32:394\\u0026ndash;399\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYano JM, Yu K, Donaldson GP et al (2015) Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161:264\\u0026ndash;276. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.cell.2015.02.047\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.cell.2015.02.047\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSiegel GJ (1999) Basic neurochemistry: molecular, cellular, and medical aspects, 6th edn. Lippincott Williams \\u0026amp; Wilkins, Philadelphia\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBinder MD, Hirokawa N, Windhorst U (2009) Encyclopedia of neuroscience. Springer, Berlin\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWu X, Kushwaha N, Albert PR, Penington NJ (2002) A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels. J Physiol 538:41\\u0026ndash;51. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1113/jphysiol.2001.012668\\u003c/span\\u003e\\u003cspan address=\\\"10.1113/jphysiol.2001.012668\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGilmore J, Fedirchuk B (2004) The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres. J Physiol 558:213\\u0026ndash;224. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1113/jphysiol.2004.064717\\u003c/span\\u003e\\u003cspan address=\\\"10.1113/jphysiol.2004.064717\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJacobs BL, Fornal CA (2010) CHAPTER 2.1 - Activity of Brain Serotonergic Neurons in Relation to Physiology and Behavior. In: M\\u0026uuml;ller CP, Jacobs BL (eds) Handbook of Behavioral Neuroscience. Elsevier, pp 153\\u0026ndash;162\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eT\\u0026ouml;rk I (1990) Anatomy of the serotonergic system. Ann N Y Acad Sci 600:9\\u0026ndash;34 discussion 34\\u0026ndash;35. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1111/j.1749-6632.1990.tb16870.x\\u003c/span\\u003e\\u003cspan address=\\\"10.1111/j.1749-6632.1990.tb16870.x\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWalther DJ, Bader M (2003) A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 66:1673\\u0026ndash;1680. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/s0006-2952(03)00556-2\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/s0006-2952(03)00556-2\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWalther DJ, Peter J-U, Bashammakh S et al (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1126/science.1078197\\u003c/span\\u003e\\u003cspan address=\\\"10.1126/science.1078197\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZill P, B\\u0026uuml;ttner A, Eisenmenger W et al (2007) Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study. J Psychiatr Res 41:168\\u0026ndash;173. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jpsychires.2005.05.004\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jpsychires.2005.05.004\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFomina T, Weichwald S, Synofzik M et al (2017) Absence of EEG correlates of self-referential processing depth in ALS. PLoS ONE 12:e0180136. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1371/journal.pone.0180136\\u003c/span\\u003e\\u003cspan address=\\\"10.1371/journal.pone.0180136\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDupuis L, Spreux-Varoquaux O, Bensimon G et al (2010) Platelet serotonin level predicts survival in amyotrophic lateral sclerosis. PLoS ONE 5:e13346. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1371/journal.pone.0013346\\u003c/span\\u003e\\u003cspan address=\\\"10.1371/journal.pone.0013346\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHolecek V, Rokyta R (2018) Possible etiology and treatment of amyotrophic lateral sclerosis. Neuro Endocrinol Lett 38:528\\u0026ndash;531\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKoschnitzky JE, Quinlan KA, Lukas TJ et al (2014) Effect of fluoxetine on disease progression in a mouse model of ALS. J Neurophysiol 111:2164\\u0026ndash;2176. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1152/jn.00425.2013\\u003c/span\\u003e\\u003cspan address=\\\"10.1152/jn.00425.2013\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGurney ME, Pu H, Chiu AY et al (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264:1772\\u0026ndash;1775. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1126/science.8209258\\u003c/span\\u003e\\u003cspan address=\\\"10.1126/science.8209258\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHenriques A, Pitzer C, Schneider A (2010) Characterization of a novel SOD-1(G93A) transgenic mouse line with very decelerated disease development. PLoS ONE 5:e15445. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1371/journal.pone.0015445\\u003c/span\\u003e\\u003cspan address=\\\"10.1371/journal.pone.0015445\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRosen DR, Siddique T, Patterson D et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59\\u0026ndash;62. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/362059a0\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/362059a0\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhou Y, Lu Y, Fang X et al (2015) An astrocyte regenerative response from vimentin-containing cells in the spinal cord of amyotrophic lateral sclerosis\\u0026rsquo;s disease-like transgenic (G93A SOD1) mice. Neurodegener Dis 15:1\\u0026ndash;12. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1159/000369466\\u003c/span\\u003e\\u003cspan address=\\\"10.1159/000369466\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKnippenberg S, Thau N, Dengler R, Petri S (2010) Significance of behavioural tests in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Behav Brain Res 213:82\\u0026ndash;87. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.bbr.2010.04.042\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.bbr.2010.04.042\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eScott S, Kranz JE, Cole J et al (2008) Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler 9:4\\u0026ndash;15. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1080/17482960701856300\\u003c/span\\u003e\\u003cspan address=\\\"10.1080/17482960701856300\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eXu R, Wu C, Tao Y et al (2008) Nestin-positive cells in the spinal cord: a potential source of neural stem cells. Int J Dev Neurosci 26:813\\u0026ndash;820. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.ijdevneu.2008.06.002\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.ijdevneu.2008.06.002\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eXu R, Wu C, Tao Y et al (2010) Description of distributed features of the nestin-containing cells in brains of adult mice: a potential source of neural precursor cells. J Neurosci Res 88:945\\u0026ndash;956. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1002/jnr.22263\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/jnr.22263\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Amyotrophic lateral sclerosis, 5-hydroxytryptamine neuron, 5-hydroxytryptamine receptor, spinal cord, brainstem, pathogenesis\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3939628/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3939628/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, the accurate pathogenesis of ALS hasn\\u0026rsquo;t been found up to now. The previous studied results revealed that the abnormal alterations of some non-motor neurons (MN) were one of potential pathogenesis of MN death in ALS. Therefore, we studied the altered features of 5-hydroxytryptamine (5-HT) distribution and expression in the spinal cord and brainstem of both Tg(SOD1*G93A)1Gur (TG) and wild-type (WT) mice through the fluorescent immunohistochemistry and Western blot methods using the biomarkers of 5-HT neuron and synapse (both 5-HT and Tryptophan hydroxylase 2). Our results revealed that 5-HT synapses mainly distributed in the funiculus lateralis, the anterior horn, the posterior horn, the central lateral column and the around central canal in the cervical, thoracic and lumbar segments of spinal cord, as well as both the raphe nucleus and the lateral paragigantocellular nucleus of brainstem, and gradually reduced following by the age increase in WT mice. However, both 5-HT synapses and 5-hydroxytryptamine receptor 1A (5-HTR1A), but not 5-HTR2A, in spinal cord and 5-HT neurons in brainstem gradually increased following by the progression of disease and presented the significantly negative correlation between the increased distribution of both 5-HT synapses and neurons and neural cell death at the onset and/or progression stages of TG mice. Therefore, it is speculated that the distribution changes of 5-HT synapses in spinal cord and 5-HT neurons in brainstem are closely associated with neuron death, is a potential pathogenesis of ALS.\\u003c/p\\u003e\",\"manuscriptTitle\":\"5-hydroxytryptamine Distribution Alterations in both Neurons and Synapses: A Potential Pathogenesis of Neuron Death in Tg(SOD1*G93A)1gur Mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-02-19 18:58:15\",\"doi\":\"10.21203/rs.3.rs-3939628/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"43286760-b88b-48f2-ac70-54d7f500dde8\",\"owner\":[],\"postedDate\":\"February 19th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-05-13T01:53:45+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-02-19 18:58:15\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3939628\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3939628\",\"identity\":\"rs-3939628\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}