Electroacupuncture Alleviates Memory Deficits in APP/PS1 Mice by Targeting Serotonergic Neurons in Dorsal Raphe Nucleus

Objective Alzheimer’s disease(AD)has become a significant global concern,but effective drugs able to slow down AD progression is still lacked.Electroacupuncture(EA)has been demonstrated to ameliorate cognitive impairment in individuals with AD.However,the underlying mechanisms remains poorly understood.This study aimed at examining the neuroprotective properties of EA and its potential mechanism of action against AD.Methods APP/PS1 transgenic mice were employed to evaluate the protective effects of EA on Shenshu(BL 23)and Baihui(GV 20).Chemogenetic manipulation was used to activate or inhibit serotonergic neurons within the dorsal raphe nucleus(DRN).Learning and memory abilities were assessed by the novel object recognition and Morris water maze tests.Golgi staining,western blot,and immunostaining were utilized to determine EA-induced neuroprotection.Results EA at Shenshu(BL 23)and Baihui(GV 20)effectively ameliorated learning and memory impairments in APP/PS1 mice.EA attenuated dendritic spine loss,increased the expression levels of PSD95,synaptophysin,and brain-derived neurotrophic factor in hippocampus.Activation of serotonergic neurons within the DRN can ameliorate cognitive deficits in AD by activating glutamatergic neurons mediated by 5-HT1B.Chemogenetic inhibition of serotonergic neurons in the DRN reversed the effects of EA on synaptic plasticity and memory.Conclusion EA can alleviate cognitive dysfunction in APP/PS1 mice by activating serotonergic neurons in the DRN.Further study is necessary to better understand how the serotonergic neurons-related neural circuits involves in EA-induced memory improvement in AD.

Overexpression of Sirt6 ameliorates sleep deprivation induced-cognitive impairment by modulating glutamatergic neuron function

Sleep benefits the restoration of energy metabolism and thereby suppo rts neuronal plasticity and cognitive behaviors.Sirt6 is a NAD+-dependent protein deacetylase that has been recognized as an essential regulator of energy metabolism because it modulates various transcriptional regulators and metabolic enzymes.The aim of this study was to investigate the influence of Sirt6 on cerebral function after chronic sleep deprivation(CSD).We assigned C57BL/6J mice to control or two CSD groups and subjected them to AAV2/9-CMV-EGFP or AAV2/9-CMV-Sirt6-EGFP infection in the prelimbic cortex(PrL).We then assessed cerebral functional connectivity(FC) using resting-state functional MRI,neuron/astrocyte metabolism using a metabolic kinetics analysis;dendritic spine densities using sparse-labeling;and miniature excitato ry postsynaptic currents(mEPSCs) and action potential(AP) firing rates using whole-cell patchclamp recordings.In addition,we evaluated cognition via a comprehensive set of behavioral tests.Compared with controls,Sirt6 was significantly decreased(P<0.05) in the PrL after CSD,accompanied by cognitive deficits and decreased FC between the PrL and accumbens nucleus,piriform cortex,motor co rtex,somatosensory co rtex,olfactory tubercle,insular cortex,and cerebellum.Sirt6 ove rexpression reve rsed CSD-induced cognitive impairment and reduced FC.Our analysis of metabolic kinetics using [1-13C] glucose and [2-13C] acetate showed that CSD reduced neuronal Glu4and GABA2synthesis,which could be fully restored via forced Sirt6 expression.Furthermore,Sirt6 ove rexpression reversed CSD-induced decreases in AP firing rates as well as the frequency and amplitude of mEPSCs in PrL pyramidal neurons.These data indicate that Sirt6 can improve cognitive impairment after CSD by regulating the PrL-associated FC network,neuronal glucose metabolism,and glutamatergic neurotransmission.Thus,Sirt6 activation may have potential as a novel strategy for treating sleep disorder-related diseases.

Chemogenetic activation of sublaterodorsal tegmental glutamatergic neurons alleviates rapid eye movement sleep behavior disorder symptoms in a chronic rat model of Parkinson disease

Rapid eye movement(REM)sleep behavior disorder(RBD)is a parasomnia that is featured by elevated motor behaviors and dream enactments during REM sleep.Clinical observations show that RBD bears significant relevance with several synucleinopathies such as Lewy body dementia and Parkinson disease(PD),and often develops prior to their diagnosis.Being a potential biomarker of PD,investigating the relationship of RBD symptoms and their emergence in developing PD would provide insight intoits pathogenesis.Here,in a chronic model of PD,rats with daily rotenone treatment exhibited key RBD features,including elevated sleep muscle tone,sleep fragmentation and EEG slowing at different time points.Based on detectedearly alpha synuclein aggregation and neural apoptosis in the sublaterodorsal tegmental nucleus(SLD),an area known to promote REM sleep and maintain sleep muscle atonia,the possible involvement of SLD glutamatergic neurons was interrogated.Via chemogenetic activation of SLD glutamatergic neurons,key RBD symptoms and EEG slowing in REM sleep were alleviated.These results are consistent with a progressive degeneration in REM sleep promoting pathways.Our findings provide a foundation for further studies into RBD and its relationship to neurodegenerative diseases.

Sodium Leak Channel in Glutamatergic Neurons of the Lateral Parabrachial Nucleus Helps to Maintain Respiratory Frequency Under Sevoflurane Anesthesia

The lateral parabrachial nucleus(PBL)is implicated in the regulation of respiratory activity.Sodium leak channel(NALCN)mutations disrupt the respiratory rhythm and influence anesthetic sensitivity in both rodents and humans.Here,we investigated whether the NALCN in PBL glutamatergic neurons maintains respiratory function under general anesthesia.Our results showed that chemogenetic activation of PBL glutamatergic neurons increased the respiratory frequency(RF)in mice;whereas chemogenetic inhibition suppressed RF.NALCN knockdown in PBL glutamatergic neurons but not GABAergic neurons significantly reduced RF under physiological conditions and caused more respiratory suppression under sevoflurane anesthesia.NALCN knockdown in PBL glutamatergic neurons did not further exacerbate the respiratory suppression induced by propofol or morphine.Under sevoflurane anesthesia,painful stimuli rapidly increased the RF,which was not affected by NALCN knockdown in PBL glutamatergic neurons.This study suggested that the NALCN is a key ion channel in PBL glutamatergic neurons that maintains respiratory frequency under volatile anesthetic sevoflurane but not intravenous anesthetic propofol.

MDGA2 Constrains Glutamatergic Inputs Selectively onto CA1 Pyramidal Neurons to Optimize Neural Circuits for Plasticity,Memory,and Social Behavior

Synapse organizers are essential for the development,transmission,and plasticity of synapses.Acting as rare synapse suppressors,the MAM domain containing glycosylphosphatidylinositol anchor(MDGA)proteins contributes to synapse organization by inhibiting the formation of the synaptogenic neuroligin-neurexin complex.A previous analysis of MDGA2 mice lacking a single copy of Mdga2 revealed upregulated glutamatergic synapses and behaviors consistent with autism.However,MDGA2 is expressed in diverse cell types and is localized to both excitatory and inhibitory synapses.Differentiating the network versus cell-specific effects of MDGA2 loss-of-function requires a cell-type and brain region-selective strategy.To address this,we generated mice harboring a conditional knockout of Mdga2 restricted to CA1 pyramidal neurons.Here we report that MDGA2 suppresses the density and function of excitatory synapses selectively on pyramidal neurons in the mature hippocampus.Conditional deletion of Mdga2 in CA1 pyramidal neurons of adult mice upregulated miniature and spontaneous excitatory postsynaptic potentials,vesicular glutamate transporter 1 intensity,and neuronal excitability.These effects were limited to glutamatergic synapses as no changes were detected in miniature and spontaneous inhibitory postsynaptic potential properties or vesicular GABA transporter intensity.Functionally,evoked basal synaptic transmission and AMPAR receptor currents were enhanced at glutamatergic inputs.At a behavioral level,memory appeared to be compromised in Mdga2 cKO mice as both novel object recognition and contextual fear conditioning performance were impaired,consistent with deficits in long-term potentiation in the CA3-CA1 pathway.Social affiliation,a behavioral analog of social deficits in autism,was similarly compromised.These results demonstrate that MDGA2 confines the properties of excitatory synapses to CA1 neurons in mature hippocampal circuits,thereby optimizing this network for plasticity,cognition,and social behaviors.

Activation of glutamatergic neurons in the somatosensory cortex promotes remyelination in ischemic vascular dementia

Chronic cerebral hypoperfusion can cause progressive demyelination as well as ischemic vascular dementia,however no effective treatments are available.Here,based on magnetic resonance imaging studies of patients with white matter damage,we found that this damage is associated with disorganized cortical structure.In a mouse model,optogenetic activation of glutamatergic neurons in the somatosensory cortex significantly promoted oligodendrocyte progenitor cell(OPC)proliferation,remyelination in the corpus callosum,and recovery of cognitive ability after cerebral hypoperfusion.The therapeutic effect of such stimulation was restricted to the upper layers of the cortex,but also spanned a wide time window after ischemia.Mechanistically,enhancement of glutamatergic neuron-OPC functional synaptic connections is required to achieve the protection effect of activating cortical glutamatergic neurons.Additionally,skin stroking,an easier method to translate into clinical practice,activated the somatosensory cortex,thereby promoting OPC proliferation,remyelination and cognitive recovery following cerebral hypoperfusion.In summary,we demonstrated that activating glutamatergic neurons in the somatosensory cortex promotes the proliferation of OPCs and remyelination to recover cognitive function after chronic cerebral hypoperfusion.It should be noted that this activation may provide new approaches for treating ischemic vascular dementia via the precise regulation of glutamatergic neuron-OPC circuits.

Glutamatergic neurons in the paraventricular nucleus of the hypothalamus participate in the regulation of visceral pain induced by pancreatic cancer in mice

Background:Visceral pain induced by pancreatic cancer seriously affects patients’quality of life,and there is no effective treatment,because the mechanism of its neural circuit is unknown.Therefore,the aim of this study is to explore the main neural circuit mechanism regulating visceral pain induced by pancreatic cancer in mice.Methods:The mouse model of pancreatic cancer visceral pain was established on C57BL/6N mice by pancreatic injection of mPAKPC-luc cells.Abdominal mechanical hyperalgesia and hunch score were performed to assess visceral pain;the pseudorabies virus(PRV)was used to identify the brain regions innervating the pancreas;the c-fos co-labeling method was used to ascertain the types of activated neurons;in vitro electrophysiological patch-clamp technique was used to record the electrophysiological activity of specific neurons;the calcium imaging technique was used to determine the calcium activity of specific neurons;specific neuron destruction and chemogenetics methods were used to explore whether specific neurons were involved in visceral pain induced by pancreatic cancer.Results:The PRV injected into the pancreas was detected in the paraventricular nucleus of the hypothalamus(PVN).Immunofluorescence staining showed that the majority of c-fos were co-labeled with glutamatergic neurons in the PVN.In vitro electrophysiological results showed that the firing frequency of glutamatergic neurons in the PVN was increased.The calcium imaging results showed that the calcium activity of glutamatergic neurons in the PVN was enhanced.Both specific destruction of glutamatergic neurons and chemogenetics inhibition of glutamatergic neurons in the PVN alleviated visceral pain induced by pancreatic cancer.Conclusions:Glutamatergic neurons in the PVN participate in the regulation of visceral pain induced by pancreatic cancer in mice,providing new insights for the discovery of effective targets for the treatment of pancreatic cancer visceral pain.

Using transcription factors for direct reprogramming of neurons in vitro

Cell therapy offers great promises in replacing the neurons lost due to neurodegenerative diseases or injuries.However,a key challenge is the cellular source for transplantation which is often limited by donor availability.Direct reprogramming provides an exciting avenue to generate specialized neuron subtypes in vitro,which have the potential to be used for autologous transplantation,as well as generation of patient-specific disease models in the lab for drug discovery and testing gene therapy.Here we present a detailed review on transcription factors that promote direct reprogramming of specific neuronal subtypes with particular focus on glutamatergic,GABAergic,dopaminergic,sensory and retinal neurons.We will discuss the developmental role of master transcriptional regulators and specification factors for neuronal subtypes,and summarize their use in promoting direct reprogramming into different neuronal subtypes.Furthermore,we will discuss up-and-coming technologies that advance the cell reprogramming field,including the use of computational prediction of reprogramming factors,opportunity of cellular reprogramming using small chemicals and microRNA,as well as the exciting potential for applying direct reprogramming in vivo as a novel approach to promote neuro-regeneration within the body.Finally,we will highlight the clinical potential of direct reprogramming and discuss the hurdles that need to be overcome for clinical translation.

Optogenetic activation of glutamatergic neu⁃rons in somatosensory cortex promotes remy⁃elination in ischemic vascular dementia

OBJECTIVE Chronic cerebral hy⁃poperfusion can lead to progressive demyelin⁃ation and ischemic vascular dementia,yet there are no effective treatments.METHODS Magnetic resonance imaging was employed in patients with white matter damage,and optogenetics and skin stroking were exerted to activate glutamater⁃gic neurons in the somatosensory cortex in a clas⁃sical mouse model of ischemia vascular dementia.RESULTS White matter damage was correlated with disrupted cortical structure from MRI results.In a mouse model,activating glutamatergic neu⁃rons in the somatosensory cortex promotes prolif⁃eration of OPCs and remyelination to rescue cog⁃nitive impairment after chronic cerebral hypoper⁃fusion.Such therapeutic action was limited to stimulation with moderate intensity at the upper layers of the cortex,but was achieved over a wide time window after ischemia.Mechanistically,enhanced glutamatergic neuron-OPC functional synaptic connections are required for protection from activation of cortical glutamatergic neurons.Finally,skin stroking activation of the somatosen⁃sory cortex,an easier approach for clinical trans⁃lation,promoted OPC proliferation and remyelin⁃ation as well as cognitive recovery after cerebral hypoperfusion.CONCLUSION Activation of gluta⁃matergic neurons in the somatosensory cortex may serve as novel approaches for treating isch⁃emic vascular dementia through precise modula⁃tion of glutamatergic neuron-OPC circuits.

蓝斑核谷氨酸能神经元参与调节小鼠体重

下丘脑是参与进食调控的重要脑区,具有复杂的环路调控机制。然而,是否存在下丘脑以外同样发挥体重调节功能的神经核团尚不清楚。本试验利用神经环路示踪技术,鉴定向背肩胛棕色脂肪组织(interscapular brown adipose tissue, IBAT)发送神经元投射的神经元类型和分布,探究中枢神经元及神经环路调节能量稳态的形态学基础。首先通过报告基因和逆行示踪识别囊泡型谷氨酸转运体2(vesicular glutamate transporter 2, VGlut2)和细胞外调节蛋白激酶(extracellular regulated protein kinase, ERK)神经元,顺行示踪试验拓展VGlut2神经元发送神经支配的大脑区域。化学遗传技术验证VGlut2免疫阳性神经元的体重和采食调控作用。结果显示:LC^(VGlut2::ERK)神经元向IBAT发送密集的神经支配信号。ERK免疫阳性神经元富集表达在LC,可作为LC特异性标记物。LC^(VGlut2)神经元向纹状体(neurons project to the striatum, CPu)、第二运动皮层(secondary motor cortex, M2)、下丘脑腹内侧核(ventromedial hypothalamic nucleus, VMH)和迷走神经背核(dorsal motor nucleus of the vagus, DMV)发送神经元投射。化学遗传激活LC^(VGlut2)神经元显著降低小鼠体重(P=0.016 5)和采食(P=0.029 0)。综上,LC^(VGlut2)神经元参与小鼠的体重调节过程。鉴定除下丘脑室旁核(paraventricular nucleus of hypothalamus, PVN)以外的谷氨酸能神经元及调节体重的下游神经环路,将进一步加深对于摄食行为和摄食过程中神经调控机制的了解,为研究和干预肥胖相关的疾病提供新思路。

五行音乐对孕期恐应激子代社交行为的影响及机制研究

目的探究五行音乐对孕期恐应激子代社交行为的影响和机制,为临床胎源性情志疾病的早期防治提供依据。方法将45只孕鼠随机分为空白组、模型组和五行音乐组。其中,模型组、五行音乐组均采用旁观电击法造模,五行音乐组在孕期的每天17:00~19:00播放五行音乐中的宫调。孕第19天对各组孕鼠采用ELISA法检测血清中促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)和皮质醇(corticosterone,CORT)的含量进行造模评价。仔鼠延续母鼠的分组,于8周龄时进行三箱社交实验观察社交行为;免疫荧光双标法检测仔鼠内侧前额叶皮层(medial prefrontal cortex,mPFC)谷氨酸能神经元活性的表达;高效液相色谱法检测仔鼠mPFC区谷氨酸(glutamate,Glu)总含量;高尔基染色观察仔鼠mPFC区神经元树突棘的变化。结果与空白组相比,模型组孕鼠血清中ACTH、CORT含量显著上升,模型组仔鼠社交交互时间和社会新奇偏好指数显著降低,谷氨酸能神经元活性、谷氨酸含量及神经元树突棘显著下降。与模型组相比,五行音乐组孕鼠血清中ACTH、CORT的含量降低及仔鼠社交行为、谷氨酸能神经元活性、谷氨酸含量及神经元树突棘的状态得到改善。结论五行音乐干预能够有效改善孕期恐应激所致子代的社交行为障碍,其机制可能与增强mPFC区谷氨酸能神经元的活性有关。

内侧前额叶皮质谷氨酸能神经元参与CD1小鼠攻击行为的机制研究

目的研究CD1小鼠发动攻击行为的内在神经机制。方法通过驻地入侵实验筛选出具有攻击行为的CD1小鼠,攻击配对偏好进一步验证后使用c-Fos标记全脑激活的脑区,通过免疫荧光双标实验分析攻击行为激活了哪种类型的神经元,最后使用光遗传学调控该类神经元的活性,观察其对攻击行为的影响。结果通过c-Fos筛选,约82%的CD1小鼠表现出攻击行为;攻击行为发生后主要激活内侧前额叶皮质(medial prefrontal cortex,mPFC),免疫荧光双标结果显示mPFC脑区c-Fos阳性神经元主要是谷氨酸能神经元;最后,我们通过光遗传激活mPFC脑区谷氨酸能神经元,发现其能够抑制CD1小鼠的攻击行为;相反,光遗传抑制mPFC脑区谷氨酸能神经元,能够促进CD1小鼠的攻击行为。结论mPFC脑区谷氨酸能神经元是调控CD1小鼠发动攻击行为的重要组成部分。

抑制dmPAG区谷氨酸能神经元可减轻创伤后应激障碍小鼠的过度防御反应

目的探索背内侧中脑导水管周围灰质(dmPAG)谷氨酸能神经元对创伤后应激障碍(PTSD)小鼠过度防御行为的调控作用。方法84只雄性C57BL/6J小鼠随机分为12组(7只/组)。研究PTSD后过度防御反应时,分为CON组和PTSD组;研究激活dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响,分为CON NS组,CON mCherry组,PTSD mCherry组和PTSD hM3Dq组;研究抑制dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响,分为CON NS组,CON mCherry组,PTSD mCherry组和PTSD hM4Di组。构建单效长时程应激模型(SPS)模拟PTSD状态,通过视觉本能恐惧实验、触须刺激实验、条件恐惧实验测试小鼠的防御行为,免疫荧光染色观察dmPAG区谷氨酸能神经元活性变化。结果与CON组相比,PTSD组回巢潜伏期缩短(P<0.001),巢内停留时间百分比增加(P<0.01),防御评分增加(P<0.001),僵直时间百分比增加(P<0.01)。免疫荧光染色结果可见PTSD小鼠防御行为过程中,dmPAG区c-fos阳性的谷氨酸能神经元占比明显增加(P<0.001)。与PTSD mCherry组相比,激活dmPAG区谷氨酸能神经元后,PTSD hM3Dq组回巢潜伏期、巢内停留时间百分比无差异,防御评分升高(P<0.05),僵直时间百分数增加(P<0.01)。与PTSD mCherry组相比,抑制dmPAG区谷氨酸能神经元后,PTSD hM4Di组回巢潜伏期延长(P<0.05),巢内停留时间百分比降低(P<0.05),防御评分降低(P<0.01),僵直时间百分比降低(P<0.01)。结论抑制dmPAG区谷氨酸能神经元活性可以明显减轻PTSD的过度防御行为。

How do lateral septum projections to the ventral CA1 influence sociability?

Social dysfunction is a risk factor for several neuropsychiatric illnesses.Previous studies have shown that the lateral septum(LS)-related pathway plays a critical role in mediating social behaviors.Howeve r,the role of the connections between the LS and its downstream brain regions in social behavio rs remains unclea r.In this study,we conducted a three-chamber test using electrophysiological and chemogenetic approaches in mice to determine how LS projections to ventral CA1(vCA1)influence sociability.Our res ults showed that gamma-aminobutyric acid(GABA)-e rgic neuro ns were activated following social experience,and that social behavio rs were enhanced by chemogenetic modulation of these neurons.Moreover,LS GABAergic neurons extended their functional neural connections via vCA1 glutamatergic pyramidal neurons,and regulating LSGABA→vCA1Gluneural projections affected social behaviors,which were impeded by suppressing LSprojecting vCA1 neuronal activity or inhibiting GABAAreceptors in vCA1.These findings support the hypothesis that LS inputs to the vCA1 can control social prefe rences and social novelty behaviors.These findings provide new insights rega rding the neural circuits that regulate sociability.

基底外侧杏仁核谷氨酸能神经元参与异氟醚麻醉觉醒的调控

目的探讨基底外侧杏仁核(BLA)谷氨酸能神经元在异氟醚麻醉中的调控作用。方法采用健康雄性8~12周龄Vglut2-Cre转基因小鼠。15只小鼠随机分为光遗传激活组(ChR2组)、抑制组(GtACR组)和对照组(mCherry组),每组5只,分别在BLA核团立体定位注射兴奋性光遗传病毒rAAV2/9-EF1α-DIO-hChR2-mCherry-WPRE-hGH pA、抑制性光遗传病毒rAAV2/9-EF1α-DIO-GtACR-mCherry-WPRE-hGH pA或对照病毒rAAV2/9-EF1α-mCherry-WPRE-hGH pA,并埋置陶瓷插芯。待病毒表达3周后,对待试小鼠进行14 mL/L异氟醚麻醉,并全程记录小鼠皮层脑电(EEG),爆发抑制比(BSR)稳定时光遗传调控BLA谷氨酸神经元,比较光刺激前2 min与刺激2 min时的BSR。21只Vglut2-Gre小鼠随机分为化学遗传激活组(hM3Dq组)、抑制组(hM4Di组)和对照组(mCherry组),每组7只,分别在双侧BLA核团注射化学遗传病毒,3周后进行化学遗传调控实验,比较稳定深度麻醉状态中同一时间点各组EEG频谱及各频段百分比总功率以及翻正反射消失及恢复(LORR/RORR)时间。结果与光刺激前相比,光刺激期间ChR2组BSR降低(P<0.05),GtACR组BSR增加(P<0.05),而mCherry组BSR无统计学差异(P>0.05)。化学遗传实验中,与mCherry组相比,深度麻醉状态下,hM4Di组皮层EEG的α、β、γ频段百分比总功率明显增加(P<0.05),hM3Dq组EEG的α、β、γ频段百分比总功率无统计学差异(P>0.05);hM3Dq组和hM4Di组LORR的时间无统计学差异(P>0.05),而hM3Dq组RORR的时间缩短,hM4Di组RORR的时间延长(P<0.01)。结论BLA谷氨酸神经元参与调控小鼠异氟醚麻醉的觉醒过程,激活BLA谷氨酸神经元,可以显著促进小鼠从异氟醚麻醉状态中觉醒。

Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Modulate the Anesthetic Potency of Isoflurane in Mice

The lateral hypothalamic area(LHA)plays a pivotal role in regulating consciousness transition,in which orexinergic neurons,GABAergic neurons,and melanin-concentrating hormone neurons are involved.Glutamatergic neurons have a large population in the LHA,but their anesthesia-related effect has not been explored.Here,we found that genetic ablation of LHA glutamatergic neurons shortened the induction time and prolonged the recovery time of isoflurane anesthesia in mice.In contrast,chemogenetic activation of LHA glutamatergic neurons increased the time to anesthesia and decreased the time to recovery.Optogenetic activation of LHA glutamatergic neurons during the maintenance of anesthesia reduced the burst suppression pattern of the electroencephalogram(EEG)and shifted EEG features to an arousal pattern.Photostimulation of LHA glutamatergic projections to the lateral habenula(LHb)also facilitated the emergence from anesthesia and the transition of anesthesia depth to a lighter level.Collectively,LHA glutamatergic neurons and their projections to the LHb regulate anesthetic potency and EEG features.

Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds

Establishing an effective three-dimensional(3D) in vitro culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane(PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system,we further developed an in vitro disease-like model of traumatic brain injury(TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D in vitro human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases.

Sinoatrial node pacemaker cells share dominant biological properties with glutamatergic neurons

Activation of the heart normally begins in the sinoatrial node(SAN).Electrical impulses spontaneously released by SAN pacemaker cells(SANPCs)trigger the contraction of the heart.However,the cellular nature of SANPCs remains controversial.Here,we report that SANPCs exhibit glutamatergic neuron-like properties.By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse,we found that SANPCs co-clustered with cortical neurons.Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system,expressing genes encoding glutamate synthesis pathway(G/s),ionotropic and metabotropic glutamate receptors(Grina,Gria3,Grm1 and Grm5)t and glutamate transporters(Slc17a7).SANPCs highly expressed cell markers of glutamatergic neurons(Snap25 and S/-c17a7)t whereas Gad1,a marker of GABAergic neurons,was negative.Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca2+transients frequency in single SANPC.Collectively,our work suggests that SANPCs share dominant biological properties with glutamatergic neurons,and the glutamatergic neurotransmitter system may act as an intrinsic regulation module of heart rhythm,which provides a potential intervention target for pacemaker cell-associated arrhythmias.

Distinct neuronal excitability alterations of medial prefrontal cortex in early-life neglect model of rats

Object:Early-life neglect has irreversible emotional effects on the central nervous system.In this work,we aimed to elucidate distinct functional neural changes in me-dial prefrontal cortex(mPFC)of model rats.Methods:Maternal separation with early weaning was used as a rat model of early-life neglect.The excitation of glutamatergic and GABAergic neurons in rat mPFC was recorded and analyzed by whole-cell patch clamp.Results:Glutamatergic and GABAergic neurons of mPFC were distinguished by typi-cal electrophysiological properties.The excitation of mPFC glutamatergic neurons was significantly increased in male groups,while the excitation of mPFC GABAergic neurons was significant in both female and male groups,but mainly in terms of rest membrane potential and amplitude,respectively.Conclusions:Glutamatergic and GABAergic neurons in medial prefrontal cortex showed different excitability changes in a rat model of early-life neglect,which can contribute to distinct mechanisms for emotional and cognitive manifestations.

Glutamatergic CYLD deletion leads to aberrant excitatory activity in the basolateral amygdala:association with enhanced cued fear expression

Neuronal activity,synaptic transmission,and molecular changes in the basolateral amygdala play critical roles in fear memory.Cylindromatosis(CYLD)is a deubiquitinase that negatively regulates the nuclear factor kappa-B pathway.CYLD is well studied in non-neuronal cells,yet underinvestigated in the brain,where it is highly expressed.Emerging studies have shown involvement of CYLD in the remodeling of glutamatergic synapses,neuroinflammation,fear memory,and anxiety-and autism-like behaviors.However,the precise role of CYLD in glutamatergic neurons is largely unknown.Here,we first proposed involvement of CYLD in cued fear expression.We next constructed transgenic model mice with specific deletion of Cyld from glutamatergic neurons.Our results show that glutamatergic CYLD deficiency exaggerated the expression of cued fear in only male mice.Further,loss of CYLD in glutamatergic neurons resulted in enhanced neuronal activation,impaired excitatory synaptic transmission,and altered levels of glutamate receptors accompanied by over-activation of microglia in the basolateral amygdala of male mice.Altogether,our study suggests a critical role of glutamatergic CYLD in maintaining normal neuronal,synaptic,and microglial activation.This may contribute,at least in part,to cued fear expression.