Anxiety is a remarkably common condition among patients with pharyngitis, but the relationship between these disorders has received little research attention, and the underlying neural mechanisms remain unknown. Here, we show that the densely innervated pharynx transmits signals induced by pharyngeal inflammation to glossopharyngeal and vagal sensory neurons of the nodose/jugular/petrosal (NJP) superganglia in mice. Specifically, the NJP superganglia project to norepinephrinergic neurons in the nucleus of the solitary tract (NTSNE). These NTSNE neurons project to the ventral bed nucleus of the stria terminalis (vBNST) that induces anxiety-like behaviors in a murine model of pharyngeal inflammation. Inhibiting this pharynx→NJP→NTSNE→vBNST circuit can alleviate anxiety-like behaviors associated with pharyngeal inflammation. This study thus defines a pharynx-to-brain axis that mechanistically links pharyngeal inflammation and emotional response.

OBJECTIVE: To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons.
METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope.
RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups.
CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.
目的: 从背根节（DRG）神经元水平说明内脏病变与相应体表穴位敏化产生的神经生物学机制。方法: 皮肤伊文思蓝（EB）外渗实验：BALB/c小鼠随机分为对照组和结肠炎组，每组4只。2，4，6-三硝基苯磺酸直结肠灌注7 d制备结肠炎模型。采用尾静脉注射EB检测体表神经源性炎性反应，观察渗出点的位置及面积。痛行为实验：C57BL/6J小鼠随机分为对照组和结肠炎组，每组8只，造模方法同上，观察下背部和足部Von Frey丝机械刺激诱发的回避或缩足反应次数。小鼠在体DRG钙成像实验：Pirt-GCaMP6s转基因小鼠随机分为对照组和结肠炎组，每组12只，造模方法同上，暴露腰（L）6或L4 DRG，在共聚焦荧光显微镜下观察神经元对直结肠扩张刺激（CRD）、下背部或后爪机械刺激的反应。结果: 与对照组比较，结肠炎组小鼠下背部及后爪神经源性炎性EB渗出较多（P<0.05）；同时结肠炎组小鼠下背部、后爪对机械刺激的回避或缩足反应次数增加（P<0.01，P<0.05）；CRD 60 mm Hg诱发内脏痛引起的L6 DRG神经元激活数量占总数的百分比均较对照组显著增加（P<0.01），其中中型神经元数量增加更为明显（P<0.01）。与对照组相比，结肠炎组小鼠L6 DRG神经元对下背部毛刷刺激反应荧光强度增加（P<0.001），不同直径神经元的荧光强度均增强（P<0.01，P<0.001，P<0.05）。于小鼠后爪施加毛刷、钳夹压力刺激，均引起与结肠不同水平的L4 DRG神经元反应总体数量百分比较对照组显著增加（P<0.01，P<0.05）。结论: 结肠炎可以引起同节段和近节段体表穴位敏化，同时DRG神经元激活数量和反应性增加。其中与直结肠同水平的L6 DRG神经元表现为神经元激活数量百分比和钙荧光信号强度增加，而与内脏邻近水平的L4 DRG神经元表现为激活数量百分比增加，提示可能存在不同的外周神经元敏化机制。.

Duplications of MECP2-containing genomic segments led to severe autistic symptoms in male. Transgenic mice overexpressing the human MECP2 gene exhibit autistic-like behaviors. Neural circuits underlying social defects in MECP2 transgenic (MECP2-TG) mice remain unknown. To observe neural activity of MECP2-TG mice in vivo, we performed calcium imaging by implantation of microendoscope in the hippocampal CA1 regions of MECP2-TG and wild type (WT) mice. We identified neurons whose activities were tightly associated with social interaction, which activity patterns were compromised in MECP2-TG mice. Strikingly, we rescued the social-related neural activity in CA1 and social defects in MECP2-TG mice by deleting the human MECP2 transgene using the CRISPR/Cas9 method during adulthood. Our data points to the neural circuitry responsible for social interactions and provides potential therapeutic targets for autism in adulthood.

Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

General anesthesia has been widely utilized since the 1840s, but its underlying neural circuits remain to be completely understood. Since both general anesthesia and sleep are reversible losses of consciousness, studies on the neural-circuit mechanisms affected by general anesthesia have mainly focused on the neural nuclei or the pathways known to regulate sleep. Three advanced technologies commonly used in neuroscience, in vivo calcium imaging, chemogenetics, and optogenetics, are used to record and modulate the activity of specific neurons or neural circuits in the brain areas of interest. Recently, they have successfully been used to study the neural nuclei and pathways of general anesthesia. This article reviews these three techniques and their applications in the brain nuclei or pathways affected by general anesthesia, to serve as a reference for further and more accurate exploration of other neural circuits under general anesthesia and to contribute to other research fields in the future.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is \"wind-up,\" in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting.

The emergence of advanced and powerful neuroscientific technologies has greatly pushed forward the development of neurobiology in the last decade. Although neurotoxicology is an interdisciplinary subject sharing a mass of technologies with neurobiology, the implementation of these advanced technologies in neurotoxicology is merely seen. Here we describe the detailed methods and materials of some emerging neuroscientific technologies, including optogenetics, fiber photometry, in vivo two-photon Imaging, in vivo calcium imaging, and in vivo electrophysiological recording, hoping that the integration of technologies from neurotoxicology and neuroscience can lend weight to the development of neurotoxicology.