BACKGROUND: Exaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice.
METHODS: We used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos.
RESULTS: We demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons.
CONCLUSIONS: This further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Parkinson\'s disease (PD) is marked by degeneration in the nigrostriatal dopaminergic pathway, affecting motor control via complex changes in the cortico-basal ganglia-thalamic motor network, including the primary motor cortex (M1). The modulation of M1 neuronal activity by dopaminergic inputs, particularly from the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), plays a crucial role in PD pathophysiology. This study investigates how nigrostriatal dopaminergic degeneration influences M1 neuronal activity in rats using in vivo calcium imaging. Histological analysis confirmed dopaminergic lesion severity, with high lesion level rats showing significant motor deficits. Levodopa treatment improved fine motor abilities, particularly in high lesion level rats. Analysis of M1 calcium signals based on dopaminergic lesion severity revealed distinct M1 activity patterns. Animals with low dopaminergic lesion showed increased calcium events, while high lesion level rats exhibited decreased activity, partially restored by levodopa. These findings suggest that M1 activity is more sensitive to transient fluctuations in dopaminergic transmission, rather than to chronic high or low dopaminergic signaling. This study underscores the complex interplay between dopaminergic signaling and M1 neuronal activity in PD symptoms development. Further research integrating behavioral and calcium imaging data can elucidate mechanisms underlying motor deficits and therapeutic responses in PD.

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

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.

Due to the assumed plasticity of immature brain, early in life brain alterations are thought to lead to better recoveries in comparison to the mature brain. Despite clinical needs, how neuronal networks and associated behaviors are affected by early in life brain stresses, such as pediatric concussions, have been overlooked. Here we provide first evidence in mice that a single early in life concussion durably increases neuronal activity in the somatosensory cortex into adulthood, disrupting neuronal integration while the animal is performing sensory-related tasks. This represents a previously unappreciated clinically relevant mechanism for the impairment of sensory-related behavior performance. Furthermore, we demonstrate that pharmacological modulation of the endocannabinoid system a year post-concussion is well-suited to rescue neuronal activity and plasticity, and to normalize sensory-related behavioral performance, addressing the fundamental question of whether a treatment is still possible once post-concussive symptoms have developed, a time-window compatible with clinical treatment.

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。.

Calcium imaging is commonly used to visualize neural activity in vivo. In particular, mesoscale calcium imaging provides large fields of view, allowing for the simultaneous interrogation of neuron ensembles across the neuraxis. In the field of Developmental Neuroscience, mesoscopic imaging has recently yielded intriguing results that have shed new light on the ontogenesis of neural circuits from the first stages of life. We summarize here the technical approaches, basic notions for data analysis and the main findings provided by this technique in the last few years, with a focus on brain development in mouse models. As new tools develop to optimize calcium imaging in vivo, basic principles of neural development should be revised from a mesoscale perspective, that is, taking into account widespread activation of neuronal ensembles across the brain. In the future, combining mesoscale imaging of the dorsal surface of the brain with imaging of deep structures would ensure a more complete understanding of the construction of circuits. Moreover, the combination of mesoscale calcium imaging with other tools, like electrophysiology or high-resolution microscopy, will make up for the spatial and temporal limitations of this technique.

Itch is a distinctive sensation that causes a specific affection and scratching reaction. The anterior cingulate cortex (ACC) has been linked to itch sensation in numerous studies; however, its precise function in processing pruritic inputs remains unknown. Distinguishing the precise role of the ACC in itch sensation can be challenging because of its capacity to conduct heterologous neurophysiological activities. Here, we used in vivo calcium imaging to examine how ACC neurons in free-moving mice react to pruritogenic histamine. In particular, we focused on how the activity of the ACC neurons varied before and after the scratching response. We discovered that although the change in neuronal activity was not synchronized with the scratching reaction, the overall activity of itch-responsive neurons promptly decreased after the scratching response. These findings suggest that the ACC does not directly elicit the feeling of itchiness.

The perception of pain is a multidimensional sensory and emotional/affective experience arising from distributed brain activity. However, the involved brain regions are not specific for pain. Thus, how the cortex distinguishes nociception from other aversive and salient sensory stimuli remains elusive. Additionally, the resulting consequences of chronic neuropathic pain on sensory processing have not been characterized. Using in vivo miniscope calcium imaging with cellular resolution in freely moving mice, we elucidated the principles of nociceptive and sensory coding in the anterior cingulate cortex, a region essential for pain processing. We found that population activity, not single-cell responses, allowed discriminating noxious from other sensory stimuli, ruling out the existence of nociception-specific neurons. Additionally, single-cell stimulus selectivity was highly dynamic over time, but stimulus representation at the population level remained stable. Peripheral nerve injury-induced chronic neuropathic pain led to dysfunctional encoding of sensory events by exacerbation of responses to innocuous stimuli and impairment of pattern separation and stimulus classification, which were restored by analgesic treatment. These findings provide a novel interpretation for altered cortical sensory processing in chronic neuropathic pain and give insights into the effects of systemic analgesic treatment in the cortex.

Plants must adapt to environmental constraints. For this, they are able to perceive several types of stress in isolation or in combination manner. At the cellular level, after the perception of stress, cell signaling is set up to allow the establishment of the specific response. The calcium ion is known to be one of the ubiquitous second messengers which is involved in most of the stresses perceived by the plant. Changes of free cytosolic calcium but also in other cellular compartments are able to activate or inactivate several mechanisms involved in the cell to cope with the changes of environmental conditions. Several calcium reporters have been intensively used to visualize calcium signals in different conditions. In this chapter, we will present only genetically encoded fluorescent reporters for calcium imaging in living plant tissues to measure variations in calcium at several scales. The FRET (fluorescence resonance energy transfer) YC3.60 and the intensiometric GCamP3 sensors will be used in this method chapter. The image analyses will be also detailed for fluorescence quantification of calcium variation.