in-vivo-calcium-imaging-neuronal-ensembles-networks-primary-sensory

<em>In Vivo</em> Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

<em>In Vivo</em> Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

Calcium imaging is an important tool for monitoring signaling mechanisms that either influx Ca2+ into the cytosol or release Ca2+ from intracellular Ca2+ ...

University of Texas Health Science Center at San Antonio

University of Alabama at Birmingham

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca response in  DRG and in  spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca response because of degeneration of primary sensory nerve fibers in the skin. Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to  models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary  populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca activity and postoperative pain.

 Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Reduced skin blood flow has been reported in neuropathic pain patients as well as various peripheral neuropathic pain model animals. We have previously shown that vasodilators, which improves reduced skin blood flow, correlatively alleviate neuropathic pain in chronic constriction injury (CCI) mice, a model of neuropathic pain from peripheral nerve injury. Here, we sought to elucidate the mechanism underlying the reduced skin blood flow in CCI rats. The skin blood flow of the ipsilateral plantar arteries was significantly reduced compared to that of the contralateral ones 4 weeks after loose ligation of the sciatic nerve. The contraction induced by noradrenaline, serotonin, and U46619, a thromboxane receptor agonist, in the isolated ipsilateral plantar arteries was significantly enhanced compared to that in the contralateral ones. KB-R7943, a Na/Ca exchanger (NCX) inhibitor, shifted the concentration-response curves of noradrenaline to the left in the contralateral arteries but had no effect on the ipsilateral side. There was no significant difference in concentration-response curves of noradrenaline between the ipsilateral and contralateral arteries in the presence of KB-R7943. Amiloride, a non-specific inhibitor of Na channels and transporters, comparably shifted concentration-response curves of noradrenaline to the left in both the contralateral and ipsilateral arteries. One hundred nM of noradrenaline induced intracellular Ca elevation in the ipsilateral arteries, which was significantly larger than that induced by 300-nM noradrenaline in the contralateral arteries. These results suggest that reduced peripheral blood flow after nerve injury is due to Na-dependent inactivation of NCX in the ipsilateral plantar arteries.

Na-dependent inactivation of vascular Na/Ca exchanger responsible for reduced peripheral blood flow in neuropathic pain model.

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3-L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3-L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1 and CGRP TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

cytoNet: Spatiotemporal network analysis of cell communities.

Chemotherapy-induced peripheral neuropathy (CIPN) affects about 68% of patients undergoing chemotherapy, causing debilitating neuropathic pain and reducing quality of life. Cisplatin is a commonly used platinum-based chemotherapeutic drug known to cause CIPN, possibly by causing oxidative stress damage to primary sensory neurons. Metabotropic glutamate receptors (mGluRs) are widely hypothesized to be involved in pain processing and pain mitigation. Meclizine is an H1 histamine receptor antagonist known to have neuroprotective effects, including an anti-oxidative effect. Here, we used a mouse model of cisplatin-induced CIPN using male and female mice to test agonists of mGluR8 and group II mGluR as well as meclizine as interventions to reduce cisplatin-induced pain. We performed behavioral pain tests, and we imaged Ca activity of the large population of DRG neurons  For the latter, we used a genetically-encoded Ca indicator, Pirt-GCaMP3, which enabled us to monitor different drug interventions at the level of the intact DRG neuronal ensemble. We found that CIPN increased spontaneous Ca activity in DRG neurons, increased number of Ca transients, and increased hyper-responses to mechanical, thermal, and chemical stimuli. We found that mechanical and thermal pain caused by CIPN was significantly attenuated by the mGluR8 agonist, (S)-3,4-DCPG, the group II mGluR agonist, LY379268, and the H1 histamine receptor antagonist, meclizine. DRG neuronal Ca activity elevated by CIPN was attenuated by LY379268 and meclizine, but not by (S)-3,4-DCPG. Furthermore, meclizine and LY379268 attenuated cisplatin-induced weight loss. These results suggest that group II mGluR agonist, mGluR8 agonist, and meclizine are promising candidates as new treatment options for CIPN, and studies of their mechanisms are warranted.Chemotherapy-induced peripheral neuropathy (CIPN) is a painful condition that affects most chemotherapy patients and persist several months or longer after treatment ends. Research on CIPN mechanism is extensive but has produced only few clinically useful treatments. Utilizing  GCaMP Ca imaging in live animals over 1800 neurons/DRG at once, we have characterized the effects of the chemotherapeutic drug, cisplatin and three treatments that decrease CIPN pain. Cisplatin increases sensory neuronal Ca activity and develops various sensitization. Metabotropic glutamate receptor agonist, LY379268 or the H1 histamine receptor antagonist, meclizine decreases cisplatin's effects on neuronal Ca activity and reduces pain hypersensitivity. Our results and experiments provide insights into cellular effects of cisplatin and drugs preventing CIPN pain.

Meclizine and metabotropic glutamate receptor agonists attenuate severe pain and Ca activity of primary sensory neurons in chemotherapy-induced peripheral neuropathy.

Rehabilitation from alcohol addiction or abuse is hampered by withdrawal symptoms including severe headaches, which often lead to rehabilitation failure. There is no appropriate therapeutic option available for alcohol-withdrawal-induced headaches. Here, we show the role of the mast-cell-specific receptor MrgprB2 in the development of alcohol-withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces headache behaviors, including facial allodynia, facial pain expressions, and reduced movement, which are symptoms often observed in humans. Those behaviors were absent in MrgprB2-deficient mice during alcohol withdrawal. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia (TG) neurons in alcohol-withdrawal WT mice, but not in alcohol-withdrawal MrgprB2-deficient mice. Increased mast cell degranulation by alcohol withdrawal in dura mater was dependent on the presence of MrgprB2. The results indicate that alcohol withdrawal causes headache via MrgprB2 of mast cells in dura mater, suggesting that MrgprB2 is a potential target for treating alcohol-withdrawal-related headaches.

Mast-cell-specific receptor mediates alcohol-withdrawal-associated headache in male mice.

Patients with temporomandibular disorders (TMDs) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca 2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo, specifically in mouse models of TMJ injury and inflammation. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of mice with TMJ injection of complete Freund adjuvant or with forced mouth opening (FMO). An inhibitor of the calcitonin gene-related peptide receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of calcitonin gene-related peptide antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca 2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain, bringing us closer to understanding the pathophysiological processes underlying TMJ pain after TMJ injury. Our study also illustrates the utility of in vivo GCaMP3 Ca 2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMJ pain.

Elucidation of neuronal activity in mouse models of temporomandibular joint injury and inflammation by in vivo GCaMP Ca 2+ imaging of intact trigeminal ganglion neurons.

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. Recent studies have suggested that external or intrinsic trauma to the temporomandibular joint (TMJ) is associated with the onset of painful TMD in patients. Here, we investigated the effects of TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors in both sexes. Forced-mouth opening increased mechanical pain as assessed by the von Frey test, with spontaneous pain-like behaviors assessed using the mouse grimace scale and anxiety-like behaviors assessed using the open-field test. Changes in pain-like behaviors were not different between male and female mice. However, in vivo GCaMP Ca 2+ imaging of intact trigeminal ganglia (TG) showed modality- and sex-dependent changes. Forced-mouth opening increased spontaneous Ca 2+ responses and mechanical hypersensitivity of TG neurons compared to the sham group, which was more pronounced in male mice. Forced-mouth opening also increased Ca 2+ responses evoked by cold, heat, and capsaicin stimuli, which was not different between the sexes. In retrogradely labeled trigeminal TMJ afferents, FMO induced an increase in small-sized neuronal proportions with increased colocalization with calcitonin gene-related peptides and transient receptor potential vanilloid subtype 1, which was modestly sex dependent. These results suggest that TMJ injury leads to persistent posttraumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors with distinct sex dependency.

Posttraumatic hyperalgesia and associated peripheral sensitization after temporomandibular joint injury in mice.

Calcium ions (Ca) and Ca channels are pivotal in the regulation of pain pathways and serve as key regulators of neuronal excitability and neurotransmitter release. We review the different types of Ca channels involved in pain processing, including voltage-gated Ca channels (VGCCs), such as L-, N-, P/Q-, and T-type channels. Each subtype is intricately involved in different aspects of pain perception, from acute pain signaling to the development and maintenance of chronic pain states. In addition, the roles of transient receptor potential (TRP) channels, particularly TRPV1 and TRPA1, are discussed in the context of their contribution to chronic pain. Advances in Ca imaging techniques, particularly through genetically encoded Ca indicators (GECIs), such as GCaMPs, have provided unprecedented insight into the dynamic role of Ca channels in pain pathways. These efforts have deepened our understanding of Ca channels and suggest novel therapeutic targets for more effective pain management strategies within Ca channels.

Regulatory Action of Calcium and Calcium Channels in Pain Pathways.

Primary sensory neurons convert external stimuli into electrical signals, yet how heterogeneous neurons encode distinct sensations remains unclear. In vivo dorsal root ganglia (DRG) imaging with genetically-encoded Ca indicators (GECIs) enables mapping of neuronal activity from over 1800 neurons per DRG in live mice, offering high spatial and populational resolution. However, GECIs' slow Ca response kinetics limit the temporal accuracy of neuronal electrical dynamics. Genetically-encoded voltage indicators (GEVIs) provide real-time voltage tracking but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a bright and fast positively tuned GEVI, to dissect temporal dynamics of DRG neuron responses to mechanical, thermal, or chemical stimulation in live male and female mice. ASAP4.4-Kv revealed previously unrecognized cell-to-cell electrical synchronization and robust dynamic transformations in sensory coding following tissue injury. Combining GEVI and GECI imaging empowers spatiotemporal analysis of sensory signal processing and integration mechanisms in vivo.

ArticleTotal : 2	Year
Publication title Cited by 1	2023
Publication title	2025

Article	Year
Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 34452938	2021
Na-dependent inactivation of vascular Na/Ca exchanger responsible for reduced peripheral blood flow in neuropathic pain model. European journal of pharmacology\| PubMed ID: 34454926	2021
Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system. Scientific reports\| PubMed ID: 34497285	2021
cytoNet: Spatiotemporal network analysis of cell communities. PLoS computational biology\| PubMed ID: 35696439	2022
Meclizine and metabotropic glutamate receptor agonists attenuate severe pain and Ca activity of primary sensory neurons in chemotherapy-induced peripheral neuropathy. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 35772967	2022
Mast-cell-specific receptor mediates alcohol-withdrawal-associated headache in male mice. Neuron\| PubMed ID: 37909038	2024
Elucidation of neuronal activity in mouse models of temporomandibular joint injury and inflammation by in vivo GCaMP Ca 2+ imaging of intact trigeminal ganglion neurons. Pain\| PubMed ID: 39365648	2024
Posttraumatic hyperalgesia and associated peripheral sensitization after temporomandibular joint injury in mice. Pain\| PubMed ID: 39715145	2024
Regulatory Action of Calcium and Calcium Channels in Pain Pathways. International journal of biological sciences\| PubMed ID: 40520015	2025
Imaging sensory transmission and neuronal plasticity in primary sensory neurons with a positively tuned voltage indicator. Nature communications\| PubMed ID: 40640176	2025

John Shannonhouse

Biography

JoVE Journal Publications

Other Publications