In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

John Shannonhouse; Hyeonwi Son; Yan Zhang; Eungyung Kim; Deoksoo Han; Ruben Gomez; Joon Tae Park; Yu Shin Kim

doi:10.3791/68284

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Method Article

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

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DOI:

10.3791/68284

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August 1st, 2025

John Shannonhouse¹^,² , Hyeonwi Son¹^,² , Yan Zhang¹^,² , Eungyung Kim¹^,² , Deoksoo Han¹^,² , Ruben Gomez¹^,² , Joon Tae Park²^,³ , Yu Shin Kim¹^,²^,⁴

¹Department of Oral & Maxillofacial Surgery, School of Dentistry, University of Texas Health Science Center at San Antonio, ²Department of Endodontics, School of Dentistry, University of Alabama at Birmingham, ³Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, ⁴Programs in Integrated Biomedical Sciences, Translational Sciences, Biomedical Engineering, Radiological Sciences, University of Texas Health Science Center at San Antonio

Summary

This protocol describes in vivo GCaMP calcium imaging of intact trigeminal ganglion (TG) neurons. This description includes TG exposure surgery, in vivo confocal microscopy imaging of TG neurons, application of somatic stimuli during TG neurons microscopic imaging, and analysis of in vivo GCaMP calcium imaging data.

Abstract

Calcium imaging is an important tool for monitoring signaling mechanisms that either influx Ca²⁺ into the cytosol or release Ca²⁺ from intracellular Ca²⁺ stores. The use of Pirt-GCaMP3 mice, which have the Ca²⁺sensor GCaMP3 inserted downstream of the Pirt promoter region and expressed in nearly all (>95%) primary sensory neurons, allows for simultaneous monitoring of the activity of >3,000 neurons via Ca²⁺ imaging of trigeminal ganglion (TG) primary sensory neurons. This allows investigators to study neural network responses to noxious stimuli and somatosensory and hormonal responses in vivo, which would otherwise be very difficult to detect using other methods.

Specifically, the ability to monitor the TG area innervating the face and other areas of the head separately allows for monitoring primary sensory neurons activated by stimuli of the face area before later layers of neuron processing. The number of neurons generating Ca²⁺ transients and the amplitude of Ca²⁺ transients, which indicate their sensitivity to different sensory modalities, can also be observed. Additionally, investigators can measure the diameter of neurons, which is an indicator of the type of fiber activated (non-noxious mechano-fibers vs. noxious nociceptive fibers, Aβ, Aδ, and C fibers). Therefore, Pirt-GCaMP3 calcium imaging of TG would be a powerful tool to dissect specific sensory modalities and neuronal cell types to study trigeminal origin pain (orofacial and craniofacial, dental or/tooth pain, headache, migraine, temporomandibular joint pain, trigeminal neuralgia), itch and touch and any other somatosensations, as well as hormonal responses.

Introduction

Primary sensory neurons directly innervate tissues, including the skin, and relay sensory signals to the central nervous system (spinal cord or brainstem). Trigeminal ganglion (TG) neurons innervate the head and respond to sensory stimuli, including pain from the teeth, periorbital region, orofacial and craniofacial regions, temporomandibular joint¹^,², and meninges³^,⁴^,⁵. TG neurons vary in size, level of myelination, and gene expression profiles⁶^,⁷^,....