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

Summary

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca²⁺ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca²⁺ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Abstract

Ca²⁺ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca²⁺ entry into the cytoplasm or the release of intracellular Ca²⁺ stores. Pirt-GCaMP3-based Ca²⁺ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca²⁺ transients as well as the amplitude of Ca²⁺ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, Aβ, Aδ, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca²⁺ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

Introduction

Primary sensory neurons directly innervate the skin and carry somatosensory information back to the central nervous system^1,2. Dorsal root ganglia (DRGs) are cell body clusters of 10,000-15,000 primary sensory neurons^3,4. DRG neurons present diverse size, myelination levels, and gene and receptor expression patterns. Smaller diameter neurons include pain-sensing neurons and larger diameter neurons typically respond to non-painful mechanical stimuli^5,6. Disorders in the primary sensory neurons s....

Protocol

All procedures described here were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.

NOTE: Once started, animal surgery (step 1) and imaging (step 2) must be completed in a continuous manner. Data analysis (step 3) may be performed later.

1. Surgery and securing the animal for right side L5 DRG imaging

NOTE: Bo.......

Discussion

Persistent pain is present in a wide range of disorders, debilitating and/or reducing the quality of life for about 8% of people²⁹. Primary sensory neurons detect noxious stimuli on the skin, and their plasticity contributes to persistent pain⁸. While neurons can be studied in cell culture and explants, doing so removes them from their normal physiological context. Surgical exposure of the DRG, followed by Pirt-GCaMP3 Ca²⁺ imaging, permits the study of primary se.......

Materials

Name	Company	Catalog Number	Comments
Anased Injection (Xylazine)	Covetrus, Akorn	33197
C Epiplan-Apochromat 10x/0.4 DIC	Cal Zeiss	422642-9900-000
Cotton Tipped Applicators	McKesson	24-106-1S
Curved Hemostat	Fine Science Tools	13007-12
DC Temperature Controller	FHC	40-90-8D
DC Temperature Controller Heating Pad	FHC	40-90-2-05
Dumont Ceramic Coated Forceps	Fine Science Tools	11252-50
FHC DC Temperature Controller	FHC	40-90-8D
Fluriso (Isoflurane)	MWI Animal Health, Piramal Group	501017
Friedman-Pearson Rongeurs	Fine Science Tools	16221-14
GelFoam	Pfizer	09-0353-01
Ketaset (Ketamine)	Zoetis	KET-00002R2
Luminescent Green Stage Tape	JSITON/ Amazon	B803YW8ZWL
Matrx VIP 3000 Isoflurane Vaporizer	Midmark	91305430
Micro dissecting scissors	Roboz	RS-5882
Micro dissecting spring scissors	Fine Science Tools	15023-10
Micro dissecting spring scissors	Roboz	RS-5677
Mini Rectal Thermistor Probe	FHC	40-90-5D-02
Operating scissors	Roboz	RS-6812
Pirt-GCaMP3 C57BL/6J mice	Johns Hopkins University	N/A	Either sex can be imaged equally well. Mice should be at least 8 weeks old due to weak or intermittent Pirt promoter expression in younger mice.
SMALGO small animal algometer	Bioseb In vivo Research Instruments	BIO-SMALGO
Stereotaxic frame	Kopf Model 923-B	923-B
td-Tomato C57BL/6J mice	Jackson Laboratory	7909
Top Plate, 6 in x 10 in	Newport	290-TP
TrpV1-Cre C57BL/6J mice	Jackson Laboratory	17769
Zeiss LSM 800 confocal microscope	Cal Zeiss	LSM800
Zeiss Zen 2.6 Blue Edition Software	Cal Zeiss	Zen (Blue Edition) 2.6