Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3–7 days after injection into the rat gastrocnemius muscle

Wei, Bai; Cui, Jingjing; Wang, Jia; Xu, Dongsheng; Wu, Shuang; Guo, Yating; Su, Yuxin; Liu, Yihan; Wang, Yuqing; Jing, Xiang-Hong

doi:10.4103/1673-5374.337055

Cited by 12 publications

(3 citation statements)

References 44 publications

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“…Mice were euthanized with 5% isoflurane 3–6 days after the tracer injections as previously reported to be optimal time period for maximal intensity ( Christianson et al, 2006 ; Cui et al, 2022 ), then perfused transcardiacally with 0.9% saline, and the colon was removed. Then, the perfusion was switched to 4% paraformaldehyde in 0.1 M phosphate buffer 2 mL/min for 15 min.…”

Section: Methodsmentioning

confidence: 99%

The parasympathetic and sensory innervation of the proximal and distal colon in male mice

Wang,

Taché

2024

Front. Neuroanat.

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IntroductionThe distributions of extrinsic neurons innervating the colon show differences in experimental animals from humans, including the vagal and spinal parasympathetic innervation to the distal colon. The neuroanatomical tracing to the mouse proximal colon has not been studied in details. This study aimed to trace the locations of extrinsic neurons projecting to the mouse proximal colon compared to the distal colon using dual retrograde tracing.MethodsThe parasympathetic and sensory neurons projecting to colon were assessed using Cholera Toxin subunit B conjugated to Alexa-Fluor 488 or 555 injected in the proximal and distal colon of the same mice.ResultsRetrograde tracing from the proximal and distal colon labeled neurons in the dorsal motor nucleus of the vagus (DMV) and the nodose ganglia, while the tracing from the distal colon did not label the parasympathetic neurons in the lumbosacral spinal cord at L6-S1. Neurons in the pelvic ganglia which were cholinergic projected to the distal colon. There were more neurons in the DMV and nodose ganglia projecting to the proximal than distal colon. The right nodose ganglion had a higher number of neurons than the left ganglion innervating the proximal colon. In the dorsal root ganglia (DRG), the highest number of neurons traced from the distal colon were at L6, and those from the proximal colon at T12. DRG neurons projected closely to the cholinergic neurons in the intermediolateral column of L6 spinal cord. Small percentages of neurons with dual projections to both the proximal and distal colon existed in the DMV, nodose ganglia and DRG. We also observed long projecting neurons traced from the caudal distal colon to the transverse and proximal colon, some of which were calbindin immunoreactive, while there were no retrogradely labeled neurons traced from the proximal to distal colon.DiscussionThese data demonstrated that the vagal motor and motor and sensory neurons innervate both the proximal and distal colon in mice, and the autonomic neurons in the intermediate zone of the lumbosacral spinal cord do not project directly to the mouse colon, which differs from that in humans.

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Section: Methodsmentioning

confidence: 99%

The parasympathetic and sensory innervation of the proximal and distal colon in male mice

Wang,

Taché

2024

Front. Neuroanat.

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“…VANs exhibit significant cellular heterogeneity, which is observed as varying cellular functions such as differential electrophysiological response to distinct effector molecules (Girardi et al 2023;Simasko et al 2002). It is plausible that the heterogeneity also manifests itself in uptake and transport of proteins, including CTB, thereby resulting in a fraction of cells exhibiting transport of CTB, as also observed in in vivo tracing studies (Wang et al 1998;Cailotto et al 2012;Cui et al 2022). Overall, the retrograde transport of CTB demonstrated here suggests that compartmentalized VANs can be used as a model to study the transport of other molecules, such as alphasynuclein, a molecule which is hypothesized to migrate from the gut to the CNS resulting in neurodegeneration (Kim et al 2019;Bindas et al 2021).…”

Section: Molecular Transport and Distal Stimulationmentioning

confidence: 99%

Microfluidic compartmentalization of rat vagal afferent neurons to model gut-brain axis

Girardi,

Zumpano,

Raybould

et al. 2024

Bioelectron Med

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Background Vagal afferent neurons represent the key neurosensory branch of the gut-brain axis, which describes the bidirectional communication between the gastrointestinal system and the brain. These neurons are important for detecting and relaying sensory information from the periphery to the central nervous system to modulate feeding behavior, metabolism, and inflammation. Confounding variables complicate the process of isolating the role of the vagal afferents in mediating these physiological processes. Therefore, we developed a microfluidic model of the sensory branch of the gut-brain axis. We show that this microfluidic model successfully compartmentalizes the cell body and neurite terminals of the neurons, thereby simulates the anatomical layout of these neurons to more accurately study physiologically-relevant processes. Methods We implemented a primary rat vagal afferent neuron culture into a microfluidic platform consisting of two concentric chambers interconnected with radial microchannels. The microfluidic platform separated cell bodies from neurite terminals of vagal afferent neurons. We then introduced physiologically-relevant gastrointestinal effector molecules at the nerve terminals and assessed their retrograde transport along the neurite or capacity to elicit an electrophysiological response using live cell calcium imaging. Results The angle of microchannel outlets dictated the probability of neurites growing into a chamber versus tracking along chamber walls. When the neurite terminals were exposed to fluorescently-labeled cholera toxin subunit B, the proteins were taken up and retrogradely transported along the neurites over the course of 24 h. Additionally, mechanical perturbation (e.g., rinsing) of the neurite terminals significantly increased intracellular calcium concentration in the distal soma. Finally, membrane-displayed receptor for capsaicin was expressed and trafficked along newly projected neurites, as revealed by confocal microscopy. Conclusions In this work, we developed a microfluidic device that can recapitulate the anatomical layout of vagal afferent neurons in vitro. We demonstrated two physiologically-relevant applications of the platforms: retrograde transport and electrophysiological response. We expect this tool to enable controlled studies on the role of vagal afferent neurons in the gut-brain axis.

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“…To prevent leakage, the microsyringe remained in place for 1 min after injection, and was then removed. 20…”

Section: Microinjection Of Neural Tracermentioning

confidence: 99%

The Effect of Pre-Electroacupuncture on Nociceptive Discharges of Spinal Wide Dynamic Range Neurons in Rat

Yu¹,

Cao²,

Wang³

et al. 2023

JPR

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Spinal wide dynamic range (WDR) neurons are well studied in pain models and they play critical roles in regulating nociception. Evidence has started to accumulate that acupuncture produces a good analgesic effect via activating different primary fibers with distinct intensities. The purpose of the present study was to compare the distinct intensities of pre-electroacupuncture (pre-EA) at local muscular receptive fields (RFs), adjacent or contralateral non-RFs regulating the nociceptive discharges of spinal WDR neurons evoked by hypertonic saline (HS). Materials and Methods: Spinal segments of electrophysiological recording were identified by neural tracers applied at the left gastrocnemius muscle. The thresholds of Aβ (T Aβ ), Aδ (T Aδ ) and C (T C ) components of WDR neurons were measured to determine the intensity of pre-EA by extracellular recording. The discharges of WDR neurons induced by distinct intensities of pre-EA and 200 µL HS (6%) injection in left gastrocnemius muscle of rats were observed by extracellular recording. Results:The spinal segments of WDR neurons were confirmed in lumbar (L)5-6 area according to the projective segments of dorsal root ganglion. T Aβ , T Aδ and T C of WDR neurons was determined to be 0.5, 1, and 2 mA, respectively. The pre-EA with intensities of T Aβ (P < 0.05), T Aδ (P < 0.05), T C (P < 0.05) or 2T C (P < 0.01) at ipsilateral adjacent non-RFs significantly reduced the discharges of WDR neurons, while at local RFs only pre-EA of T Aδ (P < 0.05), T C (P < 0.05) and 2T C (P < 0.01) could inhibit the nociceptive discharges. In addition, intensity of pre-EA at contralateral non-RFs should reach at least T C to effectively inhibit the firing rates of WDR neurons (P < 0.01). Conclusion: Pre-EA could suppress nociceptive discharges of WDR neurons and the inhibitory effects were dependent on the distinct intensities and locations of stimulation.

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Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3–7 days after injection into the rat gastrocnemius muscle

Cited by 12 publications

References 44 publications

The parasympathetic and sensory innervation of the proximal and distal colon in male mice

The parasympathetic and sensory innervation of the proximal and distal colon in male mice

Microfluidic compartmentalization of rat vagal afferent neurons to model gut-brain axis

The Effect of Pre-Electroacupuncture on Nociceptive Discharges of Spinal Wide Dynamic Range Neurons in Rat

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