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, 706, 182-188

Purinergic Receptor Expression and Function in Rat Vagal Sensory Neurons Innervating the Stomach

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Purinergic Receptor Expression and Function in Rat Vagal Sensory Neurons Innervating the Stomach

Emily N Blanke et al. Neurosci Lett.

Abstract

The nodose ganglion (NG) is the main parasympathetic ganglion conveying sensory signals to the CNS from numerous visceral organs including digestive signals such as gastric distension or the release the gastrointestinal peptides. The response characteristics of NG neurons to ATP and ADP and pharmacological interrogation of purinergic receptor subtypes have been previously investigated but often in NG cells of undetermined visceral origin. In this study, we confirmed the presence of P2X3 and P2Y1 receptors and characterized P2X and P2Y responses in gastric-innervating NG neurons. Application of ATP-evoked large inward currents and cytosolic Ca2+ increases in gastric-innervating NG neurons. Despite the expression of P2Y1 receptors, ADP elicited only minor modulation of voltage-gated Ca2+ channels. Considering the sensitivity of NG neurons to comorbidities associated with disease or neural injury, purinergic modulation of gastric NG neurons in disease- or injury-states is worthy of further investigation.

Keywords: Ca(2+)channels; Nodose ganglion; P2X3 receptor; P2Y1 receptor; stomach; vagal afferents.

Figures

Fig. 1.
Fig. 1.
Immunohistochemical labeling of P2X3 and P2Y1 in isolated gastric-innervating NG neurons (n = 4 rats). (A) Confocal image of immunopositive P2X3 gastric-innervating NG neurons (15 cells analyzed). P2X3-immunopositive expression (480 nm excitation, upper left); DiI (530 nm excitation, upper right); Merged P2X3 and DiI channels (lower left); and merged channels with DIC to delineate the entire cell (lower right). (B) Confocal image of immunopositive P2Y1 gastric-innervating NG neurons (12 cells analyzed). P2Y1-immunopositive expression (480 nm excitation, upper left); DiI (530 nm excitation, upper right); Merged P2Y1 and DiI channels (lower left); and merged channels with DIC to delineate the entire cell (lower right). Scale bar = 50 μm.
Fig. 2.
Fig. 2.
ATP-stimulated Ca2+ responses and P2X currents (IATP) in DiI labeled NG neurons (n = 7 rats, cells for each analysis in parenthesis). (A) A brightfield DIC image of an isolated DiI labeled (red) NG neuron loaded with Calbryte 520 [Ca2+-sensitive indicator dye] (upper left panel). Corresponding pseudocolor images (1–7) from the same cell plotted in B. (B) A plot of the fiuorescence intensity over time from an ROI drawn over the soma of the isolated DiI labeled NG neuron in A. The arrows correspond to the images shown in A: 1, control; 2, 10 μM BzATP containing superfusate; 3, wash; 4, elevated ([K+]o = 72 mM) [K+]o containing super-fusate; 5, wash; 6, 10 μM ATP containing superfusate; and 7, wash. The numbers with arrows indicate the time point when the image was captured during the experiment. (C) A summary scatter plot of the stimulus-evoked Ca2+ increases in basal fiuorescence. Percent increases over the basal fiuorescence levels measured in DiI labeled NG neurons in response to a stimulus: 10μM BzATP (n = 9, *p = 0.0052); 72 mM [K+]° (n = 13, *p = 0.0002): 10 μM ATP (n=16, *p < 0.0001). Lines and error bars represent the mean ± SEM (D) in DiI labeled NG neurons. Sample trace of native P2X receptor currents stimulated by ATP (10 μM) exposure indicated by the bar above the current trace. The holding potential for the neurons was −80 mV. (E) Summary scatter plot showing the mean ± SEM. P2X current density mediated by ATP in NG neurons. Number in parenthesis indicates the number of neurons tested. Scale bar = 50 μm.
Fig. 3.
Fig. 3.
ADP partially inhibits the K+-evoked-depolarization in DiI labeled NG neurons (n = 7 rats, yielding 14 cells for analysis). (A) A brightfield DIC image of an isolated DiI labeled (red) NG neuron loaded with Calbryte 520 [Ca2+-sensitive indicator dye] (upper left panel). Corresponding pseudocolor images (1–7) from the same cell plotted in B. (B) A plot of the fiuorescence intensity over time from an ROI drawn over the soma of an isolated DiI labeled NG neuron. The arrows correspond to the images shown in A: 1, control, normal ([K+]o =2.5 mM) superfusate; 2, elevated ([K+]o = 72 mM) [K+]o containing super-fusate; 3, wash or normal superfusate; 4, ADP (10 μM) and elevated [K+]o superfusate; 5, wash, or normal [K+]o superfusate; 6, elevated [K+]o containing superfusate; and 7, wash, or normal [K+]o superfusate. The numbers with arrows indicate the time point when the image was captured during the experiment. (C) A scatter plot of the fractional ADP-mediated Ca2+ response to a K+-evoked depolarization in DiI labeled NG neurons (n = 14, *p = 0.002). Line and error bars represent the mean ± SEM. Scale bar = 50 μm.
Fig. 4.
Fig. 4.
Time courses of peak Ca2+ current amplitude acquired from the application of ADP (10 μM) and DAMGO (10 μM) in control(A) and a P2Y12R-transfected NG neuron (B). Ca2+ currents were evoked every 10 sec with a depolarizing test pulse (right). The numbered Ca2+ current traces in A and B are shown to the right, where 1 and 3 represent control currents (before agonist exposure), and 2 and 4 represent currents in the presence of ADP or DAMGO, respectively. (C) Summary scatter plot showing the mean ± SEM. Ca2+ current inhibition mediated by ADP and DAMGO in control (circles) and P2Y12-expressing (triangles) NG neurons. *p < 0.05 compared to control, paired t-test. (n = 5, numbers in parenthesis indicate the number of neurons analyzed).

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