Immunoreactive localisation of P2Y1 receptors within the rat and human nodose ganglia and rat brainstem: comparison with [α33P]deoxyadenosine 5′-triphosphate autoradiography

doi:10.1016/S0306-4522(02)00237-3

Neuroscience

Volume 113, Issue 4, 10 September 2002, Pages 809-823

https://doi.org/10.1016/S0306-4522(02)00237-3 Get rights and content

Abstract

The present study employed standard peroxidase immunohistochemistry to map the distribution of P2Y₁ receptors in the rat brainstem and nodose ganglia and characterised the binding profile of [α³³P]dATP. Binding of [α³³P]dATP was fully displaceable by adenosine 5′-triphosphate (ATP), and was found on both human and rat nodose ganglia, and throughout the rat brainstem, including the nucleus tractus solitarius and ventrolateral medulla. [α³³P]dATP binding in the human nodose ganglia was significantly displaced by both 2-methylthio ATP and α,β-methylene ATP, but not by uridine 5′-triphosphate, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, 8,8′-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphtalenetrisulfonic) acid (NF279) or N-ethylcarboxamidoadenosine. [α³³P]dATP binding in the rat nodose ganglia and brainstem was significantly displaced by only 2-methylthio ATP, suggesting that [α³³P]dATP is binding to P2Y receptors in the rat. Binding of [α³³P]dATP was also significantly displaced by α,β-methylene adenosine 5′-diphosphate, suggesting a component of the binding is to endogenous ecto-5′-nucleotidase, however, almost all binding could be displaced by a combination of receptor agonists (2-methylthio ATP, uridine 5′-triphosphate and α,β-methylene ATP), suggesting preferential binding to receptors. Immunoreactivity to P2Y₁ receptor (P2Y₁-IR) exhibited similar distribution patterns to [α³³P]dATP binding, with a clear topographic profile. Particularly dense P2Y₁-IR labeling was evident in cells and fibres of the dorsal vagal complex. Immunolabeling was also present in the dorsal motor nucleus of the vagus and nucleus ambiguus, indicating the possibility of P2Y₁ receptors on vagal efferents. Unilateral vagal ligation was also performed to examine the transport of P2Y₁ receptor, using both immunohistochemistry and [α³³P]dATP autoradiography. Accumulations of both P2Y₁-IR and [α³³P]dATP binding were apparent adjacent to both ligatures, suggesting bi-directional transport of P2Y₁ receptors along the rat vagus nerve. This current study represents the first description of P2Y₁ receptor distribution within the rodent brainstem and nodose ganglion and also characterises [α³³P]dATP binding to P2Y receptors.

Section snippets

Experimental procedures

All experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986 and under the guidelines of the National Health and Medical Research Council (NH&MRC) Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. Efforts were made to minimise animal suffering and the number of animals used.

Human inferior vagal ganglion

Punctate binding of [α³³P]dATP was observed over sections of human inferior vagal ganglia (Fig. 1A). [α³³P]dATP binding (188±16 DPM/mm²) was fully displaced by ATP (1 mM). There was no significant displacement of binding by NECA, PPADS, NF279 or UTP (Fig. 1B); however, α,βMeATP (Fig. 1C) and 2MeSATP (Fig. 1D) could partially displace [α³³P]dATP binding (Table 1).

Rat nodose ganglia

Binding of [α³³P]dATP was present on the rat nodose ganglia and the vagus nerve (Fig. 2A). Binding of [α³³P]dATP (185±38 DPM/mm²) was

Discussion

The current study provides the first clear evidence of widespread P2Y₁-IR receptor in the adult rat medulla oblongata; furthermore, this P2Y₁-IR is also subject to bi-directional transport along the rat vagus nerve. In addition, observations from this study suggest the potential use of [α³³P]dATP as a radioligand for P2Y receptor autoradiography. Taken together, our observations further extend the findings of previous autoradiographic studies, which identified a number of P2Y-like binding sites