Immunoreactivity to P2X 6 receptors in the rat hypothalamo-neurohypophysial system: an ultrastructural study with extravidin and colloidal gold-silver labelling

Abstract

The distribution of the purine receptor P2X 6 subtype was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were visualised with ExtrAvidin peroxidase conjugate and immunogold-silver pre-embedding immunocytochemistry using a polyclonal antibody against an intracellular domain of the receptor. Application of ExtrAvidin labelling revealed P2X 6 receptors in subpopulations of: (i) neurosecretory cell bodies, neurosecretory and non-neurosecretory axons and dendrites of neurones in the paraventricular and supraoptic nuclei; and (ii) pituicytes and neurosecretory axons of the neurohypophysis. Some of the neurosecretory granules observed in the supraoptic and paraventricular nuclei neurone cell bodies, dendrites and axons as well as those in neurohypophysial axons were also positive for the P2X 6 receptors. In the paraventricular nucleus, some axons and dendrites of non-neurosecretory neurones positive for P2X 6 receptors formed synapses between themselves. Using the immunogold-silver method, the electron-dense particles labelling P2X 6 receptors were found in neurosecretory cell bodies of the supraoptic and paraventricular nuclei, in relation to the cytoplasm, endoplasmic reticulum, Golgi complex and neurosecretory granules. The particles indicative of P2X 6 receptors were also located in neurosecretory and non-neurosecretory axons including axonal buttons making synapses with P2X 6-negative dendrites. In the neurohypophysis, the electron-dense particles were localised in a subpopulation of pituicytes and neurosecretory axons. In neurohypophysial axons, particles were at times seen over the membrane of some neurosecretory granules (immunogold label) or microvesicles (immunoperoxidase label). We speculate that the P2X 6 receptors at the neurohypophysial level may be implicated not only in hormone release from the axon terminals, but also in membrane recycling of the granular vesicles and microvesicles.