Localization of vanilloid receptor 1 (TRPV1/VR1)-like immunoreactivity in goldfish and zebrafish retinas: Restriction to photoreceptor synaptic ribbons

Abstract

The vanilloid receptor type 1 (TRPV1/VR1) is a non-specific calcium-permeable ionotropic cation channel expressed in the peripheral sensory system as well as in the central nervous system. An endogenous ligand for TRPV1 is arachidonoyl ethanolamide (anandamide), which also activates the metabotropic cannabinoid receptor 1 (CB1). Previous studies in this laboratory reported CB1 receptors and CB1-mediated effects on voltage-gated currents in goldfish cones and bipolar cells. In this study, we show TRPV1-like-immunoreactivity (TRPV1-L-IR) by immunoblot analysis of goldfish retina and rat brain homogenates with a guinea pig polyclonal antibody against the C-terminus of the rat TRPV1. Light-level immunocytochemistry showed restriction of the guinea pig-TRPV1 antibody to a very narrow band in the outer plexiform layer in goldfish and zebrafish retinas. However, no immunoreactivity was detected using rabbit-polyclonal antibodies against the C or N-termini of the rat TRPV1. Pre and post-embedding electron microscopy (EM) immunocytochemistry revealed that TRPV1-L-IR (using the guinea pig antibody) was restricted to synaptic ribbons of all cones and many rods, but never was observed at the synaptic ribbons of bipolar cells. While pre-embedded tissue showed diffuse label associated only with photoreceptor-synaptic ribbons, analysis of post-embedded tissue showed label tightly restricted to synaptic ribbons of all cones and many rods. Oblique sections showed that immunogold particles were confined to the outer electron dense region of the ribbons, with few or no gold particles in the ribbon core or associated with tethers or vesicles. Although TRPV1-L-IR described here, does not necessarily represent TRPV1 antigen associated with synaptic ribbons, these data provide an unequivocal label with which to study the functional dynamics of ribbon formation and degradation in teleost photoreceptors.

‘TRPing’ Synaptic Ribbon Function in the Rat Pineal Gland: Neuroendocrine Regulation Involves the Capsaicin Receptor TRPV1

“…This receptor was recently shown in the retina to be restricted to the photoreceptor SR [15]. The appearance of both immunoreactions in pineal sections (which is shown here for the first time) and their colocalization makes it highly likely that the observed immunoreactivities in fact label SRs in the pineal gland.…”

“…Immunofluorescence was performed by free-floating incubation of pineal sections with a monoclonal mouse antibody directed against KIN (1:100 in phosphate-buffered saline, PBS; Covance, Berkeley, Calif., USA) [12], visualized by Cy3-conjugated F(ab) 2 -fragment of anti-mouse IgG (1:400; Jackson Immuno-Research, West Grove, Pa., USA). Double immunofluorescence was conducted with the kinesin antibody and, additionally, with either a polyclonal anti-TH antibody (raised in rabbit, 1:100 in PBS; Eugene Tech International, Ramsey, N.J., USA) or a guinea pig polyclonal vanilloid receptor type1 antibody against the C-terminus of TRPV1 (1:500 in PBS, Chemicon International, Temecula, Calif., USA; [15,27]). These were visualized by Cy2 coupled to F(ab) 2 fragments of anti-rabbit or anti-guinea pig IgG (1:200 in PBS; Jackson Immuno-Research), respectively.…”

“…The kinesin motor KIF3A – a component of the microtubule motor kinesin II (KIN) – is part of presynaptic ribbons in the retina and pineal [11,12,13]. An interesting aspect of possible SR function came up by the demonstration that KIF3A is colocalized to the transient receptor potential channel of the vanilloid receptor family, subtype 1 (TRPV1, formerly known as the vanilloid receptor, VR1) [14] at the photoreceptor SR [15]. TRPV1 is a member of the TRP channel family and forms non-selective cation channels that have a high permeability for calcium.…”

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

“…23,[25][26][27][28][29][30][31][32][33] Because of this complexity, discerning how TRPV1 exerts its influence in vivo is more difficult. We discovered recently in testing our initial hypothesis that both knock-out and pharmacological antagonism of TRPV1 accelerated RGC degeneration with exposure to elevated intraocular pressure in an inducible model of glaucoma.…”

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells

“…The preparation of goldfish retina for light-and electron-microscopical immunocytochemistry was similar to that described by Klooster et al (2001) and Zimov and Yazulla (2004). Light-adapted fish were briefly darkadapted for 5 min to facilitate removal of the retinal pigment epithelium.…”