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

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 microm in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.

Vanilloid receptor 1 (TRPV1/VR1) co-localizes with fatty acid amide hydrolase (FAAH) in retinal amacrine cells.

Fatty acid amide hydrolase (FAAH) is the degradative enzyme for anandamide (AEA), an endogenous ligand for the vanilloid receptor (TRPV1) and cannabinoid receptor 1. As FAAH and TRPV1 are integral membrane proteins, FAAH activity could modulate the availability of AEA for TRPV1 activation. Previous studies in this laboratory reported an extensive endocannabinoid system in goldfish retina. Immunocytochemistry was used to determine the relative distributions of FAAH-immunoreactivity (IR) and TRPV1-IR in goldfish retina. Here, we show the first example in an intact neural system in which TRPV1-IR co-localizes in subpopulations of FAAH-immunoreactive neurons, in this case amacrine cells. These cells are rare and consist of three subtypes: 1. ovoid cell with granular-type dendrites restricted to sublamina a, 2. pyriform cell with smooth processes in sublamina b, and 3. fusiform cell with smooth processes that project to sublaminae a and b. The varied appearances of reaction product in the dendrites suggest different subcellular localization of TRPV1, and hence function of FAAH activity regarding TRPV1 stimulation among the cell types. Ovoid and pyriform amacrine cells, but not fusiform cells, labeled with GAD-IR and constituted subsets of GABAergic amacrine cells. TRPV1 amacrine cells, though rare, are represented in the ON, OFF and ON/OFF pathways of the retina. As TRPV1 stimulation increases intracellular calcium with numerous downstream effects, co-localization of TRPV1 and FAAH suggests an autoregulatory function for anandamide. Due to the rarity of these cells, the three vanilloid amacrine cell types may be involved in global effects rather than feature extraction, for example: sampling of ambient light or maintaining homeostasis.

Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide.

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

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

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.