ABSTRACT
Mutation of the gene encoding carbonic anhydrase-related protein VIII (CAVIII) results in motor coordination deficits in mice and humans, due to loss of this protein in Purkinje cells of the cerebellum. Recent studies have indicated that the CAVIII gene, Car8, is also expressed in rod bipolar cells (RBCs), a critical glutamatergic neuron for scotopic vision. We investigated the localization of CAVIII in the mouse and macaque retina, and utilized the wdl mouse, which has a null mutation in the Car8 gene, to determine how the loss of CAVIII affects retinal signaling. CAVIII immunoreactivity was observed in RBCs, with particularly high staining intensity in the axon terminals. In addition, weaker staining was observed in a subset of cone bipolar cells and γ-aminobutyric acid (GABA)ergic amacrine cells. Light-evoked current and voltage responses of RBCs were not altered in the wdl mutant. However, light-evoked current responses from the AII-amacrine cell, a postsynaptic partner at the RBC ribbon synapse, were significantly larger, and more prolonged than in control mice. These changes could not be attributed to alterations in calcium current activation or inactivation, or to changes in the density of RBCs. Furthermore, no gross synaptic alterations were evident in the wdl mutant at the light or ultrastructural level. These data provide evidence that the CAVIII protein, which is highly conserved in vertebrates, is selectively expressed within neural circuits, and may be important for modulating retinal neurotransmission.
Subject(s)
Amacrine Cells/physiology , Biomarkers, Tumor/metabolism , Light Signal Transduction/physiology , Nerve Tissue Proteins/metabolism , Retina/cytology , Retinal Bipolar Cells/physiology , Synapses/physiology , Alcohol Oxidoreductases , Analysis of Variance , Animals , Animals, Newborn , Biomarkers, Tumor/genetics , Biophysics , Calcium/metabolism , Cell Count , Co-Repressor Proteins , DNA-Binding Proteins/metabolism , Electric Stimulation/methods , Excitatory Postsynaptic Potentials/genetics , Excitatory Postsynaptic Potentials/physiology , Eye Proteins/metabolism , In Vitro Techniques , Light , Light Signal Transduction/genetics , Macaca mulatta , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Transmission , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Retinal Rod Photoreceptor Cells/physiology , Synapses/genetics , Synapses/ultrastructure , Visual Pathways/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
Extracellular ATP acts as a neurotransmitter in the retina, via the activation of ionotropic P2X receptors and metabotropic P2Y receptors. The expression of various P2X and P2Y receptor subtypes has been demonstrated in the retina, but the localization of P2Y receptors and their role in retinal signaling remains ill defined. In this study, we were interested in determining the localization of the P2Y(4) receptor subtype in the rat retina, and using the electroretinogram (ERG) to assess whether activation of these receptors modulated visual transmission. Using light and electron microscopy, we demonstrated that P2Y(4) receptors were expressed pre-synaptically in rod bipolar cells and in processes postsynaptic to cone bipolar cells. Furthermore, we show that the expression of P2Y(4) receptors on rod bipolar cell axon terminals is reduced following dark adaptation, suggesting receptor expression may be dependent on retinal activity. Finally, using the electroretinogram, we show that intravitreal injection of uridine triphosphate, a P2Y receptor agonist, decreases the amplitude of the rod PII, supporting a role for P2Y receptors in altering inner retinal function. Taken together, these results suggest a role for P2Y(4) receptors in the modulation of inner retinal signaling.
Subject(s)
Receptors, Purinergic P2/metabolism , Retina/physiology , Adaptation, Ocular/physiology , Animals , Dark Adaptation/physiology , Electroretinography/methods , Microscopy, Immunoelectron/methods , Nerve Tissue Proteins/metabolism , Principal Component Analysis , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/ultrastructure , Retina/drug effects , Retina/ultrastructure , Uridine Triphosphate/pharmacologyABSTRACT
P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.
