Cellular and subcellular expression of monocarboxylate transporters in the pigment epithelium and retina of the rat.

The cellular and subcellular expression of the monocarboxylate transporters MCT1, MCT2 and MCT4 [corresponding to MCT3 of Price N. T. et al. (1998) Biochem. J. 329, 321-328] were investigated in the pigment epithelium and outer retina of rats. Immunofluorescence and postembedding immunogold analyses revealed strong MCT1 labelling in the apical membrane of the pigment epithelial and no detectable signal in the basolateral membrane. In contrast, antibodies to the glucose transporter GLUT1 produced intense labelling in both membranes. Neither MCT1 nor GLUT1 was enriched in intracellular compartments. The monocarboxylate transporter MCT4 was very weakly expressed in the retinal pigment epithelium of adult animals, but occurred at higher concentrations at this site in 14-day-old rats. However, even at the latter stage, the immunolabelling of MCT4 was weak compared to that of MCT1. In the neural retina, the data were consistent with a predominant glial localization of MCT1. Specifically, immunogold particles signalling MCT1 occurred in Müller cell microvilli and in the velate processes between the photoreceptors. No labelling was obtained with antibodies to MCT2. Taken together with previous biochemical analyses, the present findings indicate that MCT1 is involved in the outward transport of lactate through the retinal pigment epithelial cells, and in the transfer of lactate between Müller cells and photoreceptors.

Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in Müller cells and fibrous astrocytes.

The water permeability of cell membranes differs by orders of magnitude, and most of this variability reflects the differential expression of aquaporin water channels. We have recently found that the CNS contains a member of the aquaporin family, aquaporin-4 (AQP4). As a prerequisite for understanding the cellular handling of water during neuronal activity, we have investigated the cellular and subcellular expression of AQP4 in the retina and optic nerve where activity-dependent ion fluxes have been studied in detail. In situ hybridization with digoxigenin-labeled riboprobes and immunogold labeling by a sensitive postembedding procedure demonstrated that AQP4 and AQP4 mRNA were restricted to glial cells, including MHller cells in the retina and fibrous astrocytes in the optic nerve. A quantitative immunogold analysis of the MHller cells showed that these cells exhibited three distinct membrane compartments with regard to AQP4 expression. End feet membranes (facing the vitreous body or blood vessels) were 10-15 times more intensely labeled than non-end feet membranes, whereas microvilli were devoid of AQP4. These data suggest that MHller cells play a prominent role in the water handling in the retina and that they direct osmotically driven water flux to the vitreous body and vessels rather than to the subretinal space. Fibrous astrocytes in the optic nerve similarly displayed a differential compartmentation of AQP4. The highest expression of AQP4 occurred in end feet membranes, whereas the membrane domain facing the nodal axolemma was associated with a lower level of immunoreactivity than the rest of the membrane. This arrangement may allow transcellular water redistribution to occur without inducing inappropriate volume changes in the perinodal extracellular space.

Dopaminergic neurons in the rat retina express dopamine D2/3 receptors.

The localization of dopamine D2 and D3 receptors in the rat retina was studied using a polyclonal antibody raised against a peptide sequence common to the dopamine D2 and D3 receptors (D2/3). The D2/3 receptor antibody labelled a small number of somata in the innermost part of the inner nuclear layer and in the ganglion cell layer and a small number of photoreceptor outer segments. Processes in both plexiform layers were also labelled. Double-labelling experiments with the antibody against the D2/3 receptor and an antibody against tyrosine hydroxylase to label dopaminergic neurons resulted in the co-localization of the two antibodies. This demonstrates directly that dopaminergic neurons in the retina express D2/3 receptors. As previous biochemical and physiological studies have demonstrated that activation of D2-like receptors inhibits the release of dopamine in the retina, the present results suggest that the D2/3 receptors on dopaminergic neurons function as autoreceptors.

AII amacrine cells express functional NMDA receptors.

NMDA and non-NMDA receptors mediate the majority of fast excitatory synaptic transmission in the CNS. AII amacrine cells in the mammalian retina receive glutamatergic input from rod bipolar cells and are known to express non-NMDA receptors. We have investigated the expression of NMDA receptors in these cells by recording responses to exogenously applied NMDA in whole-cell recordings in slices of rat retina. Most cells displayed clear responses to NMDA. The responses could be blocked by a specific NMDA receptor antagonist and were characterized by voltage-dependent block with outward rectification. These results suggest that NMDA receptors could play a role in mediating excitatory synaptic input to AII amacrine cells.

Immunohistochemical localization of dopamine D1 receptors in rat retina.

We have localized the dopamine D1 receptor in rat retina using a subtype-specific monoclonal antibody. Immunolabelling can be detected in the inner and outer plexiform layers and in a number of cells in the inner nuclear layer. In the inner plexiform layer, labelled processes form four distinct horizontal bands and a series of patches. In order further to characterize the labelling pattern of the D1 receptor antibody, double-labelling experiments were performed with antibodies against population-specific neuronal markers in the retina. Antibodies against tyrosine hydroxylase, choline acetyltransferase, calretinin, calbindin, the glutamate transporter GLT-1, protein kinase C, recoverin and parvalbumin were co-applied with the D1 receptor antibody. With these cell markers we demonstrate that horizontal cells, at least three types of cone bipolar cells and a small number of amacrine cells are immunolabelled for the D1 receptor. In the inner plexiform layer, processes labelled by the D1 receptor antibody are co-stratified with processes labelled by the GLT-1 antibody. D1 receptor-labelled processes are not co-localized with the processes of amacrine cells and ganglion cells labelled by antibodies against tyrosine hydroxylase, choline acetyltransferase or calretinin. Our results indicate that dopamine D1 receptors are localized predominantly to horizontal cells and cone bipolar cells. Furthermore, the spatial disparity between dopaminergic processes and the site of the majority of D1 receptors supports the idea that in the retina dopamine acts as a neuromodulator that diffuses through extracellular space. The localization of D1 receptors to a number of identified cell types enables future physiological work to be directed towards specific synaptic circuits within the retina.

Correlation between a bicuculline-resistant response to GABA and GABAA receptor rho 1 subunit expression in single rat retinal bipolar cells.

Using patch-clamp recording in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we show in individual bipolar cells acutely dissociated from the adult rat retina a correlation between the expression of the GABAA receptor rho 1 subunit mRNA and a bicuculline-resistant, diazepam-insensitive component of the GABA-activated whole-cell current response. This "GABAC-like" response, contributing to approximately 42% of the GABA-activated whole-cell current and displaying variable sensitivity to picrotoxin, was found in bipolar cells but not in any of the ganglion cells examined. Expression profiling of GABAA receptor subunit mRNAs in individual electrophysiologically tested retinal neurons revealed that, while both bipolar cells and ganglion cells may express numerous GABAA receptor subunit isoforms, including that of rho 2, the expression of the rho 1 subunit was strictly limited to bipolar cells. We propose a possible link between the presence of a receptor with GABAC-like pharmacological profile and the expression of the retina-specific rho 1 subunit isoform. The results presented in this study constitute the first direct demonstration of such a correlation at the single-cell level.

Vasoactive intestinal polypeptide modulates GABAA receptor function through activation of cyclic AMP.

Vasoactive intestinal polypeptide (VIP) has been shown to potentiate current responses elicited by activation of the GABAA receptor (IGABA) in freshly dissociated ganglion cells of the rat retina. Here we tested the hypothesis that this heteroreceptor cross talk is mediated by an intracellular cascade of events that includes the sequential activation of a stimulatory guanine nucleotide binding (Gs) protein and adenylate cyclase, the subsequent increase in levels of cyclic AMP and, finally, the action of the cyclic AMP-dependent protein kinase (PKA). Intracellular dialysis of freshly dissociated ganglion cells with GTP gamma s irreversibly potentiated IGABA, while GDP beta s either decreased or had no effect on IGABA. Additionally, GDP beta s blocked the potentiation of IGABA by VIP. Cholera toxin rendered VIP ineffective in potentiating IGABA, while pertussis toxin had no effect on the VIP-induced potentiation of IGABA. Extracellular application of either forskolin or 8-bromo-cyclic AMP potentiated IGABA, as did the introduction of cyclic AMP directly into the intracellular compartment through the recording pipet. Intracellular application of cyclic AMP-dependent protein kinase (PKA) potentiated IGABA, while a PKA inhibitor blocked the potentiating effect of VIP. These results lead us to conclude that activation of a cyclic AMP-dependent second-messenger system mediates the modulation of GABAA receptor function by VIP in retinal ganglion cells.

Vasoactive intestinal polypeptide modulates GABAA receptor function in bipolar cells and ganglion cells of the rat retina.

1. The effect of vasoactive intestinal polypeptide (VIP) on bipolar cells and ganglion cells freshly dissociated from the rat retina was studied under voltage clamp with the use of patch-clamp recording in the whole-cell configuration. 2. Application of VIP (1-100 microM) by itself resulted in no detectable current response in either bipolar cells or ganglion cells. However, gamma-aminobutyric acid (GABA)-activated macroscopic current responses elicited in both neuronal populations were potentiated on superimposed exposure to the neuropeptide. 3. GABA-activated chloride currents and muscimol-induced current responses were similarly potentiated on exposure to VIP, suggesting a synergistic interaction between VIP and GABAA receptor mechanisms. 4. We postulate that VIP plays a neuromodulatory role by regulating the excitability of inner retinal neurons and in this way modulates the efficacy of synaptic transmission in the retina.