Pu Qin

Localization of AMPA-selective glutamate receptor subunits in the cat retina: A light- and electron-microscopic study

The distribution of AMPA-selective glutamate receptor subunits was studied in the cat retina using antisera against GluR1 and GluR2/3. Both antisera were localized in postsynaptic sites in the outer plexiform layer (OPL) as well as the inner plexiform layer (IPL). Immunoreactivity for GluR1 was seen in a subpopulation of OFF cone bipolar cells and a number of amacrine and ganglion cells. Within the IPL, processes staining for GluR1 received input from OFF and ON cone bipolar cells but not from rod bipolars. Labeling for GluR2/3 was seen in horizontal cells, an occasional cone bipolar cell, and numerous amacrine and ganglion cells. In the IPL, GluR2/3 staining was postsynaptic to cone bipolar cells in both sublaminae. AII amacrine cells which receive rod bipolar input were also labeled for GluR2/3. With both antisera, staining was limited to a single member of the bipolar dyad complex, providing morphological evidence for functional diversity in glutamatergic pathways.

Distribution of AMPA-selective glutamate receptor subunits in the cat retina

Immunocytochemical techniques were used to localize AMPA-selective glutamate receptor subunits in the cat retina. The antisera employed recognize GluR1, GluR2/3 or GluR4 subunits. Each antiserum produced a distinctive staining pattern which included horizontal cells, cone bipolar cells, and amacrine and ganglion cells. Some cells such as alpha ganglion cells expressed multiple subunits whereas amacrine cells were typically immunoreactive with only one of the antisera.

Immunocytochemical localization of three vesicular glutamate transporters in the cat retina.

Vesicular transporters play an essential role in the packaging of glutamate for synaptic release and so are of particular importance in the retina, where glutamate serves as the neurotransmitter for photoreceptors, bipolar cells, and ganglion cells. In the present study, we have examined the distribution of the three known isoforms of vesicular glutamate transporter (VGLUT) in the cat retina. VGLUT1 was localized to all photoreceptor and bipolar cells, whereas VGLUT2 was found in ganglion cells. This basic pattern of complementary distribution for the two transporters among known populations of glutamatergic cells is similar to previous findings in the brain and spinal cord. However, the axon terminals of S‐cone photoreceptors were found to express both VGLUT1 and VGLUT2 and some ganglion cells labeled for both VGLUT2 and VGLUT3. Such colocalizations suggest the existence of dual modes of regulation of vesicular glutamate transport in these neurons. Staining for VGLUT2 was also present in a small number of varicose processes, which were seen to ramify throughout the inner plexiform layer. These fibers may represent axon collaterals of ganglion cells. The most prominent site of VGLUT3 immunoreactivity was in a population of amacrine cells; the axon terminals of B‐type horizontal cells were also labeled at their contacts with rod spherules. The presence of the VGLUT3 transporter at sites not otherwise implicated in glutamate release may indicate novel modes of glutamate signaling or additional roles for the transporter molecule. J. Comp. Neurol. 475:518–530, 2004.

Immunocytochemical localization of kainate-selective glutamate receptor subunits GluR5, GluR6, and GluR7 in the cat retina

Localizations of the kainate-selective glutamate receptor subunits GluR5, 6, and 7 were studied in the cat retina by light and electron microscopic immunocytochemistry. GluR5 immunoreactivity was observed in the cell bodies and dendrites of numerous cone bipolar cells and ganglion cells. The labeled cone bipolar cells make basal or flat contacts with cone pedicles in the outer plexiform layer, leading to their identification as OFF-center bipolar cells. Reaction product within the inner plexiform layer was observed in processes of ganglion cells at their sites of input from cone bipolar cells. Staining for GluR6 was localized to A- and B-type horizontal cells, numerous amacrine cells, and an occasional cone bipolar cell. The larger ganglion cells were also immunoreactive. As with other GluR molecules, labeling was usually confined to one of the two postsynaptic elements at a cone bipolar dyad contact. Immunoreactivity for GluR7 was very limited and was seen only in a few amacrine and displaced amacrine cells. Findings of this study are consistent with a major role for kainate receptors in mediating OFF pathways in the outer retina with participation in both OFF and ON pathways in the inner retina.

Identification of a cone bipolar cell in cat retina which has input from both rod and cone photoreceptors

It has been generally accepted that rod photoreceptor cells in the mammalian retina make synaptic contact with only a single population of rod bipolar cells, whereas cone photoreceptors contact a variety of cone bipolar cells. This assumption has been challenged in rodents by reports of a type of cone bipolar cell which receives input from both rods and cones. Questions remained as to whether similar pathways are present in other mammals. We have used an antiserum against the glutamate transporter GLT1‐B to visualize a population of cone bipolar cells in the cat retina which make flat contacts with axon terminals of both rod and cone photoreceptor cells. These cells are identified as OFF‐cone bipolar cells and correspond morphologically to type cb1 (CBa2) cone bipolar cells which are a major source of input to OFF‐beta ganglion cells in the cat retina. The GLT1‐B transporter was also localized to processes making flat contacts with photoreceptor terminals in rat and rabbit retinas. Examination of tissue processed for the GluR1 glutamate receptor subunit showed that cb1 cone bipolar cells, like their rodent counterparts, express this α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐selective receptor at their contacts with rod spherules. Thus, a direct excitatory pathway from rod photoreceptors to OFF‐cone bipolar cells appears to be a common feature of mammalian retinas. J. Comp. Neurol. 464:104–113, 2003.

Cellular and subcellular distribution of NMDA receptor subunit NR2b in the retina

Immunocytochemical studies showed the presence of staining for the N‐methyl‐D‐aspartate (NMDA)‐R2B glutamate receptor subunit at multiple sites in the cat retina. Reaction product in photoreceptor cells was localized at the inner/outer segment junction and in the axon terminals. Staining within the inner retina was limited to ganglion cells and their dendrites ramifying throughout the inner plexiform layer. These cells were seen to receive synaptic input from cone bipolar cells in both sublaminae. As with other glutamate receptor subunits, this immunoreactivity was typically confined to a single postsynaptic element at a cone bipolar dyad complex. Immunocytochemical localization of the NMDA‐R1 subunit, considered to be an essential component of functional receptors, showed a widespread distribution across the retina including all the sites where NMDA‐R2B staining was seen. Immunoprecipitation and Western blot analysis were used to confirm the presence of the NR2B receptor protein and its association with the NR1 subunit in both proximal and distal retinal layers. The findings suggest that NMDA‐R2B subunits are positioned for multiple functions within the retina. J. Comp. Neurol. 433:75–85, 2001.

Differential cellular and subcellular distribution of glutamate transporters in the cat retina

Retrieval of glutamate from extracellular sites in the retina involves at least five excitatory amino acid transporters. Immunocytochemical analysis of the cat retina indicates that each of these transporters exhibits a selective distribution which may reflect its specific function. The uptake of glutamate into Muller cells or astrocytes appears to depend upon GLAST and EAAT4, respectively. Staining for EAAT4 was also seen in the pigment epithelium. The remaining transporters are neuronal with GLT-1alpha localized to a number of cone bipolar, amacrine, and ganglion cells and GLT-1v in cone photoreceptors and several populations of bipolar cells. The EAAC1 transporter was found in horizontal, amacrine, and ganglion cells. Staining for EAAT5 was seen in the axon terminals of both rod and cone photoreceptors as well as in numerous amacrine and ganglion cells. Although some of the glutamate transporter molecules are positioned for presynaptic or postsynaptic uptake at glutamatergic synapses, others with localizations more distant from such contacts may serve in modulatory roles or provide protection against excitoxic or oxidative damage.

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina

Fast‐acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) ‐selective and kainate‐selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium‐permeable channels. Treatment with both agonists resulted in staining of A‐ and B‐type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA‐stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA‐selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate‐stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types. J. Comp. Neurol. 454:341–349, 2002.