Silke Haverkamp

Immunocytochemical analysis of the mouse retina

Transgenic mice provide a new approach for studying the structure and function of the mammalian retina. In the past, the cellular organization of the mammalian retina was investigated preferentially in primates, cats, and rats but rarely in mice. In the current study, the authors applied 42 different immunocytochemical markers to sections of the mouse retina and studied their cellular and synaptic localization by using confocal microscopy. The markers applied were from three major groups: 1) antibodies against calcium‐binding proteins, such as calbindin, parvalbumin, recoverin, or caldendrin; 2) antibodies that recognize specific transmitter systems, such as glycine, γ‐aminobutyric acid, or acetylcholine; and 3) antibodies that recognize transmitter receptors and show their aggregation at specific synapses. Only a few markers labeled only one cell type: Most antibodies recognized specific groups of neurons. These were analyzed in more detail in double‐labeling experiments with different combinations of the antibodies. In light of their results, the authors offer a list of immunocytochemical markers that can be used to detect possible changes in the retinal organization of mutant mice. J. Comp. Neurol. 424:1–23, 2000.

Types of bipolar cells in the mouse retina.

We studied the morphology of bipolar cells in fixed vertical tissue sections (slices) of the mouse retina by injecting the cells with Lucifer Yellow and Neurobiotin. Nine different cone bipolar cell types and one rod bipolar cell type were distinguished. The major criteria for classifying the cells were the branching pattern and stratification level of their axon terminals in the inner plexiform layer (IPL). To assess this, the IPL was subdivided into five strata of equal width. The slices were immunostained for calretinin, which labels three horizontal bands serving as a standard measure for the precise localization of the axon terminals. Immunostaining the retina with antibodies against the G‐protein Gγ13, a marker for ON‐bipolar cells, made it possible to separate OFF‐ and ON‐bipolar cells. At least two OFF‐cone bipolar cells (Types 1 and 2) were immunolabeled with antibodies against the neurokinin 3 receptors (NK3R). A further OFF‐ and an ON‐cone bipolar cell (Types 3 and 5) were immunostained with antibodies against the calcium‐binding protein CaB5. The bipolar cell types described here were compared with previous schemes of rat and primate bipolar cells. Homologous types between the three species are discussed. J. Comp. Neurol. 469:70–82, 2004.

Cone Contacts, Mosaics, and Territories of Bipolar Cells in the Mouse Retina

We report a quantitative analysis of the different bipolar cell types of the mouse retina. They were identified in wild-type mice by specific antibodies or in transgenic mouse lines by specific expression of green fluorescent protein or Clomeleon. The bipolar cell densities, their cone contacts, their dendritic coverage, and their axonal tiling were measured in retinal whole mounts. The results show that each and all cones are contacted by at least one member of any given type of bipolar cell (not considering genuine blue cones). Consequently, each cone feeds its light signals into a minimum of 10 different bipolar cells. Parallel processing of an image projected onto the retina, therefore, starts at the first synapse of the retina, the cone pedicle. The quantitative analysis suggests that our proposed catalog of 11 cone bipolar cells and one rod bipolar cell is complete, and all major bipolar cell types of the mouse retina appear to have been discovered.

The Primordial, Blue-Cone Color System of the Mouse Retina

Humans and old world primates have trichromatic color vision based on three spectral types of cone [long-wavelength (L-), middle-wavelength (M-), and short-wavelength (S-) cones]. All other placental mammals are dichromats, and their color vision depends on the comparison of L- and S-cone signals; however, their cone-selective retinal circuitry is still unknown. Here, we identified the S-cone-selective (blue cone) bipolar cells of the mouse retina. They were labeled in a transgenic mouse expressing Clomeleon, a chloride-sensitive fluorescent protein, under the control of the thy1 promoter. Blue-cone bipolar cells comprise only 1-2% of the bipolar cell population, and their dendrites selectively contact S-opsin-expressing cones. In the dorsal half of the mouse retina, only 3-5% of the cones express S-opsin, and they are all contacted by blue-cone bipolar cells, whereas all L-opsin-expressing cones (∼95%) are avoided. In the ventral mouse retina, the great majority of cones express both S- and L-opsin. They are not contacted by blue-cone bipolar cells. A minority of ventral cones express S-opsin only, and they are selectively contacted by blue-cone bipolar cells. We suggest that these are genuine S-cones. In contrast to the other cones, their pedicles contain only low amounts of cone arrestin. The blue-cone bipolar cells of the mouse retina and their cone selectivity are closely similar to primate blue-cone bipolars, and we suggest that they both represent the phylogenetically ancient color system of the mammalian retina.

The cone pedicle a complex synapse in the retina

Cone pedicles, the synaptic terminals of cone photoreceptors, are connected in the macaque monkey retina to several hundred postsynaptic dendrites. Using light and electron microscopy, we found underneath each cone pedicle a laminated distribution of dendritic processes of bipolar and horizontal cells. Superimposed were three strata of glutamate receptor (GluR) aggregates, including a novel layer of glutamate receptors clustered at desmosome-like junctions. They are, most likely, postsynaptic densities on horizontal cell dendrites. GABA(A) and GABA(C) receptors are aggregated on bipolar cell dendrites in a narrow band underneath the cone pedicle. Glutamate released from cone pedicles and GABA released from horizontal cell dendrites act not only through direct synaptic contacts but also (more so) through diffusion to the appropriate receptors.

Immunocytochemical Description of Five Bipolar Cell Types of the Mouse Retina

With the ever‐growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin‐3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)‐binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF‐cone bipolar cells could be identified. One type of ON‐cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Cα and CaB5. It was also shown that putative OFF‐cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON‐cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF‐cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors. J. Comp. Neurol. 455:463–476, 2003.

HCN channels are expressed differentially in retinal bipolar cells and concentrated at synaptic terminals

Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels codetermine the integrative behaviour of neurons and shape their response to synaptic stimulation. We used immunohistochemistry and patch‐clamp recording to study the composition and distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1–4) are expressed differentially in the retina. In particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This suggests that HCN channels are involved in the control of transmitter release.

Impaired Channel Targeting and Retinal Degeneration in Mice Lacking the Cyclic Nucleotide-Gated Channel Subunit CNGB1

Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B subunits. In heterologous expression systems, B subunits alone cannot form functional CNG channels, but they confer a number of channel properties when coexpressed with A subunits. To investigate the importance of the CNGB subunits in vivo, we deleted the CNGB1 gene in mice. In the absence of CNGB1, only trace amounts of the CNGA1 subunit were found on the rod outer segment. As a consequence, the vast majority of isolated rod photoreceptors in mice lacking CNGB1 (CNGB1-/-) failed to respond to light. In electroretinograms (ERGs), CNGB1-/- mice showed no rod-mediated responses. The rods also showed a slow-progressing degeneration caused by apoptotic death and concurred by retinal gliosis. Cones were primarily unaffected and showed normal ERG responses up to 6 months, but they started to degenerate in later stages. At the age of ∼1 year, CNGB1-/- animals were devoid of both rods and cones. Our results show that CNGB1 is a crucial determinant of native CNG channel targeting. As a result of the lack of rod CNG channels, CNGB1-/- mice develop a retinal degeneration that resembles human retinitis pigmentosa.

Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse

Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABAA receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABAA receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABAA receptors. Because homozygous (geph −/−) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (−/−) mouse retina. In the case of GABAA receptors, there was a significant reduction of clusters incorporating the γ2, α2, and α3 subunits; however, a substantial number of hot spots was still present in geph (−/−) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABAA receptors. In heterozygous geph (+/−) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit. J. Comp. Neurol. 427:634–648, 2000.

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N‐methyl‐D‐aspartate (NMDA) receptor subunits NR2A and NR1C2′ were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits δ1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the δ1/2 subunits are mostly localized on amacrine cell processes. J. Comp. Neurol. 447:138–151, 2002.