MAP1B is required for axon guidance and Is involved in the development of the central and peripheral nervous system.

Microtubule-associated proteins such as MAP1B have long been suspected to play an important role in neuronal differentiation, but proof has been lacking. Previous MAP1B gene targeting studies yielded contradictory and inconclusive results and did not reveal MAP1B function. In contrast to two earlier efforts, we now describe generation of a complete MAP1B null allele. Mice heterozygous for this MAP1B deletion were not affected. Homozygous mutants were viable but displayed a striking developmental defect in the brain, the selective absence of the corpus callosum, and the concomitant formation of myelinated fiber bundles consisting of misguided cortical axons. In addition, peripheral nerves of MAP1B-deficient mice had a reduced number of large myelinated axons. The myelin sheaths of the remaining axons were of reduced thickness, resulting in a decrease of nerve conduction velocity in the adult sciatic nerve. On the other hand, the anticipated involvement of MAP1B in retinal development and gamma-aminobutyric acid C receptor clustering was not substantiated. Our results demonstrate an essential role of MAP1B in development and function of the nervous system and resolve a previous controversy over its importance.

Knock out of direction selectivity in the retina.

Retinal ganglion cells show direction selectivity in their responses to moving stimuli. The circuitry necessary to generate directional selectivity in these cells has been long debated. Yoshida et al. (2001) use immunotoxin-mediated cell ablation to demonstrate that the starburst amacrine cell is at the core of this computation.

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.

The synaptic architecture of AMPA receptors at the cone pedicle of the primate retina.

Cone pedicles, the output synapses of cone photoreceptors, transfer the light signal onto the dendrites of bipolar and horizontal cells. Cone pedicles contain between 20 and 45 ribbon synapses (triads) which are the release sites for glutamate, the cone transmitter. Several hundred postsynaptic dendrites contact individual cone pedicles, and we studied the glutamate receptors expressed and clustered at these contacts, particularly the AMPA receptor subunits. Using immunocytochemistry and confocal imaging we were able to resolve individual triads within the cone pedicles by light microscopy. We studied their differences in L/M- and S-cones, and we counted the number of triads per pedicle across the retina. The presynaptic matrix protein bassoon, the synapse-associated membrane protein P84, and peanut agglutinin were used to specifically label synaptic ribbons, invaginating dendrites of horizontal cells and invaginating dendrites of ON-cone bipolar cells, respectively. Pre- and post-embedding immunocytochemistry and electron microscopy were used to localize the AMPA receptor subunits at the cone pedicle base. They were aggregated at three different postsynaptic sites: at horizontal cell invaginating contacts, at bipolar cell flat contacts, and at desmosome-like junctions underneath the cone pedicles. We also performed double-labeling experiments with the triad-specific markers and the antibodies against the AMPA receptor subunits. AMPA receptors were preferentially expressed by horizontal cells, and to a lesser extent by OFF-cone bipolar cells. We did not observe any cone-selective expression of AMPA receptor subunits postsynaptic to L/M- or S-cones, suggesting AMPA receptors are not the key to understanding trichromatic signaling in the primate retina.

Glutamate receptors in the rod pathway of the mammalian retina.

Rod bipolar (RB) cells of the mammalian retina release glutamate in a graded, light-dependent fashion from 20 to 40 ribbon synapses (dyads). At the dyads, two classes of amacrine cells, the AI and AII cells, are the postsynaptic partners. We examined the glutamate receptors (GluRs) that are expressed by AI and AII cells using immunocytochemistry with specific antibodies against GluR subunits. Sections of macaque monkey and rabbit retina were examined by confocal microscopy. AII amacrine cells were selectively labeled for calretinin, and AI cells in rabbits were labeled for 5-HT uptake. Thus, double- and triple-labeling for these markers and GluR subunits was possible. Electron microscopy using postembedding immunocytochemistry and double-labeling was applied to show the synaptic expression of GluRs. We also studied the synaptic localization of the two postsynaptic density proteins PSD-95 and glutamate receptor-interacting protein (GRIP). We found that AII amacrine cells express the AMPA receptor subunits GluR2/3 and GluR4 at the RB cell dyads, and they are clustered together with PSD-95. In contrast, AI amacrine cells express the delta1/2 subunits that appear to be associated with kainate receptor subunits and to be clustered together with GRIP. The RB cell dyad is therefore a synapse that initiates two functionally and molecularly distinct pathways: a "through conducting" pathway based on AMPA receptors and a modulatory pathway mediated by a combination of delta1/2 subunits and kainate receptors.

Synaptic currents generating the inhibitory surround of ganglion cells in the mammalian retina.

The receptive field (RF) of retinal ganglion cells (RGCs) consists of an excitatory central region, the RF center, and an inhibitory peripheral region, the RF surround. It is still unknown in detail which inhibitory interneurons (horizontal or amacrine cells) and which inhibitory circuits (presynaptic or postsynaptic) generate the RF surround. To study surround inhibition, light-evoked whole-cell currents were recorded from RGCs of the isolated, intact rabbit retina. The RFs were stimulated with light or dark spots of increasing diameters and with annular light stimuli. Direct inhibitory currents could be isolated by voltage clamping ganglion cells close to the Na(+)/K(+) reversal potential. They mostly represent an input from GABAergic amacrine cells that contribute to the inhibitory surround of ganglion cells. This direct inhibitory input and its physiological function were also investigated by recording light-evoked action potentials of RGCs in the current-clamp mode and by changing the intracellular Cl(-) concentration. The excitatory input of the ganglion cells could be isolated by voltage clamping ganglion cells at the Cl(-) reversal potential. Large light spots and annular light stimuli caused a strong attenuation of the excitatory input. Both GABA(A) receptors and GABA(C) receptors contributed to this inhibition, and picrotoxinin was able to completely block it. Together, these results show that the RF surround of retinal ganglion cells is mediated by a combination of direct inhibitory synapses and presynaptic surround inhibition.

Morphological Classification of Bipolar Cells of the Primate Retina.

Bipolar cells were studied in Golgi - Colonnier-stained whole mounts of macaque monkey retinae. A piece of retina, at 6 - 7 mm eccentricity, was particularly well stained for the analysis of the different bipolar cell types. Many midget bipolar cells were encountered and the dichotomy into flat and invaginating midget bipolars was confirmed. Six types of diffuse cone bipolar cell are distinguished. They differ in their dendritic branching pattern, in the number of cones contacted-usually between five and ten-and in the shape and branching level of their axons. The size, shape and stratification of the axons were found to be the most reliable distinguishing features for classifying diffuse cone bipolar cells. The stratification of the axons in the inner plexiform layer (IPL), whether closer to the amacrine or ganglion cells, was used to name diffuse cone bipolar cells in the order DB1 to DB6. Blue cone and rod bipolar cells were confirmed as distinct types. Axon terminals of diffuse cone bipolars were found to tile their sublamina of the IPL in a territorial manner. From this the density of each type could be estimated, and it is shown that a single cone is likely to be in contact with as many as 15 individual diffuse bipolar cells, as well as two midget bipolars. The diffuse bipolar cells observed contact all the cone pedicles in their dendritic fields. It is, therefore, unlikely that they carry a chromatic signal into the inner retina. The presence of many midget bipolar cells, which make contact with one cone pedicle only, suggests that midget bipolars provide chromatic input to ganglion cells in peripheral retina as well as in the fovea. The data show that the P- and M-cell pathways of the primate visual system are, to a significant extent, already anatomically discrete at the photoreceptor synapse.

Horizontal Cells in the Monkey Retina: Cone connections and dendritic network.

Horizontal cells of the macaque monkey retina were quantified and the number of cones converging onto an individual horizontal cell as well as the number of horizontal cells contacting a single cone were determined. This was done by combining data from individual horizontal cells stained by the Golgi method with the results of immunocytochemical staining described in the preceding paper (Röhrenbeck et al., 1989). The observation (Boycott et al., 1987) that all horizontal cells contact all cones in their dendritic field irrespective of cone type was confirmed. The particular cones contacted by the terminal aggregates of each horizontal cell were found. The dendritic fields of H1 and H2 cells increase with increasing eccentricity; close to the fovea H1 cells are smaller than H2 cells, at 6 mm eccentricity they are about the same size and in peripheral retina H1 cells are much larger than H2 cells. The density gradients of the two cell types balance their denritic field changes so that throughout the retina each and every cone synapses with 3 - 5 horizontal cells of each type. Horizontal cells of both cat (Wässle et al., 1978) and monkey retina follow the general rule that all cones in the dendritic fields are contacted, their perikarya form a regular mosaic and the boundaries of their dendritic fields are marked by the perikarya of their homologous neighbours.

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, gamma-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.

Immunocytochemical localization of glycine receptors in the mammalian retina.

The distribution of glycinergic synapses in the mammalian retina was studied with monoclonal antibodies against glycine receptors and a glycine receptor-related protein (gephyrin). Monoclonal antibody 2b is specific for the alpha 1 subunit of the glycine receptor; monoclonal antibody 4a is specific for all known alpha subunits and the beta subunit, and monoclonal antibody 7a is specific for gephyrin. The three antibodies were applied to the retina of cat, macaque monkey, rat, and rabbit. The general staining pattern is comparable in all these species and it is similar but distinct with all of the three antibodies. Labeling is characterized by a punctate appearance indicating that it occurs at synapses. In the inner plexiform layer, labeling is concentrated in two bands. One band is located close to the inner nuclear layer; the other band is located in the middle of the inner plexiform layer. In the outer plexiform layer, sparse punctate labeling is seen. The distribution of gephyrin was also studied at the ultrastructural level in cat and monkey retina. Gephyrin is present on the postsynaptic membrane of amacrine cells and ganglion cells. The presynaptic profile to gephyrin immunoreactivity is always of an amacrine cell. The AII amacrine cell, the crucial glycinergic interneuron of the rod pathway, is presynaptic to gephyrin immunoreactivity in the OFF-sublamina and is itself gephyrin-positive at an input synapse from another (possibly GABAergic) amacrine cell in the ON-sublamina.

Immunocytochemical identification of cone bipolar cells in the rat retina.

We studied the morphology of bipolar cells in fixed vertical tissue sections of the rat retina by injecting the cells with Lucifer Yellow and neurobiotin. In addition to the rod bipolar cell, nine different putative cone bipolar cell types were distinguished according to the position of their somata in the inner nuclear layer and the branching pattern and stratification level of their axon terminals in the inner plexiform layer. Some of these bipolar cell populations were labeled immunocytochemically in vertical and horizontal sections using antibodies against the calcium-binding protein recoverin, the glutamate transporter GLT-1, the alpha isoform of the protein kinase C, and the Purkinje cell marker L7. These immunocytochemically labeled cell types were characterized in terms of cell density and distribution. We found that rod bipolar cells and GLT-1-positive cone bipolar cells occur at higher densities in a small region located in the upper central retina. This area probably corresponds to the central area, which is the region of highest ganglion cell density. A second peak of rod bipolar cell density in the lower temporal periphery matches the retinal area of binocular overlap. The population densities of the immunocytochemically characterized bipolar cells indicate that at least 50% of all bipolar cells are cone bipolar cells. The variety and total number of cone bipolar cells is surprising because the retina of the rat contains 99% rods. Our findings suggest that cone bipolar cells may play a more important role in the visual system of the rat than previously thought.

The cat optic nerve: fibre total count and diameter spectrum.

An electron microscopic examination of two cat optic nerves indicates a mean total count of 193,000 fibres ranging from 0.5 mu to 13.5 mu in diameter. This count, although nearly double any previously reported, supports recent minimum estimates of the retinal ganglion cell population of the cat eye. A radial gradient of packing density exists across the nerve close to the globe; a high density "core" with a unimodal fibre diameter spectrum may be identified as the area centralis outflow and a peripheral low density region with a trimodal diameter spectrum contains the projection of the peripheral retina. Division of the peripheral fibre spectrum suggests the percentages of alpha, beta and gamma ganglion cells in the peripheral retina to be 5%, 42% and 53% respectively.

GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study.

The distribution of GABA-like immunoreactivity in the macaque monkey retina was studied by using postembedding techniques on semithin and ultrathin sections. At the light microscopic level, both inner and outer plexiform layers showed strong GABA-like immunoreactivity in the central retina. All the horizontal cells, some bipolar cells, 30-40% of amacrine cells, occasional interplexiform cells, and practically all displaced amacrine cells were labeled. In the peripheral retina (beyond 5 mm eccentricity), the outer plexiform layer and the horizontal cells were not labeled, but all other cell types showed the same labeling pattern as in the central retina. Synapses of the inner plexiform layer involving a pre- or postsynaptic GABA-labeled process were studied electron microscopically. Synapses involving a GABA-labeled presynaptic amacrine cell process made up 80% of the synapses observed. These GABA-labeled amacrine processes synapsed onto amacrine, bipolar, and ganglion cell processes as well as onto amacrine and ganglion cell bodies. Synapses involving a postsynaptic GABA-labeled process made up 20% of the synapses studied. The GABA-like immunoreactive processes were postsynaptic to bipolar cells at the dyads and to amacrine cells at conventional synapses.

Immunocytochemical studies on astroglia of the cat retina under normal and pathological conditions.

The cell density and morphology of astrocytes in whole-mounted adult cat retinae have been studied by immunocytochemical localization of glial fibrillary acidic protein (GFAP). There was a peak density at the optic nerve head of about 2,000 astrocytes/mm2 dropping to approximately 200 astrocytes/mm2 in the far periphery. In the central area there was a local minimum of astrocyte density. Whereas the processes of astroglia in the outermost retinal periphery were radially arranged, thus giving the cells a characteristic star-shaped appearance, the majority of astroglial processes in the central region of the retina were aligned in parallel with the ganglion cell axon bundles. To study the correlation between optic nerve fibres and astrocytes, ganglion cell axons were caused to degenerate following photocoagulation lesions close to the optic disc. Postlesion the processes of astrocytes in the central retina lost their directional preference and linearity, thus appearing to develop a star-shaped morphology typical of astroglia in the far periphery of normal retinae. The density of astrocytes decreased by approximately 50% on the optic disc side of the lesions and by up to 80% on the peripheral side of the lesions. The results show that the morphology and number of astrocytes vary as a function of their proximity to optic nerve fibres and to the density of these fibres.

Immunocytochemical staining of AII-amacrine cells in the rat retina with antibodies against parvalbumin.

The rod dominated rodent retina is the preferred tissue for in vitro studies of mammalian retinal physiology and pharmacology. The rod pathway through the rat retina was investigated, therefore, in order to find out whether its organization follows the mammalian "plan." AII-amacrine cells of the rat retina were injected with Lucifer Yellow to characterize the morphology of this bistratified interneuron of the rod pathway. When sections or whole mounts of the rat retina were stained with antibodies against the calcium binding protein parvalbumin (PV), two different amacrine cell types were labeled: the AII-amacrine cell and a widefield amacrine cell. They occur at a ratio of 12:1. Weak label was also observed in ganglion cells. The density of PV-labeled AII-cells decreases from approximately 7,000 cells/mm2 in upper central retina to 2,000 cells/mm2 in peripheral retina. Their cell bodies form a regular mosaic, and the dendritic arbors of three neighbouring AII-amacrine cells overlap (coverage of 3).

The retinal projection to the thalamus in the cat: a quantitative investigation and a comparison with the retinotectal pathway.

The projection of cat retinal ganglion cells to the thalamus was examined using the method of retrograde axonal transport of horseradish peroxidase (HRP). After the injection site was determined physiologically, HRP was applied by one of three methods: iontophoretic injection of minimal amounts, single pressure injections and multiple pressure injections. Iontophoretic injections into single laminae of the dorsal part of the lateral geniculate nucleus (LGNd) revealed that laminae A and A1 receive almost exclusively axon terminals from alpha and beta cells. Single pressure injections elucidated the retinotopic organization of the LGNd. Multiple injections lead to HRP uptake in the whole LGNd including parts of adjacent thalamic nuclei and revealed that at least 77% of all retinal ganglion cells project to the thalamus. This pathway is made up of all alpha cells, all beta cells and almost half of the gamma cells. The thalamus receives its visual input predominantly from the ipsilateral temporal and the contralateral nasal retina; some alpha cells were also labeled in the contralateral temporal retina. The shape of the decussation line was analyzed and its width was found to be proportional to the average ganglion cell spacing along the dorsoventral axis of the retina. From a comparison of the retinothalamic and retinotectal pathways, an estimate of the number of cells with bifurcating axons could be given. The axons of all alpha cells, 10% of the beta cells, and every second gamma cell bifurcate; this amounts to 30% of the retinal ganglion cells.

The retinal projection to the superior colliculus in the cat: a quantitative study with HRP.

The projections of cat retinal ganglion cells to the superior colliculus (SC) were examined using the method of retrograde axonal transport of horseradish peroxidase (HRP). Several injections of HRP were made in a single SC after the visual projection to the injection sites had been established physiologically. The HRP injections resulted in a homogeneous distribution of labelled ganglion cells in whole mount preparations of the retinae of both eyes. In the eye contralateral to the injected colliculus, ganglion cells with a crossed projection were labelled in both nasal and temporal retina; in the ipsilateral eye, ganglion cells with uncrossed projection were labelled only in the temporal retina. Analysis of the counterstained retinal whole mounts indicated that at least 50% of all ganglion cells in the nasal retina and 26% in the temporal retina have crossed projection to SC, and that 24% of all ganglion cells of the temporal retina have an uncrossed projection to the SC. The morphological classes of retinal ganglion cells have different patterns of crossed/uncrossed decussation and they participate in varying proportions in the retino-tectal projection. Almost all Alpha cells in the retina send axon collaterals to the SC. Probably only about 10% of the Beta cells project to the SC and at least 80% of all Gamma cells send axons to the SC.

Indoleamine-accumulating amacrine cells are presynaptic to rod bipolar cells through GABA(C) receptors.

gamma-Aminobutyric acid (GABA), is a main source of inhibitory modulation of the rod pathway in the mammalian retina. The authors previously showed that rod bipolar cells express at least three types of ionotropic GABA receptors. Here, the authors sought to determine which neurons are the presynaptic partners at these synapses in the rabbit retina. Indoleamine-accumulating amacrine cells (IACs) were immunolabeled with an antiserum against serotonin (5HT) in vertical sections and wholemounts of rabbit retinae that had been preloaded with 5HT. The tissue was double labeled for the rho subunits of the GABA(C) receptor or the alpha3 subunit of the GABA(A) receptor. Punctate immunofluorescence was observed for both receptor subunits and was found to coincide with the dendrites and varicosities of IACs. The localization of rho subunits was examined at the ultrastructural level by using postembedding techniques on slam-frozen, cryosubstituted tissue. Double labeling at the electron microscopic level revealed that 5HT-immunoreactive processes were presynaptic to rod bipolar cells through GABA(C) receptors. Intracellular injection of the two morphologic subclasses of IAC amacrine cells, S1 and S2, with Lucifer yellow followed by immunolabeling for the alpha3 or rho subunits revealed that varicosities on the dendrites of both cell types were in register with alpha3- and rho-immunoreactive puncta. Taken together, these results suggest that IACs are presynaptic to rod bipolar cells through GABA(C) receptors and possibly through GABA(A) receptors.

Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity.

An antibody directed against protein kinase C (PKC) was applied to various mammalian retinae. In the cat, rat, rabbit, and macaque monkey we found PKC-like immunoreactivity in bipolar cells which had the morphology of rod bipolar cells; in the rat some amacrine cells were also immunoreactive. In the outer plexiform layer, labeled dendrites were always the central elements of the rod spherule invagination, and in the inner plexiform layer only rod bipolar axons and their axon terminals were immunoreactive. The antibody against PKC thus can be used to distinguish rod bipolar cells from cone bipolar cells. The antibody against PKC was used to determine the densities of rods and rod bipolar cells in the cat retina. In the central retina we found a rod to rod bipolar ratio of 16 to 1, in the periphery the ratio increases to 25 to 1. In freshly dissociated retina, cells with rod bipolar morphology could be identified; these cells were also labeled with the anti-PKC antibody. Hence, PKC-like immunoreactivity can be used to recognize rod bipolar cells in vitro.

GABA-like immunoreactivity in the cat retina: light microscopy.

Semithin sections of the cat retina were stained with antibodies against GABA conjugated to bovine serum albumin with glutaraldehyde. Labelled cells were visualized by means of the peroxidase-antiperoxidase method. In the outer plexiform layer both A- and B-type horizontal cells and the B-type axon terminal system expressed GABA-like immunoreactivity. Approximately 25-30% of all amacrine cells and the whole inner plexiform layer were heavily labelled. Two types of putative GABA-ergic interplexiform cells could be distinguished. One of them also expressed tyrosine-hydroxylase-like immunoreactivity. A few bipolar cells were also GABA-immunolabelled. GABA-like immunoreactivity and 3H-muscimol uptake were colocalized in 90% of the amacrine cells labelled. However, horizontal cells did not accumulate 3H-muscimol.