The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.

In the mammalian retina, rods feed into the cone pathway through electrotonic coupling, and recent histological data suggest the involvement of connexin36 (Cx36) in this pathway. We therefore generated Cx36 null mice and monitored the functional consequences of this deficiency on early visual transmission. The homozygous mutant mice had a normally developed retina and showed no changes in the cellular organization of the rod pathway. In contrast, the functional coupling between AII amacrine cells and bipolar cells was impaired. Recordings of electroretinograms revealed a significant decrease of the scotopic b-wave in mutant animals and an increased cone threshold that is compatible with a distorted, gap junctional transmission between AII amacrine cells and cone bipolar cells. Recordings of visual evoked potentials showed extended latency in mutant mice but unaffected ON and OFF components. Our results demonstrate that Cx36-containing gap junctions are essential for normal synaptic transmission within the rod pathway.

Connexin45 (Cx45) is known to be expressed in the retina, but its functional analysis was problematic because general deletion of Cx45 coding DNA resulted in cardiovascular defects and embryonic lethality at embryonic day 10.5. We generated mice with neuron-directed deletion of Cx45 and concomitant activation of the enhanced green fluorescent protein (EGFP). EGFP labeling was observed in bipolar, amacrine, and ganglion cell populations. Intracellular microinjection of fluorescent dyes in EGFP-labeled somata combined with immunohistological markers revealed Cx45 expression in both ON and OFF cone bipolar cells. The scotopic electroretinogram of mutant mice revealed a normal a-wave but a 40% reduction in the b-wave amplitude, similar to that found in Cx36-deficient animals, suggesting a possible defect in the rod pathway of visual transmission. Indeed, neurotransmitter coupling between AII amacrine cells and Cx45-expressing cone bipolar cells was disrupted in Cx45-deficient mice. These data suggest that both Cx45 and Cx36 participate in the formation of functional heterotypic electrical synapses between these two types of retinal neurons that make up the major rod pathway.

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced by ␤-galactosidase (␤-gal). In the outer nuclear layer, ␤-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36 -EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36 -EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.Key words: gap junctions; connexin; retina; rods; cones; bipolar cells; glutamate receptors IntroductionGap junctions are intercellular connections between adjacent cells and thus provide the structural basis for metabolic and electrical coupling. Gap junction channels are encoded by the connexin (Cx) family of genes, with at least 19 and 20 members in the murine and human genome, respectively (Eiberger et al., 2001;. Six connexin proteins associate to form a connexon or hemichannel, and the docking of two hemichannels located in opposing membranes leads to the generation of a functional gap junction between the cells. Connexons can be assembled from the same (homomeric) or from different (heteromeric) types of connexin proteins, which impart variability in their biophysical properties and potential intracellular regulatory sites.Connexin-based channels are permeable to metabolites with a molecular mass of up to 1 kDa as well as to inorganic ions, forming a low-resistance pathway to current flow between cells. In the adult brain, gap junctions mediate temporal coordination of neuronal activity (Strata et al., 1997;Mann-Metzer and Yarom, 1999), and they are responsible f...

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.

Intracellular recordings and dye injections of Lucifer yellow, horseradish peroxidase, or Neurobiotin were made in bipolar, amacrine, and ganglion cells of the Pseudemys turtle retina. By using a standard light-stimulation protocol in a sample of 375 labeled neurons, we were able to identify morphological and physiological characteristics of 11 types of bipolar cell, 37 types of amacrine cell, and 24 types of ganglion cell. To make sense of these data, we have chosen to group the 72 essentially different neuron types into traditional, functionally significant pathways. In this paper we look at the neuronal types in the inner plexiform layer (IPL) in terms of their contribution to generalized luminosity responses such as sustained ON- or OFF-center and transient ON-OFF ganglion cells; in the companion paper (J. Ammermüller, J.F. Muller, and H. Kolb, 1995, J. Comp. Neurol. 358:35-62) we look at them in terms of their involvement in color opponency and directional selectivity. A functional organization of the turtle IPL into OFF sublaminae (strata 1 and 2) and ON sublaminae (strata 3, 4, and 5), as has been described for other vertebrate retinas, was quite clear for two varieties of OFF-center bipolar cells (B4 and B5) and for all four types of sustained ON-center bipolar cell (B1, B2, B6, and B7). Thus, we found no sustained ON-center bipolar cell terminating in strata 1 and 2. We did, however, see three varieties of sustained OFF-center bipolar cells (B3, B9, and B10) having axon terminals in strata 3-5 (the ON sublamina) in addition to their terminations in stratum 1 or 2 (the OFF sublamina). Monostratified sustained ON- and OFF-center amacrine and ganglion cells rigidly obeyed the border of ON and OFF sublaminae. However, multistratified and diffuse sustained amacrine and ganglion cells could be either ON-center or OFF-center, and they did not strictly obey the border: such ON-center cells always had processes in one of the ON sublaminae (strata 3-5), and the equivalent OFF-center cells always had processes in one of the OFF sublaminae (strata 1 and 2). Monostratified transient amacrine and ganglion cells were concentrated in the middle of the IPL (around stratum 3), whereas bi-, tri-, or multistratified transient amacrine or ganglion cells always had processes in both the ON and the OFF sublaminae.(ABSTRACT TRUNCATED AT 400 WORDS)

Complexins regulate the speed and Ca2+ sensitivity of SNARE-mediated synaptic vesicle fusion at conventional synapses. Two of the vertebrate complexins, Cplx3 and Cplx4, are specifically localized to retinal ribbon synapses. To test whether Cplx3 and Cplx4 contribute to the highly efficient transmitter release at ribbon synapses, we studied retina function and structure in Cplx3 and Cplx4 single- and double-knockout mice. Electroretinographic recordings from single and double mutants revealed a cooperative perturbing effect of Cplx3 and Cplx4 deletion on the b-wave amplitude, whereas most other detected effects in both plexiform synaptic layers were additive. Light and electron microscopic analyses uncovered a disorganized outer plexiform layer in the retinae of mice lacking Cplx3 and Cplx4, with a significant proportion of photoreceptor terminals containing spherical free-floating ribbons. These structural and functional aberrations were accompanied by behavioural deficits indicative of a vision deficit. Our results show that Cplx3 and Cplx4 are essential regulators of transmitter release at retinal ribbon synapses. Their loss leads to aberrant adjustment and fine-tuning of transmitter release at the photoreceptor ribbon synapse, alterations in transmission at bipolar cell terminals, changes in the temporal structure of synaptic processing in the inner plexiform layer of the retina and perturbed vision.