Johann Helmut Brandstätter

The Presynaptic Active Zone Protein Bassoon Is Essential for Photoreceptor Ribbon Synapse Formation in the Retina

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.

Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex.

The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel α1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

Glycine and GABA receptors in the mammalian retina

Molecular cloning has introduced an unexpected diversity of neurotransmitter receptors. In this study we review the types, the localization and possible synaptic function of the inhibitory neurotransmitter receptors in the mammalian retina. Glycine receptors (GlyRs) and their localization in the mammalian retina were analyzed immunocytochemically. Specific antibodies against the alpha 1 subunit of the GlyR (mAb2b) and against all subunits of the GlyR (mAb4a) were used. Both antibodies produced a punctate immunofluorescence, which was shown by electron microscopy to represent clustering of GlyRs at synaptic sites. Synapses expressing the alpha 1 subunit of the GlyR were found on ganglion cell dendrites and on bipolar cell axons. GlyRs were also investigated in the oscillator mutant mouse. The complete loss of the alpha 1 subunit was compensated for by an apparent upregulation of the other subunits of the GlyR. GABAA receptors (GABAARs) and their retinal distribution were studied with specific antibodies that recognize the alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 2 and delta subunits. Most antibodies produced a punctate immunofluorescence in the inner plexiform layer (IPL) which was shown by electron microscopy to represent synaptic clustering of GABAARs. The density of puncta varied across the IPL and different subunits were found in characteristic strata. This stratification pattern was analyzed with respect to the ramification of cholinergic amacrine cells. Using intracellular injection with Lucifer yellow followed by immunofluorescence, we found that GABAARs composed of different subunits were expressed by the same ganglion cell, however, they were clustered at different synaptic sites. The distribution of GABAC receptors was studied in the mouse and in the rabbit retina using an antiserum that recognizes the rho 1, rho 2 and rho 3 subunits. GABAC receptors were found to be clustered at postsynaptic sites. Most, if not all of the synapses were found on rod and cone bipolar axon terminals. In conclusion we find a great diversity of glycine and GABA receptors in the mammalian retina, which might match the plethora of morphological types of amacrine cells. This may also point to subtle differences in synaptic function still to be elucidated.

Diversity of glutamate receptors in the mammalian retina

The main neurotransmitters in the vertebrate retina are glutamate, GABA and glycine. Their localization in the different cell types in the retina is well known. In addition, there exists a number of neuropeptides and other neuroactive substances that are only expressed by sparse populations of neurons. In recent years, molecular biology has led to the discovery of a rapidly increasing number of neurotransmitter receptors and the apparent simplicity of neurotransmitters in the mammalian retina is contrasted by the expression of a plethora of neurotransmitter receptors and receptor subunits (not mentioning receptor isoforms). This article will concentrate on glutamate receptors with the intention of reviewing some of the recent data on glutamate receptor expression in the mammalian retina and their possible involvement in retinal function.

Expression of NMDA and High-affinity Kainate Receptor Subunit mRNAs in the Adult Rat Retina

The expression patterns of nine genes encoding the N‐methyl‐d‐aspartate (NMDA) receptor subunits NR1 and NR2A, NR2B, NR2C and NR2D, and the high‐affinity kainate receptor subunits KA1, KA2, GluR6 and GluR7, were studied in the adult rat retina by in situ hybridization. Hybridization with [35S]dATP‐labelled oligonucleotide probes revealed the expression of four of the NMDA receptor subunits (NR1, NR2A, NR2B and NR2C) and three of the high‐affinity kainate receptor subunits (KA2, GluR6 and GluR7) in the retina. The NMDA receptor subunit NR2D and the high‐affinity kainate receptor subunit KA1 could not be detected. In the ganglion cell layer, virtually every ganglion cell or displaced amacrine cell expressed the receptor subunits NR1, NR2A, NR2B, NR2C, KA2 and GluR7. The GluR6 subunit was expressed in a more restricted manner in the ganglion cell layer. In the inner nuclear layer, the receptor subunits NR1 and KA2 were homogeneously distributed, and therefore are most likely expressed by all cell types in this layer. The GluR6, NR2A, NR2B and NR2C subunits were expressed by subsets of amacrine cells. Labelling for NR2C was also found above the middle of the inner nuclear layer, corresponding to the location of bipolar cell somata. The GluR7 subunit was expressed by most amacrine and bipolar cells. These findings suggest that NMDA and high‐affinity kainate receptor subunits could be present at a majority of glutamatergic retinal synapses.

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

Synaptic localization of NMDA receptor subunits in the rat retina

The distribution and synaptic clustering of N‐methyl‐D‐aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD‐95, PSD‐93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2` subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals. J. Comp. Neurol. 420:98–112, 2000.

Expression of GABA Receptor ρ1 and ρ2 Subunits in the Retina and Brain of the Rat

We have investigated the distribution of GABA receptor ρ1 and ρ2 subunits in the rat central nervous system. Cloning of rat ρ1 and ρ2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (ρ1) and 88% (ρ2) at the protein level. Whereas the human ρ2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse‐transcribed total RNA (RT‐PCR) from different brain tissues revealed that transcript for the ρ1 subunit was present in the retina only. The ρ2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that ρ1 and ρ2 transcripts are present in the inner nuclear layer. RT‐PCR and in situ hybridization of isolated retinal cells showed that both ρ subunits are present in rod bipolar cells. Since these cells express bicuculline‐insensitive GABA receptors, our results further support the idea that ρ subunits are part of the GABAC receptor.

Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina

Bassoon is a 420‐kDa presynaptic protein which is highly concentrated at the active zones of nerve terminals of conventional synapses, both excitatory glutamatergic and inhibitory GABAergic, in rat brain. It is thought to be involved in the organization of the cytomatrix at the site of neurotransmitter release. In the retina, there are two structurally and functionally distinct types of synapses: ribbon and conventional synapses. Antibodies against bassoon were applied to sections of rat and rabbit retina. Strong punctate immunofluorescence was found in the outer and inner plexiform layers. Using pre‐ and post‐embedding immunostaining and electron microscopy, bassoon was localized in the outer plexiform layer at ribbon synapses formed by rods and cones but was absent from basal synaptic contacts formed by cones. In the inner plexiform layer a different picture emerged. As in the brain, bassoon was found at conventional inhibitory GABAergic synapses, made by amacrine cells, but it was absent from the bipolar cell ribbon synapses. These data demonstrate differences in the molecular composition of the presynaptic apparatuses of outer and inner plexiform layer ribbon synapses. Thus, differential equipment with cytomatrix proteins may account for the functional differences observed between the two types of ribbon synapses in the retina.

Synaptic clustering of GABAC receptor ρ-subunits in the rat retina

Polyclonal antibodies which recognize the ρ‐subunits of the GABAC receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABAC receptors at synaptic sites. During postnatal development diffuse ρ‐immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the ρ‐immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the ρ‐antiserum were performed. The experiments showed that ρ‐immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABAC receptor ρ‐subunits coassemble with GABAA receptor subunits, double‐labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABAC receptor expressing puncta did not coincide with GABAA receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABAA and GABAC receptor subunits are coassembled. Similar double‐labelling experiments were also performed to find out whether GABAC receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABAC receptors consist of ρ‐subunits and are not coassembled with GABAA‐ or glycine‐receptor subunits.