The neurosciences at the Max Planck Institute for Biophysical Chemistry in Göttingen.

The Max Planck Institute (MPI) for Biophysical Chemistry (Karl-Friedrich Bonhoeffer Institute) was founded in 1971 in Göttingen. Two of the 11 departments at the institute had a neuroscientific focus. Otto D. Creutzfeldt (1927-1992) and Victor P. Whittaker (1919-2016) were directors of the Neurobiological and Neurochemical Departments, respectively. Creutzfeldt's department researched the structure and function of the cerebral cortex, and Whittaker's department concentrated on the biochemical analysis of synapses and synaptic vesicles. Creutzfeldt and Whittaker were already internationally respected scientists when they were appointed to Göttingen. The next generation of departmental directors, Erwin Neher and Bert Sakmann, were "home-grown" researchers from the institute and, during their time as junior group leaders, they developed the so-called patch clamp technique, with which they were able to measure single ion channels in nerve cells. This technique revolutionized neurophysiology, and Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology or Medicine for their work in this area. Neher was appointed director of the Membrane Biophysics Department in 1983 and, since then, his department has mainly examined the role of Ca2+ in the release of neurotransmitters at synapses and in the secretion of catecholamines from chromaffin cells. From 1985, Sakmann was director of the Cell Physiology Department, and his laboratory concentrated on the molecular and physiological characterization of transmitter receptors in postsynaptic membranes. In 1989, he was appointed to the MPI for Medical Research in Heidelberg. Reinhard Jahn became director of the Neurobiology Department in 1997, researching the molecular mechanisms of the release of neurotransmitters from the presynaptic terminals, and he discovered several proteins associated with the synaptic vesicles. With their work, Neher, Sakmann, and Jahn have made the MPI for Biophysical Chemistry one of the world's leading research centers for the transmission of signals at synapses.

Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells.

The progression of rod and cone degeneration in retinally degenerate (rd) mice ultimately results in a complete loss of photoreceptors and blindness. The inner retinal neurons survive and several recent studies using genetically targeted, light activated channels have made these neurons intrinsically light sensitive. We crossbred a transgenic mouse line expressing channelrhodopsin2 (ChR2) under the control of the Thy1 promoter with the Pde6b(rd1) mouse, a model for retinal degeneration (rd1/rd1). Approximately 30-40% of the ganglion cells of the offspring expressed ChR2. Extracellular recordings from ChR2-expressing ganglion cells in degenerated retinas revealed their intrinsic light sensitivity which was approximately 7 log U less sensitive than the scotopic threshold and approximately 2 log U less sensitive than photopic responses of normal mice. All ChR2-expressing ganglion cells were excited at light ON. The visual performance of rd1/rd1 mice and ChR2 rd1/rd1 mice was compared. Behavioral tests showed that both mouse strains had a pupil light reflex and they were able to discriminate light fields from dark fields in the visual water task. Cortical activity maps were recorded with optical imaging. The ChR2rd1/rd1 mice did not show a better visual performance than rd1/rd1 mice. In both strains the residual vision was correlated with the density of cones surviving in the peripheral retina. The expression of ChR2 under the control of the Thy1 promoter in retinal ganglion cells does not rescue vision.

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.

Glycinergic input of widefield, displaced amacrine cells of the mouse retina.

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRalpha1 (Glra1(spd-ot), 'oscillator' mouse), the GlyRalpha2 (Glra2(-/-)) and the GlyRalpha3 subunit (Glra3(-/-)). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, tau approximately 25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1(spd-ot) and Glra3(-/-) mice (tau approximately 25 ms); however, glycinergic currents were absent in group II cells of Glra2(-/-) mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (tau approximately 50-70 ms). We propose that GlyRs of group II cells are dominated by the alpha2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the alpha4 subunit.

Receptive field properties of ON- and OFF-ganglion cells in the mouse retina.

There are two subclasses of alpha cell in the mammalian retina, which are morphologically identical in plain view but have opposite responses to a luminance change: one is ON center and the other is OFF center. Recent studies have shown that the neural circuitries, which underlie light responses in such ON- and OFF-ganglion cell pairs, are not mirror symmetric with respect to the ON and OFF pathways (Pang et al., 2003; Zaghloul et al., 2003; Murphy & Rieke, 2006). This study examines alpha-cell homologues in the mouse retina and elucidates the synaptic mechanisms that generate their light responses. Morphological analysis of recorded cells revealed three subclasses that were essentially identical in plan view but had distinct vertical stratification levels. We refer to these cells as the sustained ON (ON-S), sustained OFF (OFF-S), and transient OFF (OFF-T) cells (Murphy & Rieke, 2006; Margolis & Detwiler, 2007). Both ON-S and OFF-S cells were largely driven through the ON pathway via changes in excitatory and inhibitory inputs, respectively. Light responses of OFF-T cells were driven by transient changes in excitatory and inhibitory inputs. Light responses of OFF-S cells were also measured in connexin 36 knockout mice in order to dissect glycinergic input arising from AII amacrine cells. At photopic/mesopic intensities, peak glycinergic input to OFF-S cells in the connexin 36 knockout mouse was reduced by ~85% compared to OFF-S cells in the wild-type retina. This is consistent with the idea that AII cells receive their input from ON-cone bipolar cells through gap junctions and in turn provide glycinergic inhibition to OFF-S cells.

Diversity of glycine receptors in the mouse retina: localization of the alpha4 subunit.

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Whereas the retinal distributions of glycine receptor (GlyR) subunits alpha1, alpha2, and alpha3 have been mapped, the role of the alpha4 subunit in retinal circuitry remains unclear. A rabbit polyclonal antiserum was raised against a peptide that comprises the C-terminal 14 amino acids of the mouse GlyR alpha4 subunit. Using immunocytochemistry, we localized the alpha4 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by double-labeling sections for GlyR alpha4 and synaptic markers (bassoon, gephyrin). Double-labeling sections for GlyR alpha4 and the other GlyR alpha subunits shows that they are mostly clustered at different synapses; however, approximately 30% of the alpha4-containing synapses also express the alpha2 subunit. We also studied the pre- and postsynaptic partners at GlyR alpha4-containing synapses and found that displaced (ON-) cholinergic amacrine cells prominently expressed the alpha4 subunit. The density of GlyR alpha4-expressing synapses in wildtype, Glra1(ot/ot), and Glra3(-/-) mouse retinas did not differ significantly. Thus, there is no apparent compensation of the loss of alpha1 or alpha3 subunits by an upregulation of alpha4 subunit gene expression; however, the alpha2 subunit is moderately upregulated.

A Collection of Brain Sections of "Euthanasia" Victims: The Series H of Julius Hallervorden.

Julius Hallervorden, a distinguished German neuropathologist, admitted on several occasions that he had received some five hundred brains of "euthanasia" victims from the Nazi killing centres for the insane. He investigated the brains in the summer of 1942; however, their traces were subsequently lost. The present study shows, that the Series H, which was part of the Hallervorden collection of brain sections in the Max Planck Institute for Brain Research, comprises the brain sections of the above mentioned five hundred euthanasia victims. The provenance of 105 patients could be reconstructed and 84 are for sure euthanasia victims. Most of them were killed in Bernburg or in Sonnenstein-Pirna. Hallervorden used the brain sections of Series H until 1956 for his studies and never publicly regretted this abuse of the brains of euthanasia victims.

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 (approximately 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.

Expression of the vesicular glutamate transporter vGluT2 in a subset of cones of the mouse retina.

Cone photoreceptors have a continuous release of glutamate that is modulated by light. Vesicular glutamate transporters (vGluT) play an essential role for sustaining this release by loading synaptic vesicles in the cone synapse, the so-called cone pedicle. In the present study mouse retinas were immunostained for vGluT1 and vGluT2. vGluT1 was localized to all cone pedicles and rod spherules, whereas vGluT2 was found in only 10% of the cone pedicles. The vGluT2-expressing cones were characterized in more detail. They are distributed in a regular array, suggesting they are a distinct type. Their proportion does not differ between dorsal (L-cone-dominated) and ventral (S-cone-dominated) retina, and they are not the genuine blue cones of the mouse retina. During development, vGluT1 and vGluT2 expression in cones starts at around P0 and right from the beginning vGluT2 is only expressed in a subset of cones. Bipolar cells contact the vGluT2-expressing cones and other cones nonselectively. The possible functional role of vGluT2 expression in a small fraction of cones is discussed.

AII amacrine cells express L-type calcium channels at their output synapses.

AII amacrine cells play a critical role in the high-fidelity signal transmission pathways involved with nighttime vision. The temporal properties of the light responses strongly depend on the transfer function at different synaptic stages and consequently on presynaptic calcium influx. AII light responses are complex waveforms generated by graded input, they comprise Na+-based spikes as well as a sustained component, and they are transferred to graded cone bipolar cells. It is, therefore, of interest to determine the properties of AII voltage-dependent calcium channels (VDCCs) to establish whether these cells express N-type and/or P/Q-type VDCCs, characteristic of spiking neurons, or whether they are more like graded neurons, which mostly use L-type VDCCs. We combined electrophysiological, molecular biological, and imaging techniques to characterize calcium currents and their sites of origin in mouse AII amacrine cells. Calcium currents activated at potentials more positive than -60 mV (maximally between -50 and -20 mV) and inactivated slowly. These currents were blocked by dihydropyridine (DHP) antagonists and were enhanced by the DHP agonist BayK 8644. Single-cell RT-PCR analysis of mRNA encoding for different calcium channel alpha subunits in AIIs revealed a consistent expression of the alpha1-D subunit. Calcium imaging of AII cells showed that the greatest change in intracellular calcium occurred in the lobular appendages, with minor changes being observed in the arboreal dendrites. Depolarization-induced calcium rises were also modulated by DHPs, suggesting that a particular kind of L-type VDCC, mainly localized to the lobular appendages, enables these spiking-capable neurons to release neurotransmitter in a sustained manner onto OFF-cone bipolar cells.

Horizontal Cells in the Monkey Retina: Immunocytochemical staining with antibodies against calcium binding proteins.

Immunocytochemistry with antibodies against the calcium binding proteins parvalbumin (PV) and calbindin (CaBP-28kD) was used to study horizontal cells in the macaque monkey retina. Both morphological types (H1 and H2) are stained by PV immunocytochemistry. Horizontal cells in the centre of the fovea are described for the first time. From a minimum of 250/mm2 at the foveal centre, total horizontal cell density rapidly increases to a peak of 23,000/mm2 in an annulus of 0.6 mm radius around the fovea. Horizontal cell density then drops continuously to approximately 800 - 1000 cells/mm2 in peripheral retina. The density ratio of cones to horizontal cells is 1.5 in central and 4 in peripheral retina. The cones were stained with antibodies against CaBP-28kD. Thus the spatial correlation between the cone inner segments and the cone pedicles could be measured to show that they are in perfect register. There is no reorganization of the spatial array of cone outer segments to produce chromatically specific connections between cone pedicles and horizontal cells.

Characterization of an amacrine cell type of the mammalian retina immunoreactive for vesicular glutamate transporter 3.

Immunocytochemical staining of vertical sections through rat, mouse, and macaque monkey retinae with antibodies against the vesicular glutamate transporter vesicular glutamate transporter 3 (vGluT3) showed a sparse population of amacrine cells. The labeled cells had similar appearances in the three species and probably represent homologous types. They were studied in detail in the rat retina. The thin varicose dendrites of vGluT3 amacrine cells formed a convoluted dendritic tree of approximately 100 microm in diameter that was bistratified in the center of the inner plexiform layer. The dendrites of vGluT3 cells were squeezed between the two strata of cholinergic dendrites. The density of vGluT3 cells was measured in retinal wholemounts and increased from 200/mm2 in peripheral retina to 400/mm2 in central retina, accounting for about 1% of all amacrine cells in the rat retina. The vGluT3 cells had a two- to threefold dendritic overlap, and their cell bodies formed a regular mosaic, suggesting they represent a single type of amacrine cell. The vGluT3 amacrine cells expressed glycine and glycine transporter 1 (GlyT1) but not the vesicular glycine transporter (vesicular inhibitory amino acid transporter). They also expressed glutamate; hence, there is the possibility that, comparable to cholinergic amacrine cells, they are "dual transmitter" amacrine cells. The synaptic input of vGluT3 cells was studied by electron microscopy. They received input from bipolar cells at ribbon synapses and from other amacrine cells at conventional synapses. The types of bipolar cells possibly involved with vGluT3 cells were demonstrated by double labeling sections for vGluT3 and the calcium-binding protein CaB5. The axon terminals of type 3 and 5 bipolar cells costratified with vGluT3 dendrites, and it is possible that vGluT3 cells have ON and OFF light responses.

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 delta 1/2 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 delta 1/2 subunits are mostly localized on amacrine cell processes.

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 Ggamma13, 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.

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 Calpha 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.

Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit.

Gamma-aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the retina, glycine being produced in approximately half of all amacrine cells. Whereas retinal cell types expressing the glycine receptor (GlyR) alpha1 and alpha3 subunits have been mapped, the role of the alpha2 subunit in retinal circuitry remains unclear. By using immunocytochemistry, we localized the alpha2 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by doubly labeling sections for GlyR alpha2 and bassoon (a presynaptic marker) or gephyrin (a postsynaptic marker). Synapses containing GlyR alpha2 were rarely found on ganglion cell dendrites but were observed on bipolar cell axon terminals and on amacrine cell processes. Recently, an amacrine cell type has been described that is immunopositive for glycine and for the vesicular glutamate transporter vGluT3. The processes of this cell type were presynaptic to GlyR alpha2 puncta, suggesting that vGluT3 amacrine cells release glycine. Double labeling of sections for GlyR alpha1 and GlyR alpha2 subunits showed that they are clustered at different synapses. In sections doubly labeled for GlyR alpha2 and GlyR alpha3, approximately one-third of the puncta were colocalized. The most abundant GlyR subtype in retina contains alpha3 subunits, followed by those containing GlyR alpha2 and GlyR alpha1 subunits.

Diversity of glycine receptors in the mouse retina: localization of the alpha3 subunit.

Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in wild-type and GlyR alpha3 subunit-deficient (Glra3(-/-)) mouse retinae. GlyRs are localized in the inner plexiform layer in brightly fluorescent puncta, which are likely to represent postsynaptically clustered GlyRs. Approximately one third of the clusters were found to contain the alpha1 subunit, and half possessed the alpha3 subunit. However, these two GlyR isoforms were localized at different glycinergic synapses. In the Glra3(-/-) mouse, alpha3 subunit clusters were completely eliminated, although the total number of GlyR clusters was only slightly reduced. This finding indicates that other GlyR subunits (such as alpha2 or alpha4) may have compensated for the loss of the alpha3 subunit. Characteristic expression patterns of the alpha1 and alpha3 subunits within the synaptic circuits of the retina were revealed by double labeling sections for GlyRs and markers that define specific retinal neurons. The alpha1 subunit mediates signal transfer in the rod pathway between AII amacrine cells and OFF-cone bipolar cells. In contrast, the alpha3 subunit appears to be predominantly involved with the cone pathways. Thus, expression of different GlyR alpha subunit genes correlates with anatomically defined connectivities.

Vesicular gamma-aminobutyric acid transporter expression in amacrine and horizontal cells.

The vesicular gamma-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preembedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release.

Spontaneous synaptic activity in an organotypic culture of the mouse retina.

PURPOSE: Many strains of mutant mice die at birth, when the retina is still very immature. The retinas of such mice can be studied in organotypic cultures. After a preceding anatomic study of the synaptic development, the electrical activity of the synaptic circuits within such cultures was studied in wild-type and gephyrin-deficient mice. METHODS: Organotypic cultures of newborn mouse retinas were grown for 14 days in vitro. Spontaneous postsynaptic currents (sPSCs) of amacrine cells were measured by using the whole-cell configuration of the patch-clamp technique. GABAergic and glycinergic currents that were isolated with specific antagonists, and retinas from wild-type (geph(+/+)) and gephyrin-deficient (geph(-/-)) mice were compared. RESULTS: Rapidly decaying sPSCs that were blocked by kynurenic acid were mediated by ionotropic glutamate receptors, whereas sPSCs with significantly higher peak amplitudes and slow-decay kinetics were identified as spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) and glycine receptors (GlyRs). In gephyrin-deficient (geph(-/-)) cultures, we found no sIPSCs mediated by GlyRs. sIPSCs mediated by GABA(A)Rs expressed in amacrine cells of geph(-/-) retinas decayed significantly faster than GABAergic sIPSCs recorded in amacrine cells of geph(+/+) retinas. CONCLUSIONS: The different decay kinetics of GABA(A)Rs expressed in amacrine cells of geph(+/+) and of geph(-/-) retinas suggests that these cells express at least two types of GABA(A)R subtypes. In amacrine cells of geph(-/-) mice, a specific GABA(A)R subtype that may contain the alpha2 subunit, is impaired by the absence of gephyrin, whereas other GABA(A)Rs appear to function normally.

Glycinergic transmission in the Mammalian retina.

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Glycinergic amacrine cells are small-field amacrine cells with vertically oriented dendrites and comprise more than 10 different morphological types. The retinal distributions of glycine receptor (GlyR) alpha1, alpha2, alpha3 and alpha4 subtypes have been mapped with subunit-specific antibodies. GlyRs were clustered at postsynaptic hot spots which showed selective distributions for the different subunits. As a rule, only one alpha subunit was expressed at a given postsynaptic site. The kinetic properties of GlyRs were measured by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from identified retinal neurons in wild-type, Glra1(spd-ot), Glra2 and Glra3 knockout mice. From observed differences of sIPSCs in wild-type and mutant mice, the cell-type specific subunit composition of GlyRs could be defined. OFF-cone bipolar cells and A-type ganglion cells receive prominent glycinergic input with fast kinetics that is mainly mediated by alpha1beta GlyRs (decay time constant tau approximately 5 ms). By contrast, AII amacrine cells express alpha3beta GlyRs with medium fast kinetics (tau approximately 11 ms). Narrow-field (NF) and wide-field amacrine cells contain predominantly alpha2beta GlyRs with slow kinetics (tau approximately 27 ms). Lastly, ON-starburst, narrow-field and wide-field amacrine cells in Glra2 knockout mice express alpha4beta GlyRs with very slow kinetics (tau approximately 70 ms).