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Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. We present a 1-photon light-sheet imaging system that can measure the activity in hundreds of neurons in the ex vivo retina over a large field of view while presenting visual stimuli. This allows for a reliable functional classification of different retinal cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, large field of view, and fast image acquisition make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches.

Editorial on the Research Topic How do we see? Morphology and physiology of retinal neurons.

Glaucoma is the leading cause of irreversible vision loss worldwide. Glaucoma is a neurodegenerative disease characterised by damage to the optic nerve and the selective death of retinal ganglion cells. The leading risk factors for developing glaucoma are increased age and elevated intraocular pressure (IOP). Currently, lowering IOP remains the only treatment strategy for glaucoma and if treated early, it is possible to slow disease progression. However, glaucoma diagnosis often occurs late in disease when 25 – 35% of retinal ganglion cells are already lost. Retinal dysfunction has been found to precede retinal ganglion cell loss and early stages of retinal ganglion cell dysfunction may provide a therapeutic window of opportunity where retinal ganglion cell damage is reversible. This has necessitated a better understanding of retinal dysfunction in early disease. To study the early, reversible stages of retinal dysfunction, the murine acute IOP elevation model was developed. Acute IOP elevation induces a loss of inner retinal function without causing extensive retinal ganglion cell death, which allowed us to investigate early disease mechanisms. Furthermore, retinal ganglion cell dysfunction following acute IOP elevation is reversible, which allowed us to study the mechanisms by which retinal ganglion cells may recover in glaucoma. Finally, functional recovery following IOP elevation is age dependent. Retinal ganglion cell function recovers within 7-days in young mice, while older mice experience more persistent retinal ganglion cell dysfunction, which allowed us to study the effects of increased age on retinal ganglion cell injury and recovery. Little is known about the cellular changes responsible for the loss and recovery of retinal ganglion cell function following IOP elevation. Acute IOP elevation causes no change in retinal ganglion cell output, as measured by action potential firing and visual evoked potential latency. This suggests that retinal ganglion cell

PurposeLoss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors.MethodsBipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation.ResultsImmunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively.ConclusionsThese results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

From the bipolar cells to higher brain visual centers, signals in the vertebrate visual system are transmitted along parallel on and off pathways. These two pathways are spatially segregated along the depth axis of the retina. Yet, to our knowledge, there is no way to directly assess this anatomical stratification in vivo. Here, employing ultrahigh resolution visible light Optical Coherence Tomography (OCT) imaging in humans, we report a stereotyped reflectivity pattern of the inner plexiform layer (IPL) that parallels IPL stratification. We characterize the topography of this reflectivity pattern non-invasively in a cohort of normal, young adult human subjects. This proposed correlate of IPL stratification is accessible through non-invasive ocular imaging in living humans. Topographic variations should be carefully considered when designing studies in development or diseases of the visual system.

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+ /cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+ . As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.

Cav1.4 Calciumkanäle vom L-Typ werden überwiegend in den Photorezeptoren der Netzhaut exprimiert, wo sie eine entscheidende Rolle für die Lichtsignalübertragung spielen. Mutationen im Gen, das den Cav1.4-Kanal codiert, sind mit angeborener stationärer Nachtblindheit Typ 2 (CSNB2) assoziiert. Es wurde zuvor berichtet, dass Mäuse, die die Cav1.4-Punktmutation I745T (Cav1.4-IT) tragen, als gutes Modell für den funktionellen Phänotyp beim Menschen dienen (Knoflach et al., 2013). Neben dem stäbchengesteuerten skotopischen Sehen wurden bei CSNB2-Patienten auch kegelgesteuerte photopische Reaktionen stark beeinflusst, die zugrunde liegenden Mechanismen sind jedoch noch nicht bekannt. In meiner Diplomarbeit untersuchte ich die kegelgesteuerte Signalübertragung in Cav1.4-IT-Mäusen mithilfe von Multielektrodenarray- und Patch-Clamp-Aufzeichnungen. In MEA-Aufzeichnungen sezierte ich lichtinduzierte Ganglienzellenreaktionen, die durch die Aktivierung von Stäbchen und Zapfen ausgelöst wurden. In der Cav1.4-IT-Retina zeigten nur 26% der Ganglienzellen, die auf eine scotopische Stimulation reagierten, auch eine Reaktion auf photopische Stimuli, was auf eine schwerwiegende Beeinträchtigung des Kegelwegs hinweist. Zapfen in Cav1.4-IT-Netzhäuten zeigen morphologische Aberrationen wie z. axonale Äste und vergrößertes axonales Terminal, aber im Gegensatz zu Stäbchen zeigen sie im Alter von 2-3 Monaten keine axonale Retraktion und keinen Zelltod. Wenn wir horizontale Zell-Patch-Clamp-Aufzeichnungen als Auslesung für die Kegelexozytose verwendeten, schien die Fähigkeit zur Freisetzung von Glutamat erhalten zu bleiben, wie durch eine mit dem Wildtyp vergleichbare Freisetzungshäufigkeit angezeigt. Während der Stabstimulation in MEA-Aufzeichnungen war die bipolare Übertragung von Stab und Zapfen von Zelle zu Ganglienzelle verzögert, aber intakt. Die pharmakologische Isolierung des OFF-Kegel-BC-Weges in Gegenwart von 50 uM des mGluR-Agonisten L-AP4 deutete jedoch auf einen postsynaptischen b, Cav1.4 L-type calcium channels are predominantly expressed in the retinal photoreceptors where they play a crucial role for light signal transmission. Mutations in the gene encoding Cav1.4 channel are associated with congenital stationary night blindness type 2 (CSNB2). It was previously reported that mice carrying the Cav1.4 point mutation I745T (Cav1.4-IT) serve as a good model for the functional phenotype in humans (Knoflach et al., 2013). Besides rod-driven scotopic vision also cone-driven photopic responses were severely effected in CSNB2 patients but the underlying mechanisms are still elusive. In my thesis, I investigated the cone-driven signal transmission in Cav1.4-IT mice using multielectrode array and patch-clamp recordings. In MEA recordings, I dissected light induced ganglion cell responses triggered by rod and cone activation. In the Cav1.4-IT retina only 26% of the ganglion cells responding to scotopic stimulation showed also a response to photopic stimuli indicative of a major impairment in the cone pathway. Cones in Cav1.4-IT retinas show morphological aberrations like e.g. axonal branches and enlarged axonal terminal, but in contrast to rods, they do not show axonal retraction and cell death at 2-3 months of age. When we used horizontal cell patch-clamp recordings as readout for cone exocytosis the ability to release glutamate seemed preserved as indicated by a release frequency comparable to wild type. During rod stimulation in MEA recordings both rod and cone bipolar cell-to-ganglion cell transmission was delayed but intact. Pharmacological isolation of the OFF-cone BC pathway in the presence of 50 µM of the mGluR-agonist L-AP4, however, suggested a postsynaptic cone bipolar cell defect. This hypothesis was supported by the fact that in immunohistochemical stainings, performed by my colleague M.Sc. Irem Kilicarslan, cone bipolar cells showed elongation of dendritic sprouts that didn´t contact displaced rod photoreceptor terminals. During my PhD, Mag. rer. nat. Lucia Zanetti, Dissertation University of Innsbruck 2020

Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit's output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.

Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit's output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.

The retina is a thin layer of neural tissue that lines the back of the eye and transforms the visual scene into an electrical signal that is sent to the brain via the optic nerve. The synapse between photoreceptors, the light sensitive neurons, and bipolar cells is the first connection in the retinal circuitry. Loss of photoreceptors during retinal degeneration results in permanent visual loss. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of bipolar cells to establish new functional synapses with photoreceptors is poorly understood. In my work, I investigate if deafferented bipolar cells, i.e. the ones that lost photoreceptors, in the adult mammalian retina can rewire with new photoreceptors. I use focal laser photocoagulation to selectively ablate a small patch of photoreceptors while leaving the rest of the retinal neurons intact. I use electrophysiology recordings of retinal response to visual stimuli to show that the neighboring healthy photoreceptors shift into the ablation zone, thus returning visual sensitivity to the previously deafferented bipolar cells. Furthermore, I use immunohistochemistry to determine if the new synapses of the deafferented bipolar cells are selective for appropriate pre-synaptic partners and investigate the structural mechanisms bipolar cells use to find new partners. I find that deafferented bipolar cells are capable of rewiring correctly with new photoreceptors, but different bipolar cell types use different rewiring strategies. I also find that the new synapses may not be as selective as those in the healthy retina. These findings support the idea that bipolar cells might be able to synapse with reintroduced photoreceptors, thereby restoring vision in patients blinded by retinal degeneration. However, the diversity of responses to deafferentation between bipolar cell types shows that different parallel pathways have access to different plasticity mechanisms, suggestin

The retina is a thin layer of neural tissue that lines the back of the eye and transforms the visual scene into an electrical signal that is sent to the brain via the optic nerve. The synapse between photoreceptors, the light sensitive neurons, and bipolar cells is the first connection in the retinal circuitry. Loss of photoreceptors during retinal degeneration results in permanent visual loss. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of bipolar cells to establish new functional synapses with photoreceptors is poorly understood. In my work, I investigate if deafferented bipolar cells, i.e. the ones that lost photoreceptors, in the adult mammalian retina can rewire with new photoreceptors. I use focal laser photocoagulation to selectively ablate a small patch of photoreceptors while leaving the rest of the retinal neurons intact. I use electrophysiology recordings of retinal response to visual stimuli to show that the neighboring healthy photoreceptors shift into the ablation zone, thus returning visual sensitivity to the previously deafferented bipolar cells. Furthermore, I use immunohistochemistry to determine if the new synapses of the deafferented bipolar cells are selective for appropriate pre-synaptic partners and investigate the structural mechanisms bipolar cells use to find new partners. I find that deafferented bipolar cells are capable of rewiring correctly with new photoreceptors, but different bipolar cell types use different rewiring strategies. I also find that the new synapses may not be as selective as those in the healthy retina. These findings support the idea that bipolar cells might be able to synapse with reintroduced photoreceptors, thereby restoring vision in patients blinded by retinal degeneration. However, the diversity of responses to deafferentation between bipolar cell types shows that different parallel pathways have access to different plasticity mechanisms, suggestin

The blind mole rat, Spalax ehrenbergi, can, despite severely degenerated eyes covered by fur, entrain to the daily light/dark cycle and adapt to seasonal changes due to an intact circadian timing system. The present study demonstrates that the Spalax retina contains a photoreceptor layer, an outer nuclear layer (ONL), an outer plexiform layer (OPL), an inner nuclear layer (INL), an inner plexiform layer (IPL), and a ganglion cell layer (GCL). By immunohistochemistry, the number of melanopsin (mRGCs) and non-melanopsin bearing retinal ganglion cells was analyzed in detail. Using the ganglion cell marker RNA-binding protein with multiple splicing (RBPMS) it was shown that the Spalax eye contains 890 ± 62 RGCs. Of these, 87% (752 ± 40) contain melanopsin (cell density 788 melanopsin RGCs/mm2). The remaining RGCs were shown to co-store Brn3a and calretinin. The melanopsin cells were located mainly in the GCL with projections forming two dendritic plexuses located in the inner part of the IPL and in the OPL. Few melanopsin dendrites were also found in the ONL. The Spalax retina is rich in rhodopsin and long/middle wave (L/M) cone opsin bearing photoreceptor cells. By using Ctbp2 as a marker for ribbon synapses, both rods and L/M cone ribbons containing pedicles in the OPL were found in close apposition with melanopsin dendrites in the outer plexus suggesting direct synaptic contact. A subset of cone bipolar cells and all photoreceptor cells contain recoverin while a subset of bipolar and amacrine cells contain calretinin. The calretinin expressing amacrine cells seemed to form synaptic contacts with rhodopsin containing photoreceptor cells in the OPL and contacts with melanopsin cell bodies and dendrites in the IPL. The study demonstrates the complex retinal circuitry used by the Spalax to detect light, and provides evidence for both melanopsin and non-melanopsin projecting pathways to the brain.

The blind mole rat, Spalax ehrenbergi, can, despite severely degenerated eyes covered by fur, entrain to the daily light/dark cycle and adapt to seasonal changes due to an intact circadian timing system. The present study demonstrates that the Spalax retina contains a photoreceptor layer, an outer nuclear layer (ONL), an outer plexiform layer (OPL), an inner nuclear layer (INL), an inner plexiform layer (IPL), and a ganglion cell layer (GCL). By immunohistochemistry, the number of melanopsin (mRGCs) and non-melanopsin bearing retinal ganglion cells was analyzed in detail. Using the ganglion cell marker RNA-binding protein with multiple splicing (RBPMS) it was shown that the Spalax eye contains 890 ± 62 RGCs. Of these, 87% (752 ± 40) contain melanopsin (cell density 788 melanopsin RGCs/mm2). The remaining RGCs were shown to co-store Brn3a and calretinin. The melanopsin cells were located mainly in the GCL with projections forming two dendritic plexuses located in the inner part of the IPL and in the OPL. Few melanopsin dendrites were also found in the ONL. The Spalax retina is rich in rhodopsin and long/middle wave (L/M) cone opsin bearing photoreceptor cells. By using Ctbp2 as a marker for ribbon synapses, both rods and L/M cone ribbons containing pedicles in the OPL were found in close apposition with melanopsin dendrites in the outer plexus suggesting direct synaptic contact. A subset of cone bipolar cells and all photoreceptor cells contain recoverin while a subset of bipolar and amacrine cells contain calretinin. The calretinin expressing amacrine cells seemed to form synaptic contacts with rhodopsin containing photoreceptor cells in the OPL and contacts with melanopsin cell bodies and dendrites in the IPL. The study demonstrates the complex retinal circuitry used by the Spalax to detect light, and provides evidence for both melanopsin and non-melanopsin projecting pathways to the brain.

Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels.

Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels.

Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels.

This report results from a contract tasking The Netherlands Ophthalmic Research Institute as follows: The final study demonstrated how cones compress natural stimuli into a dynamic range the rest of the visual system can cope with. Furthermore, the study demonstrated how horizontal cells, that store global stimulus parameters such as spectral composition and e-vector orientation of the global stimulus, adjust the gains of the cone synapse such that it suits the global stimulus conditions. It was also found that bipolar cells process these responses and how interaction between inputs to bipolar cells enhances their sensitivity to changes of intensity, color and presumably e-vector orientation. We identified an additional level of gain control in bipolar cells. Finally additional opponent processing of horizontal cells could not be revealed at the ganglion cell level. This indicates that just as in the spectral domain, opponency in horizontal cells is an efficient way of information coding and does not reflect a critical analysis step in 3-vector processing. Horizontal cell system is the memory of the global spectral composition and e-vector orientation of the animal., The original document contains color images.

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degener- ation in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm2. In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degener- ation in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm2. In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm2. In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities

—To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription– polymerase chain reaction, we measured detectable levels of the isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.

Several classes of second-order retinal neurons have been studied electrophysiologically in European eel (Anguilla anguilla) from two different localities, Lake Seliger in Russia and the coastal waters of the Adriatic Sea in Montenegro. The majority of L-horizontal cells (68 explored) had both rod and cone inputs, an uncommon phenomenon among teleosts. Pronounced color-opponent properties, often taken as pointing to the capacity of color vision, were identified in one amacrine cell, apparently of the "blue/yellow" (or "blue/ green") type. Microspectrophotometric measurements revealed two different spectral classes of cones with absorption maxima at about 525 and 434 nm. The existence of green-sensitive and blue-sensitive cone units was thus revealed by both electrophysiological and microspectrophotometric techniques.

—Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.

Several classes of second-order neurons have been electrophysiologically explored in immature European eels (Anguilla anguilla) from two distant and ecologically different localities tin Russia and Yugoslavia). The majority of L-horizontal cells (58 explored) had both rod and cone inputs, an uncommon phenomenon among teleosts. Spectral sensitivity characteristics of a number of horizontal and bipolar cells indicated that yellow-sensitive and green-sensitive cones coexist in the retina of the European eel, and that rods and green-sensitive cones contain similar visual pigments. Pronounced color-opponent properties, often taken as the capacity of color vision, were identified in one amacrine cell, apparently of the B/Y (or B/G) type. Differences in retinal structure and responsiveness between eels from the two localities, presumably due to differences in local conditions for growth, were less important than between eels of the yellow and silver stage.

Calbindin-D28K and calretinin are homologous cytosolic calcium binding proteins localized in many retinal neurons from different species. In this report, location of cells immunoreactive to both proteins was investigated in the retina of the lamprey, Lampetra fluviatilis. This organism constitutes one of the older representative vertebrates and possesses a peculiar organization, probably unique: two-thirds of the ganglion cells are in the classical amacrine cell layer and the nerve fiber layer is located in the scleral part of the inner plexiform layer. Calbindin-like immunoreactivity was demonstrated in large bipolar cells and in cell bodies located in the inner retina. Although the distinction between labelled ganglion cells and labelled amacrine cells was rendered difficult, we hypothesized that the majority of calbindin-immunoreactive cells observed in the inner retina are ganglion cells, because of the high number of labelled fibers in the nerve fiber layer. Calretinin-like immunoreactivity was detected in both large and small bipolar cells, and also in cells located in the inner retina. Since few calretinin-immunoreactive fibers were observed in the nerve fiber layer, we assume that the latter category of cells are amacrine cells. Horizontal cells were both negative for calbindin and calretin-like immunoreactivities. Calbindin and calretinin, which are present in cones from many species, could not be detected in the photoreceptor layer favouring the rod-dominated lamprey retina. Although their distribution differs from those observed in most vertebrates, the present results indicate the good conservation of both calcium binding proteins in the retina during the vertebrate evolution., Journal Article, Research Support, Non-U.S. Gov't, FLWNA, SCOPUS: ar.j, info:eu-repo/semantics/published

Calbindin-D28K and calretinin are homologous cytosolic calcium binding proteins localized in many retinal neurons from different species. In this report, location of cells immunoreactive to both proteins was investigated in the retina of the lamprey, Lampetra fluviatilis. This organism constitutes one of the older representative vertebrates and possesses a peculiar organization, probably unique: two-thirds of the ganglion cells are in the classical amacrine cell layer and the nerve fiber layer is located in the scleral part of the inner plexiform layer. Calbindin-like immunoreactivity was demonstrated in large bipolar cells and in cell bodies located in the inner retina. Although the distinction between labelled ganglion cells and labelled amacrine cells was rendered difficult, we hypothesized that the majority of calbindin-immunoreactive cells observed in the inner retina are ganglion cells, because of the high number of labelled fibers in the nerve fiber layer. Calretinin-like immunoreactivity was detected in both large and small bipolar cells, and also in cells located in the inner retina. Since few calretinin-immunoreactive fibers were observed in the nerve fiber layer, we assume that the latter category of cells are amacrine cells. Horizontal cells were both negative for calbindin and calretin-like immunoreactivities. Calbindin and calretinin, which are present in cones from many species, could not be detected in the photoreceptor layer favouring the rod-dominated lamprey retina. Although their distribution differs from those observed in most vertebrates, the present results indicate the good conservation of both calcium binding proteins in the retina during the vertebrate evolution., Journal Article, Research Support, Non-U.S. Gov't, FLWNA, SCOPUS: ar.j, info:eu-repo/semantics/published