Search

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

Glutamate is an essential neurotransmitter for signal processing in the vertical pathway of the mammalian retina, where it is involved in the distribution of visual information into several parallel channels. The excitatory effects of glutamate are mediated by AMPA-, kainate-, and NMDA-type ionotropic glutamate receptors (iGluRs). The expression patterns of these receptors in the vertebrate retina have been investigated so far with mainly immunocytochemical, in-situ hybridization, and electrophysiological/pharmacological techniques. Here, we have used scRNA sequencing data from chicken, mouse, macaque, and human retina to describe and compare the profile of iGluR expression in major retinal cell types across species. Our results suggest that major retinal cell types each express a unique set of iGluRs with substantial differences between non-mammalian and mammalian retinae. Expression of iGluRs has been investigated in more detail for amacrine and bipolar cell types of the human retina, each showing minor variations of a common pattern. The differential expression of iGluRs is likely to convey unique signal processing properties to individual elements of the retinal circuitry.

Bassoon (BSN) is a presynaptic cytomatrix protein ubiquitously present at chemical synapses of the central nervous system, where it regulates synaptic vesicle replenishment and organizes voltage‐gated Ca2+ channels. In sensory photoreceptor synapses, BSN additionally plays a decisive role in anchoring the synaptic ribbon, a presynaptic organelle and functional extension of the active zone, to the presynaptic membrane. In this study, we functionally and structurally analyzed two mutant mouse lines with a genetic disruption of Bsn—Bsngt and Bsnko—using electrophysiology and high‐resolution microscopy. In both Bsn mutant mouse lines, full‐length BSN was abolished, and photoreceptor synaptic function was similarly impaired, yet synapse structure was more severely affected in Bsngt/gt than in Bsnko/ko photoreceptors. The synaptic defects in Bsngt/gt retina coincide with remodeling of the outer retina—rod bipolar and horizontal cell sprouting, formation of ectopic ribbon synaptic sites—and death of cone photoreceptors, processes that did not occur in Bsnko/ko retina. An analysis of Bsngt/ko hybrid mice revealed that the divergent retinal phenotypes of Bsngt/gt and Bsnko/ko mice can be attributed to the expression of the Bsngt allele, which triggers cone photoreceptor death and neurite sprouting in the outer retina. These findings shed new light on the existing Bsn mutant mouse models and might help to understand mechanisms that drive photoreceptor death.

Aim Off cone bipolar cells of the mammalian retina connect to cone photoreceptor synaptic terminals via non-invaginating flat contacts at a considerable distance from the only established neurotransmitter release site so far, the synaptic ribbon. Diffusion from the ribbon synaptic active zone is considered the most likely mechanism for the neurotransmitter glutamate to reach postsynaptic receptors on the dendritic tips of Off cone bipolar cells. We used a mutant mouse with functionally impaired photoreceptor ribbon synapses to investigate the importance of intact ribbon synaptic active zones for signal transmission at Off cone bipolar cell contacts. Methods Whole-cell patch-clamp recordings from Off cone bipolar cells in a horizontal slice preparation of wildtype (Bsnwt ) and mutant (BsnΔEx4/5 ) mouse retina were applied to investigate signal transmission between cone photoreceptors and Off cone bipolar cells. The distribution of postsynaptic glutamate receptors in Off cone bipolar cell dendrites was studied using multiplex immuncytochemistry. Results Tonic synaptic activity and evoked release were significantly reduced in mutant animals. Vesicle replenishment rates and the size of the readily releasable pool were likewise decreased. The precisely timed transient current response to light offset changed to a sustained response in the mutant, exemplified by random release events only loosely time-locked to the stimulus. The kainate receptor distribution in postsynaptic Off cone bipolar cell dendritic contacts in BsnΔEx4/5 mice was largely disturbed. Conclusion Our results suggest a major role of functional ribbon synaptic active zones for signal transmission and postsynaptic glutamate receptor organization at flat Off cone bipolar cell contacts.

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 μW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

Angiogenesis due to hypoxic-ischemic (HI) injury represents a crucial compensatory mechanism of the developing brain that is mainly regulated by hypoxia-inducible transcription factors (HIF). Pharmacological stimulation of HIF is suggested as a neuroprotective option, however, studies of its effects on vascular development are limited. We analyzed the influence of the prolyl-4-hydroxylase inhibitor (PHI), FG-4497, and erythropoietin (rhEPO) on post-hypoxic angiogenesis (angiogenic growth factors, vessel structures) in the developing mouse brain (P7) assessed after a regeneration period of 72 h. Exposure to systemic hypoxia (8% O2, 6 h) was followed by treatment (i.p.) with rhEPO (2500/5000 IU/kg) at 0, 24 and 48 h or FG-4497 (60/100 mg/kg) compared to controls. In response to FG-4497 treatment cortical and hippocampal vessel area and branching were significantly increased compared to controls. This was associated with elevated ANGPT-2 as well as decreased ANGPT-1 and TIE-2 mRNA levels. In response to rhEPO, mildly increased angiogenesis was associated with elevated ANGPT-2 but also TIE-2 mRNA levels in comparison to controls. In conclusion, present data demonstrate a differential regulation of the angiopoietin/TIE-2 system in response to PHI and rhEPO in the post-hypoxic developing brain pointing to potential functional consequences for vascular regeneration and vessel development.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca(2+) channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1β are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM–Munc13–RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis. SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca(2+) channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

As all visual information is represented in the spatio-temporal dynamics of transmitter release from photoreceptors and the combined postsynaptic responses of second-order neurons, appropriate synaptic transfer functions are fundamental for a meaningful perception of the visual world. The functional contribution of horizontal cells to gain control and organization of bipolar and ganglion cell receptive fields can only be evaluated with an in-depth understanding of signal processing in horizontal cells. Therefore, a horizontal slice preparation of the mouse retina was established to record from horizontal cell bodies with their dendritic fields intact and receiving functional synaptic input from cone photoreceptors. Horizontal cell bodies showed spontaneous excitatory currents (spEPSCs) of monophasic and more complex multi-peak waveforms. spEPSCs were induced by quantal release of glutamate from presynaptic cones with a unitary amplitude of 3 pA. Non-stationary noise analysis revealed that spEPSCs with a monoexponential decay were mediated by 7–8 glutamate receptors with a single-channel amplitude of 1.55 pA. Responses to photopic full-field illumination were characterized by reduction of a tonic inward current or hyperpolarization, inhibition of spEPSCs, followed by a fast and transient inward current at light offset. The response to periodic dark/light transitions of different frequencies was dependent on the adaptational status of the cell with a limiting frequency of 10 Hz. Both on and off components of the light response were mediated by AMPA and kainate receptors. Detailed analysis of horizontal cell synaptic physiology is a prerequisite for understanding signal coding and processing at the photoreceptor ribbon synapse.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue. To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows (1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites. Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

Schlusselkonzepte 1. Der Transport der Atemgase im Korper erfolgt durch einfache Diffusion und Konvektion. 2. Respiratorische Pigmente wie Hamoglobin erhohen die Transportkapazitat des Blutes oder der Hamolymphe fur Sauerstoff (O2). 3. Kohlenstoffdioxid wird im Blut physikalisch gelost als CO2, als Hydrogencarbonat und kovalent an Hamoglobin gebunden transportiert. 4. Kiemen sind Ausstulpungen der Korperoberflache, deren respiratorische Funktion auf einer unidirektionalen Ventilation und dem Gegenstromprinzip beruht. 5. Die evolutionare Entwicklung von Lungen als Einstulpungen der Korperoberflache ermoglicht die Verwendung von Luft als Atemmedium und damit die Besiedlung terrestrischer Lebensraume. 6. Wasser ist ein ungunstigeres Atemmedium als Luft, da es weniger O2 enthalt, mehr Energie zur Ventilation benotigt und langere Diffusionszeiten erfordert. 7. Das Tracheensystem von Insekten bringt die Austauschflache zu den Korperzellen; Atemgastransport durch ein Kreislaufsystem ist nicht erforderlich. 8. Die Atmung bei Wirbeltieren wird durch einen Rhythmusgenerator im Stammhirn reguliert. 9. Abweichungen des CO2-Partialdrucks und des pH-Werts im Blut, aber auch Anderungen des O2-Partialdrucks bewirken eine erhohte Aufnahme von O2.

Schlusselkonzepte 1. Die durch die Kontraktion erzeugten Druckdifferenzen zwischen Eingang und Ausgang des Herzens sind Voraussetzung fur einen gerichteten Blutstrom durch das Kreislaufsystem. 2. Schrittmacherzellen konnen aufgrund ihrer spezifischen Ausstattung mit Ionenkanalen autonom und spontan Aktionspotenziale erzeugen. 3. Die Schrittmacheraktivitat gibt eine konstante Herzfrequenz vor. Die Anpassung der Herzleistung an veranderte Bedingungen erfolgt durch erregende und hemmende neuronale Einflusse. 4. Das Aktionspotenzial einer Herzmuskelzelle dauert genauso lange wie ihre mechanische Kontraktion. Das Myokard ist daher im Gegensatz zur Skelettmuskulatur nicht tetanisierbar. 5. Gap Junctions sind die strukturelle Grundlage fur niederohmige interzellulare Verbindungen und ermoglichen eine verzogerungsfreie elektrische Kommunikation zwischen Zellen des Myokards. 6. Die Kontraktionskraft einer Herzmuskelfaser wird masgeblich durch die Vordehnung ihrer Sarkomere bestimmt. 7. Ca\({}^{2+}\)-Ionen verknupfen eine elektrische Spannungsanderung uber der Plasmamembran mit der Verschiebung von Actin- und Myosinfilamenten im Inneren einer Muskelzelle als Voraussetzung fur ihre mechanische Kontraktion.

Schlusselkonzepte 1. Ein Neuron ist die grundlegende anatomische, physiologische, genetische und metabolische Einheit des Nervensystems. 2. Gliazellen regulieren als Astrozyten die Homoostase im Nervensystem, bilden als Oligodendrozyten oder Schwann-Zellen Myelin und spielen als Mikroglia eine wichtige Rolle im angeborenen und erworbenen Immunsystem. 3. Ionenkanale sind Transmembranproteine, die den Fluss von Na\({}^{+}\), K\({}^{+}\), Ca\({}^{2+}\) und Cl\({}^{-}\) uber die hydrophobe Zellmembran ermoglichen. 4. Der Selektivitatsfilter der Ionenkanale basiert energetisch und strukturell auf dem Ersatz der Hydrathulle durch ein polares, hochkonserviertes Peptidsegment. 5. Ionenkanale werden in einem als Gating bezeichneten Prozess durch chemische, mechanische und elektrische Signale reguliert.

Schlusselkonzepte 1. Die Evolution der Lichtwahrnehmung verlauft nach visuellen Aufgaben zunehmender Komplexitat. Eine steigende Informationsmenge erfordert immer leistungsfahigere Nervensysteme. 2. Alle Formen der Lichtwahrnehmung sind fur das von ihnen unterstutzte Verhalten optimiert. 3. Die Entwicklung von Linsensystemen mit fokussierender Optik ermoglicht hochauflosendes raumliches Sehen. 4. Photorezeptoren absorbieren Photonen und wandeln die elektromagnetische Energie der Lichtquanten in ein zellulares elektrisches Signal um. 5. Ziliare Photorezeptoren hyperpolarisieren auf einen Lichtreiz, wahrend rhabdomerische Photorezeptoren depolarisieren. Die Amplitude dieses Rezeptorpotenzials codiert die Starke des Lichtreizes. 6. Ein effizienter intrazellularer Verstarkungsmechanismus ermoglicht die Detektion einzelner Photonen. 7. Die raumliche Auflosung von Komplexaugen entspricht der Anzahl ihrer Ommatidien, diejenige der Kameraaugen dem Durchmesser der Ausensegmente ihrer Photorezeptoren. 8. Die Signalverarbeitung in der Retina beruht auf einer Dekonstruktion des visuellen Stimulus in seine grundlegenden Eigenschaften. 9. In hoheren visuellen Zentren des Gehirns integrieren unterschiedliche signalverarbeitende Systeme Prozesse mit hoher zeitlicher und raumlicher Auflosung zu einem koharenten Abbild der auseren Welt.

Schlusselkonzepte 1. Die Nahrung versorgt heterotrophe Organismen mit Energie, Baustoffen, Vitaminen und Elektrolyten. 2. Tiere erhalten ihre Nahrung durch Aufnahme weniger groser oder vieler kleiner Individuen oder mithilfe von symbiotischen Mikroorganismen. 3. Verdauung und Resorption bestimmen den ernahrungsphysiologischen Wert der Nahrung. 4. Das Verdauungssystem der Wirbeltiere besteht aus einem tubularen System mit unterschiedlichen Funktionen, in das akzessorische Drusen munden. 5. Die Steuerung von Korpergewicht, Nahrungsaufnahme und Verdauungsprozessen erfolgt durch neuronale, endokrine und parakrine Mechanismen.

Schlusselkonzepte 1. Horen und Gleichgewichtssinn basieren auf einer mechanoelektrischen Transduktion, bei der das Anspannen von Proteinfaden Ionenkanale offnet. 2. Die evolutionaren Vorteile einer mechanoelektrischen Transduktion liegen in ihrer auserordentlichen Schnelligkeit und hohen Empfindlichkeit. 3. Lautstarke und Tonhohe auditorischer Signale werden durch die Amplitude des Rezeptorpotenzials bzw. durch eine Ortstransformation auf die Basilarmembran der Cochlea codiert. 4. Die Frequenzzusammensetzung eines Schallereignisses wird uber die gesamte Horbahn tonotop reprasentiert. 5. Haarzellen besitzen zwei Regionen mit unterschiedlichen Gleichgewichtspotenzialen fur K\({}^{+}\). Daher fuhrt das Offnen von K\({}^{+}\)-Kanalen in den Stereovilli zu einem Ausstrom von K\({}^{+}\), das Offnen basolateraler K\({}^{+}\)-Kanale zu einem Einstrom. 6. Die horizontale Lokalisation einer Schallquelle beruht auf der Auswertung von Laufzeit- und Intensitatsunterschieden zwischen beiden Ohren. Die hohe Prazision setzt eine binaurale Verschaltung auf einer fruhen Ebene der Horbahn voraus. 7. Das Vestibularorgan registriert Drehbeschleunigungen und lineare Beschleunigungen als Grundlage fur den Gleichgewichtssinn und die raumliche Orientierung des Korpers relativ zur Schwerkraft.

Schlusselkonzepte 1. Die Stoffwechselrate sowie die Konformation und Funktionalitat von Proteinen und Zellmembranen hangen von der Temperatur ab. 2. Temperaturunterschiede zwischen der Umgebung und dem Korper eines Tieres fuhren zu einer Aufnahme oder Abgabe von Warme. 3. Endotherme Organismen sind in der Lage, ihre Korpertemperatur durch metabolische Warmebildung konstant zu halten, wahrend ektotherme Tiere ihre Korpertemperatur an die Umgebungstemperatur anpassen. 4. Die Stoffwechselrate ektothermer Organismen steigt exponentiell mit der Temperatur bis zu einem Optimum, danach fallt sie steil ab. 5. Bei endothermen Tieren ist die Stoffwechselrate in der Thermoneutralzone konstant, wahrend sie bei niedrigeren und hoheren Temperaturen ansteigt. 6. Zahlreiche mittel- und langfristige Strategien sowie evolutionare Prozesse dienen der optimalen Anpassung an vorherrschende Temperaturen.

Aim A key feature of the mammalian retina is the segregation of visual information in parallel pathways, starting at the photoreceptor terminals. Cone photoreceptors establish synaptic contacts with On bipolar and horizontal cells at invaginating, ribbon-containing synaptic sites, whereas Off bipolar cells form flat, non-ribbon-containing contacts. The cytomatrix protein Bassoon anchors ribbons at the active zone, and its absence induces detachment of ribbons from the active zone. In this study we investigate the impact of a missing Bassoon on synaptic transmission at the first synapse of the visual system. Methods Release properties of cone photoreceptors were studied in wild-type and mutant mouse retinae with a genetic disruption of the presynaptic cytomatrix protein Bassoon using whole-cell voltage-clamp recordings. Light and electron microscopy revealed the distribution of Ca2+ channels and synaptic vesicles, respectively, in both mouse lines. Results Whole-cell recordings from postsynaptic horizontal cells of the two mouse lines showed that the presence of Bassoon (and a ribbon) enhanced the rate of exocytosis during tonic and evoked release by increasing synaptic vesicle pool size and replenishment rate, while at the same time slowing synaptic vesicle release. Furthermore, the number of Cav 1.4 channels and synaptic vesicles was significantly higher at wild-type than at Bassoon mutant synaptic sites. Conclusion The results of our study demonstrate that glutamate release from cone photoreceptor terminals can occur independent of a synaptic ribbon, but seems restricted to active zones, and they show the importance of a the synaptic ribbon in sustained and spatially and temporally synchronized neurotransmitter release.

The amino acid glycine acts as a neurotransmitter at both inhibitory glycinergic and excitatory glutamatergic synapses predominantly in caudal regions of the central nervous system but also in frontal brain regions and the retina. After its presynaptic release and binding to postsynaptic receptors at caudal glycinergic synapses, two high-affinity glycine transporters GlyT1 and GlyT2 remove glycine from the extracellular space. Glycinergic neurons express GlyT2, which is essential for the presynaptic replenishment of the transmitter, while glial-expressed GlyT1 was shown to control the extracellular glycine concentration. Here we show that GlyT1 expressed by glycinergic amacrine cells of the retina does not only contribute to the control of the extracellular glycine concentration in the retina but is also essential for the maintenance of the glycinergic transmitter phenotype of this cell population. Specifically, loss of GlyT1 from the glycinergic AII amacrine cells impairs AII-mediated glycinergic neurotransmission and alters regulation of the extracellular glycine concentration, without changes in the overall distribution and/or size of glycinergic synapses. Taken together, our results suggest that GlyT1 expressed by amacrine cells in the retina combines functions covered by neuronal GlyT2 and glial GlyT1 at caudal glycinergic synapses.

Complexins (Cplxs) are SNARE complex regulators controlling the speed and Ca(2+) sensitivity of SNARE-mediated synaptic vesicle fusion. We have shown previously that photoreceptor ribbon synapses in mouse retina are equipped with Cplx3 and Cplx4 and that lack of both Cplxs perturbs photoreceptor ribbon synaptic function; however, Cplx3/4 function in photoreceptor synaptic transmission remained elusive. To investigate Cplx3/4 function in photoreceptor ribbon synapses, voltage-clamp recordings from postsynaptic horizontal cells were performed in horizontal slice preparations of Cplx3/4 wild-type (WT) and Cplx3/4 double knock-out (DKO) mice. We measured tonic activity in light and dark, current responses to changes in luminous intensity, and electrically evoked postsynaptic responses. Cplx3/4 decreased the frequency of tonic events and shifted their amplitude distribution to smaller values. Light responses were sustained in the presence of Cplx3/4, but transient in their absence. Finally, Cplx3/4 increased synaptic vesicle release evoked by electrical stimulation. Using electron microscopy, we quantified the number of synaptic vesicles at presynaptic ribbons after light or dark adaptation. In Cplx3/4 WT photoreceptors, the number of synaptic vesicles associated with the ribbon base close to the release site was significantly lower in light than in dark. This is in contrast to Cplx3/4 DKO photoreceptors, in which the number of ribbon-associated synaptic vesicles remained unchanged regardless of the adaptational state. Our results indicate a suppressing and a facilitating action of Cplx3/4 on Ca(2+)-dependent tonic and evoked neurotransmitter release, respectively, and a regulatory role in the adaptation-dependent availability of synaptic vesicles for release at photoreceptor ribbon synapses. SIGNIFICANCE STATEMENT Synaptic vesicle fusion at active zones of chemical synapses is executed by SNARE complexes. Complexins (Cplxs) are SNARE complex regulators and photoreceptor ribbon synapses are equipped with Cplx3 and Cplx4. The absence of both Cplxs perturbs ribbon synaptic function. Because we lack information on Cplx function in photoreceptor synaptic transmission, we investigated Cplx function using voltage-clamp recordings from postsynaptic horizontal cells of Cplx3/4 wild-type and Cplx3/4 double knock-out mice and quantified synaptic vesicle number at the ribbon after light and dark adaptation using electron microscopy. The findings reveal a suppressing action of Cplx3/4 on tonic neurotransmitter release, a facilitating action on evoked release, and a regulatory role of Cplx3/4 in the adaptation-dependent availability of synaptic vesicles at mouse photoreceptor ribbon synapses.

Postsynaptic to photoreceptors, horizontal cells face prolonged exposure to glutamate in the dark. Therefore, efficient hyperpolarizing mechanisms are crucial to keep horizontal cells within an operating range and to reduce glutamate-induced excitotoxicity. Combining electrophysiology, single-cell reverse transcriptase polymerase chain reaction, and immunocytochemistry, we found that horizontal cell bodies but not their axon terminals express the ether-à-gogo-related gene isoform 1 (erg1) K(+) channel. Erg1-mediated outward currents displayed voltage-dependent activation and C-type inactivation. Recovery from inactivation involved a transient open state. Gating of erg1 channels kept the voltage response to glutamate brief and at physiological amplitudes. With erg1 channels blocked, the response of horizontal cells to the onset of darkness was significantly enhanced. These results indicate a functional dichotomy between horizontal cell bodies and axon terminals in the processing of photoreceptor signals. The dark response thus reflects a finely tuned balance determined by the successive gating of ionotropic glutamate receptors and erg1 channels.

The identification of the proteins that make up the gap junction channels between rods and cones is of crucial importance to understand the functional role of photoreceptor coupling within the retinal network. In vertebrates, connexin proteins constitute the structural components of gap junction channels. Connexin36 is known to be expressed in cones whereas extensive investigations have failed to identify the corresponding connexin expressed in rods. Using immunoelectron microscopy, we demonstrate that connexin36 (Cx36) is present in gap junctions of cone but not rod photoreceptors in the mouse retina. To identify the rod connexin, we used nested reverse transcriptase polymerase chain reaction and tested retina and photoreceptor samples for messenger RNA (mRNA) expression of all known connexin genes. In addition to connexin36, we detected transcripts for connexin32, connexin43, connexin45, connexin50, and connexin57 in photoreceptor samples. Immunohistochemistry showed that connexin43, connexin45, connexin50, and connexin57 proteins are expressed in the outer plexiform layer. However, none of these connexins was detected at gap junctions between rods and cones as a counterpart of connexin36. Therefore, the sought-after rod protein must be either an unknown connexin sequence, a connexin36 splice product not detected by our antibodies, or a protein from a further gap junction protein family.

In the mammalian retina, rods and cones connect to distinct sets of bipolar cells. Rods are presynaptic to a single type of rod bipolar cell, whereas cones connect to different types of cone bipolar cells. Synaptic rewiring between cone photoreceptor terminals and rod bipolar cell dendrites has been described as a general result of photoreceptor degeneration. To investigate whether cone bipolar cells also show synaptic plasticity in the absence of cone input, we studied the connectivity of cone bipolar cell dendrites inCNGA3−/−mice, a model with specific loss of cone photoreceptor function. Dendritic connections of ON and OFF cone bipolar cells were visualized using specific cell markers or by intracellular injection with fluorescent dyes. The results show that cone bipolar cells inCNGA3−/−mice form ectopic synapses with rods. In contrast, cone bipolar cells do not form ectopic synapses with rods inCNGA3−/−Rho−/−mice, in which both types of photoreceptors are nonfunctional. In analogy with these results, we found that input-deprived rod bipolar cells form ectopic synapses with functional cones inRho−/−mice but not with inoperable cones in theCNGA3−/−Rho−/−mouse. Our data indicate that the formation of ectopic bipolar cell synapses in the outer plexiform layer requires a functional presynaptic photoreceptor.

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.

Dopaminergic local circuit neurons in the retina (DA cells) show robust, spontaneous, tetrodotoxin-sensitive pacemaking. To investigate the mechanism underlying this behavior, we characterized the sodium current and a subset of the potassium currents in the cells in voltage-clamp experiments. We found that there is a persistent component of the sodium current in DA cells which activates at more depolarized potentials than the transient component of the current. The transient component was completely inactivated at −50mV, but DA cells remained able to fire spontaneous action potentials when potassium channels were partially blocked and the membrane potential remained above −40mV. Based on these electrophysiological data, we developed a reduced computer model that reproduced the major features of DA cells. In simulations at the physiological resting potential, the persistent component of the sodium current was both necessary and sufficient to account for spontaneous activity, and the major contribution of the transient component of the sodium current was to initiate the depolarization of the model cell during the interspike interval. When tonic inhibition was simulated by lowering the input impedance of the model cell, the transient component played a larger role.

We have studied the expression pattern of neuronal connexin36 (Cx36) in the mouse and rat retina. In vertical sections of both retinas, a polyclonal antibody directed against Cx36 produced punctate labeling in the inner plexiform layer (IPL). Intense immunoreactivity was localized to the entire OFF sublamina of the IPL, and much weaker staining could be observed in the ON sublamina. Double-labeling experiments in the rat retina with antibodies directed against parvalbumin indicate that Cx36 is expressed on dendrites of AII amacrine cells. Cx36-like immunoreactivity in sublamina a of the IPL did not overlap with lobular appendages or cell bodies of AII amacrine cells. In a mouse retinal slice preparation, AII amacrine and ON cone bipolar cells were intracellularly injected with Neurobiotin and counterstained with antibody against Cx36. Punctate labeling appeared to be in register with dendritic arborization of AII amacrines and cone bipolar cells in the ON sublamina of the IPL. Whereas AII amacrine cells isolated from the rat retina clearly displayed Cx36-like immunoreactivity, isolated ON cone bipolar cells were negative for Cx36. Axon terminals of rod bipolar cells were decorated with Cx36-positive contacts but did not express Cx36 themselves.These results indicate that Cx36 is expressed by AII amacrine cells in homologous and heterologous gap junctions made with AII amacrines and cone bipolar cells, respectively. The heterologous gap junctions appear to be heterotypic, because ON cone bipolar cells do not express Cx36.

When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABAAreceptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABAAreceptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABAAtype because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC50value of 7.4 μM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn2+with an IC50of 58.9 ± 8.9 μM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC50of 340 ± 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 μM) and by 107% (10 μM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABAAreceptor subunits (α1, α3, α4, β1, β3, γ1, γ2S, and γ2L) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least α1, β3 and γ2 subunits are assembled into functional receptors.

GABAA and GABAC receptors were studied on cultured or freshly isolated rat retinal bipolar cells. The cells displayed GABA-induced whole-cell currents, which were only partially blocked by high concentrations (100 microM) of the GABAA receptor antagonist bicuculline. The bicuculline-resistant (GABAC) component was insensitive to the GABAA receptor modulators flunitrazepam (1 microM) and pentobarbital (50 microM). The bicuculline-sensitive portion of the current was strongly augmented by both drugs, indicating that it was mediated by conventional GABAA receptors. The GABAC and GABAA receptor subtypes displayed a 7-fold difference in their binding affinity for GABA, the EC50 values being 4.2 microM and 27.1 microM, respectively. The Hill coefficient was approximately 2 for both receptors. The bicuculline-insensitive GABAC receptors were markedly blocked by 100 microM picrotoxinin, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR-95531) and gamma-hexachlorocyclohexane, drugs known to be antagonists of GABAA receptors. Examination of single-channel currents indicated main-state conductances of 7.9 pS and 29.6 pS for GABAC and GABAA receptors, respectively. The pore diameter of open GABAC receptor channels was 5.1 A, i.e. close to the value of 5.6 A reported for the GABAA receptor. These results demonstrate that rod bipolar cells possess two populations of pharmacologically distinct GABA receptors, GABAA and novel-type GABAC receptors, which might subserve different physiological functions in controlling visual transduction in the retina.

The gamma-aminobutyric acid type A (GABAA) receptor is the predominant Cl(-)-channel protein mediating inhibition in the retina and elsewhere in the mammalian brain. We have observed a time-dependent increase of GABA-induced whole-cell currents when dopamine was applied externally to rat retinal amacrine cells. After 20 min, the peak current was increased to 208% +/- 10% of its initial value. A comparable effect was observed with the dopamine D1 receptor agonist (+)-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) but not with the D2 agonist bromocryptine. The action of dopamine involved phosphorylation of GABAA receptors by protein kinase A, as evident from intracellular application of protein kinase A, cAMP, and forskolin. Both guanosine 5'-[gamma-thio]triphosphate and cholera toxin augmented the GABA response, indicating a role for the guanosine 5'-triphosphate-binding protein Gs in the transduction cascade. Phosphorylation of GABAA receptors shifted the half-maximally effective GABA concentration from 71 microM to 47 microM without affecting the maximal response amplitude. The elevated binding affinity for GABA was caused by an increase of the open probability of the channels from 0.09 to 0.33 (2 microM GABA); conductance and mean open time did not change. Several other receptor agonists such as adenosine, histamine, somatostatin, enkephalin, and vasoactive intestinal peptide were found to couple to the same intracellular phosphorylation pathway. Since some of these cotransmitters colocalize with GABA in amacrine cells, they may fine-tune GABAergic inhibition in the retina.

In the mammalian retina, two types of catecholaminergic amacrine cells have been described. Although dopaminergic type 1 cells are well characterized, the physiology of type 2 cells is, so far, unknown. To target type 2 cells specifically, we used a transgenic mouse line that expresses green fluorescent protein under the control of the tyrosine hydroxylase promoter. Type 2 cells are GABAergic and have an extensive dendritic arbor, which stratifies in the middle of the inner plexiform layer. Our data suggest that type 2 cells comprise two subpopulations with identical physiological properties: one has its somata located in the inner nuclear layer and the other in the ganglion cell layer. Immunostaining with bipolar cell markers suggested that type 2 cells receive excitatory inputs from type 3 OFF and type 5 ON bipolar cells. Consistently, patch-clamp recordings showed that type 2 cells are ON–OFF amacrine cells. Blocking excitatory inputs revealed that different rod and cone pathways are active under scotopic and mesopic light conditions. Blockade of inhibitory inputs led to membrane potential oscillations in type 2 cells, suggesting that GABAergic and glycinergic amacrine cells strongly influence type 2 cell signaling. Among the glycinergic amacrine cells, we identified the VGluT3-immunoreactive amacrine cell as a likely candidate. Collectively, light responses of type 2 cells were remarkably uniform over a wide range of light intensities. These properties point toward a general function of type 2 cells that is maintained under scotopic and mesopic conditions.

Vertical slices of 6-day postnatal (P6) rat retina were cut at a thickness of 100 μm and cultured using the roller-tube technique. After 14–21 daysin vitrothere was significant distortion of normal retinal architecture, but localized areas of the slices showed the typical pattern of layering of mature retina. The following immunocytochemical markers were used to characterize the different retinal cell types: antibodies against protein kinase C (PKC), calcium binding protein (CabP 28kD), neurofilaments (NF), glia-specific antibodies (GFAP, vimentin), and transmitter-specific antibodies (GABA, TH). The expression of these markers was compared in P6 retina, adult retina, and slice culture. To further characterize the cultured cells, patch-clamp recordings were performed in combination with intracellular injection of Lucifer Yellow (LY). Transmitter-and voltage-gated membrane currents were recorded from morphologically identified neurons. The experiments show that a mammalian slice culture can be used to study differentiation and function of retinal cell types.

γ-AMlNOBUTYRlC acid (GABA), a major inhibitory neurotransmitter in the mammalian nervous system, is known to operate bicuculline-sensitive CI− channels through GABAA receptors and bicuculline-insensitive cation channels through GABAB receptors1,2. Recent observations indicate that the retina may contain GABA receptors with unusual pharmacological properties3–5. Here we report that GABA gates bicuculline-insensitive CI− channels in rod bipolar cells of the rat retina, which were not modulated by flunitrazepam, pentobarbital and alphaxalone and were only slightly blocked by picrotoxinin. Moreover, the GABAB receptor agonist baclofen, and the antagonist 2-hydroxysaclofen had no effect. The underlying single-channel conductance was 7 pS and the open time 150 ms. These values are clearly different from those obtained for GABAA receptor channels recorded in other neurons of the same preparation, and in other parts of the brain1,6–8. The bicuculline- and baclofen-insensitive GABA receptors were activated selectively by the GABA analogue cis-4-aminocrotonic acid (CACA)9. Hence they may be similar to those receptors termed GABAC receptors10.

Protein kinase (PKC)-α is abundant in retinal bipolar cells. This study was performed to explore its role in visual processing.PKCα-knockout (Prkca(-/-)) mice and control animals were examined by using electroretinography (ERG), light microscopy, and immunocytochemistry.The Prkca(-/-) mice showed no signs of retinal degeneration up to 12 months of age, but ERG measurements indicated a decelerated increase in the ascending limb of the scotopic (rod-sensitive) b-wave as well as a delayed return to baseline. These results suggest that PKCα is an important modulator that affects bipolar cell signal transduction and termination. Confocal microscopy of retinal sections showed that PKCα co-localized with calbindin, which indicates a PKCα localization in close proximity to the horizontal cell terminals. In addition, the implicit time of the ERG c-wave originating from the retinal pigment epithelium (RPE) and the recovery of photoreceptors from bleaching conditions were substantially faster in the knockout mice than in the wild-type control animals.These results suggest that PKCα is a modulator of rod-bipolar cell function by accelerating glutamate-driven signal transduction and termination. This modulation is of importance in the switch between scotopic and photopic vision. Furthermore, PKCα seems to play a role in RPE function.

PURPOSE Successful regeneration and re-establishment of synaptic connections in the adult central nervous system is a complex process determined by both the exterior environment and the endogenous neural activity of the regenerating growth cones. The purpose of this study was to determine the expression and properties of voltage-gated sodium channels (Na(v)) expressed by regenerating growth cones. METHODS Na(v) channels were studied in an organotypic explant culture of the adult rat retina by immunocytochemistry and whole-cell, patch-clamp recordings. RESULTS Regenerating axons and growth cones, but not glial processes, expressed Na(v) channels. Whole-cell, current-clamp recordings from growth cones displayed a high input resistance of 1.29 GOmega and a resting membrane potential of -69.0 mV. All growth cones responded to depolarizing voltage steps with fast, transient, inward currents mediated by Na(+) ions, followed by slow, sustained outward K(+) currents. Half-maximum activation clustered in two groups, suggesting the presence of at least two Na(v) channel isoforms. Steady state inactivation and recovery from fast inactivation were characterized by a half-maximum value of -69.7 mV and by a time constant of 3.64 ms, respectively. Injection of depolarizing current steps larger than threshold (-29.3 mV) consistently induced a single action potential, whereas ganglion cell bodies responded to above-threshold stimulation with a series of fast, all-or-none action potentials. CONCLUSIONS These experiments describe for the first time the biophysical properties of Na(v) channels recorded from the growth cones of regenerating retinal ganglion cells and contrast their properties with those of adult retinal ganglion cells.

Injury to the mature primate and subprimate optic nerve results in irreversible impairment and loss of vision, because the retinal ganglion cells (RGCs) fail to regenerate their cut axons within the optic nerve interior. This study was performed to examine whether aging monkey RGCs retain the ability to regenerate their axons in organ culture and whether axonal regeneration is associated with specific proteomic profile. Retinal stripes obtained from marmoset eyes (C. jacchus) were cultured between the day of birth and adult stages on different substrates like laminin-1, laminin-2, collagen, matrigel and poly-D-lysine. No neurotrophic factors were added to the medium. Axonal growth was monitored with microscopy and immunohistochemistry. Onset and rate of growth was examined with time-lapse videography. Vigorous regeneration of axons occurred from identifiable morphological types of RGCs throughout all stages of life, although the numbers of axons decreased with age. Axonal growth occurred virtually only on laminin-1. Growth correlated with re-expression of the laminin-1 receptor alpha6-integrin and sustained staining for GAP-43 as shown by immunohistochemistry and immunoblotting. At proteomic level, there is a maturation-dependent change in the protein immunostaining within the retina. When retinal slices of the same age were compared, regeneration-specific protein staining included calmodulin, fatty acid binding protein, alpha-crystallin, IFN-gamma, cyclin-dependent kinase inhibitor (p21), beta-hemoglobin, 60s-ribosomal protein, GAP-DH and ADP-ribosylation factor (ARF). To our knowledge these data are the first from subhuman animals to suggest that axonal regeneration of injured RGCs is correlated to expression of identifiable proteins within the retina.

A combination of transgenic technology and single-cell reverse transcription polymerase chain reaction (RT-PCR)has been used to study gene expression in dopaminergic amacrine (DA)cells of the mouse retina. Because there are only 900 DA cells, and they cannot be distinguished from neighboring neurons on the basis of their morphology, we labeled them with human placental alkaline phosphatase (PLAP)by introducing into the mouse genome PLAP cDNA under the control of the promoter of the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis. Because PLAP is an enzyme that resides on the outer surface of the cell membrane, we can identify DA cells after dissociation of the retina by immunocytochemistry in the living state. Cells are then patch clamped and harvested for single-cell RT-PCR analysis of gene expression. Here, we describe the preparation of the fluorescent antibody E6-Cy3 to specifically detect PLAP-expressing cells, methods to obtain short-term cultures of solitary neurons from mouse retinas, and techniques to detect gene expression in individual neurons. Properties and pitfalls of single-cell RT-PCR are described and discussed.

Horizontal cells modulate the output of the photoreceptor to bipolar cell synapse, thereby providing the first level of lateral information processing in the vertebrate retina. Because horizontal cells do not generate sodium-based action potentials, calcium is likely to play an important role for graded potential changes as well as for intracellular events involved in the modulatory role of horizontal cells within the retinal network. Therefore we wanted to determine how the activation of glutamate receptors, voltage-gated calcium channels, and release of calcium from internal stores shape the calcium signal in horizontal cells. All horizontal cells responded to depolarizing voltage steps with sustained inward currents, which activated at around –20 mV, reached a peak amplitude of –79.1 pA at 5 mV, and reversed sign at around 66 mV. The current was insensitive to tetrodotoxin, and it was partially blocked by the L-type channel antagonists verapamil and nifedipine. The N-type channel blocker ω-conotoxin GVIA induced an additional reduction of current amplitudes. Calcium influx through ionotropic glutamate receptors was mediated by both AMPA and kainate but not by N-methyl-d-aspartate receptors. Two agonists at group I metabotropic glutamate receptor, trans-1-amino-1,3-cyclopentanedicarboxylic acid and quisqualate, had no effect. However, intracellular calcium was increased by caffeine, indicating release of calcium from internal stores via ryanodine receptors. These data show that intracellular calcium in horizontal cells is regulated by voltage-dependent L- and N-type calcium channels, ionotropic AMPA and kainate receptors, and release of calcium from internal stores after activation of ryanodine receptors.

GABA-induced currents have been characterized in isolated horizontal cells from lower vertebrates but not in mammalian horizontal cells. Therefore horizontal cells were isolated after enzymatical and mechanical dissociation of the adult mouse retina and visually identified. We recorded from horizontal cell bodies using the whole cell and outside-out configuration of the patch-clamp technique. Extracellular application of GABA induced inward currents carried by chloride ions. GABA-evoked currents were completely and reversibly blocked by the competitive GABAAreceptor antagonist bicuculline (IC50= 1.7 μM), indicating expression of GABAAbut not GABACreceptors. Their affinity for GABA was moderate (EC50= 30 μM), and the Hill coefficient was 1.3, corresponding to two GABA binding sites. GABA responses were partially reduced by picrotoxin with differential effects on peak and steady-state current values. Zinc blocked the GABA response with an IC50value of 7.3 μM in a noncompetitive manner. Furthermore, GABA receptors of horizontal cells were modulated by extracellular application of diazepam, zolpidem, methyl 6,7-dimethoxy-4-ethyl-β-carboxylate, pentobarbital, and alphaxalone, thus showing typical pharmacological properties of CNS GABAAreceptors. GABA-evoked single-channel currents were characterized by a main conductance state of 29.8 pS and two subconductance states (20.2 and 10.8 pS, respectively). Kinetic analysis of single-channel events within bursts revealed similar mean open and closed times for the main conductance and the 20.2-pS subconductance state, resulting in open probabilities of 44.6 and 42.7%, respectively. The ratio of open to closed times, however, was significantly different for the 10.8-pS subconductance state with an open probability of 57.2%.