Photoreceptor Synaptic Terminals Research Articles

Size variations in synaptic terminals among different types of photoreceptors and across the zebrafish retina

Photoreceptor synaptic terminals are responsible for transmitting visual information to downstream neurons. In vertebrate retinas, photoreceptor synaptic terminals are of different sizes and structures. The molecular mechanisms that underlie photoreceptor synaptic development are not clearly understood. Here, we have systematically examined the size variations in the synaptic terminals of cone and rod photoreceptors in the adult zebrafish retina. We reveal that the average cone pedicle sizes expand in the order of UV, blue, green, and red cones, echoing the increasing maximally sensitive wavelengths of the opsins expressed in the corresponding cone types. In addition, rod spherules are smaller than all cone pedicles. The terminals of each photoreceptor type also display distinct regional variations across the retina and between males and females. These findings establish the basis for using the zebrafish retina to study the molecular mechanisms that regulate the sizes and structures of photoreceptor terminals for proper visual functions.

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

The absence of functional bassoon at cone photoreceptor ribbon synapses affects signal transmission at Off cone bipolar cell contacts in mouse retina.

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. 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 immunocytochemistry. 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. 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.

Critical Decrease in the Level of Axon Guidance Receptor ROBO1 in Rod Synaptic Terminals Is Followed by Axon Retraction.

PurposeTo define remodeling of photoreceptor synaptic terminals and second-order retinal neurons in canine X-linked progressive retinal atrophy 1 caused by a five-nucleotide deletion in the RPGR exon ORF15.MethodsRetinas of normal and mutant dogs were used for gene expression, Western blot, and immunohistochemistry. Cell-specific markers were used to examine disease-dependent retinal remodeling.ResultsIn mutant retinas, a number of rod axon terminals retract into the outer nuclear layer. This neuritic atrophy preceded significant loss of rods and was evident early in disease. Rod bipolar and horizontal cell processes were found to extend into the outer nuclear layer, where they seemed to form contacts with the spherules of rod photoreceptors. No ectopic rewiring was observed. Because cytoskeletal reorganization was previously shown to underlie photoreceptor axon retraction, we examined normal and mutant retinas for expression of axon guidance receptors ROBO1 and ROBO2, which are known to regulate actin cytoskeleton dynamics. We found that the overall expression of both ROBO1 and ROBO2 is retained at the same level in premature and fully developed normal retinas. However, analysis of predisease and early disease retinas identified markedly decreased levels of ROBO1 in rod spherules compared with controls. In contrast, no differences in ROBO1 signals were noted in cone pedicles in normal and mutant retinas, where ROBO1 levels remained similarly low.ConclusionsDepletion of ROBO1 in rod synaptic terminals correlates with the remodeling of axonal and dendritic processes in the outer retina of dogs with X-linked progressive retinal atrophy 1 and may play a role in the retraction of rod axons.

Photoreceptor degeneration in a new Cacna1f mutant mouse model

The Cacna1f gene encodes the α1F subunit of an L-type voltage-gated calcium channel, Cav1.4. In photoreceptor synaptic terminals, Cav1.4 channels mediate glutamate release and postsynaptic responses associated with visual signal transmission. We have discovered a new Cacna1f mutation in nob9 mice, which display more severe phenotypes than do nob2 mice. To characterize the nob9 phenotype at different ages, we examined the murine fundus, applied retinal optical coherence tomography, measured flash electroretinograms (ERGs) in vivo, and analyzed the retinal histology in vitro. After identifying the X-linked recessive inheritance trait, we sequenced Cacna1f as the candidate gene. Mutations in this gene were detected by polymerase chain reaction (PCR) and confirmed by restriction fragment length polymorphism. Morphologically, an early-onset of retinal disorder was detected, and the degeneration of the outer plexiform layers progressed rapidly. Moreover, the mutant mice showed drastically reduced scotopic ERGs with increasing age. In 14-month-old nob9 retinas, immunostaining of cone opsins demonstrated a reduction in the number of short-wavelength opsins (S-opsins) to 54% of wild-type levels, and almost no middle-wavelength opsins (M-opsins) were observed. No cone ERGs could be detected from residual cones, in which S-opsins abnormally migrated to inner segments of the photoreceptors. The mutations of the Cacna1f gene in nob9 mice involved both a single nucleotide G to A transition and a 10-nucleotide insertion, the latter resulting in a frame-shift mutation in exon 14.

Splicing of an automodulatory domain in Cav1.4 Ca2+ channels confers distinct regulation by calmodulin.

Ca2+ influx through Cav1.4 L-type Ca2+ channels supports the sustained release of glutamate from photoreceptor synaptic terminals in darkness, a process that is critical for vision. Consistent with this role, Cav1.4 exhibits weak Ca2+-dependent inactivation (CDI)-a negative feedback regulation mediated by Ca2+-bound calmodulin (CaM). CaM binds to a conserved IQ domain in the proximal C-terminal domain of Cav channels, but in Cav1.4, a C-terminal modulatory domain (CTM) disrupts interactions with CaM. Exon 47 encodes a portion of the CTM and is deleted in a Cav1.4 splice variant (Cav1.4Δex47) that is highly expressed in the human retina. Cav1.4Δex47 exhibits CDI and enhanced voltage-dependent activation, similar to that caused by a mutation that is associated with congenital stationary night blindness type 2, in which the CTM is deleted (K1591X). The presence of CDI and very negative activation thresholds in a naturally occurring variant of Cav1.4 are perplexing considering that these properties are expected to be maladaptive for visual signaling and result in night blindness in the case of K1591X. Here we show that Cav1.4Δex47 and K1591X exhibit fundamental differences in their regulation by CaM. In Cav1.4Δex47, CDI requires both the N-terminal (N lobe) and C-terminal (C lobe) lobes of CaM to bind Ca2+, whereas CDI in K1591X is driven mainly by Ca2+ binding to the C lobe. Moreover, the CaM N lobe causes a Ca2+-dependent enhancement of activation of Cav1.4Δex47 but not K1591X. We conclude that the residual CTM in Cav1.4Δex47 enables a form of CaM N lobe regulation of activation and CDI that is absent in K1591X. Interaction with the N lobe of CaM, which is more sensitive to global elevations in cytosolic Ca2+ than the C lobe, may allow Cav1.4Δex47 to be modulated by a wider range of synaptic Ca2+ concentrations than K1591X; this may distinguish the normal physiological function of Cav1.4Δex47 from the pathological consequences of K1591X.

Loss of the Drosophilam-AAA mitochondrial protease paraplegin results in mitochondrial dysfunction, shortened lifespan, and neuronal and muscular degeneration

The progressive accumulation of dysfunctional mitochondria is implicated in aging and in common diseases of the elderly. To oppose this occurrence, organisms employ a variety of strategies, including the selective degradation of oxidatively damaged and misfolded mitochondrial proteins. Genetic studies in yeast indicate that the ATPase Associated with diverse cellular Activities (AAA+) family of mitochondrial proteases account for a substantial fraction of this protein degradation, but their metazoan counterparts have been little studied, despite the fact that mutations in the genes encoding these proteases cause a variety of human diseases. To begin to explore the biological roles of the metazoan mitochondrial AAA+ protease family, we have created a CRISPR/Cas9 allele of the Drosophila homolog of SPG7, which encodes an inner membrane-localized AAA+ protease known as paraplegin. Drosophila SPG7 mutants exhibited shortened lifespan, progressive locomotor defects, sensitivity to chemical and environmental stress, and muscular and neuronal degeneration. Ultrastructural examination of photoreceptor neurons indicated that the neurodegenerative phenotype of SPG7 mutants initiates at the synaptic terminal. A variety of mitochondrial defects accompanied the degenerative phenotypes of SPG7 mutants, including altered axonal transport of mitochondria, accumulation of electron-dense material in the matrix of flight muscle mitochondria, reduced activities of respiratory chain complexes I and II, and severely swollen and dysmorphic mitochondria in the synaptic terminals of photoreceptors. Drosophila SPG7 mutants recapitulate key features of human diseases caused by mutations in SPG7, and thus provide a foundation for the identification of Drosophila paraplegin substrates and strategies that could be used to ameliorate the symptoms of these diseases.

An experimental study to evaluate safety/toxicity of intravitreal natalizumab.

Purpose:The purpose of this prospective experimental study was to evaluate the safety/toxicity of α4β1 integrin blockade in rabbit retina using its monoclonal antibody (Natalizumab).Methods:Twelve New Zealand albino rabbits were divided into three groups (n = 4). Unilateral intravitreal injections of three different concentrations of natalizumab were performed in every rabbit of each group (Group A: 0.625 mg, Group B: 1.25 mg, and Group C: 2.5 mg). Baseline electroretinogram (ERG) and fundus photography were performed prior to injection. At days 1, 7, and 21 postinjection, ERG and fundus photography of each eye were performed. At last follow-up, Group C animals with highest drug concentration were sacrificed and the enucleated eyes were evaluated for retinal toxicity using transmission electron microscopy (TEM).Results:No difference in ERG responses was observed in eyes injected with low and intermediate concentration of natalizumab between day 0 and day 21. Furthermore, rabbits injected intravitreally with highest dose showed reduction in amplitude of “a” wave (P = 0.0017) and a reduction in amplitude of “b” wave of ERG at day 21 (P = 0.0117). TEM revealed changes in the outer plexiform layer and inner nuclear layer, suggestive of toxicity primarily to the photoreceptor synaptic terminals and bipolar cells.Conclusion:Low-dose (0.625 mg) and intermediate-dose (1.25 mg) intravitreal injection of natalizumab appears safe for rabbit retina. However, functional and anatomical changes were observed in rabbit retina following a high-dose (2.5 mg) intravitreal injection of a monoclonal antibody blocking α4β1 integrin.

Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A)

ABSTRACTCongenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α1F subunit of a L-type voltage-gated calcium channel, Cav1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our ‘G305X’ mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function – measured by immunohistochemistry, optokinetic response and electroretinography – deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas – ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Cav1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Cav1.4 in photopic vision, it also suggests a potential new target for restorative therapy.

The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses.

L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Cav1.2, Cav1.3, and Cav1.4) expressed in the retina. While Cav1.2 is expressed in all retinal cells including the Müller glia and neurons, Cav1.3 and Cav1.4 are expressed in the retinal neurons with Cav1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Cav1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Cav1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Cav1.3 are not associated with severe vision impairment in humans or in Cav1.3-null (Cav1.3−/−) mice. However, a failure to regulate Cav1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Cav1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Cav1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Cav1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Cav1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Cav1.3−/− mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Cav1.3−/− mice retinas. Hence, Cav1.3 plays a more prominent role in retinal physiology and function than previously reported.

Specific Alleles of CLN7/MFSD8, a Protein That Localizes to Photoreceptor Synaptic Terminals, Cause a Spectrum of Nonsyndromic Retinal Dystrophy

Recessive mutations in CLN7/MFSD8 usually cause variant late-infantile onset neuronal ceroid lipofuscinosis (vLINCL), a poorly understood neurodegenerative condition, though mutations may also cause nonsyndromic maculopathy. A series of 12 patients with nonsyndromic retinopathy due to novel CLN7/MFSD8 mutation combinations were investigated in this study. Affected patients and their family members were recruited in ophthalmic clinics at each center where they were examined by retinal imaging and detailed electrophysiology. Whole exome or genome next generation sequencing was performed on genomic DNA from at least one affected family member. Immunofluorescence confocal microscopy of murine retina cross-sections were used to localize the protein. Compound heterozygous alleles were identified in six cases, one of which was always p.Glu336Gln. Such combinations resulted in isolated macular disease. Six further cases were homozygous for the variant p.Met454Thr, identified as a founder mutation of South Asian origin. Those patients had widespread generalized retinal disease, characterized by electroretinography as a rod-cone dystrophy with severe macular involvement. In addition, the photopic single flash electroretinograms demonstrated a reduced b- to a-wave amplitude ratio, suggesting dysfunction occurring after phototransduction. Immunohistology identified MFSD8 in the outer plexiform layer of the retina, a site rich in photoreceptor synapses. This study highlights a hierarchy of MFSD8 variant severity, predicting three consequences of mutation: (1) nonsyndromic localized maculopathy, (2) nonsyndromic widespread retinopathy, or (3) syndromic neurological disease. The data also shed light on the underlying pathogenesis by implicating the photoreceptor synaptic terminals as the major site of retinal disease.

Kinetics of Inhibitory Feedback from Horizontal Cells to Photoreceptors: Implications for an Ephaptic Mechanism.

Inhibitory feedback from horizontal cells (HCs) to cones generates center-surround receptive fields and color opponency in the retina. Mechanisms of HC feedback remain unsettled, but one hypothesis proposes that an ephaptic mechanism may alter the extracellular electrical field surrounding photoreceptor synaptic terminals, thereby altering Ca(2+) channel activity and photoreceptor output. An ephaptic voltage change produced by current flowing through open channels in the HC membrane should occur with no delay. To test for this mechanism, we measured kinetics of inhibitory feedback currents in Ambystoma tigrinum cones and rods evoked by hyperpolarizing steps applied to synaptically coupled HCs. Hyperpolarizing HCs stimulated inward feedback currents in cones that averaged 8-9 pA and exhibited a biexponential time course with time constants averaging 14-17 ms and 120-220 ms. Measurement of feedback-current kinetics was limited by three factors: (1) HC voltage-clamp speed, (2) cone voltage-clamp speed, and (3) kinetics of Ca(2+) channel activation or deactivation in the photoreceptor terminal. These factors totaled ∼4-5 ms in cones meaning that the true fast time constants for HC-to-cone feedback currents were 9-13 ms, slower than expected for ephaptic voltage changes. We also compared speed of feedback to feedforward glutamate release measured at the same cone/HC synapses and found a latency for feedback of 11-14 ms. Inhibitory feedback from HCs to rods was also significantly slower than either measurement kinetics or feedforward release. The finding that inhibitory feedback from HCs to photoreceptors involves a significant delay indicates that it is not due to previously proposed ephaptic mechanisms. Lateral inhibitory feedback from horizontal cells (HCs) to photoreceptors creates center-surround receptive fields and color-opponent interactions. Although underlying mechanisms remain unsettled, a longstanding hypothesis proposes that feedback is due to ephaptic voltage changes that regulate photoreceptor synaptic output by altering Ca(2+) channel activity. Ephaptic processes should occur with no delay. We measured kinetics of inhibitory feedback currents evoked in photoreceptors with voltage steps applied to synaptically coupled HCs and found that feedback is too slow to be explained by ephaptic voltage changes generated by current flowing through continuously open channels in HC membranes. By eliminating the proposed ephaptic mechanism for HC feedback regulation of photoreceptor Ca(2+) channels, our data support earlier proposals that synaptic cleft pH changes are more likely responsible.

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit.

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels.

Stability and Safety of an AAV Vector for Treating RPGR-ORF15 X-Linked Retinitis Pigmentosa.

Our collaborative successful gene replacement therapy using AAV vectors expressing a variant of human RPGR-ORF15 in two canine models provided therapeutic proof of concept for translation into human treatment. The ORF15 sequence contained within this AAV vector, however, has ORF15 DNA sequence variations compared to the published sequence that are likely due to its unusual composition of repetitive purine nucleotides. This mutability is a concern for AAV vector production and safety when contemplating a human trial. In this study, we establish the safety profile of AAV-hIRBP-hRPGR and AAV-hGRK1-hRPGR vectors used in the initial canine proof-of-principle experiments by demonstrating hRPGR-ORF15 sequence stability during all phases of manipulation, from plasmid propagation to vector production to its stability in vivo after subretinal administration to animals. We also evaluate potential toxicity in vivo by investigating protein expression, retinal structure and function, and vector biodistribution. Expression of hRPGR is detected in the inner segments and synaptic terminals of photoreceptors and is restricted to the connecting cilium when the vector is further diluted. Treated eyes exhibit no toxicity as assessed by retinal histopathology, immunocytochemistry, optical coherence tomography, fundoscopy, electroretinogram, and vector biodistribution. Therefore, the hRPGR-ORF15 variant in our AAV vectors appears to be a more stable form than the endogenous hRPGR cDNA when propagated in vitro. Its safety profile presented here in combination with its proven efficacy supports future gene therapy clinical trials.

Characterization of Cav1.4 Complexes (α11.4, β2, and α2δ4) in HEK293T Cells and in the Retina

In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 α1, β2, and α2δ4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, and α2δ4 were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Cav1.4 α1 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by β2a and β2X13 variants.

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina.

Calcium operates by several mechanisms to regulate glutamate release at rod and cone synaptic terminals. In addition to serving as the exocytotic trigger, Ca2+ accelerates replenishment of vesicles in cones and triggers Ca2+-induced Ca2+ release (CICR) in rods. Ca2+ thereby amplifies sustained exocytosis, enabling photoreceptor synapses to encode constant and changing light. A complete picture of the role of Ca2+ in regulating synaptic transmission requires an understanding of the endogenous Ca2+ handling mechanisms at the synapse. We therefore used the "added buffer" approach to measure the endogenous Ca2+ binding ratio (κendo ) and extrusion rate constant (γ) in synaptic terminals of photoreceptors in retinal slices from tiger salamander. We found that κendo was similar in both cell types-∼25 and 50 in rods and cones, respectively. Using measurements of the decay time constants of Ca2+ transients, we found that γ was also similar, with values of ∼100 s(-1) and 160 s(-1) in rods and cones, respectively. The measurements of κendo differ considerably from measurements in retinal bipolar cells, another ribbon-bearing class of retinal neurons, but are comparable to similar measurements at other conventional synapses. The values of γ are slower than at other synapses, suggesting that Ca2+ ions linger longer in photoreceptor terminals, supporting sustained exocytosis, CICR, and Ca2+ -dependent ribbon replenishment. The mechanisms of endogenous Ca2+ handling in photoreceptors are thus well-suited for supporting tonic neurotransmission. Similarities between rod and cone Ca2+ handling suggest that neither buffering nor extrusion underlie differences in synaptic transmission kinetics.

Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

Remodelling of retinal on‐ and off‐bipolar cells following experimental retinal detachment

To study the response of ON and OFF bipolar cells in experimental retinal detachment. Domestic cat retinas were detached for 7 days. The retinas were prepared for immunocytochemical staining with antibodies to Go alpha (α), glutamate transporter GLT-1, protein kinase C and rod opsin, which serve as markers for ON bipolar cells, OFF bipolar cells, rod bipolar cells and rod photoreceptors, respectively. Both sections and whole-mounts were labelled with antibodies to Goα and GLT-1. Following 7 days of detachment, ON bipolar cell processes extended into the outer nuclear layer and had neurites extending beyond their target layer into the inner plexiform layer. In contrast, OFF bipolar cell processes were reduced in the outer plexiform layer following detachment. ON and OFF bipolar cells undergo significant remodelling of their processes in response to retinal detachment, and the ON and OFF pathways may be differentially affected. The remodelling may be due to morphological changes that have previously been shown to occur in photoreceptor synaptic terminals or as a result of loss of synaptic connections due to photoreceptor cell death.

Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

Targeted Expression of Anoctamin Calcium-Activated Chloride Channels in Rod Photoreceptor Terminals of the Rodent Retina

In the vertebrate retina, calcium-activated chloride channels are expressed in photoreceptor synaptic terminals. These channels are involved in the control of transmitter release in the dark. The search for their molecular identity has recently lead to the localization of the protein anoctamin 2 (also TMEM16B) in the outer plexiform layer of the rodent retina. Since both rod and cone photoreceptors have their terminals in this layer, it was not clear which of these express anoctamin 2. Here, we examine rod spherules and cone pedicles for expression of anoctamin 2. Expression of anoctamin genes was studied in the rat eye using RT-PCR. Immunohistochemical experiments were used to localize anoctamins and chloride transporters with their regulatory kinases. Photoreceptor synaptic proteins, as well as the lectins Peanut agglutinin and Griffonia simplicifolia agglutinin, were used to distinguish retinal structures. Anoctamin 1, 2, and 10 were found to be expressed in the eye. Anoctamin 2 was expressed as a splice variant that includes exon 15 of the genomic structure. The protein is exclusively expressed in rod terminals and is not present in cone pedicles. Expression is not clustered at the ribbon complex, but spread across the presynaptic membrane where it colocalizes with the plasma membrane calcium pump. The electroneutral chloride transporter NKCC1 is expressed in photoreceptor terminals, together with its regulatory kinases SPAK and OSR1. Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.