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Background: Spinocerebellar ataxias (SCA) are a group of neurodegenerative disorders characterized by neuronal degeneration leading to loss of motor coordination. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is an age-related cerebellar neurodegenerative disorder caused by mutations in the fatty acid elongase-4 (ELOVL4). The ELOVL4 is an essential enzyme that mediates biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons) that are critical for the normal function of brain, skin, retina, Meibomian glands, and testes in which ELOVL4 is expressed. Global deletion or homozygous expression of truncated mutant ELOVL4 that lack VLC-SFA and VLC-PUFA biosynthesis cause severe skin disorders, seizures and neonatal mortality in rodents and humans. Methods: To understand role of ELOVL4 and its products in neuronal function and to evaluate the consequences of ELOVL4 mutations in pathogenesis of age-related SCA34, we generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. We performed biochemical, neuroanatomical and behavioral analyses by rotorod to measure motor function. We used electrophysiological recordings from cerebellar slices to determine the impact of the W246G mutation on neuronal function. Results were analyzed using GraphPad Prism Statistical software. Results: Heterozygous and homozygous rats carrying the W246G mutation developed impaired motor deficits by two months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum up 6 months of age showed normal cytoarchitectural organization despite the early-onset motor deficits and defects in synaptic plasticity. Conclusions: Our results point to ELOVL4 and its products being essential for motor function and cerebellar synaptic plasticity. The results further suggest that in SCA34 patients, ataxia arises from primary impairment of synaptic plasticity and cerebellar network desynchronization that precedes cerebellar degeneration and loss of motor coordination with aging.

Cognitive decline is a debilitating aspect of aging and neurodegenerative diseases such as Alzheimer's disease are closely associated with mitochondrial dysfunction, increased reactive oxygen species, neuroinflammation, and astrogliosis. This study investigated the effects of decreased mitochondrial antioxidant response specifically in astrocytes on cognitive performance and neuronal function in C57BL/6J mice using a tamoxifen-inducible astrocyte-specific knockout of manganese superoxide dismutase (aSOD2-KO), a mitochondrial matrix antioxidant that detoxifies superoxide generated during mitochondrial respiration. We reduced astrocyte SOD2 levels in male and female mice at 11–12 months of age and tested in an automated home cage (PhenoTyper) apparatus for diurnal patterns, spatial learning, and memory function at 15 months of age. aSOD2-KO impaired hippocampal-dependent spatial working memory and decreased cognitive flexibility in the reversal phase of the testing paradigm in males. Female aSOD2-KO showed no learning and memory deficits compared with age-matched controls despite significant reduction in hippocampal SOD2 expression. aSOD2-KO males further showed decreased hippocampal long-term potentiation, but paired-pulse facilitation was unaffected. Levels of d-serine, an NMDA receptor coagonist, were also reduced in aSOD2-KO mice, but female knockouts showed a compensatory increase in serine racemase expression. Furthermore, aSOD2-KO mice demonstrated increased density of astrocytes, indicative of astrogliosis, in the hippocampus compared with age-matched controls. These data demonstrate that reduction in mitochondrial antioxidant stress response in astrocytes recapitulates age-related deficits in cognitive function, d-serine availability, and astrogliosis. Therefore, improving astrocyte mitochondrial homeostasis may provide a therapeutic target for intervention for cognitive impairment in aging. SIGNIFICANCE STATEMENT Diminished antioxidant response is associated with increased astrogliosis in aging and in Alzheimer's disease. Manganese superoxide dismutase (SOD2) is an antioxidant in the mitochondrial matrix that detoxifies superoxide and maintains mitochondrial homeostasis. We show that astrocytic ablation of SOD2 impairs hippocampal-dependent plasticity in spatial working memory, reduces long-term potentiation of hippocampal neurons and levels of the neuromodulator d-serine, and increases astrogliosis, consistent with defects in advanced aging and Alzheimer's disease. Our data provide strong evidence for sex-specific effects of astrocytic SOD2 functions in age-related cognitive dysfunction.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3(f/f(iCre75)) and Stx3(f/f(CRX-Cre))] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Mild traumatic brain injury (mTBI) diagnoses have increased due to aggressive sports and blast-related injuries, but the cellular mechanisms and pathology underlying mTBI are not completely understood. Previous reports indicate that Nociceptin Orphanin/FQ (N/OFQ), an endogenous neuropeptide, contributes to post-injury ischemia following mechanical brain injury, yet its specific role in cerebral hypoxia, vestibulomotor function and injury marker expression following blast-induced TBI is not known. This study is the first to identify a direct association of N/OFQ and its N/OFQ peptide (NOP) receptor with TBI-induced changes following a single 80psi head blast exposure in male rats. N/OFQ and NOP receptor expression increased in brain tissue and plasma following TBI, concurrent with vestibular dysfunction but preceding hypoxia and appearance of injury markers compared to sham rats. A single post-blast treatment with the NOP receptor antagonist, SB-612111, transiently improved acute vestibulomotor performance. It also prevented increases in markers of TBI-induced hypoxia, pro-apoptotic proteins and injury seen 8-10days post-blast. This study reveals an apparent role for the N/OFQ-NOP receptor system in blast TBI and suggests potential therapeutic utility of NOP receptor antagonists for mTBI.

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥ 28 carbons) in the brain are unknown. The enzyme ELOngation of Very Long-chain fatty acids-4 (ELOVL4) catalyzes the rate-limiting step in the biosynthesis of VLC-FA (Agbaga et al., Proc Natl Acad Sci USA 105(35): 12843–12848, 2008; Logan et al., J Lipid Res 55(4): 698–708, 2014), which we identified in the brain as saturated fatty acids (VLC-SFA). Homozygous mutations in ELOVL4 cause severe neuropathology in humans (Ozaki et al., JAMA Neurol 72(7): 797–805, 2015; Mir et al., BMC Med Genet 15: 25, 2014; Cadieux-Dion et al., JAMA Neurol 71(4): 470–475, 2014; Bourassa et al., JAMA Neurol 72(8): 942–943, 2015; Aldahmesh et al., Am J Hum Genet 89(6): 745–750, 2011) and are post-natal lethal in mice (Cameron et al., Int J Biol Sci 3(2): 111–119, 2007; Li et al., Int J Biol Sci 3(2): 120–128, 2007; McMahon et al., Molecular Vision 13: 258–272, 2007; Vasireddy et al., Hum Mol Genet 16(5): 471–482, 2007) from dehydration due to loss of VLC-SFA that comprise the skin permeability barrier. Double transgenic mice with homozygous knock-in of the Stargardt-like macular dystrophy (STDG3; 797-801_AACTT) mutation of Elovl4 with skin-specific rescue of wild-type Elovl4 expression (S + Elovl4 mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S + Elovl4 mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S + Elovl4 mut/mut mice exhibited accelerated synaptic release kinetics. Supplementation of VLC-SFA to cultured hippocampal neurons from mutant mice rescued defective synaptic release to wild-type rates. Together, these studies establish a critical, novel role for ELOVL4 and its VLC-SFA products in regulating synaptic release kinetics and epileptogenesis. Future studies aimed at understanding the molecular mechanisms by which VLC-SFA regulate synaptic function may provide new targets for improved seizure therapies.

To dissect the role of vascular endothelial growth factor receptor-2 (VEGFR2) in Müller cells and its effect on neuroprotection in diabetic retinopathy (DR), we disrupted VEGFR2 in mouse Müller glia and determined its effect on Müller cell survival, neuronal integrity, and trophic factor production in diabetic retinas. Diabetes was induced with streptozotocin. Retinal function was measured with electroretinography. Müller cell and neuronal densities were assessed with morphometric and immunohistochemical analyses. Loss of VEGFR2 caused a gradual reduction in Müller glial density, which reached to a significant level 10 months after the onset of diabetes. This observation was accompanied by an age-dependent decrease of scotopic and photopic electroretinography amplitudes and accelerated loss of rod and cone photoreceptors, ganglion cell layer cells, and inner nuclear layer neurons and by a significant reduction of retinal glial cell line–derived neurotrophic factor and brain-derived neurotrophic factor. Our results suggest that VEGFR2-mediated Müller cell survival is required for the viability of retinal neurons in diabetes. The genetically altered mice established in this study can be used as a diabetic animal model of nontoxin-induced Müller cell ablation, which will be useful for exploring the cellular mechanisms of neuronal alteration in DR.

Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (∼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.

To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1(-/-) /Tpst2 (-/-) ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.

The Pde6b(rd10) (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6b(rd10) (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well-known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5.

Voltage-gated sodium channels (Nav channels) in retinal neurons are known to contribute to the mammalian flash electroretinogram (ERG) via activity of third-order retinal neurons, i.e. amacrine and ganglion cells. This study investigated the effects of tetrodotoxin (TTX) blockade of Nav channels on the b-wave, an ERG wave that originates mainly from activity of second-order retinal neurons. ERGs were recorded from anaesthetized Brown Norway rats in response to brief full-field flashes presented over a range of stimulus energies, under dark-adapted conditions and in the presence of steady mesopic and photopic backgrounds. Recordings were made before and after intravitreal injection of TTX (∼3 μm) alone, 3–6 weeks after optic nerve transection (ONTx) to induce ganglion cell degeneration, or in combination with an ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 200 μm) to block light-evoked activity of inner retinal, horizontal and OFF bipolar cells, or with the glutamate agonist N-methyl-d-aspartate (NMDA, 100–200 μm) to reduce light-evoked inner retinal activity. TTX reduced ERG amplitudes measured at fixed times corresponding to b-wave time to peak. Effects of TTX were seen under all background conditions, but were greatest for mesopic backgrounds. In dark-adapted retina, b-wave amplitudes were reduced only when very low stimulus energies affecting the inner retina, or very high stimulus energies were used. Loss of ganglion cells following ONTx did not affect b-wave amplitudes, and injection of TTX in eyes with ONTx reduced b-wave amplitudes by the same amount for each background condition as occurred when ganglion cells were intact, thereby eliminating a ganglion cell role in the TTX effects. Isolation of cone-driven responses by presenting test flashes after cessation of a rod-saturating conditioning flash indicated that the TTX effects were primarily on cone circuits contributing to the mixed rod–cone ERG. NMDA significantly reduced only the additional effects of TTX on the mixed rod–cone ERG observed under mesopic conditions, implicating inner retinal involvement in those effects. After pharmacological blockade with CNQX, TTX still reduced b-wave amplitudes in cone-isolated ERGs indicating Nav channels in ON cone bipolar cells themselves augment b-wave amplitude and sensitivity. This augmentation was largest under dark-adapted conditions, and decreased with increasing background illumination, indicating effects of background illumination on Nav channel function. These findings indicate that activation of Nav channels in ON cone bipolar cells affects the b-wave of the rat ERG and must be considered when analysing results of ERG studies of retinal function.

Current models of synaptic transmission require retrieval of membrane from the presynaptic terminal following neurotransmitter exocytosis. Dynamin, a GTPase, is thought to be critical for this retrieval process. At ribbon synapses of retinal bipolar neurons, however, compensatory endocytosis does not require GTP hydrolysis, suggesting that endocytosis mechanisms may differ among synapses. To understand better the synaptic vesicle recycling at conventional and ribbon synapses, the distributions of dynamin and two associated proteins, amphiphysin and clathrin, were examined in the retinas of goldfish and mouse by using immunocytochemical methods. Labeling for dynamin, clathrin, and amphiphysin was distributed differentially among conventional and ribbon synapses in retinas of both species. Ribbon synapses of photoreceptors and most bipolar cells labeled only weakly for dynamin relative to conventional synapses. Amphyiphysin labeling was strong at many ribbon synapses, and labeling in rod terminals was stronger than in cone terminals in the mouse retina. Clathrin labeling was heterogeneous among ribbon synapses. Similarly to the case with amphiphysin, mouse rod terminals showed stronger clathrin labeling than cone terminals. Among conventional synapses, there was heterogeneous labeling for all three endocytic proteins. Some labeling for each protein might have been associated with postsynaptic terminals. The differential distribution of labeling for these proteins among identified synapses in the retina suggests considerable heterogeneity in the molecular mechanisms underlying synaptic membrane retrieval, even among synapses with similar active zone ultrastructure. Thus, as with exocytosis, mechanisms of synaptic membrane retrieval may be tuned by the precise complement of proteins expressed within the synaptic terminal. J. Comp. Neurol. 484:440–457, 2005. © 2005 Wiley-Liss, Inc.

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1-immunoreactivity (-IR); no VGLUT2-IR was present in any synapses of the developing or adult retina. VGLUT1-IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4-P5. Rod terminals showed VGLUT1-IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1-IR around P8, 2-3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6-P8, most did not show VGLUT1-IR until P8-P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca2+-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis. J. Comp. Neurol. 460:106–122, 2003. © 2003 Wiley-Liss, Inc.

Synaptotagmin I is the leading candidate for the calcium sensor that triggers exocytosis at conventional synapses. However, physiological characterization of the calcium sensor for phasic release at the ribbon-style synapses of the goldfish Mb1 bipolar cell demonstrates a lower than predicted affinity for calcium, suggesting that a modified or different sensor triggers exocytosis at this synapse. We examined synaptotagmin immunolabeling in goldfish retina using two different antibodies directed against synaptotagmin epitopes that specifically labeled the expected 65-kDa protein on western blots of goldfish and mouse retinal membranes. The first antiserum strongly labeled conventional synapses in the inner plexiform layer (IPL), but did not label the ribbon-style synapse-containing synaptic terminals of goldfish Mb1 bipolar cells or photoreceptors. The second antibody also specifically labeled the expected 65-kDa protein on western blots but did not label any synapses in the goldfish retina. A third synaptotagmin antibody that performed poorly on western blots selectively labeled goldfish photoreceptor terminals. These results suggest that synaptotagmin may exist in at least three distinct “forms” in goldfish retinal synapses. These forms, which are differentially localized to conventional synapses, bipolar cell, and photoreceptor terminals, may represent differences in isoform, posttranslational modifications, epitope availability, and protein-binding partners. Labeling with these antibodies in the salamander and mouse retina revealed species-specific differences, indicating that synaptotagmin epitopes can vary across species as well as among synapses.

Regeneration of functionally normal synapses is required for functional recovery after degenerative central nervous system insults and requires proper expression and targeting of presynaptic proteins by regenerating neurons. The reconstitution of presynaptic terminals by regenerating adult neurons is poorly understood, however. We examined the intrinsic ability of regenerating adult retinal photoreceptors to reconstitute properly differentiated presynaptic terminals in the absence of target contact. The expression and localization of vesicle-associated membrane protein (VAMP), synaptic vesicle protein 2 (SV2), synaptophysin, synapsin I, and synaptosomal-associated protein of 25 kDa (SNAP-25) was assessed immunocytochemically. Photoreceptor terminals in the intact retina contain VAMP, SV2, synaptophysin, and SNAP-25, but not synapsin I. Isolated, regenerating adult photoreceptors intrinsically expressed the proper complement of synaptic vesicle proteins in the absence of target contact: VAMP, SV2, and synaptophysin were present at all stages of regenerative growth; synapsin I was never expressed. At early stages of regenerative growth, VAMP, SV2, and synaptophysin were diffusely localized in the cell, with prominent VAMP labeling distributed along the plasma membrane. SV2 and synaptophysin rapidly localized to regenerated terminals, but VAMP accumulated much more slowly, indicating that these proteins are trafficked independently. In contrast, labeling for SNAP-25, which is associated with the presynaptic plasma membrane, was undetectable in regenerating photoreceptors, suggesting that SNAP-25 expression is target-regulated. Thus, regenerating photoreceptors can intrinsically regulate the expression of the proper set of synaptic vesicle proteins. Proper expression of other presynaptic proteins, such as SNAP-25, and proper subcellular localization of synaptic proteins such as VAMP, however, may require extrinsic cues such as target contact.

The gene encoding Elongation of Very Long Chain Fatty Acids-4 (ELOVL4) is mutated in patients with autosomal dominant Stargardt's Macular Dystrophy Type 3 (STDG3). ELOVL4 catalyzes the initial condensation step in the elongation of polyunsaturated fatty acids (PUFA) containing more than 26 carbons (26C) to very long chain PUFA (VLC-PUFA; C28 and greater). To investigate the role of VLC-PUFA in rod photoreceptors, we generated mice with rod-specific deletion of Elovl4 (RcKO). The mosaic deletion of rod-expressed ELOVL4 protein resulted in a 36 % lower amount of VLC-PUFA in the retinal phosphatidylcholine (PC) fraction compared to retinas from wild-type mice. However, this reduction was not sufficient to cause rod dysfunction at 7 months or photoreceptor degeneration at 9 or 15 months.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts. J. Comp. Neurol. 431:424‐436, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: synaptobrevin; synapse; photoreceptor; bipolar cell; amacrine cell

The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779). We further investigated the hypothesis that glutamate is the excitatory neurotransmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural-pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural-pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural-pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no gamma-aminobutyric acid or glycine immunoreactivity was observed in the pleural-pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high-affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221-1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779)) establish glutamate as an excitatory neurotransmitter of the sensorimotor synapse.

Aims: Human γ-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize γ-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX3C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell. Antioxid. Redox Signal. 19, 1877–1888.

Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.

Melatonin is a chemical signal of darkness that is produced by retinal photoreceptors and pinealocytes. In the retina, melatonin diffuses from the photoreceptors to bind to specific receptors on a variety of inner retinal neurons to modify their activity. Potential target cells for melatonin in the inner retina are amacrine cells, bipolar cells, horizontal cells, and ganglion cells. Melatonin inhibits the release of dopamine from amacrine cells and increases the light sensitivity of horizontal cells. Melatonin receptor subtypes show differential, cell-specific patterns of expression that are likely to underlie differential functional modulation of specific retinal pathways. Melatonin potentiates rod signals to ON-type bipolar cells, via activation of the melatonin MT2 (Mel1b) receptor, suggesting that melatonin modulates the function of specific retinal circuits based on the differential distribution of its receptors. The selective and differential expression of melatonin receptor subtypes in cone circuits suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons and thus promote dark adaptation.

Melatonin is a chemical signal of darkness that is produced by retinal photoreceptors and pinealocytes. In the retina, melatonin diffuses from the photoreceptors to bind to specific receptors on a variety of inner retinal neurons to modify their activity. Potential target cells for melatonin in the inner retina are amacrine cells, bipolar cells, horizontal cells, and ganglion cells. Melatonin inhibits the release of dopamine from amacrine cells and increases the light sensitivity of horizontal cells. Melatonin receptor subtypes show differential, cell-specific patterns of expression that are likely to underlie differential functional modulation of specific retinal pathways. Melatonin potentiates rod signals to ON-type bipolar cells, via activation of the melatonin MT2 (Mel1b) receptor, suggesting that melatonin modulates the function of specific retinal circuits based on the differential distribution of its receptors. The selective and differential expression of melatonin receptor subtypes in cone circuits suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons and thus promote dark adaptation.

Regenerating adult central nervous system (CNS) neurons must re-establish synaptic circuits in an environment very different from that present during development. However, the complexity of CNS circuitry has made it extremely difficult to assess the selectivity and mechanisms of synaptic regeneration at the cellular level in vivo. The synaptic preferences of adult photoreceptors were examined by using a defined cell culture system known to support regenerative process growth, presynaptic varicosity formation, and establishment of functional synapses. Immunolabeling for synaptic vesicle protein 2 and ultrastructural analysis demonstrated that cell-cell contacts made by photoreceptors were synaptic in nature. Target selectivity was determined by quantitative analysis of contacts onto normal and novel target cell types in cultures in which opportunities to contact all retinal cell types were present. Target cells were identified by morphology and immunolabeling for the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine. Regenerating photoreceptors showed a strong preference for novel multipolar cell targets (amacrine and ganglion cells) over normal photoreceptor, horizontal, and bipolar cell targets. Additionally, photoreceptors were selective for targets containing the transmitter GABA. These results indicate first, that the normal synaptic partners for photoreceptors are not intrinsically the optimal targets for regenerative synapse formation, and second, that GABA may modulate synaptic targeting by adult photoreceptors.

Rod and cone photoreceptors degenerate under a variety of pathologic conditions, including those caused by bright light exposure and a wide array of hereditary retinal diseases, such as retinitis pigmentosa (RP), macular degeneration (MD), and cone-rod dystrophies. Defects in a large number of genes are linked to inherited retinal degenerative disorders (see more information at http://www.sph.uth.tmc.edu/RetNet/disease.htm). These genes include those encoding phototransduction-related proteins, such as rhodopsin, subunits of cyclic nucleotide phosphodiesterase (PDE), and cyclic nucleotide-gated (CNG) channel subunits, as well as genes encoding photoreceptor outer segment structural proteins, such as peripherin/rds. Secondary, or nonautonomous photoreceptor degeneration occurs when a disease gene expressed specifically in one type of photoreceptor (e.g., rods) leads to loss of photoreceptors that do not express the disease gene. For example, RP arising from defects in the rod-specific PDE gene leads to secondary loss of cones. Death of cones after rod death in RP is a characteristic of secondary photoreceptor degeneration associated with disease progression, and is also found in other human photoreceptor degenerations. Indeed, cone degeneration secondary to rod death has been studied intensively in human RP patients and in animal models of RP.1–4 In contrast, our understanding of the impact of cone degeneration on rod function and survival is very limited. Though early reports suggested normal or nearly normal rod function in patients with cone degenerations, such as achromatopsia and cone dystrophy,5–7 several recent studies have reported reduced rod electroretinographic (ERG) responses and disrupted rod photoreceptor mosaic in such disorders,8–10 suggesting that secondary impairment of rod function and viability may be a common consequence of primary cone degeneration. CNG channels, which are localized to the plasma membrane of the outer segment of rods and cones, play a pivotal role in phototransduction. In darkness, rod CNG channels are activated by binding of cyclic guanosine monophosphate (cGMP), allowing a steady inward cation (Na+ and Ca2+) current. Light triggers a sequence of enzymatic reactions that leads to the hydrolysis of cGMP, resulting in CNG channel closure, reduction in the inward cation current, and membrane hyperpolarization.11–14 A similar transduction scheme exists in cones. However, the CNG channels of rods and cones are formed from different A and B subunits, leading to profound differences in cGMP sensitivity, Ca2+ permeation, and functional modulation.15,16 The rod CNG channel is formed from CNGA1 and CNGB1 subunits, while the cone CNG channel is formed from CNGA3 and CNGB3 subunits. Heterologous expression studies have shown that the A subunits are responsible for the ion-conducting activity of the channel, whereas the B subunits function as modulators. Mutations in the rod-specific CNGA1 and CNGB1 genes are associated with RP,17,18 while mutations in the cone-specific CNGA3 and CNGB3 genes are linked to achromatopsia, cone dystrophy, and some maculopathies.6,19 Indeed, over 70 disease-associated mutations have been identified in CNGA3 and CNGB3,6,19–21 and these mutations account for over 70% of achromatopsia patients.6,22–24 Achromatopsia is a devastating hereditary visual disorder characterized by reduced cone-mediated ERG responses, color blindness, visual acuity loss, pendular nystagmus, extreme light sensitivity, and daytime blindness. As the disease is primarily caused by mutations in CNG channel subunits, achromatopsia is often referred to as a “channelopathy.” Cone degeneration in patients with CNG channel deficiency has been documented by optical coherence tomography (OCT) studies.25–27 Loss of cone function and progressive cone degeneration also has been documented in CNGA3−/− and CNGB3−/− mouse models.28–30 The current studies were designed to explore the secondary effects of cone degeneration on rods by characterizing rod function, structural integrity and survival after loss of cones in CNGA3−/− mice, a model for human achromotopsia. CNGA3−/− mice have no detectable cone ERG responses and develop early-onset cone degeneration,28,29 which is detected as early as the second postnatal week, with complete loss of cones from the ventral retina after the third postnatal month.29 In this report, we show secondary, age-dependent effects on rod-driven electrophysiological function in CNGA3−/− mice. The scotopic ERG b-wave was reduced as early as 1 month, while a reduced ERG a-wave only appeared much later, at 9 months. We also show a reduced outer nuclear layer (ONL) thickness and reduced expression of rod-specific proteins in CNGA3−/− mice at 12 months. Cone terminals in the CNGA3−/− retina showed a progressive loss of neurochemical and ultrastructural integrity, accompanied by disorganized rod terminal ultrastructure by 12 months. The appearance of scotopic ERG b-wave defects before a-wave deficits, together with the progressive loss of neurochemical and ultrastructural integrity of cone photoreceptor terminals, suggest that compromised CNG channel function and phototransduction lead to early impairment of synaptic terminal function and structural integrity. Our findings also indicate that impaired cone function leads to deleterious secondary effects on rod function, structure, and survival.

Golgi-impregnated amacrine cells in the all-cone lizard retina ( Anolis carolinensis ) were characterized on the bases of dendritic and somatic criteria. Four major cell categories, comprising 23 types were identified: three non-stratified, 13 monostratified, five bistratified, and two tristratified types. Four of the cell types comprised two to four subtypes based on stratification of their dendrites within the inner plexiform layer (IPL). Golgi impregnation strongly favoured monostratified amacrine cells with cell bodies at the proximal margin of the inner nuclear layer. The neurotransmitter content of each of the 23 amacrine cell types was examined by combined Golgi-immunocytochemistry after morphological classification. Putative neurotransmitters examined included gamma-aminobutyric acid (GABA), glycine (GLY) and aspartate (ASP). Seventeen cell types showed GABA-immunoreactivity (IR), three cell types showed GLY-IR, and four cell types showed neither GABA-IR nor GLY-IR. No cell types showed ASP-IR. Each cell type had a characteristic neurochemical signature, with the exception of one monostratified cell type that showed three different neurochemical signatures. Postembedding immunocytochemistry on conventionally processed retinas confirmed the localization of glutamic acid decarboxylase, the synthetic enzyme for GABA, to cells similar to several of the GABA-IR Golgi-stained types. Postembedding immunocytochemistry for tyrosine hydroxylase (the synthetic enzyme for catecholamines) and GABA on serial sections demonstrated colocalization of GABA and a catecholamine,probably dopamine, in a bistratified amacrine cell type. We conclude that GABA-IR amacrine cell types are more numerous and morphologically heterogeneous than GLY-IR amacrine cells. The morphological heterogeneity and, with one exception, exclusivity of GABA-IR and GLY-IR amacrine cell types indicate that both neurotransmitters play a variety and different functional roles in the lizard inner retina.

Neurons likely to utilize glycine (GLY) as a neurotransmitter were identified immunocytochemically in the “all-cone” lizard retina and the basic anatomical organization of the retinal GLY and gamma-aminobutyric acid (GABA) systems was compared. Four types of GLY-immunoreactive (GLY-IR) neurons were identified. Most GLY-IR cells were amacrine cells, which comprised at least two types. GLY-IR interplexiform cells and ganglion cells also were identified. The first GLY-IR amacrine cell type was characterized by a small pyriform soma, located distal to the border of the inner plexiform layer (IPL), and fine dendrites. Most GLY-IR amacrine cells were of this type and several subtypes may exist within this group. The second amacrine cell type was characterized by a large, distally located soma and a large descending process. This amacrine cell type showed colocalization of GLY-IR and GABA-IR and comprised about 4% of the total GLY-IR amacrine cell population.Comparison of GLY-IR and GABA-IR on serial sections showed that GLY and GABA were present in largely separate neuronal populations. Generally, GLY-IR amacrine cells were smaller, more distally located in the inner nuclear layer and had finer dendrites than GABA-IR amacrine cells. Distribution of GLY-IR and GABA-IR in the outer plexiform layer and the inner plexiform layer differed considerably.Based on the segregated distribution of GLY-IR and GABA-IR in the synaptic layers of the lizard retina, GLY and GABA may have fundamentally different roles in retinal processing.

The morphology and axon terminal arrangement of Golgi stained goldfish bipolar cells were examined to understand better the organization of bipolar cells in the inner plexiform layer (IPL) of the retina. Fifteen morphological bipolar cell types were identified, representing two major cell classes: mixed input cells that receive input from rod and cone photoreceptors, and cone bipolar cells that receive input from cones only. Mixed input bipolar cells comprised six types, including two new types, characterized by large somas and terminals. The terminals of mixed input bipolar cells terminated strictly within sublamina a or b of the IPL. Cone bipolar cells comprised nine subtypes, including seven new types, characterized by small somas and from one to four small terminal bulbs along the length of the axon, each having a characteristic termination depth in the IPL. The cone bipolar cell system had a complex multilaminar organization of terminals in the IPL, but maintained a high degree of anatomical symmetry about sublamina a and b. Cone bipolar cells could be divided into three groups: cells terminating within sublamina a and having an anatomically symmetrical counterpart terminating in sublamina b; cells with anatomically similar terminals in both sublamina a and b; and cells having no anatomically symmetrical counterpart or having anatomically dissimilar terminals in sublamina a and b. Based on bipolar cell terminal arrangement, we suggest that each bipolar cell type probably has a unique set of synaptic targets in the IPL, and that several bipolar cell types may be involved in functionally equivalent circuits at more than one level in the IPL.

To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1(-/-) /Tpst2 (-/-) ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.

Immunocytochemical and autoradiographic methods were used to identify neurons in the pure cone retina of the lizard (Anolis carolinensis) that are likely to employ glutamate (GLU) or aspartate (ASP) as a neurotransmitter.GLU immunocytochemistry demonstrated high levels of endogenous GLU in all cone types and numerous bipolar cells. Moderate GLU levels were found in horizontal and ganglion cells. Müller cells and most amacrine cells had very low GLU levels. GLU immunoreactivity (GLU-IR) in the cones was present from the inner segment to the synaptic pedicle. A large spherical cell type with moderate GLU-IR was identified in the proximal inner plexiform layer (IPL). These cells also contain ASP and have been tentatively identified as amacrine cells. Uptake of [3H]-L-GLU labeled all retinal layers. All cone types and Müller cells sequestered [3H]-D-ASP, a substrate specific for the GLU transporter.Anti-ASP labeling was observed in cones, horizontal cells, amacrine cells, and cells in the ganglion cell layer. ASP immunoreactivity (ASP-IR) in the cones was confined to the inner segment. One ASP-containing pyriform amacrine cell subtype ramifying in IPL sublamina b was identified.Analysis of GLU-IR, ASP-IR, and GABA-IR on serial sections indicated that there were two distinct populations of horizontal cells in the Anolis retina: one containing GABA-IR, GLU-IR, and ASP-IR; and another type containing only GLU-IR and ASP-IR. Light GLU-IR was frequently found in GABA-containing amacrine cells but ASP-IR was not.The distinct distributions of GLU and ASP may indicate distinctly different roles for these amino acids. GLU, not ASP, is probably the major neurotransmitter in the cone-biploar-ganglion cell pathway of the Anolis retina. Both GLU and ASP are present in horizontal cells and specific subpopulations of amacrine cells, but it is unclear if GLU or ASP have a neurotransmitter role in these cells.

In formal logic there is a premium on clever paraphrase, for it subsumes troublesome inferences under a familiar theory. (A paradigm is Davidson's analysis 1967 of inferences like ‘He buttered his toast with a knife; so, he buttered his toast’.) But the need for paraphrase in formal logic runs deeper than the odd recalcitrant inference, and thus, I shall argue, commits logicians to some interesting consequences. First, the thesis that arguments are valid in virtue of their form must be severely qualified (§4). And second, it is misleading to view a formal logical theory as a standard for justifying and criticizing inference (§7). The latter point depends on the nature and role of paraphrase, which permit a range of conflicting logical theories. Conflicting logical theories arise from the conflicting goals of logical theorists and the promiscuous nature of paraphrase makes reconciliation impossible.

Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells.

Immunoreactivities of two monoclonal antibodies (MAbs) that recognize cone photopigments were tested in the retinas of congenitally blind retinal degenerate ( rd ) chicks and compared to normally sighted carrier chicks, heterozygous for the mutation. MAb OS-2 had been previously determined to label rod and most cone outer segment membranes in normal chick retinas and is believed to bind to an epitope that is common to several photopigments in chickens. MAb COS-1 labels specifically middle-to-long-wavelength-sensitive cone photopigments in a number of vertebrate species. In rd chicks MAb OS-2 labeled the same number of rod outer segments at the same densities as carrier chicks. However, cone outer segments were less frequently and significantly less heavily labeled with this MAb at all ages tested (1 day, 1 week and 2 weeks post hatching). MAb COS-1 labeled the same number of cone outer segments in both rd and carrier retinas at 1 day of age, however, those outer segments that were labeled in rd specimens had significantly fewer gold particles on them. At both 1 week and 2 weeks of age, rd chick retinas had a significant reduction in numbers of cone outer segments labeled by COS-1. These findings support the hypothesis that the cone photopigment protein is abnormal in the rd chick model of hereditary blindness and retinal degeneration.

Recent studies indicate the presence of functional voltage-gated sodium channels (Na(v) channels) in the distal retina in several species. This study examined the distribution of Na(v) channels in the outer plexiform layer (OPL) of rat, mouse, and rabbit retinas.Immunohistochemical and electroretinographic approaches were used.Antibodies specific for Na(v)1 alpha-subunits appropriately labeled retinal ganglion cells, their axons, and amacrine cells that are known to have tetrodotoxin (TTX)-sensitive Na(v) channels. Pan-Na(v), Na(v)1.2, and Na(v)1.6 labeling was found in horizontal cells and processes in all three species. Weaker Na(v)1.1 labeling was observed in rodent horizontal cells, but some rabbit horizontal cells and processes were prominently labeled. Additional labeling for Na(v)1.1, Na(v)1.2, and Na(v)1.6 that was not attributable to horizontal cells was also present in the OPL. Much of this labeling was diffusely distributed. Some of the additional Na(v)1.1 labeling was associated with photoreceptor terminals. By exclusion using photoreceptor and horizontal cell markers, some of this labeling could have been associated with bipolar cell dendrites, although colocalization was not directly established due to the diffuse nature of the labeling and limits on anatomical resolution. No Na(v)1 alpha-subunit labeling was observed in bipolar cell bodies. Testing for functional Na(v) channels was performed by recording full field flash electroretinograms from dark-adapted rats before and after intravitreal injections of TTX, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or TTX+CNQX. TTX and CNQX+TTX, but not CNQX alone, greatly attenuated the dark-adapted cone-driven b-waves.Horizontal cells from three different mammalian retinas showed prominent labeling for Na(v)1 alpha-subunits. Some additional diffuse Na(v)1 alpha-subunit labeling in the OPL was associated with photoreceptor terminals. Na(v)1 alpha-subunit labeling also may have been present on bipolar cell dendrites, although it was not possible to establish this localization unequivocally by immunostaining. However, cone-driven b-waves in rats were reduced in maximum amplitude by TTX in the presence of CNQX which blocks synaptic input to horizontal, amacrine, and ganglion cells. This finding is consistent with TTX effects on the b-wave being due to blockade of Na(v) channels in cone bipolar cell dendrites in the OPL. The role of Na(v) channels in horizontal cells remains to be determined.

Background Syntaxins 1 through 4 are SNAP receptor (SNARE) proteins that mediate vesicular trafficking to the plasma membrane. In retina, syntaxins 1 and 3 are expressed at conventional and ribbon synapses, respectively, suggesting that synaptic trafficking functions differ among syntaxin isoforms. To better understand syntaxins in synaptic signaling and trafficking, we further examined the cell- and synapse-specific expression of syntaxins 1 through 4 in the mouse retina by immunolabeling and confocal microscopy. Results Each isoform was expressed in the retina and showed a unique distribution in the synaptic layers of the retina, with little or no colocalization of isoforms. Syntaxin 1 was present in amacrine cell bodies and processes and conventional presynaptic terminals in the inner plexiform layer (IPL). Syntaxin 2 was present in amacrine cells and their processes in the IPL, but showed little colocalization with syntaxin 1 or other presynaptic markers. Syntaxin 3 was found in glutamatergic photoreceptor and bipolar cell ribbon synapses, but was absent from putative conventional glutamatergic amacrine cell synapses. Syntaxin 4 was localized to horizontal cell processes in the ribbon synaptic complexes of photoreceptor terminals and in puncta in the IPL that contacted dopaminergic and CD15-positive amacrine cells. Syntaxins 2 and 4 often were apposed to synaptic active zones labeled for bassoon. Conclusion These results indicate that each syntaxin isoform has unique, non-redundant functions in synaptic signaling and trafficking. Syntaxins 1 and 3 mediate presynaptic transmitter release from conventional and ribbon synapses, respectively. Syntaxins 2 and 4 are not presynaptic and likely mediate post-synaptic trafficking.

Leukemia inhibitory factor (LIF) belongs to the interleukin-6 cytokine family, all members of which signal through the common gp130 receptor. Neurotrophic members of this cytokine family are known to arrest photoreceptor maturation and are likely to regulate maturation of other retinal neurons as well. We have used transgenic mice that constitutively express LIF beginning in embryonic development to determine its effects on synaptic organization and molecular maturation of all classes of retinal neurons. LIF reduced the numbers of cells showing markers characteristic of mature cells of all neuronal classes and caused synaptic ectopia. The net effect was disrupted morphological development and disturbed synaptic organization. Our study suggests that cytokines signaling through gp130 are capable of regulating many aspects of neuronal differentiation in the retina, including synaptic targeting.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.

Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system.

Many proteins associated with synaptic vesicle exocytosis are differentially distributed among synapses in the retina and elsewhere in the central nervous system. The synapse-specific distribution of these proteins and their isoforms is thought to contribute to synapse-specific functional differences. Vesicle-associated membrane protein (VAMP, also known as synaptobrevin) is an integral synaptic vesicle membrane protein that is part of the fusion core complex needed for docking and fusing of synaptic vesicles at the synaptic active zone. Two VAMP isoforms have been identified that are considered to be synaptic, VAMP-1 and VAMP-2, however their distributions among the various synapses in the mammalian retina have not been characterized.Single- and double-labeling immunocytochemistry was used to investigate the distribution of the synaptic VAMP isoforms, VAMP-1 and VAMP-2, in the mouse retina.VAMP-2 was the predominant isoform in both synaptic layers. Double-labeling studies using conventional and ribbon-synapse-specific markers showed that VAMP-2 was broadly distributed among conventional and ribbon synapses. In contrast, the distribution of VAMP-1 was very limited. In the outer retina, only weak labeling was present in photoreceptor terminals. In the inner retina, labeling for VAMP-1 was found in the dendrites, cell bodies, and axons of some ganglion cells, as demonstrated by double labeling with the ganglion cell markers, microtubule-associated protein-1 and Brn-3a. VAMP-1 labeling did not colocalize with amacrine or bipolar cell markers, nor did it colocalize with other pre-synaptic markers, suggesting that VAMP-1 is not associated directly with neurotransmitter release in the inner retina. Labeling for VAMP-1 identified a set of large ganglion cells that ramified in the mid-IPL (inner plexiform layer), suggesting that they may show ON-OFF responses. Some of these cells had cell bodies displaced to the inner nuclear layer. The dendrites of the large VAMP-1-immunoreactive ganglion cells did not co-stratify with the cholinergic plexuses of the starburst amacrine cells (labeled for choline acetyltransferase) and therefore are unlikely to show directional selectivity. However, these cells are likely to receive input from bipolar cells and a population of putative glutamatergic amacrine cells.VAMP-1 and VAMP-2 are differentially distributed among the synapses of the mouse retina. VAMP-2 is the predominant isoform and is widely expressed at ribbon and conventional synapses in both plexiform layers. VAMP-1 expression in the mouse retina is much more limited and is not restricted to presynaptic terminals. In the OPL, VAMP-1 is co-expressed with VAMP-2 presynaptically in photoreceptor terminals. However, VAMP-1 expression in the IPL is associated with ganglion cells and does not appear to be localized to presynaptic terminals. VAMP-1 is a specific marker for a set of large ganglion cells and displaced ganglion cells that ramify in the mid-IPL and are likely to have ON-OFF physiology.

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.

Retinal bipolar cells show neurochemical changes in response to retinal insult, which could have profound effects on retinal function or recovery after insult however, the neurochemistry of bipolar cells is incompletely understood. METHODS The distribution of the amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA), and glycine, and the metabolic amino acids, aspartate and glutamine, was examined immunocytochemically in the retina of the tiger salamander, a major model for the study of retinal anatomy and function.Each amino acid showed a unique distribution among retinal bipolar cells. All bipolar cells appear to contain glutamate and, under appropriate conditions, may also contain aspartate. GABA and glycine were restricted to a subset of conventionally placed bipolar cells located in the inner nuclear layer, but were not present in displaced bipolar cells located in the outer nuclear layer. In contrast, high levels of glutamine were almost exclusively restricted to displaced bipolar cells. Analysis of co-localization patterns revealed several different amino acid signatures within the bipolar cell population.The striking neurochemical heterogeneity among bipolar cells suggests important differences in signaling and metabolism that could affect cell survival, plasticity, and functional recovery from retinal insult in a cell-type-specific manner.