Alejandro Vila

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release. Mechanistically, we show that intracellular S1P promotes deoxygenated Hb anchoring to the membrane, enhances the release of membrane-bound glycolytic enzymes to the cytosol, induces glycolysis and thus the production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific glycolytic intermediate, which facilitates O2 release. Altogether, we reveal S1P as an intracellular hypoxia-responsive biolipid promoting erythrocyte glycolysis, O2 delivery and thus new therapeutic opportunities to counteract tissue hypoxia.

COMPARISON OF THE ONTOGENY OF THE VESICULAR GLUTAMATE TRANSPORTER 3 (VGLUT3) WITH VGLUT1 AND VGLUT2 IN THE RAT RETINA

Glutamate is the major excitatory neurotransmitter in the retina, and most glutamatergic neurons express one of the three known vesicular glutamate transporters (VGLUT1, 2, or 3). However, the expression profiles of these transporters vary greatly in the retina. VGLUT1 is expressed by photoreceptor and bipolar cell terminals, and VGLUT2 appears to be predominately expressed by ganglion cells, and perhaps Müller cells, cone photoreceptor terminals, and horizontal cells in some species. The discovery of a third vesicular glutamate transporter, VGLUT3, has brought about speculation concerning its role and function based on its expression in amacrine cells. To address this we studied the postnatal development of VGLUT3 from day 0 through adult in the rat retina, and compared this with the expression patterns of VGLUT1 and VGLUT2. VGLUT3 expression was restricted to a population of amacrine cells. Expression of VGLUT3 was first observed at postnatal day 10 (P10) in the soma and some processes, which extensively arborized in both the ON and OFF sublamina of the IPL by P15. In contrast, VGLUT1 and VGLUT2 expression appeared earlier than VGLUT3; with VGLUT1 initially detected at P5 in photoreceptor terminals and P6 in bipolar terminals, and VGLUT2 immunoreactivity initially detected at P0 in ganglion cell bodies, and remained prominent throughout all stages of development. Interestingly, VGLUT3 has extensive somatic expression throughout development, which could be involved in non-synaptic modulation by glutamate in developing retina, and could influence trophic and extra-synaptic neuronal signaling by glutamate in the inner retina.

Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes.

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity.

A Thy1 -CFP DBA/2J mouse line with cyan fluorescent protein expression in retinal ganglion cells

A DBA/2J (D2) transgenic mouse line with cyan fluorescent protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1-CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1-CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1-CFP transgene. CFP-fluorescent ganglion cells, 6-20 microm in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.

Adenosine inhibits voltage-dependent Ca2+ influx in cone photoreceptor terminals of the tiger salamander retina.

Endogenous adenosine has already been shown to inhibit transmitter release from the rod synapse by suppressing Ca2+ influx through voltage‐gated Ca2+ channels. However, it is not clear how adenosine modulates the cone synapse. Cone photoreceptors, like rod photoreceptors, also possess L‐type Ca2+ channels that regulate the release of L‐glutamate. To assess the impact of adenosine on Ca2+ influx though voltage‐gated Ca2+ channels in cone terminals, whole‐cell perforated‐patch clamp recording and Ca2+ imaging with fluo‐4 were used on isolated cones and salamander retinal slices. Synaptic markers (VAMP and piccolo) and activity‐dependent dye labeling revealed that tiger salamander cone terminals contain a broad, vesicle‐filled cytoplasmic extension at the base of the somatic compartment, which is unlike rod terminals that contain one or more thin axons, each terminating in a large bulbous synaptic terminal. The spatiotemporal Ca2+ responses of the cone terminals do not differ significantly from the Ca2+ responses of the soma or inner segment like that observed in rods. Whole‐cell recording of cone ICa and Ca2+ imaging of synaptic terminals in cones demonstrate that adenosine inhibited both ICa and the depolarization‐evoked Ca2+ increase in cone terminals in a dose‐dependent manner from 1 to 50 μM. These results indicate that, as in rods, adenosines ability to suppress voltage‐dependent Ca2+ channels at the cone synapse will limit the amount of L‐glutamate released. Therefore, adenosine has an inhibitory effect on L‐glutamate release at the first synapse, which likely favors elevated adenosine levels in the dark or during dark‐adapted conditions.

Synaptic vesicle dynamics in mouse rod bipolar cells.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of 7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Histamine Receptors of Cones and Horizontal Cells in Old World Monkey Retinas

In primates the retina receives input from histaminergic neurons in the posterior hypothalamus that are active during the day. In order to understand how this input contributes to information processing in Old World monkey retinas, we have been localizing histamine receptors (HR) and studying the effects of histamine on the neurons that express them. Previously, we localized HR3 to the tips of ON bipolar cell dendrites and showed that histamine hyperpolarizes the cells via this receptor. We raised antisera against synthetic peptides corresponding to an extracellular domain of HR1 between the 4th and 5th transmembrane domains and to an intracellular domain near the carboxyl terminus of HR2. Using these, we localized HR1 to horizontal cells and a small number of amacrine cells and localized HR2 to puncta closely associated with synaptic ribbons inside cone pedicles. Consistent with this, HR1 mRNA was detected in horizontal cell perikarya and primary dendrites and HR2 mRNA was found in cone inner segments. We studied the effect of 5 μM exogenous histamine on primate cones in macaque retinal slices. Histamine reduced Ih at moderately hyperpolarized potentials, but not the maximal current. This would be expected to increase the operating range of cones and conserve ATP in bright, ambient light. Thus, all three major targets of histamine are in the outer plexiform layer, but the retinopetal axons containing histamine terminate in the inner plexiform layer. Taken together, the findings in these three studies suggest that histamine acts primarily via volume transmission in primate retina. J. Comp. Neurol., 2012;520:528–543.

Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas

The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.