Cloning, expression, and localization of a rat brain high-affinity glycine transporter

A cDNA clone encoding a glycine transporter has been isolated from rat brain by a combined PCR and plaque-hybridization strategy. mRNA synthesized from this clone (designated GLYT1) directs the expression of sodium-and chloride-dependent, high-affinity uptake of [3H]glycine by Xenopus oocytes. [3H]Glycine transport mediated by clone GLYT1 is blocked by sarcosine but is not blocked by methylaminoisobutyric acid or L-alanine, a substrate specificity similar to that described for a previously identified glycine-uptake system called system Gly. In situ hybridization reveals that GLYT1 is prominently expressed in the cervical spinal cord and brainstem, two regions of the central nervous system where glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame of the GLYT1 cDNA predicts a protein containing 633 amino acids with a molecular mass of ≈70 kDa. The primary structure and hydropathicity profile of GLYT1 protein reveal that this protein is a member of the sodium- and chloride-dependent superfamily of transporters that utilize neurotransmitters and related substances as substrates

Prox1 Is a Marker for AII Amacrine Cells in the Mouse Retina

The transcription factor Prox1 is expressed in multiple cells in the retina during eye development. This study has focused on neuronal Prox1 expression in the inner nuclear layer (INL) of the adult mouse retina. Prox1 immunostaining was evaluated in vertical retinal sections and whole mount preparations using a specific antibody directed to the C-terminus of Prox1. Strong immunostaining was observed in numerous amacrine cell bodies and in all horizontal cell bodies in the proximal and distal INL, respectively. Some bipolar cells were also weakly immunostained. Prox1-immunoreactive amacrine cells expressed glycine, and they formed 35 ± 3% of all glycinergic amacrine cells. Intracellular Neurobiotin injections into AII amacrine cells showed that all gap junction-coupled AII amacrine cells express Prox1, and no other Prox1-immunostained amacrine cells were in the immediate area surrounding the injected AII amacrine cell. Prox1-immunoreactive amacrine cell bodies were distributed across the retina, with their highest density (3887 ± 160 cells/mm2) in the central retina, 0.5 mm from the optic nerve head, and their lowest density (3133 ± 350 cells/mm2) in the mid-peripheral retina, 2 mm from the optic nerve head. Prox1-immunoreactive amacrine cell bodies comprised ~9.8% of the total amacrine cell population, and they formed a non-random mosaic with a regularity index (RI) of 3.4, similar to AII amacrine cells in the retinas of other mammals. Together, these findings indicate that AII amacrine cells are the predominant and likely only amacrine cell type strongly expressing Prox1 in the adult mouse retina, and establish Prox1 as a marker of AII amacrine cells

Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter

We report that activators and inhibitors of protein kinase C (PKC) and protein phosphatases regulate the activity of a cloned rat brain gamma- aminobutyric acid (GABA) transporter (GAT1) expressed in Xenopus oocytes. Four compounds known to activate PKC increased GABA uptake 2- 3.5-fold over basal control levels. Inhibition of PKC by bisindolylmaleimide reduced basal GABA uptake 80% and blocked the phorbol 12-myristate 13-acetate (PMA)-induced stimulation of transport. Okadaic acid, a protein phosphatase inhibitor, stimulated transport 2.5- fold; a 4-fold increase in GABA uptake occurred when oocytes were treated with cyclosporin A, a specific inhibitor of protein phosphatase 2B. Modulation resulted in changes to Vmax but not to Km and was influenced by the functional expression level of the transporter protein; as expression level increased, the ability to up-regulate transporter activity decreased. Down-regulation of transporter activity was independent of expression level. Modulation did not occur through phosphorylation of the three consensus PKC sites predicted by the primary protein sequence since their removal had no effect on the susceptibility of the transporter to modulation by PMA or bisindolylmaleimide. Subcellular fractionation of oocyte membranes demonstrated that under basal level conditions, the majority of GAT1 was targeted to a cytoplasmic compartment corresponding to the trans- Golgi or low density vesicles. Stimulation of PKC with PMA resulted in a translocation of transporters from this compartment to the plasma membrane. At higher expression levels of GAT1 protein, a larger portion of GAT1 was found on the plasma membrane during basal level conditions and treatment with bisindolylmaleimide resulted in removal of these transporters from the plasma membrane. At expression levels demonstrated to be resistant to modulation by PMA, PMA-treatment still resulted in translocation of transporters from the cytoplasm to the plasma membrane. Thus, the inability of PMA to increase uptake at high expression of the GAT1 protein is due to saturation at a step subsequent to translocation. These findings 1) demonstrate the presence of a novel regulated secretory pathway in oocytes and 2) suggest a modulatory mechanism for neurotransmitter transporters that could have significant effects upon synaptic function

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells

Calcium Binding Protein-Like Immunoreactivity Labels the Terminal Field of Nucleus Laminaris of the Barn Owl

Nucleus laminaris (NL) is the site at which the timing of sounds arriving in the 2 ears is compared in the auditory system of the barn owl. Earlier studies have reported vitamin D-dependent calcium binding protein (CaBP)-like immunoreactivity in the somata of NL. We report here that CaBP-like immunoreactivity stains the terminal field of NL. The specific CaBP immunoreactivity is localized to a dense plexus of fibers that have bouton-like swellings, usually around unstained somata. This type of immunoreactivity is found in a restricted portion of the central nucleus of the inferior colliculus (ICc), in the anterior division of the ventral lateral lemniscal complex (VLVA), and in the superior olivary nucleus (SO), all of which have been shown by anterograde transport of 3H-proline to be innervated by NL. The immunoreactivity is absent from the posterior division of ventral lateral lemniscal complex and from the region that surrounds the portion of ICc innervated by NL. A restricted lesion in NL results in a localized deficit in immunoreactivity in those regions of ICc and VLVA that are known to be innervated by the lesioned area of NL. In adjacent sections processed by the Fink-Heimer method, degenerating axons are present in the region of the deficit in immunoreactivity

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.National Institutes of Health (U.S.) (K08 Award NS41411)Howard Hughes Medical Institute (Investigator

Modulation of Voltage-Gated Ion Channels in Rat Retinal Ganglion Cells Mediated by Somatostatin Receptor Subtype 4

Somatostatin (somatotropin release-inhibiting factor [SRIF]) is known to modulate the excitability of retinal ganglion cells, but the membrane mechanisms responsible and the extent to which intracellular calcium signaling is affected have not been determined. We show that somatostatin receptor subtype 4 (sst4) is expressed specifically in rat ganglion cells and that the generation of repetitive action potentials by isolated ganglion cells is reduced in the presence of L-803,087, a selective sst4 agonist (10 nM). Under voltage clamp, L-803,087 increased outward K+ currents by 51.1 ± 13.1% at 0 mV and suppressed Ca2+ channel currents by 32.5 ± 9.4% at −10 mV in whole cell patch-clamped ganglion cells. The N-type Ca2+ channel blocker ω-conotoxin GVIA (CTX, 1 μM) reduced L-type Ca2+ current (ICa) in ganglion cells by 43.5 ± 7.2% at −10 mV, after which addition of L-803,087 further reduced ICa by 28.0 ± 16.0% . In contrast, ganglion cells treated first with nifedipine (NIF, 10 μM), which blocked 46.1 ± 3.5% of the control current at −10 mV, did not undergo any further reduction in ICa in the presence of L-803,087 (−3.5 ± 3.8% vs. NIF), showing that stimulation of sst4 reduces Ca2+ influx through L-type Ca2+ channels. To assess the effects of sst4 stimulation on intracellular Ca2+ levels ([Ca2+]i) in ganglion cells, fura-2 was used to measure changes in [Ca2+]i in response to depolarization induced by elevated [K+]o. [Ca2+]i was increased to a lesser extent (86%) in the presence of L-803,087 compared with recordings made in the absence of the sst4 agonist and this effect was blocked by NIF (10 μM). Suppression of spiking and Ca2+ signaling via sst4 may contribute to the reported neuroprotective actions of somatostatin and promote ganglion cell survival following ischemia and axonal trauma

Differential calcium signaling mediated by voltage-gated calcium channels in rat retinal ganglion cells and their unmyelinated axons.

Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury

Differential calcium signaling mediated by voltage-gated calcium channels in rat retinal ganglion cells and their unmyelinated axons.

Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury