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Scholarly Works (14 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2014)

ABSTRACT OF DISSERTATION

Localization and contribution of voltage-gated calcium channels in retinal ganglion cells and their unmyelinated axons

by

Allison Michelle Sargoy

Doctor of Philosophy in Neurobiology

University of California, Los Angeles

Dr. Nicholas C. Brecha, Chair

Retinal ganglion cell (RGC) death has been attributed to aberrant calcium signaling in injury and disease. Calcium has a dual nature in mediating both homeostatic and apoptotic signaling pathways that modulate cell survival and cell death, respectively. The regulation of excessive calcium signaling through the inhibition of voltage-gated calcium channels (VGCCs) provides a potential strategy to reduce the loss of RGCs in injury and disease. My dissertation outlines the identification and contribution of the VGCCs, their modulation through the use of pharmacological blockers and the identification of an injury-resistant subtype of RGCs to provide a platform for future studies to investigate the expression and functional properties of VGCCs that may contribute to the superior ability to withstand injury.

Calcium channel expression studies that utilized immunohistochemical techniques localized the L-, P/Q- and N-type VGCCs to the RGCs and the L-type VGCCs to the RGC axons. Weak immunostaining of the N-type VGCC was detected in the RGC axons. Likewise, calcium imaging studies investigating the functional contributions of the VGCCs provided evidence for the L-, P/Q-, N- and T-type VGCCs to the RGCs and the L-type VGCCs to the RGC axons. Patch clamp analysis further confirmed the presence of T-type VGCCs in RGCs. Lastly, the survival of RGCs and their axons that underwent optic nerve transection was investigated to identify a RGC type that is more resistant to injury than any other RGC type in the retina. Immunohistochemical analysis further confirmed the M1 RGC as the most resistant RGC type in the retina to injury.

    Cover page: Localization and contribution of voltage-gated calcium channels in retinal ganglion cells and their unmyelinated axons
    • Thesis
    • Peer Reviewed
    UCLA Electronic Theses and Dissertations (2015)

    Amacrine cells form the most diverse group of interneurons in the retina. There are at least 30 identified types, which are differentiated based on their stratification patterns, neurotransmitter content, and soma and dendritic field sizes. Despite knowing the morphology of each amacrine cell type, our understanding of the connectivity, intrinsic, and functional properties of a majority of the amacrine cell types remain unclear. This study aims to investigate the intrinsic properties of neuropeptide-expressing amacrine cells, as well as their role in modulating inner retinal microcircuits. These studies will focus on two neuropeptide-expressing amacrine cells examined are the somatostatin-expressing (SRIF) and vasoactive intestinal polypeptide-expressing (VIP) amacrine cells.

    Previous studies have shown SRIF and VIP amacrine cells play a role in regulating dopamine levels or modulating GABA signaling in the inner retina. In this study we tested how SRIF amacrine cells can modulate the cells that comprise the light adaptation network: dopamine-expressing (DA) amacrine cells and melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). In addition, we used a novel transgenic mouse line to map the intrinsic electrophysiological properties and synaptic partners of VIP amacrine cells.

    In order to address these questions about SRIF- and VIP-amacrine cells in the network of cells in the inner retina, I employed a combination of anatomical, pharmacological, and electrophysiological manipulations in multiple transgenic mouse lines. The 3D modeling generated from antibody-labeled whole mount retinas showed the relationship of the processes of SRIF- and DA-amacrine cells, as well as the processes between SRIF amacrine cells and melanopsin ipRGCs. Using pharmacology and whole patch clamp protocols I showed SRIF, acting through specific SRIF receptor subtypes (sst2A and sst4), effectively increases K+ currents, decreases Ca2+ currents, as well as regulate the spontaneous firing rate of both cell types. In addition, SRIF can directly inhibit the intrinsic light response of melanopsin ipRGCs.

    Using the VIP-tdTomato transgenic mouse line we detail the ion channel composition of VIP- amacrine cells, which include delayed inward rectifying K+ channels, verapamil-sensitive L-type Ca2+ channels, a hyperpolarizing activated K= channel (Ih), and TTX-sensitive Na+ channel currents. The recorded VIP amacrine cells showed varying combination of these ion channels, suggesting there may be multiple subtypes of VIP amacrine cells. Finally, using a puff protocol, we showed VIP amacrine cells receive inhibitory inputs mediated by GABA and glycine. In addition, they receive excitatory input from type 2 OFF- and type 6 ON-cone bipolar cells, likely through activation of an ionotropic glutamate receptor subtype, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor.

      Cover page: Neuropeptide releasing amacrine cells modulate microcircuits in the inner retina
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (1996)
      The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT-1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.
        Cover page: Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cellsCreative Commons 'BY' version 4.0 license
        • Thesis
        • Peer Reviewed
        UCLA Electronic Theses and Dissertations (2012)

        In the retina, somatostatin (SRIF) is an inhibitory neuropeptide that influences multiple cell types, including bipolar, amacrine and ganglion cells. SRIF is reported to have multiple cellular actions including modulation of the release of dopamine (DA) from tyrosine hydroxylase (TH)-containing amacrine cells; however, the cellular basis of this interaction is unknown. Using immunohistochemistry I showed SRIF- and TH-containing wide-field amacrine cells co-stratify in the OFF sublamina of the inner plexiform layer (IPL) adjacent to the inner nuclear layer (INL). These processes form a dense network, which co-ramify and make numerous contacts along their processes and at varicosities. SRIF- and TH-immunoreactive (IR) cell bodies and processes also contain GABA and vesicular GABA transporter (VGAT) immunoreactivity, and express the GABAA α3 receptor subunit. TH-IR cells also express the SRIF receptor subtype 2A (sst2A) and SRIF-IR cells express the D1 receptor. Calcium imaging recordings of Fluo-4 labeled TH-red fluorescent protein (TH-RFP) processes exhibited a ~40% decrease in fluorescent intensity following 60 mM [K+] depolarization in the presence of SRIF (100nM-100 μM) and L054264 (1-10μM), a selective sst2A agonist. These findings suggest a reciprocal relationship between these two amacrine cell types mediated by both feedback and feed forward actions. SRIF amacrine cells act at TH-containing amacrine cells by a paracrine mechanism at sst2A, as well as directly at GABAA receptors. TH amacrine cells likely act at SRIF-containing amacrine cells at D1 and GABAA receptors. This retinal microcircuit defines a novel modulatory relationship for SRIF and demonstrates its broad influence on multiple visual processes.

          Cover page: Heterogeneous Functional Organization of Somatostatin- and Dopamine-containing Wide-field Amacrine Cells in Mouse Retina
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Increasing spike rates drive greater neuronal energy demand. In turn, mitochondrial ATP production leads to the generation of reactive oxygen species (ROS) that can modulate ion channel gating. Does ROS production autoregulate the excitability of a neuron? We investigated the links between retinal ganglion cell (RGC) excitability and spike activity-driven ROS production in male and female mice. Changes to the light-evoked and current-evoked spike patterns of functionally identified αRGC subtypes, along with their NaV channel-gating properties, were recorded during experimentally induced decreases and increases of intracellular ROS. During periods of highest spike rates (e.g., following light onset in ON sustained RGCs and light offset in OFF sustained RGCs), these αRGC subtypes responded to reductions of ROS (induced by catalase or glutathione monoethyl ester) with higher spike rates. Increases in ROS (induced by mercaptosuccinate, antimycin-A, or H2O2) lowered spike rates. In ON and OFF transient RGCs, there were no changes in spike rate during ROS decreases but increased ROS increased spiking. This suggests that endogenous ROS are intrinsic neuromodulators in RGCs having high metabolic demands but not in RGCs with lower energy needs. We identified ROS-induced shifts in the voltage-dependent gating of specific isoforms of NaV channels that account for the modulation of ON and OFF sustained RGC spike frequency by ROS-mediated feedback. ROS-induced changes to NaV channel gating, affecting activation and inactivation kinetics, are consistent with the differing spike pattern alterations observed in RGC subtypes. Cell-autonomous generation of ROS during spiking contributes to tuning the spike patterns of RGCs.SIGNIFICANCE STATEMENT Energy production within retinal ganglion cells (RGCs) is accompanied by metabolic by-products harmful to cellular function. How these by-products modulate the excitability of RGCs bears heavily on visual function and the etiology of optic neuropathies. A novel hypothesis of how RGC metabolism can produce automodulation of electrical signaling was tested by identifying the characteristics and biophysical origins of changes to the excitability of RGCs caused by oxidizing by-products in the retina. This impacts our understanding of the pathophysiology of RGC dysfunction, supporting an emerging model in which increases in oxidizing chemical species during energy production, but not necessarily bioenergetic failure, lead to preferential degeneration of specific subtypes of RGCs, yielding loss of different aspects of visual capacity.
            Cover page: Transient and Sustained Ganglion Cell Light Responses Are Differentially Modulated by Intrinsically Produced Reactive Oxygen Species Acting upon Specific Voltage-Gated Na+ Channel Isoforms.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2015)
            An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst(2A) and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH-RFP mice and M1 ipRGCs in OPN4-EGFP mice. SRIF increases K(+) currents, decreases Ca(2+) currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst(2A) agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4'-piperidine]-1'-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N(2)-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-L-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain.
              Cover page: Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2014)
              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.
                Cover page: Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2020)
                How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears to be mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control photoreceptor synaptic gain and enhance key aspects of temporal and spatial center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, the next key task in visual processing is the transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via horizontal cell feedback, which acts as a thermostat to keep the synaptic transmission in an optimal range during changes to light patterns and intensities. Novel findings of a recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse are reviewed. This novel inter-neuronal messaging system carries feedback signals using two separate, but interwoven regulated systems. The complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, has obscured it's being defined. The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential, a function of stimulus intensity and size, regulates inhibition of photoreceptor voltage-gated Ca2+ channels, and strong artificial pH buffering eliminates this action. This review compares and contrasts models of how these foundations are linked, focusing on a recent report in mammals that shows tonic horizontal cell release of GABA activating Cl- and HCO3 - permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated HCO3 - efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization in darkness and contributing to cleft acidification via NHE-mediated H+ efflux. This model challenges interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.
                  Cover page: Horizontal Cell Feedback to Cone Photoreceptors in Mammalian Retina: Novel Insights From the GABA-pH Hybrid Model
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2020)
                  Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors.
                    Cover page: Vesicular Release of GABA by Mammalian Horizontal Cells Mediates Inhibitory Output to Photoreceptors
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2011)
                    To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.
                      Cover page: Conical Tomography of a Ribbon Synapse: Structural Evidence for Vesicle Fusion
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