Date on Master's Thesis/Doctoral Dissertation

12-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Degree Program

Anatomical Sciences and Neurobiology, PhD

Committee Chair

McCall, Maureen

Committee Co-Chair (if applicable)

Bickord, Martha

Committee Member

Bickord, Martha

Committee Member

DeMarco, Paul

Committee Member

Gregg, Ronald

Committee Member

Krimm, Robin

Committee Member

Mellen, Nicholas

Author's Keywords

ganglion cell; electrophysiology; bipolar cell; LRIT3; vision; nyctalopin

Abstract

The retina processes light information through parallel pathways in order to extract and encode the visual scene. Light information is transmitted to the brain through approximately 30 ganglion cells (GCs), the retinal output neurons. Trp channels modulate the responses of retinal neurons within specific pathways. The study of the expression and function of the majority of Trp channels in the retina is largely in its infancy. My dissertation first investigated the expression and function of the transient receptor potential vanilloid-1 (TRPV1) receptor/channel in the retina. TRPV1, the first cloned and most highly studied Trp channel in the peripheral nervous system, is a non-selective cation channel with an affinity for Ca2+. The channel can be activated by capsaicin, acid, endovanilloids, noxious heat or pressure (Moreira et al., 2012). Located on the peripheral and central terminals of nociceptive fibers in the PNS and in limited areas of the CNS (Cavanaugh et al, 2011b). TRPV1 plays a role in inflammation, chronic pain, nociceptor sensitization and desensitization, long-term depression and potentiation, and apoptosis. The role of TRPV1 in the retina is not known. Using the electroretinogram (ERG), a mass potential that assesses the function of photoreceptors and bipolar cells, the TRPV1 knockout mouse appears normal. However, TRPV1 is thought to play a role in calcium regulation and glaucoma (Sappington et al., 2009 & Leonelli et al., 2010) so we investigated its role in normal visual transduction in the inner retina. To investigate TRPV1 modulation, I recorded GC spiking responses to light stimuli from mice which either express or lack TRPV1 protein. I found that TRPV1 is critical for: 1. GC responses to dim light. 2. Sustained responses to light 3. Surround suppression of GCs to large spots. Further, I investigate the specific retinal cells that express TRPV1. I used TRPV1cre mice with genetic or viral methods to fluorescently label neurons that express TRPV1. I determined TRPV1 is expressed in four classes of amacrine and three classes of ganglion cells in the inner retina. My results indicate TRPV1 activity in the amacrine cells enhances the sustained spiking responses in GCs. In this way, TRPV1 likely enhances the perception of subtle details in the visual world. TRPV1 also is expressed in subsets of intrinsically photosensitive GCs, which are known to play a role in circadian photoentrainment. TRPV1 therefore has the potential to modulate circadian photoentrainment or other non-image forming visual functions as well. The role of TRPM1 in the retina is well known. It is required for signaling through the ON pathway, which detects light increments. Responses through the vii ON pathway are initiated by synapses between rod and cone photoreceptors with ON bipolar cells (BCs). The human disease, complete congenital stationary night blindness (cCSNB) results from a disruption in signaling within the ON BC mGluR6 G-protein coupled cascade, which culminates in the opening of the TRPM1 channel and signaling through ON BCs. I helped expand our understanding of the role of TRPM1 in the retina by investigating the expression and function of leucine rich repeat immunoglobulin like transmembrane protein 3 (LRIT3), a novel protein component in the mGluR6-TRPM1 signalplex that was found mutated within cCSNB patients and a knockout mouse (Zeitz et al., 2013; Neuillé et al., 2014). The function of LRIT3 within the cascade remains unknown. To better understand the role of LRIT3, we examined retinal structure and function. We compared the structure of the pre and postsynaptic elements in the OPL of WT and Lrit3-/- mice using a variety of antibodies and with confocal microscopy. We assessed overall retinal function with ERG and GC spontaneous and visually evoked activity with single cell and multielectrode array recordings. The overall laminar structure of the Lrit3-/- retina is similar to WT. Consistent with published results and other cCSNB mouse models, Lrit3-/- mouse dark- and lightadapted ERGs have a normal a-wave, but lack a b-wave. The dendritic terminals of Lrit3-/- ON BCs lack expression of nyctalopin and TRPM1. Lrit3-/- mice significantly differ from other cCSNB mutants. Cone ON BCs lack expression of mGluR6, GPR179 and RGS11, whereas rod BCs maintain expression of these proteins. LRIT3 is necessary for expression and localization of nyctalopin and TRPM1 to the ON BC dendrites. As expected there are no ON responses, but surprisingly very few (~22%) Lrit3-/- GCs have even OFF responses. Lrit3-/- OFF BCs express functional kainate glutamate receptors. However, Lrit3-/- OFF BC and OFF GCs have significantly smaller response to light decrements than WT. Like all other mouse models of cCSNB, LRIT3 is critical to signaling in ON BCs, however, unlike all other cCSNB models, LRIT3 also has a trans-synaptic role in enhancing glutamate transmission from cones to BCs.

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