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The Heterogeneity of intrinsically photosensitive Retinal Ganglion Cells (ipRGCs)Ben Loreto & Kris Cronise

OutlineipRGCsFunctionHeterogeneity of ipRGCsAnatomy and PhysiologyModelFuture GoalsReferences and Acknowledgements

SIMPLIFY

ipRGCsClassical Photoreceptors (rods and cones)

Horizontal Cells,Bipolar Cells,And Amacrine Cells

Ganglion Cells

ipRGCs = intrinically photosensitive retinal ganglion cellsSource: http://www.skidmore.edu/~hfoley/images/Retina.jpg

pathway = LIGHT PHOTORECEPTORS HORIZONTAL BIPOLAR AMACRINE GANGLION OPTIC NERVE

Photoreceptors at the back of the eye2 types: the rods and cones. Their outer segment disks are the site of phototransduction. LIGHT HITS THIS FIRST Bipolar Cellstransfer all signals between photoreceptors and ganglionsHorizontal Cellstheir processes enable lateral interactions between photoreceptors and bipolar cells that maintain the visual systems sensitivity to luminance (AKA contrast)Amacrine cellsprocesses are postsynaptic to bipolar cell terminals and presynaptic to dendrites of ganglion cellsGanglion Cellstheir signals feed into the optic nerve

Some of the ganglion cells mentioned are intrinsically photosensitive, and thus make up a third class of photoreceptors - known as ipRGCs

Melanopsin Amination here

Like rods and cones which express their own photopigment, rhodopsin, ipRGCs use the photopigment melanopsin

ActivationLight converts 11-cis retinal in melanopsin to trans configurationthis allows for binding of a g protein (GP), in its inactive form bound to GDPit then exchanges GDP for GTP, thus activating the g proteinactivated g protein dissociates its alpha subunit from its beta and gamma subunits, and is then free to interact with phospholipase c (PLC)phospholipase c is then able to cleave pip2 into a second messengerthis second messenger is then able bind to calcium channels and allow them to open, resulting in an influx of extracellular calcium ions which depolarize the cellDeactivationIn response to a rise in calcium concentration, a kinase phosphorylates the melanopsin tail, thus allowing arrestin to bind with arrestin bound, the inactive g protein is unable to bind to melanopsin, thus stopping the cascade

Heterogeneity of ipRGCs5 major classes of ipRGCs

Each classified by certain differences in anatomy and physiology

Adapted from: http://www.nature.com/nature/journal/v415/n6871/images/415493a-f1.2.jpg

Ask about how diversity among iprgcs discoveredThis is a photoreceptive net made up of ipRGCs, obtained with immunoflouresence using antibodies labeled against melanopsinBack when Melanopsin was first discovered, it was believed that only one type of ipRGC existed, so this picture was believed to compose of only one type But as scientists have made more and more discoveries. It is now believed that this is a Mosaic of just 2 of the 5 types (M1 and M2) of Melanopsin containing ipRGCseach type of ipRGC is classified by a few of its differences in its anatomy and physiology (structure and function

Soma SizeSoma size = Size of cell body

Both figures modified from: Schmidt et al. (2011)

The comparative sizes of each class. M1=M5 is the smallest cell body, M4 is the largest. M2 & M3 intermediateOn the left is a picture of some examples of ipRGCs, m1, m2 and m4, captured using an Intracellular dye GFP,

Dendritic Stratification and FieldDendritic Stratification = Where signal is received in the retinaDendritic field = Amount of area covered by dendrites

Both figures modified from: Ecker. (2011)

Looking at this graph, we can tell the morphological differences between each melanopsin class:RGCs with dendrites stratifying in the outer sublamina of the IPL receive excitatory synaptic inputs from bipolar cells that respond to light decrements (OFF)whereas RGCs with dendrites stratifying in the inner sublamina of the IPL receive excitatory synaptic inputs from bipolar cells that respond to light increments (ON)Where each ipRGC class ramifies its dendrites.M1 stratify in the outermost sublamina of the IPL (OFF), M2 in the innermost sublamina (ON)M3 stratifies in both the inner and outer sublamina (OFF/ON)M4 and M5 stratify in the innermost sublamina (ON)Whats interesting is that M1 receives majority of ON signals, even when stratified in the off sublamina

Molecular IdentityMolecular Identity = Level of Gene ExpressionMelanopsin gene = e.g. Opn4

Both figures modified from: Schmidt et al. (2011)

level of OPN 4 expression amount of melanopsin expressionStrong Opn4+ Opntau-lacZ+ OpnCre+ Brn3b+/-Weak Opn4+ Opntau-lacZ- OpnCre+ Brn3b+Weak Opn4+ Opntau-lacZ+ OpnCre+ Brn3b+Opn4- Opntau-lacZ- OpnCre+ Brn3b+Opn4- Opntau-lacZ- OpnCre+ Brn3b+because of this M1 is the most well studied, because it expresses the most melanopsin

Intrinsic Light ResponseEvaluation of response to light

Traits include sensitivity based on both size of peak and duration

Modified from: Schmidt et al (2011)

corresponding whole cell voltage clamp recordings below, measured in pico amps, It measures 1 flash, for 20 seconds after flash.the logs correspond to intensity of logs, with 0 log having the highest intensityM1 cells have largest, most sensitive intrinsic light-evoked responseswhereas M2 cells have significantly smaller light responses that are at least one logarithmic unit less sensitive to light M1 cells have higher input resistance, a more depolarized resting membrane potential, and spike at lower frequencies than M2 cellsIn-depth electrophysiological analyses of the rare M3 cells demonstrated that these cells are remarkably invariable, which is surprising given the dendritic variability within this subtypeM4 and M5 have the smallest and least sensitive intrinsic light responses of the ipRGCsthey could be considered insensitive

Role in VisionRods and Cones = IF Vision

ipRGCs = NIF Vision

Adapted from: http://www.photobiology.info/Sengupta.html

IF vision = colors, shapes, textures, distanceNIF vision = brightness, and darkness

M1 ipRGCs innervate only the shell of the OPN, Non-M1 cells supply signals to the core of the OPN for PLRNon-M1 cells also contribute substantial synaptic input to the dorsal lateral geniculate nucleus (dLGN), a structure involved in image-forming vision, and the superior colliculus (SC), also involved in image forming vision

What Has Been DoneMethods:Chemical reactions based on hypothesized pathway

Example:

Conversion of chemical reactions to differential equations using mass action (and Michaelis-Menten kinetics)Example:

System of differential equations was solved in Matlab and the solution for the concentration of open channels was plottedParameters (rate constants) were adjusted to fit experimental data with the help of MATLAB function fminunc.

Based on the hypothesized melanopsin signaling pathway, Abbey and Jess came up with a list of chemical reactionsThey then converted these chemical reactions into differential equations using the law of mass action, and Michaelis Menten kineticsSolved the system of differential equations using Matlab, and plotted the solution for the concentration of open channelsThey then adjusted the parameters (or rate constants and initial conditions) to fit the experimental data

Law of Mass Action in chemical kinetics states that the rate at which a chemical is produced is proportional to the concentration of the reactants

What Has Been DoneResults: Note: Response of M1 cells

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Electrophysiological data obtained from Jess and AbbyElectrophysiological data obtained from R.L. Brown

(Left) Single flash HEK cellsHuman embryonic kidney (HEK) cells were transfected with mouse melanopsin gene.A calcium-sensitive dye was added to the cells and they were exposed to a flash of light, and they measured fluorescencemodel parameters (rate constants) then adjusted to fit this data(Right) Single flash ipRGCsElectrophysiological data obtained from collaborator R. L. BrownMeasured current across cell membrane of an ipRGC

Results:Model fits well with WT experimental data for a single flash.The same parameters are sensitive in both models

What Were Doing

Single flash data we obtained for HEK cells transfected with the murine (mouse) melanopsin gene we have yet to fit a model to our data

Further DirectionsComplete literature review of non-M1 ipRGCs

Compile differences among different ipRGC classes

Incorporate those differences into the model

ReferencesReferences:Schmidt. "Melanopsin-Positive Intrinsically Photosensitive Retinal Ganglion Cells: From Form to Function." The Journal of Neuroscience, 9 Nov. 2011. Web. 20 Oct. 2014.Wong. "Photoresponse Diversity among the Five Types of Intrinsically Photosensitive Retinal Ganglion Cells." The Journal of Physiology, 3 Feb. 2014. Web. 20 Oct. 2014.Hattar et al. "Melanopsin-Expressing Retinal Ganglion-Cell Photoreceptors: Cellular Diversity and Role in Pattern Vision." Neuron (2010): 49-60. Print.

AcknowledgementsWe would like to thank:UBM @ UMBCDrs. Phyllis Robinson Kathleen Hoffman, and Hye-Won KangAbigail Jackson and Jessica OrtegaPrevious CohortsThe Robinson Lab and its collaboratorsNSF