Neuromodulation of Ganglion Cell Photoreceptors

Home , Retinal ganglion cell, Retinohypothalamic tract

NEUROMODULATION OF

GANGLION CELL PHOTORECEPTORS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Puneet Sodhi, B.S.

Graduate Program in Neuroscience

The Ohio State University

2015

Dissertation Committee:

Dr. Andrew TE Hartwick, Advisor

Dr. Karl Obrietan

Dr. Stuart Mangel

Dr. Heather Chandler

Puneet Sodhi

2015

ABSTRACT

Intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a rare subset of ganglion cells in the mammalian retina that are primarily involved in non-image forming (NIF) visual processes. In the presence of light, ipRGC photoreceptors exhibit sustained depolarization, in contrast to the transient hyperpolarizing responses of rod and cone photoreceptors. The persistence of this response with light offset underlies the reduced temporal resolution exhibited by these ipRGCs. The overall aim of this thesis was to determine whether the unique temporal dynamics of ipRGC photoresponses are subject to modification by endogenous retinal neuromodulators. As post-synaptic photoreceptors, ipRGCs are capable of integrating photic information transmitted from pre-synaptic neurons regulated by rod- and cone-driven signaling. Given that ipRGCs possess dense dendritic nets that span the entire retina, I hypothesized that these ganglion cell photoreceptors were capable of being modulated by extrinsic input from the retinal network. Using multi-electrode array recordings on rat retinas, I demonstrated that the duration of light-evoked ipRGC spiking can be modified through an intracellular cAMP/PKA-mediated signaling pathway. Specifically, stimulation of the cAMP/PKA pathway leads to prolonged ipRGC light responses. Expanding upon these findings, I next identified an endogenous retinal neuromodulator capable of modulating ipRGC photoresponses through this signaling pathway. I demonstrated that the retinal neuromodulator adenosine suppressed light-evoked ipRGC spiking through activation of the Gi-coupled A1 receptor. These receptors were expressed by ipRGCs themselves, as

ii confirmed using immunohistochemistry and calcium imaging experiments on dissociated ipRGCs. Notably, I show that endogenous adenosine A1-mediated suppression of ipRGC photoresponses can occur during dark-adapted conditions, consistent with an elevation in retinal adenosine levels after maintenance in the dark. Dark adaptation serves to increase sensitivity and contrast detection in conventional photoreceptors.

However, in ipRGCs, dark adaptation leads to a global suppression of light-evoked responses that does not appear to be due to a reduction in sensitivity. I also showed that ipRGCs can be directly stimulated through a melanopsin-independent pathway by cholinergic activation of muscarinic receptors present on ipRGCs. The prolonged action potential firing evoked by cholinergic compounds was relatively specific to ipRGCs, as compared to other RGCs. Activation of these receptors by endogenous acetylcholine could be elicited by exposing the retina to yellow 6 Hz flickering light stimuli, as application of muscarinic antagonists blocked the robust ipRGC responses induced by this stimulus. Moreover, preliminary evidence suggests that these results could be translatable to humans as examination of pupillary constriction in two subjects revealed that 6 Hz flickering red light produces a greater mean pupil constriction than continuous red light exposure. Taken together, these studies indicate that ipRGC photoresponses are much more complex than previously appreciated. The temporal characteristics of these ganglion cell photoreceptors are not a static feature. Instead, ipRGCs are highly dynamic, capable of altering their responses to light in the presence of retinal neuromodulators such as adenosine and acetylcholine.

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DEDICATION

This dissertation is dedicated to my family and to my current and former mentors.

ACKNOWLEDGMENTS

First, I would like to acknowledge my past mentors for the tremendous guidance they provided during my undergraduate years. I thank Eva Feldman for taking the time to steer me towards Jack Parent’s lab, despite the fact that she receives hundreds of emails from undergraduate students looking for research experience and despite the lack of availability in her own lab. If it were not for her guidance, I would have never considered a career as a scientist. I would like to thank Dr. Jack Parent for his support and mentorship throughout my undergraduate studies. I would especially like to thank

Patricia Reuter-Lorenz for her mentorship throughout the duration of my time in her lab, as I completed my undergraduate thesis. She truly molded me as a researcher and inspired me to embark upon my current endeavor. I am thankful for her support to this day, as she continues to provide valuable insight.

I would truly like to thank Andrew Hartwick for his mentorship throughout my graduate studies. I greatly appreciate his zeal for scientific study and his approach to science as a whole, as I know he has shaped my growth as a scientist in a tremendous capacity. I appreciated the freedom I was afforded in the lab to pursue scientific problems that inspired me. I would also like to thank him for truly supporting my goals and career aspirations. He has molded his mentorship to fit my aspirations, which is uncommon for mentors and for that, I am very thankful. As his first graduate student, I am grateful for his support and close interactions throughout my time at Ohio State. I

v would also like to thank David Hackos for his support during my time at Genentech and for his continued mentorship and for his valuable insight as a scientist in industry.

I would like to thank my labmates, Patrick and Phil, for their support and friendship. I am especially grateful to Phil for his help with the pupil experiments presented in this thesis. Without him, these experiments would not have been possible.

I am grateful for the NGSP faculty at Ohio State for their expertise and support. I would specifically like to thank Karl Obrietan, Stuart Mangel, and Heather Chandler for taking the time to be on my committee. I thank them for their support and feedback on my work. I would especially like to thank Heather for her support and insight, both in relation to scientific matters and beyond, throughout my time at Ohio State.

I would like to thank my parents and my brother for their tremendous support and encouragement throughout the years I spent in higher education. Last but not least, I would like to thank my husband Kyle for his patience, encouragement, and insight throughout my graduate studies. As a physician scientist in training himself, he has provided invaluable insight and support during my training. I am forever grateful for his unrelenting encouragement both professionally and personally.

VITA

2005 ...... Troy High School

2009 ...... B.S. Neuroscience, Psychology (Honors),

University of Michigan

2009 to present ...... Graduate Research Associate, Department

of Neuroscience, The Ohio State University

PUBLICATIONS

1. Sodhi P and Hartwick TE (2014). Adenosine modulates light responses of rat ganglion cell photoreceptors through a cAMP-mediated pathway. Journal of Physiology, Epub PMID 25038240.

2. Sodhi P and Hartwick TE (2015). Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors. Journal of Neuroscience (in review)

FIELDS OF STUDY

Major Field: Neuroscience

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TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vi

List of Figures ...... vii

Chapters:

1. Introduction ...... 1

2. Modulation of ipRGC photoreseponses through a cAMP/PKA signaling pathway ...... 37

3. Adenosine modulates ipRGC photoresponses through a cAMP/PKA pathway ...... 60

4. Muscarinic acetylcholine receptor-mediated stimulation of ipRGCs ...... 91

5. Conclusions ...... 115

List of References ...... 133

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LIST OF FIGURES

Figure Page

1.1 Differences in vertebrate and invertebrate phototransduction…………………..35

1.2 Proposed model for ipRGC photoresponse regulation through adenosingeric and cholinergic signaling pathways ……………………………………………………………..36

2.1 Effect of spontaneous activity blockade on neonatal ipRGC light responses….54

2.2 Effect of forskolin, an adenylate cyclase stimulator, on neonatal ipRGC photoresponses……………………………………………………………………………….55

2.3 Role for cAMP and PKA in modifying neonatal ipRGC light responses………..57

2.4 Effect of synaptic activity blockade on adult ipRGC light responses…………....58

2.5 Role for cAMP in modifying adult ipRGC light responses………………………..59

3.1 Adenosine A1 receptor-mediated modulation of neonatal ipRGC light responses………………………………………………………………………………………82

3.2 Effects of adenosine on neonatal ipRGC light responses can be reversed by cAMP application……………………………………………………………………………....83

3.3 Adenosine A1 receptor-mediated modulation of adult ipRGC light responses….84

3.4 Adenosine inhibits light-evoked calicum responses from isolated ipRGCs in purified cultures………………………………………………………………………………...85

3.5 Adenosine A1 receptor expression on ipRGC somata in adult rat retinas……...... 87

3.6 Adenosine A1 receptor activation alters ipRGC photoresponses to light of varying intensities……………………………………………………………..………………………....88

3.7 Effect of presentation order, involving light stimuli of different irradiances, on ipRGC light-evoked spiking…..…………….………………………………………………...89

3.8 Extracellular endogenous adenosine induces global suppression of ipRGC light responses that can be reversed through A1 receptor antagonism……..………………...90

4.1 Carbachol-evoked spiking in rat ipRGCs…………………………………………..108

4.2 Robust carbachol-evoked spiking is specific to rat ipRGCs versus non- photosensitive RGCs………………………..………………………………………………..109

4.3 Carbachol’s effect on ipRGCs is mediated by Gq-coupled mAChR activation....110

4.4 Effect of carbachol on neonatal ipRGCs, using MEA retinal recordings and calcium imaging on isolated ipRGCs in purified cultures.………………………………..111

4.5 Effect of endogenous acetylcholine, stimulated by cholinesterase inhibition or use of flickering light stimuli, on ipRGCs ex vivo……………………………….………..……..112

4.6 Effect of continuous versus flickering light exposure on pupillary constriction in human subjects in vivo……………………………………………………………………….114

5.1 Potential neuromodulators that may regulate ipRGC light reseponses and ipRGC- driven behaviors………………………………………………………………………………132

CHAPTER 1

INTRODUCTION

Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs)

Intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a rare subset of ganglion cells in the mammalian retina that are primarily involved in non-image forming (NIF) visual processes. These cells, in addition to rods and cones, are classified as photoreceptors due to their ability to capture light and convert it into an electrical signal in a process called phototransduction. The classical view of mammalian phototransduction held to the principle that rod and cones were the only photosensitive cells in the retina. However, this view was overturned once ipRGCs were discovered. ipRGCs display duality in their ability to function as both a retinal ganglion cell (RGC), output neuron of the retina, and a photoreceptor. In order to understand the idiosyncrasies of this ganglion cell photoreceptor, I will provide an overview of ipRGCs and key concepts that relate to the neuromodulation of ipRGCs.

Discovery of an Inner Retinal Photoreceptor

Up until a little over a decade ago, scientists firmly believed that the mammalian visual pathway involved two photoreceptors— rods and cones. Despite great progress in the field of vision throughout the past century, this classical view of photoreception remained unchanged. However, there was one individual, Clyde Keeler, whose work suggested that a novel photoreceptor existed in the mammalian retina. As a graduate

1 student, Keeler bred mice in his dorm room that had a spontaneous gene mutation that caused serve degradation of rod and cone photoreceptors and rendered the mice functionally blind (Keeler, 1927). Surprisingly, when Keeler illuminated each eye, the pupils of these mice still constricted (Keeler, 1927). Given what was known about the retina at the time, there should have been no pupillary light reflex (PLR) as detection and signaling of light information could not occur if the photoreceptive cells were absent.

These astounding results led Keeler to the conclusion that there may be a yet undiscovered photoreceptor that mediated visual functions such as pupillary constriction

(Keeler, 1927, 1928).

Keeler’s work was largely ignored by the scientific community until Russell Foster illustrated that circadian rhythms, the synchronization of internal rhythms to the day/night cycle of the external environment, could be regulated independent of rod- and cone- driven signaling. Foster and colleagues utilized genetically engineered rd/rd mice, which mimicked the severe rod and cone degeneration of Keeler’s mice, to show that mice lacking classical photoreceptors were capable of circadian photoentrainment (Foster et al., 1991). Specifically, both wild-type and rd/rd mice were able to undergo circadian phase shifts, the shift from their current circadian rhythm to match the external environment, in response to photic input. While this supported Keeler’s findings, the maintenance of proper photoentrainment was assumed to be most likely due to a small population of remaining cones in the rd/rd mice. However, a follow up study demonstrated analogous photoentrainment with the use of rd/rd cl mice in which cones were selectively ablated (Freedman et al., 1999). Circadian disruption only occurred in the mice following enucleation, further supporting the existence of a novel ocular photoreceptor. These mice retained a variety of NIF visual reflexes such as the PLR,

2 suppression of melatonin secretion, and masking of locomotor behavior (Freedman et al., 1999; Lucas et al., 1999; Mrosovsky et al., 2001). Most importantly, the spectral sensitivity of the light stimuli needed to drive the PLR in rd/rd cl mice peaked near 480 nm, which did not match known murine rod (498 nm) and cone opsins (360 nm and 508 nm) (Lucas et al., 2001). The mouse studies also paralleled previous findings from a group of blind patients with advanced retinal degeneration that retained similar levels of light-induced melatonin suppression as seen in normal patients (Czeisler et al., 1995).

Taken together, these studies supported the potential existence of a novel photoreceptor in humans.

Interestingly, identification of the opsin involved in NIF functions came from studies in which Provencio and colleagues turned to Xenopus melanophores instead of the mammalian retina (Provencio et al., 1998). Non-mammalian vertebrates and invertebrates possess extra-ocular photosensitive cells. For example, several amphibians such as Xenopus express photopigments that are contained in dermal melanophores and respond to light (Oshima, 2001). Provencio and colleagues screened and isolated a novel protein from Xenopus melanophores that, like rod and cone photopigment, appeared to be a G protein-coupled opsin (Provencio et al., 1998). This opsin, named melanopsin (Opn4), was remarkably found to be more similar to invertebrate opsins and shared only 30% amino acid homology with vertebrate opsins

(Provencio et al., 1998). To extend such findings to mammalian vertebrates, melanopsin orthologs were cloned in mammals and in situ hybridization revealed melanopsin mRNA expression in a subset of RGCs in primate and murine retinas (Provencio et al., 2000).

Several novel opsins were identified in vertebrates, but of these only melanopsin was conserved in the mammalian genome (Peirson et al., 2009).

While numerous reports supported the existence of a novel photoreceptor, definitive proof remained elusive until two concurrently published studies identified a melanopsin photoreceptor in the mammalian inner retina (Berson et al., 2002; Hattar et al., 2002). Hattar and colleagues utilized genetically engineered mice to chart axonal projections from melanopsin-expressing RGCs. Specifically, mice were generated in which a construct encoding tau microtubule-associated protein and β-galactosidase supplanted the melanopsin coding region. Staining for β-galactosidase confirmed that axonal projections from melanopsin-expressing RGCs terminated in the brain, specifically in the suprachiasmatic nucleus (SCN) and regions involved in NIF visual functions (Hattar et al., 2002). Genetic approaches informed the morphological existence of melanopsin-expressing RGCs, yet electrophysiological studies examining the intrinsic light responses of these cells were crucial in establishing them as true photoreceptors

(Berson et al., 2002). Berson and colleagues recorded the light-evoked responses of

RGCs, retrogradely labeled by injecting florescent microspheres in the SCN, in the presence of a cocktail of synaptic blockers to eliminate rod- and cone-driven synaptic input. These SCN-projecting RGCs displayed an intrinsic light response that resulted in the cell depolarizing and firing action potentials, as opposed to the graded, hyperpolarizing responses seen in rods and cones (Berson et al., 2002). Furthermore, ipRGC photoresponses were found to have a peak spectral sensitivity of 480 nm, in accord with the action spectrum for the PLR in rd/rd cl mice (Lucas et al., 2001).

Heterogeneity among ipRGC Classes

Melanopsin-expressing photoreceptors exhibit a great deal of morphological and physiological diversity, which has led to the classification of these neurons into five

4 distinct subtypes— M1, M2, M3, M4, and M5. Of these, the M1-M3 are the best characterized based upon their stratification and melanopsin expression patterns. The

M4 and M5 cells are the most recently discovered with their attributes still being defined.

The M1 subtype comprises the ipRGCs initially described by Berson et al. in

2002. M1 cells are found predominately in the RGC layer, although displaced cells have been reported in the inner nuclear layer (INL) with photoresponses similar to conventionally placed M1 cells (Dumitrescu et al., 2009). M1 dendrites stratify in the outermost or OFF sublamina of the inner plexiform layer (IPL) in which OFF-bipolar cell axons reside. However, ON-bipolar cells comprise the majority of the synaptic input received by M1 cells through en passant synapses they form along M1 dendrites

(Dumitrescu et al., 2009; Hoshi et al., 2009; Grunert et al., 2011). While these cells are rare (~1% of rodent and 0.2% of human RGCs) and possess small somata (~15 μm), their dense dendrites converge to form a photoreceptive net that spans the entire retina

(Hattar et al., 2002; Provencio et al., 2002; Dacey et al., 2005; Berson et al., 2010). This dense dendritic net allows the sparse number of M1 cells to capture photons of light across a broad retinal area, in addition to receiving and integrating photic information as it traverses through rod/cone-driven retinal circuits.

Additionally, M1 cells express the greatest levels of melanopsin photopigment which explains their higher photon capture probability (Do et al., 2009) and ability to exhibit the largest intrinsic photoresponse of the ipRGC subtypes (Schmidt and Kofuji,

2009), though they are only able to fire action potentials at relatively low frequencies

(Wong et al., 2007; Schmidt and Kofuji, 2009). Greater melanopsin expression suggests that M1 cells heavily rely on intrinsic melanopsin-dependent signaling to drive their photoresponses. In support, the M1 cells of Opn4-/- mice, which are missing the

5 melanopsin photopigment, exhibit a substantial reduction in their light response while light responses of wild-type mice under synaptic blockade (which eliminates ON pathway input) remain unaffected (Schmidt and Kofuji, 2010, 2011).

While M1 and M2 cells are equally abundant (Viney et al., 2007; Baver et al.,

2008; Berson et al., 2010) and span the entire retina (Berson et al., 2010), they differ considerably in their morphology and physiological responses to light. M2 cells have larger somata (~20 μm), found in the RGC layer, and display great complexity in their dendritic arborization that spans a larger area than M1 cells (Schmidt and Kofuji, 2009;

Berson et al., 2010; Ecker et al., 2010; Muller et al., 2010). These cells stratify opposite to M1 cells in the innermost or ON sublamina of the IPL where they receive synaptic input from ON bipolar cells, as expected (Schmidt and Kofuji, 2010). M2 cells express lower levels of melanopsin than M1 cells (Do et al., 2009), which results in a one log unit reduction in sensitivity to light and a subsequently diminished intrinsic light response

(Schmidt and Kofuji, 2009). However, M2 cells are able to fire at a substantially greater frequency than M1 cells (Schmidt and Kofuji, 2009). Given the differences between M1 and M2 cells, M2 cells would appear to rely less on melanopsin-dependent signaling to drive their photoresponses and instead would be better suited for integrating synaptic input received from rod/cone-driven circuits. In support, the light responses of M2 cells in

Opn4-/- and wild-type mice are similar, indicating that rod and cone photoreceptors have a greater contribution in shaping M2 photoresponses (Schmidt and Kofuji, 2010, 2011).

M3 cells are similar to M2 cells in soma size, spiking patterns, and a reduction in their intrinsic light response as compared to M1 cells (Schmidt and Kofuji, 2011). Akin to

M2 cells, M3 cells depend on rod and cone photoreceptors to shape their light responses as these cells elicit similar photoresponses in Opn4 -/- and wild-type mice

(Schmidt and Kofuji, 2010, 2011). However, M3 cells differ from M1 and M2 cells as they are bi-stratified with dendrites that project to both the ON and OFF sublamina of the IPL

(Viney et al., 2008; Schmidt et al., 2008; Berson et al., 2010). Despite variability in their stratification patterns, M3 cells are homogenous in that ON input predominately drives their light responses (Schmidt and Kofuji, 2011). Interestingly, there is some debate as to whether M3 cells constitute a truly distinct subtype given their significantly lower cell count and gaps in their dendritic net that does not span their entire retina (Viney et al.,

2007; Berson et al., 2010). However, this criterion appears to be more relevant for IF visual processes as opposed to the NIF functions of ipRGCs (Do and Yau, 2010).

The M4 and M5 subtypes have been identified more recently as their low melanopsin expression was not detected by conventional immunostaining methods employed in the initial classification studies. Identification of these cells remained elusive until a mouse driver line expressing Cre recombinase exclusively in ipRGCs was crossed with a Cre-EGFP expressing reporter mouse line (see Ecker et al., 2010). This method revealed the M4 subtype, which stratify in the ON sublamina of the IPL and exhibit a comparable morphology to ON alpha RGCs. These cells contain larger somata and a greater area of dendritic arborization than M2 cells. The dendrites of M5 cells also stratify in the ON sublamina of the IPL, but they have more compact dendritic arborization than M4 cells. M4 and M5 ipRGCs display greater spontaneous firing at rest but weaker light-evoked inward currents than the M1-M3 subtypes (Zhao et al., 2014).

Melanopsin-dependent phototransduction

Phototransduction is a process by which light is captured and converted into an electrical signal by photoreceptors in the retina. Phototransduction varies greatly in

7 vertebrates and invertebrates, though both utilize G-protein receptor based mechanisms and analogous visual pigments that contain a vitamin A-based retinal attached to an opsin apoprotein. To appreciate ipRGC phototransduction, a thorough understanding of the intricacies of vertebrate and invertebrate phototransduction is necessary.

Vertebrates rely on ciliary photoreceptors, rods and cones, to initiate a signaling cascade that ultimately results in the closure of cyclic GMP-gated ion channels and subsequent hyperpolarization (see Lamb, 2009). In brief, photons of light are absorbed by rod or cone photopigment which causes the attached 11-cis-retinal to undergo isomerization to all-trans-retinal (see Figure 1.1a). This conformational change activates the photopigment, which in turn activates the G protein transducin that is present in photopigment-containing discs within the outer segments of the photoreceptors.

Activated transducin (Gt) then activates the effector phosphodiesterase (PDE), causing this enzyme to hydrolyze diffusible messenger cyclic GMP (cGMP) into 5’-GMP. This results in reduced cystolic cGMP levels within the photoreceptors that lead to subsequent closure of cGMP-gated channels located on the outer membranes of these cells. In the dark, there is a constant inflow of Na+2 through these channels in the photoreceptor outer segments, with a concurrent outflow of K+ through channels in the inner segments. The net result of this ‘dark current’ is a relative depolarization (-40 mV) of the resting membrane potential in the dark, as compared to a typical neuron at rest.

During light exposure, closure of cGMP-gated channels reduces Na+2 influx but outward

K+ flux remains. Thus, rods and cones hyperpolarize upon light stimulation. This hyperpolarization causes voltage-gated calcium channels to close and Ca+2 reduction results in decreased glutamate release at the axon terminal, relative to the amount released in the dark.

Conversely, in some invertebrates such as Drosophila, there are rhabdomeric photoreceptors that utilize an entirely separate signal transduction cascade. Like in vertebrates, Drosophila phototransduction is initiated when light is absorbed by the photopigment and the 11-cis-retinal chromophore is converted to all-trans-retinal (Figure

1.1b). However, in these photoreceptors, the activated photopigment activates a Gq protein (Hardie and Raghu, 2001) instead of Gt protein (transducin). Gq in turn activates phospholipase C-β (PLCβ), which catalyzes phosphatidylinositol 4,5-bisphosphate

(PIP2) into the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG).

DAG is further catalyzed into polyunsaturated fatty acid (PUFA). IP3 can increase cytosolic Ca+2 levels through Ca+2 release from stores (Yau and Hardie, 2009).

Ultimately, DAG, PUFA, and Ca+2 activate transient receptor potential (TRP) cation channels, which leads to Na+2 and Ca+2 influx and subsequent membrane depolarization

(Hardie and Minke, 1992; Phillips et al., 1992; Hardie and Raghu, 2001). However, the manner by which these intermediate factors activate TRP channels is a complex and varied process that is not yet well defined.

Interestingly, ipRGCs deviate from other vertebrate photoreceptors in terms of their phototransduction cascade. Evidence indicates a similar pathway for ipRGC phototransduction, as that described for Drosophila, consistent with the strong homology between invertebrate opsins and melanopsin (Provencio et al., 2000; Koyanagi et al.,

2005). The current view on melanopsin-dependent phototransduction involves the light- induced activation of Gq by melanopsin (Graham et al., 2008; Terakita et al., 2008) which in turn activates PLCβ4 (Graham et al., 2008; Xue et al., 2011) and ultimately leads to activation of a TRPC6/7 heterodimeric channel (Xue et al., 2011) that induces Ca+2 influx

(Warren et al. 2003; Sekaran et al., 2003, 2005; Hartwick et al., 2008) and results in

9 membrane depolarization (Berson et al., 2002; Hattar et al., 2002). In support, the light- induced current is occluded in ipRGCs when inhibiting either the Gq protein or PLCβ directly (Graham et al., 2008). However, the steps between PLCβ4 and TRPC6/7 activation remain unresolved, despite efforts by numerous groups. Until recently, there was also discrepancy among researchers as to whether TRPC3, TRPC6, TRPC7 or a combination of this family of TRPC channels represented the ipRGC light-gated channel.

Initial reports supported TRPC6 or TRPC7 (Warren et al., 2006; Hartwick et al., 2007;

Sekaran et al., 2007), though studies in heterologous systems also indicated TRPC3 channels might be involved (Melyan et al., 2005; Panda et al., 2005; Qiu et al., 2005).

However, a recent study found complete occlusion of the light-gated current in ipRGCs using a TRPC6/7-/- mouse, while partial occlusion was only observed in TRPC6-/- or

TRPC7-/- mice (Xue et al., 2011). This study provided the most compelling evidence that a hybrid TRPC6/7 channel is the ipRGC light-gated channel.

Given their distinct phototransduction cascade, it is not surprising that ipRGC photoresponses vary greatly from those of rod and cone photoreceptors. Most strikingly, melanopsin-expressing RGCs depolarize in response to light exposure (Berson et al.,

2002; Dacey et al., 2005) as opposed to rods and cones, which display graded hyperpolarizing responses characteristic of ciliary photoreceptors (Yau and Hardie,

2009). These depolarizing responses have markedly slow kinetics that are an order of magnitude slower than rod/cone responses (Berson et al., 2002). Response latency is inversely related to light brightness (Berson et al., 2002). However, under physiological conditions (37°C) ipRGC light responses are three times larger in amplitude and kinetics than at room temperature (Do et al., 2009). Thus, ipRGC recordings in this thesis were

10 performed at physiological temperature, as preparations at room temperature do not accurately represent endogenous photoresponses.

Though ipRGCs are capable of responding to a single photon of light, the threshold for activation is very high (Do et al., 2009). In addition, single-photon responses are exceptionally sluggish with response dynamics much slower than that of either invertebrate (Hardie and Postma, 2008) or vertebrate photoreceptors (20 times slower than rods, 100 times than cones) (Do et al., 2009). Therefore, ipRGCs are less sensitive than rods and cones to absolute irradiances, though their sensitivity to steady light is considerable given their long lasting responses (Dacey et al., 2005; Wong et al.,

2007; Do et al., 2009). ipRGCs are capable of light adaptation as illustrated by their response profile depicting a rise to peak firing levels followed by a plateau, a shortened time to peak in brighter light, and attenuation of photoresponses following repeated stimulation (Warren et al., 2003; Dacey et al., 2005; Wong et al., 2005; Do et al., 2009).

A key factor in the sluggish nature of ipRGC photoresponses may be attributed to the sparse and diffuse nature of melanopsin expression in the retina, which limits photon capture. Melanopsin is not concentrated within specialized structures like discs, as seen in ciliary photoreceptors. Instead, melanopsin is present in the soma, dendrites, and axons of ipRGCs (Bellingham et al., 2002). Thus, ipRGCs have adapted several compensatory mechanisms to attempt to overcome limitations due to photopigment availability. For example, the high gain or signal amplification of ipRGC photoresponses allows for more efficient responses to a single photon as compared to rods and cones

(Do et al., 2009). Moreover, ipRGCs exhibit prolonged integration times (~8 s for a single photon) which allows for the summation of photons over time yet renders these cells fairly insensitive to rapid changes in light intensity (Do et al., 2009). M1 cells are capable

11 of increasing their light-induced firing rate in response to single photon absorption (Do et al., 2009), yet are susceptible to depolarization block (Schmidt and Kofuji, 2009). Lastly, the resting membrane potential for ipRGCs is much closer to threshold than a typical neuron as evidenced by spontaneous firing in the absence of light (Do et al., 2009). By hovering near threshold, ipRGCs are “primed” to fire in response to single-photon events despite their low sensitivity. Even so, such mechanisms cannot fully compensate for diffuse melanopsin expression patterns that cause the poor ipRGC photon capture rate.

Synaptic connectivity of ipRGCs

The truly unique nature of ganglion cell photoreceptor light responses can be attributed to the duality between the role ipRGCs play as a RGC and a photoreceptor.

The light responses of ipRGCs are composed of both an intrinsic and extrinsic component. The intrinsic component is due to melanopsin-dependent phototransduction as described above. However, ipRGCs also receive and integrate extrinsic photic information transmitted from rods and cones (Goz et al., 2008; Guler et al., 2008; Hatori et al., 2008). Akin to non-photosensitive RGCs, but unlike rods and cones, ipRGCs are post-synaptic neurons that have extensive dendritic arbors (Berson et al., 2010) along which synaptic connections with retinal bipolar and amacrine cells occur (Belenky et al.,

2003; Wong et al., 2007). These properties afford ipRGCs a unique position to integrate network signals within the retina and transmit photic information regarding ambient light levels to the brain.

Bipolar and amacrine cells transmit rod and cone-driven signaling to ipRGCs in the IPL, which serves as a site of convergence for network effects in the retina (Do and

Yau, 2010). All ipRGCs receive both excitatory and inhibitory input, from bipolar and

12 amacrine cells, that is present at rest and during light exposure (Wong et al., 2007).

Environmental light is not uniform and therefore, each photoreceptor may be variably active during different lighting conditions. For example, scotopic vision occurs under low light conditions and is mediated by rods. As light levels increase, rods exhibit saturated responses. Thus, vision under more photopic conditions is instead mediated by cones.

Mesopic vision occurs in intermediate light levels in which both rods and cones contribute to visual signaling pathways. Of the three photoreceptors, ipRGCs are the least sensitive to absolute light levels and are thought to primarily function in the upper register of the photopic range. Given these distinctions, it is possible to predict that the extrinsic ipRGC light response is driven primarily by rods under more dim light and by cones as ambient light levels increase.

The cone-driven extrinsic ipRGCs photoresponses predominately originate from

ON- and OFF-type bipolar cells through excitatory glutamatergic synapses. Excitatory input to ipRGCs appears to be more sustained, as compared to transient glutamatergic input received by non-photosensitive RGCs (Wong et al., 2007). ipRGCs receive cone- driven input from ON bipolar cells, whose receptive fields span the entire dendritic arbor of these cells (Wong et al., 2007). ON bipolar cells dominate input to M1-type ipRGCs as input from OFF bipolar cells is minimal (Wong et al. 2007). In primates, bipolar cells form two times more synapses with M2 ipRGCs than M1 ipRGCs (Jusuf et al., 2007), in line with synaptic drive being more crucial for M2 cells to compensate for their lower sensitivity (Schmidt and Kofuji, 2009). In mammals, rod-driven signaling is transmitted through specific rod ON bipolar cells (Masland, 2001). In rodents, it has been shown that rod bipolar cells can form direct synaptic contact with ipRGCs (Ostergaard et al., 2007).

Inhibitory synaptic input to ipRGCs arises from the release of either GABA or glycine from amacrine cells, which induces an influx of chloride ions in ipRGCs (Wong et al., 2007). The resulting hyperpolarization can inhibit the excitatory bipolar cell input to ipRGCs. In ipRGCs, these chloride conductances are present in the dark and blockade of amacrine-driven signaling leads to an increase in basal spiking. During light exposure, inhibitory drive shapes ipRGC photoresponses as light-induced firing becomes stronger when signaling from amacrine cells is blocked. Thus, amacrine cells serve to inhibit ipRGC firing both in the dark and during light exposure. Interestingly, inhibitory drive to ipRGCs is more transient than to non-photosensitive RGCs, which contrasts the more sustained extrinsic excitatory drive to ipRGCs (Perez-Leon et al., 2006).

ipRGCs form a greater number of synapses with amacrine cells than bipolar cells

(Belenky et al., 2003; Jusuf et al., 2007). Distinct ipRGC subtypes can form synapses with specific amacrine cell subtypes. For example, some M1 cells are post-synaptic to dopaminergic amacrine cells, which release dopamine and GABA. Interestingly, M1 ipRGCs also form synapses with dopaminergic amacrine cells in which they are pre- synaptic neurons (Ostergaard et al., 2007; Viney et al., 2007; Vugler et al., 2007). On the other hand, M2 cells can be post-synaptic to mono-stratified amacrine cells (Viney et al.,

2007). ipRGCs also form electrical synapses with amacrine cells, as gap junction coupling between ipRGCs and GABAergic amacrine cells has been observed (Muller et al., 2010). Anatomical evidence that ipRGCs receive synaptic input from cholinergic starburst amacrine cells (SACs) has not been documented. However, I report that ipRGCs are capable of responding to acetylcholine, released in the retina only by SACs.

The distinct extrinsic and intrinsic components of the ipRGC light response can be discerned through electrophysiological recordings (see Wong et al., 2007). In bright

14 light, ipRGCs photoresponses exhibit an initial transient component that is driven by extrinsic synaptic input followed by prolonged, sustained firing mediated by the intrinsic melanopsin-based signaling cascade. Thus, with synaptic blockade of rod- and cone- driven signaling, only the latter component remains. Upon stimulation with a dim light that is below the threshold for melanopsin activation, only the initial burst of firing is observed. Overall, extrinsic input serves to extend the dynamic range of ipRGCs, allowing them to respond to dim and brief light stimuli beyond the range of detection required to elicit intrinsic melanopsin-dependent photoresponses. The transient nature of ipRGC photoresponses in low light reduces their susceptibility to depolarization block, further extending their dynamic range. These characteristics, and atypical sustained excitatory synaptic input, render ipRGCs to be well suited for prolonged signaling and likely aids their role as an irradiance detector.

Major Projections and Functions

The response properties of ipRGCs make these photoreceptors well suited to be irradiance detectors, as their responses correlate to ambient lights levels in the environment. As retinal output neurons, ipRGCs directly signal irradiance information to brain targets. Light-driven behaviors thought to be under ipRGC control are usually tonic and require long integration times, which is in line with the physiological properties of these RGC photoreceptors that are capable of signaling for 10 hours in vitro (Wong,

2012). Given that ipRGCs are involved in NIF functions, the vast majority of their projections terminate in brain regions that govern these processes. The most notable projections are to structures that regulate circadian photoentrainment, such as the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL), the PLR (olivary

15 pretectal nucleus, OPN), negative masking (ventral subparaventricular zone, vSPZ), and sleep (ventrolateral preoptic nucleus, VLPO) (see Sand et al., 2012). Initial reports indicated that ipRGCs sent very few fibers to image-forming visual centers such as the dorsal lateral geniculate nucleus (dLGN), unlike non-photosensitive RGCs (Hattar et al.,

2002, 2006). However, later studies have shown that ipRGCs do in fact innervate the dLGN and superior colliculus (Brown et al., 2010; Ecker et al., 2010), suggesting that ipRGCs may have a greater role in image-forming vision than previously appreciated.

Recent reports indicate ipRGCs may contribute to light brightness perception (Brown et al., 2010, 2012) and contrast detection (Schmidt et al., 2014), though extensive research is still needed to confirm these novel roles. Moreover, our understanding of the broad range of NIF functions under ipRGC control is far from complete, as these RGC photoreceptors have been found to mediate known effects of light on mood (Rockelin et al., 2009; LeGates et al., 2012), cognition (Vandewalle et al., 2009, LeGates et al.,

2012), alertness (Altimus et al., 2008), and light-aversive behaviors such as photophobia

(Johnson et al., 2010; Noseda et al., 2010; Semo et al., 2010; Matynia et al., 2012).

The pupillary light reflex (PLR) contributes to visual processing by reducing saturation of rods and cones to prolonged light exposure and by increasing depth of field and resolution (Do et al., 2010). As previously discussed, this unconscious reflex has proven to be critical in the identification of melanopsin-expressing photoreceptors and their contribution to NIF functions. The PLR is ideal as a functional readout for ipRGCs, as it is a very rapid response and non-invasive method to assess ipRGC-driven constriction. Studies have shown that ipRGCs largely contribute to the PLR under bright illumination, as would be expected. In Opn4-/- mice, the PLR is similar to wild-type mice except that these mice constrict to 80% of the initial pupil size and cannot achieve

16 complete pupil constriction (Lucas et al., 2003). In addition, the PLR in Opn4-/- mice exhibits a more transient period of peak pupil constriction before re-dilation occurs (Zhu et al., 2007), supporting the premise that the melanopsin-driven component of the PLR is necessary to produce sustained constriction. Mice that lack rods and cones display complete pupil constriction to bright light yet pupil responses to dimmer light levels are reduced or absent (Lucas et al., 2003; Panda et al., 2003; Semo et al., 2003). The PLR is abolished when signaling from all three photoreceptors is collectively disrupted (Hattar et al., 2003; Panda et al., 2003) or ipRGCs are selectively ablated (Guler et al., 2008).

These results demonstrate that all three photoreceptors contribute to the PLR, but ipRGCs are essential in serving as the conduit for the majority of the rod, cone, and melanopsin-driven light information that is conveyed to the OPN (Hattar et al., 2002;

Gooley et al., 2003). In addition, human studies provide further insight regarding the contribution of rods and cones versus ipRGCs. Upon exposure to light, rods and cones drive the initial, rapid pupil constriction (Alpern and Campbell, 1962) while ipRGCs mediate constriction during continuous light exposure (Mure et al., 2009; McDougal and

Gamlin, 2010). Collectively, these studies suggest that ipRGCs contribute to the PLR in a robust manner during sustained bright light exposures, driving complete and sustained pupil constriction

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), which serves as an endogenous clock in the brain. This clock synchronizes to environmental light cycle information conveyed to the SCN by ipRGCs. Melanopsin-expressing axons in the optic nerve form direct, glutamatergic synapses with the SCN through the retinohypothalamic tract (RHT). Projections to the SCN are composed almost entirely of melanopsin-positive axons (Baver et al., 2008; Hattar et al., 2006; Guler et al., 2008).

Overall, the magnitude of phase shifts (the shift from the current circadian cycle) increase as light becomes brighter. Melanopsin-driven signaling appears to be necessary for this process as the extent of these phase shifts reach a plateau in Opn4-/- mice, only shifting halfway (50%) to the new cycle, as compared to wild-type mice

(Panda et al., 2002; Ruby et al., 2002). In addition, rodless/coneless mice and rats display circadian photoentrainment (Barnard et al., 2004; Freedman et al., 1999; Hattar et al., 2003; Panda et al., 2003; Semo et al., 2003) which occurs with an action spectrum that is closer to ipRGCs than rods and cones (Yoshima and Ebihara, 1996). Moreover, ablation of ipRGCs eliminates photoentrainment, further supporting the need for these cells in carrying irradiance information (including rod/cone-driven signals) to the SCN

(Hatori et al., 2008). Rods and cones play a far greater role in the PLR, as they are capable of driving 80% maximal constriction. However, their contribution to photoentrainment peaks at a 50%, in terms of phase shifts (Panda et al., 2002). Taken together, these studies indicate ipRGCs are imperative for photoentrainment of the mammalian circadian system.

Modulation of Neuronal Output in the Retina

The distinction between a neurotransmitter and a neuromodulator lies in their modes of action. Neurotransmitters are chemical messengers released from synaptic vesicles in the pre-synaptic neuron, which then traverse the synaptic cleft to bind to receptors on one or two post-synaptic neurons. Conversely, neuromodulators released from a single neuron are capable of affecting multiple post-synaptic neurons due to their diffusion after release. Once bound to receptors, neuromodulators typically trigger secondary messenger cascades to produce long-lasting effects. As examples, glutamate

18 and GABA are considered common excitatory and inhibitory neurotransmitters, respectively, with tightly regulated actions occurring through ionotropic receptors. In contrast, dopamine and adenosine predominately act as neuromodulators, generating prolonged effects through metabotropic receptors.

In the retina, modulation of neuronal excitability and sensitivity to light exposure exists to extend the dynamic range of retinal neurons. Namely, light adaptation occurs in photoreceptors (Baylor and Hodgkin, 1974) and network adaptation in interneurons and

RGCs (Barlow and Levick, 1969; Sakmann and Filion, 1972; Green et al., 1975), in which a reduction in light sensitivity is seen as a function of luminance intensity. This serves to improve contrast to enable object detection amongst varying intensities of background luminance and eliminates response saturation (Shapley and Enroth-Cugell,

1984). Conversely, photoreceptor dark adaptation results in a gradual increase in neuronal sensitivity. Generally, optimal sensitivity is achieved during brief, dim pre- adapting light exposure followed by prolonged dark exposure. Interestingly, network adaptation can occur at dimmer luminance intensities and is effective for longer durations than photoreceptor light adaption (Pirenne, 1958; Sakmann and Filion, 1972;

Green et al., 1975). Overall, adaptation to ambient illumination occurs at all three retinal levels as seen in photoreceptors (Baylor and Hodgkin, 1974), interneurons (Ashmore and Falk, 1980; Piccolino et al., 1984) and RGCs (Vaquero et al., 2001).

Cyclic AMP (cAMP)-mediated modulation has been observed both at the photoreceptor and ganglion cell level. Changes in internal cAMP levels modulate the light responses of Drosophila and Limulus photoreceptors (Chyb et al., 1999; Battelle,

2002), suggesting that this may be a common feature of invertebrate photoreception.

Given the homology between melanopsin and invertebrate opsins, ipRGC light

19 responses may be subject to similar cAMP-mediated modulation. Moreover, cAMP has been shown to play a role in network adaptation at the ganglion cell level in vertebrates.

Light adaptation increases cAMP immunoreactivity in the RGC layer and increased cAMP, through dopaminergic activity, alters firing in non-photosensitive RGCs (Vaquero et al., 2001). Thus, cAMP modulates both neuronal excitability in RGCs and sensitivity to light in invertebrate photoreceptors.

Various mechanisms exist to regulate RGC excitability to allow these retinal output neurons to adjust to changes in input and avoid saturation. For example, hyperpolarization- activated channels produce Ih currents in non-photosensitive RGCs and ipRGCs that serve to boost excitability, as increased spiking is observed following hyperpolarization (Eng et al., 1990; O’Brien et al., 2002; Skaliora et al., 1993; Tabata and Ishida, 1996). Conversely, prolonged depolarization leads to accommodation or a reduction in spike frequency as a function of time. Interestingly, ipRGCs are unique in that they are retinal output neurons that are subject to modulation, yet they themselves modulate other retinal neurons. Photic information flow is generally one way, leaving the retina through RGCs that are entirely post-synaptic within the retina. However, ipRGCs have been shown to couple to GABAergic amacrine cells, participate in reciprocal dopaminergic amacrine cell modulation, and even modulate cone response (Do and

Yau, 2010). Thus, the highly dynamic bidirectional photic information flow from ipRGCs may regulate diverse functions that have yet to be discovered.

Adenosine as a Retinal Neuromodulator

Overview

The purine nucleoside adenosine is a known neuromodulator in the central nervous system (CNS). Adenosine is not considered a true neurotransmitter due to the lack of evidence demonstrating its storage in vesicles, calcium dependent release, and synapses where it acts as the primary neurotransmitter (see Latini and Pedeta, 2001 and Fredholm et al., 2001 for review). Intracellular and extracellular production of adenosine primarily occurs through the degradation of AMP by endo- and ecto- nucleotidases, respectively. The conversion of AMP to adenosine represents the rate- limiting step of adenosine triphosphate (ATP) catabolism (Dunwiddie et al., 1997). ATP is also a purine nucleoside that can be released as a transmitter or co-transmitter and is known to affect purinergic receptors partly through direct binding at ATP receptors (P2 subtype), and following conversion to adenosine, at adenosine receptors (P1 subtype)

(Nikbakht and Stone, 2000). ATP can be co-released with classical neurotransmitters such as acetylcholine, dopamine, serotonin, and norepinephrine (White, 1977; Fredholm et al., 1982) and is capable of both inhibitory and excitatory signaling in the CNS.

Adenosine accumulation in extracellular space of the brain occurs through two primary mechanisms, release through equilibritive nucleoside transporters (ENTs) (Cass et al., 1998; Baldwin et al., 1999), primarily the ENT1 subtype (Anderson et al., 1999;

Sinclair et al., 2001), or formation following enzymatic conversion of ATP by extracellular ecto-nucleotidases (Housley et al., 2009). Ectoenzymes are very rapid, converting ATP to adenosine in less than a second (Dunwiddie et al., 1997). Removal of adenosine from the extracellular space requires reuptake and subsequent intracellular breakdown of adenosine by adenosine deaminase or adenosine kinase (Dunwiddie and Masino, 2001;

Latini and Pedata, 2001). However, inhibition of adenosine deaminase has little effect on extracellular adenosine concentrations (Park et al., 1994; Zhu and Krnjevic, 1994).

Instead, reuptake through nucleoside transporters is important, as reuptake inhibitors have been shown to increase the concentration of extracellular adenosine considerably

(Dunwiddie and Diao, 1999) by occluding adenosine influx. Net flux of adenosine is typically directed inward due to the high catalytic activity of adenosine kinase, which maintains low levels of intracellular adenosine (Dunwiddie and Masino, 2001).

Purinergic adenosine receptors are G-protein coupled receptors that can be subdivided into two groups, based upon their signal transduction pathways. Activation of

A1 and A3 adenosine receptors is typically linked to inhibition of the enzyme adenylate cyclase, which causes subsequent lowering of intracellular cyclic AMP (cAMP) levels, while A2A and A2B receptor activation leads to increased cAMP generation (Fredholm et al., 2001). Adenosine receptors have a wide distribution in the CNS with the A1 receptor having the broadest distribution of the receptor subtypes. The A1 receptor is most abundant adenosine receptor in the brain. On the other hand, A2A and A2B receptors are localized to distinct regions of the brain such as the striatum (Fink et al., 1992; Marala and Mustafa, 1993). A3 receptors are also expressed throughout the brain, but have a far lower expression level and affinity for binding adenosine (see Pedata et al., 2009).

Modulation in the Central Nervous System

Adenosine alters neuronal excitability through activation of both pre- and post- synaptic adenosine receptors. Due to its pre-synaptic actions, adenosine is most commonly associated with being an inhibitory neuromodulator in the brain (Trussell and

Jackson, 1985; Gerber et al., 1989; Greene and Haas, 1991; Zimmermann, 1994;

Cunha, 2001). At pre-synaptic terminals, adenosine has been shown to modulate the release of excitatory neurotransmitters (Goodman et al., 1983) such as glutamate, acetylcholine, and serotonin (Santos et al., 2000; Okada et al., 2001; Marchi et al., 2002) and inhibitory neurotransmitters (GABA) primarily through activation of A1 receptors

(Oliet and Poulain, 1999; Poli et al., 2001). Activation of pre-synaptic A1 receptors leads to decreased neurotransmitter release, largely of glutamate (e.g. Dunwiddie and Hoffer,

1980; Kocsis et al., 1984; Obrietan et al., 1995), and attenuation of neuronal excitability through inhibition of N-type voltage-gated calcium channels (VGCCs) (Macdonald et al.,

1986). Conversely, pre-synaptic A2A receptor-induced increases in neurotransmitter release and activation of P-type VGCCs has also been observed in several brain regions

(Cunha, 2001; Solinas et al., 2002; Quarta et al., 2004). Post-synaptically, adenosine attenuates neuronal excitability through activation of A1 receptors, which in turn activate

G protein-coupled inwardly rectifying K+ channels (GIRKs) and subsequently lead to hyperpolarization of the membrane potential (Trussell and Jackson, 1985; Luscher et al.,

1997; Cunha, 2001; Ponzio and Hatton, 2005).

In the CNS, adenosine plays a prominent role in the regulation of sleep

(Shiromani et al., 1998; Pace-Schott and Hobson, 2002). Adenosine contributes to sleep-wake homeostasis, the homeostatic drive to sleep that is present upon waking and progressively increases throughout the day. This drive is thought to be due to the gradual accumulation of adenosine that peaks prior to sleep, upon which it dissipates

(Porkka-Heiskanen et al., 1997; Porkka-Heiskanen, 1999). Adenosine modulates sleep through adenosine receptor-mediated inhibition of cholinergic neurons that promote arousal or wakefulness (Rainnie et al., 1994). Overall, adenosine receptor antagonists support wakefulness (Lin et al., 1997) while agonists support sleep (Portas et al., 1997),

23 due to opposing actions on cholinergic neurons. However, non-cholinergic regulation of sleep has been shown to occur in the preoptic area as adenosine or A1 agonists can reduce sleep latency and increase sleep duration (Ticho and Radulovacki, 1991;

Mendelson, 2000). While A1 receptors are the primary receptor implicated in promoting sleep, A2A receptors may also play a minor role (Dunwiddie and Masino, 2001).

Evidence as a Retinal Modulator

Purinergic signaling has been observed in the retinas of several species

(Blazynski and Perez, 1991). Moreover, the existence of enzymes required for adenosine metabolism and expression of adenosine transporters indicates that endogenous production and release of adenosine occurs in the retina. In the retina,

Muller glial cells (Newman 2001, 2002, 2003), cholinergic ‘starburst’ amacrine cells

(Neal and Cunningham, 1994; Santos et al., 1999), and perhaps some RGCs (Newman,

2005) are the major sources of ATP. ATP released from these neurons is then rapidly converted to adenosine in the extracellular space. All four adenosine receptors are found throughout the mammalian retina, with the A1 and A3 expressed in RGCs (Braas et al.,

1987; Blazynski and Perez, 1991; Zhang et al., 2006). Studies utilizing immunohistochemistry and autoradiography demonstrated that adenosine and A1 receptors are especially prevalent in the ganglion cell layer (Braas 1986, 1987;

Blazynski, 1989; Blazynski and Perez, 1991).

In the rabbit retina, the concentration of extracellular adenosine is differentially regulated through the circadian clock and environmental lighting conditions (Ribelayga and Mangel, 2005). Environmental lighting conditions modulate extracellular adenosine levels with peak adenosine accumulation present during dark adaptation. Following dark

24 adaptation, exposure to background light in the photopic range was shown to reduce extracellular adenosine and increased light intensity heightened this inhibitory effect.

However, complete suppression of adenosine is not observed with background illumination, as basal levels of adenosine are still detected under light-adapted conditions. This is due to the fact that tonic ATP release and subsequent conversion to adenosine occurs in the extracellular retinal milieu, not the intracellular space, across variable environmental light exposure conditions. Thus, adenosine is only undetectable once ATP is completely removed. Conversely, circadian clock controlled increases in extracellular adenosine are predominately due to a reduction in the net flux of adenosine directed inward rather than an increase in the extracellular production of adenosine.

Thus, adenosine levels peak during the circadian night as compared to the circadian day, when tonic levels of adenosine are still present. Overall, the circadian clock and environmental lighting conditions work in congruence to maximize retinal adenosine levels at night and during prolonged darkness (Ribelayga and Mangel, 2005).

A substantial portion of the literature on adenosine and its effects on retinal neurons comes from developmental studies. Most notably, adenosine has been shown to regulate the spontaneous waves of neural activity that sweep across the retina during development (Stellwagen et al., 1999). These spontaneous waves of correlated RGC spiking activity are critical for proper segregation and refinement of retinal circuitry (Ford and Feller, 2012). Adenosine appears to contribute to wave activity during the earliest stage of retinal development, when waves are propagated through gap junction coupling. Specifically, adenosine activates A2 receptors on amacrine cells and RGCs, which in turn drives waves in a cAMP-mediated manner (Stellwagen et al., 1999) as evidenced by elevated intracellular cAMP in retinal neurons (Dunn et al., 2006).

Moreover, adenosine modulates electrical coupling beyond retinal development. In the mature outer retina, adenosine participates in photoreceptor coupling as a function of the circadian clock and environmental lighting conditions. Photoreceptor coupling serves to improve signal to noise and increase the dynamic range of rods during the night and under dark adapted-conditions, when coupling is maximal as opposed to during light adaptation (Wang and Mangel, 1996; Ribelayga et al., 2008). Adenosine and dopamine work in opposition to regulate coupling through gap junction phosphorylation and PKA- dependent signaling (Li et al., 2009, 2013). During the night under dark-adapted conditions, the activation of A2A receptors on photoreceptors by adenosine causes gap junction phosphorylation and subsequent photoreceptor coupling (Li et al., 2013, 2014).

The converse is true during light-adapted conditions, when dopamine activates D4 receptors to induce and maintain photoreceptor uncoupling (Li et al., 2013, 2014).

Akin to its actions in the CNS, adenosine modulates neuronal excitability following activation of pre- and post-synaptic A1 receptors in the retina. Pre-synaptic A1 receptors have been shown to modulate acetylcholine (ACh) release in cultured cholinergic amacrine-like neurons through inhibition of N-type VGCCs that regulate ACh release (Santos et al., 1999; 2000). Adenosine was found to only inhibit ACh release in these cholinergic amacrine-like neurons and not GABA, a co-transmitter released from starburst amacrine cells (Santos et al., 2000). Evidence of pre-synaptic neuromodulation has also been observed through Muller glia-mediated activity. Stimulation of Muller glia with non-hydrolysable ATP substrates results in inhibition of RGCs (Newman, 2004).

While modulation can occur through pre-synaptic mechanisms, adenosine- mediated modulation of RGC excitability predominately occurs through activation of post-synaptic A1 receptors (Sun et al., 2002; Hartwick et al., 2004; Newman, 2003; Clark

26 et al., 2009). In the inner retina, Muller glia modulate excitability through activation of post-synaptic A1 receptors expressed on RGCs (Newman, 2003, 2004; Clark et al.,

2009). Specifically, ATP released from Muller glia (Cotrina et al., 2000; Newman, 2001) activates A1 receptors on RGCs and subsequently leads to hyperpolarization (Newman,

2003) through activation of GIRK and small conductance calcium-activated potassium

(SK) channels (Clark et al., 2009). Ultimately, this results in suppression of RGC excitability as evidenced by the reduction in light- and depolarization-evoked firing (Clark et al., 2009). Furthermore, adenosine can modulate RGC excitability through inhibition of voltage-gated calcium channels (Sun et al., 2002; Hartwick et al., 2004). In rodent retinas, stimulation of A1 receptors on non-photosensitive RGCs results in the inhibition of voltage-gated calcium channels and glutamate-induced calcium inﬂux (Hartwick et al.,

2004). This previous work indicates that adenosine suppresses excitatory neurotransmission at the RGC level, but the effect of this neuromodulator on melanopsin-based irradiance detection by ipRGCs is currently unknown.

Cholinergic Regulation in the Inner Retina

Overview

Acetylcholine (ACh), the first characterized neurotransmitter, functions in the peripheral and central nervous system. ACh is synthesized from choline and acetyl-CoA by cholinergic neurons that contain the enzyme choline acetyltransferase (ChAT).

Following synthesis, ACh is packaged and stored in vesicles through uptake via a vesicular ACh transporter (VAChT). Upon stimulation, ACh is released in a calcium dependent manner that is typical of classical neurotransmission. Clearance from the extracellular space requires degradation by the enzyme acetylcholinesterase (AChE)

27 into acetate and choline. The exceptionally high catalytic rate of AChE (1 AChE molecule degrades 25,000 ACh molecules per second) allows for the rapid recycling of choline for subsequent ACh synthesis (see Taylor and Radic, 1994 for review).

Cholinergic signaling occurs through ionotropic nicotinic (nAChR) and metabotropic muscarinic (mAChR) receptors. nAChRs are non-selective, ligand-gated cation channels (Changeux et al., 1998; Clementi et al., 2000; Picciotto et al., 2001) comprised of homomeric or heteromeric configurations of α (α2-α7) and β (β2-β4) subunits (Picciotto et al., 2000). In the CNS, α4β2 and α7 nAChRs are well investigated with the α7 exhibiting a lower affinity for ACh and rapid desensitization (Alkondon and

Aluquerque, 1993). Activation of nAChRs induces Na+2 and Ca+2 influx and subsequently leads to amplification of neuronal excitability. Alternatively, mAChRs alter neuronal excitability in both a positive and negative direction, depending on activation of two distinct subclasses. Activation of the Gq-PLC coupled receptors (M1, M3, M5 ) results in increased neuronal excitability through activation of non-specific cation channels, Ca+2 release from intracellular stores, or inhibition of Ca+2-activated K+ channels (Brown,

2010). Conversely, Gi-coupled receptors (M2, M4) are linked to adenylate cyclase inhibition and subsequent cAMP reduction, which leads to suppression of neuronal excitability through activation of K+ channels or inhibition of Ca+2 channels (Wess et al.,

1997). In the CNS, mAChRs are more abundant and functionally diverse as compared to nAChRs (Brown, 2010; Picciotto et al., 2012). These receptors exhibit wide distribution patterns in terms of pre- and post-synaptic expression patterns (Picciotto et al., 2012) as well as the localization of multiple subtypes on a singular neuron (Hassel et al., 1993).

Modulation in the Central Nervous System

In the periphery, such as at the neuromuscular junction and in autonomic ganglia, ACh acts as a classical excitatory neurotransmitter (Brown, 2010). However, its role in the CNS more closely resembles that of a neuromodulator (see Picciotto et al.,

2012 for review) as ACh regulates neuronal excitability at pre- and post-synaptic sites and synchronizes firing across neuronal subpopulations (Wonnacott, 1997; Rice and

Cragg, 2004; Zhang and Sulzer, 2004; Kawai et al., 2007). At pre-synaptic terminals,

ACh participates in auto-inhibition of cholinergic neurons through activation of M2 or M4 receptors (Raiteri et al., 1984; Douglas et al., 2002), which leads to decreased glutamate release from corti-cortical and cortico-striatal synapses (Gil et al., 1997; Higley et al.,

2009) and attenuation of neuronal excitability through inhibition of VGCCs as seen in basal forebrain neurons (Allen and Brown, 1993; Allen, 1999). Post-synaptic inhibition of neuronal excitability occurs through activation of M2 receptors, which leads to activation of GIRK channels (Wickman and Clapham, 1995). Conversely, post-synaptic neuronal excitability is due to inhibition of sub-threshold K+ channels (referred to as ‘M-channels’) by M1, M3, and M5 receptors (Brown and Adams, 1980). M-channel closure results in an inward, depolarizing current which has been shown to increase neuronal excitability in cortical (McCormick and Williamson, 1989) and hippocampal pyramidal neurons

(Halliwell and Adams, 1982; McCormick and Price, 1985; Douglas et al., 2002).

In the brain, ionotropic nAChRs act in an atypical neuromodulatory fashion that is generally reserved for metabotropic receptors (Picciotto, 2003). For example, nAChRs have a wide distribution pattern with pre-synaptic (Vidal and Changeux, 1993; McGehee et al., 1995), somatic, and axonal expression (Hill et al., 1993; Arroyo-Jime’nez et al.,

1999; Kawai et al., 2007). Activation of nAChRs can modulate the release of classical

29 neurotransmitters such as dopamine, GABA, ACh, and serotonin (McGehee et al., 1995;

Wonnacott, 1997). Differential nicotinic modulation arises from subtype specific coupling in distinct brain areas, as α7 nAChRs regulate glutamate and α4β4 regulate GABA release in the ventral tegmental area (Mansvelder et al., 2002) while β2 nAChRs regulate glutamate release from thalamocortical areas (Parikh et al., 2010).

Acetylcholine in the Mammalian Retina

Cholinergic signaling through nAChRs and mAChRs has been observed in the retinas of several species (Schmidt et al., 1987; Kaneda et al., 1995; Baldridge, 1996;

Kittila and Massey, 1997; Wasselius et al., 1998; Zucker and Ehinger, 1998; Yamada et al, 2003; Liu et al. 2009; McBrien et al., 2009). Both receptor subtypes have been localized to bipolar cells, amacrine cells, and RGCs. Specifically, M2 receptors have been localized to cells in the INL and IPL (Zucker and Ehinger, 2001) while M3 expression is seen in subsets of horizontal, bipolar, and amacrine cells (Yamada et al.,

2003). In addition, M1, M2, M3, and M5 expression has been observed in subsets of

RGCs (Strang et al., 2010). M1 expression was found in the outer and inner sublamina of the IPL, where ON and OFF RGCs stratify, respectively (Strang et al., 2010).

In the retina, synthesis and release of ACh arises from a singular source, starburst amacrine cells (SACs), as conferred by their expression of acetyltransferase

(Masland and Mills, 1979; Masland et al., 1984; Pourcho and Osman, 1986; Voigt, 1986;

Brandon, 1987; Brecha et al., 1988; Kosaka et al., 1988). Two subtypes of SACs are differentiated based upon their dendritic stratification in the IPL: one stratifies to the outermost sublamina ‘a’ and releases ACh in response to light offset, and the other to the innermost sublamina ‘b’ and releases ACh during light onset (Masland and Mills,

1979; Famiglietti, 1983; Vaney, 1984). Typically, SAC somas reside within the OFF sublamina of the IPL (Perry and Walker, 1980; Vaney et al., 1981; Brandon, 1987;

Famiglietti and Tumosa, 1987). However, ON SACs are ‘displaced’ with somas that reside in the RGC layer (Hayden et al., 1980; Hughes and Vaney, 1980).

Starburst amacrine cells release acetylcholine in a tonic and light-evoked manner

(Masland and Livingstone, 1976; Peters and Masland, 1996). Synthesis and release of

ACh is markedly unregulated during photic stimulation (Masland and Livingstone, 1976).

Therefore, extracellular accumulation of ACh is predominately due to the transient, light- evoked release of ACh as opposed to the minute tonic contribution (Masland et al.,

1984). Light onset and offset induces ACh release from displaced ON and conventional

OFF SACs (Masland et al., 1984). Thus, flickering light stimuli is more efficient at inducing robust ACh release as opposed to continuous light (Masland and Livingstone,

1976; Neal and Massey, 1980). Specifically, numerous reports indicate maximal ACh release is achieved by exposing the retina to 3-10 Hz flickering light stimuli (Masland and Livingstone, 1976; Massey and Neal, 1979; Masland et al., 1984; Massey and

Redburn, 1985; O’Malley and Masland, 1993; Firth et al., 2003).

Released ACh modulates RGC activity through activation of nicotinic and muscarinic receptors (Masland and Ames, 1976; Masland et al., 1984; Schmidt et al.,

1987; Baldridge, 1996; Strang et al., 2005). Rapid effects are mediated through nicotinic receptors that induce robust excitation of RGCs (Masland and Ames I976; Ariel and Daw

1982). Specifically, α7 (Strang et al., 2003) and β2-expressing (Strang et al., 2005) nAChRs have been shown to be involved in the light-induced responses of RGCs.

Various RGC cell types alter their responses in the presence of cholinergic compounds

(Ames and Pollen 1969; Masland and Ames, I976) such as choline and physostigmine

(an AChE inhibitor). OFF RGC firing appears to be inhibited, while ON and ON-OFF

RGCs are stimulated, by choline in a muscarinic-dependent manner (Strang et al.,

2010). Overall, nAChRs and Gq-coupled mAChRs expressed on bipolar cells and RGCs mediate stimulatory effects on RGC firing while nAChRs and mAChRs expressed by

SACs or M2 and M4 receptors expressed by bipolar cells and RGCs inhibit RGC firing

(Dmitrieva et al., 2007; Strang et al., 2005, 2010, 2014).

Most notably, nAChRs (Ariel and Daw, 1982; Kittila and Massey, 1997; Strang et al., 2005, 2007) and mAChRs (Baldridge, 1996; Kaneda et al., 1995; Schmidt et al.,

1987; Strang et al., 2010) regulate directionally selective ganglion cell (DSGC) firing properties. In addition to ACh, SACs synthesize and release GABA (Brecha et al., 1998;

O’Malley and Masland, 1989) and adenosine (Blazynski, 1989). Both cholinergic and

GABAergic input regulates DSGCs and the subsequent detection of motion in our environment (see Taylor and Vaney, 2003 for review). Detection of movement in the preferred direction of a DSGC is predominantly due to fast cholinergic transmission via nAChRs (Lee et al., 2010). GABAergic inhibition informs movement in the opposite or null direction. This GABAergic input is crucial for direction-specific detection, as inhibition of GABA (Caldwell et al., 1978; Ariel and Daw, 1982), specifically GABAA (Massey et al.,

1997) receptors on DSGCs, abolishes direction selectivity while inhibition of nAChRs attenuates responses to movement in the preferred direction (Grzywacz et al., 1997;

Kittila and Massey, 1997). Complete elimination of direction selectivity is also achieved through selective ablation of SACs (He and Masland, 1997; Yoshida et al., 2001).

Overall, DSGCs receive symmetrical cholinergic excitatory drive from SACs that is then tightly regulated by asymmetric GABAergic input, due to a greater number of GABAergic

32 synapses between SACs and DSGCs on the null side (Briggman et al., 2011), to inform directional selectivity and motion in our environment.

During development, the cholinergic network plays a key role in the refinement of retinal circuitry through SAC-mediated activity and subsequent coordinated firing among

RGCs (see Ford and Feller, 2012 for review). The cholinergic network is present at birth and mediates signaling between SACs, as these cells spontaneously release ACh that binds to nAChRs and depolarizes neighboring SACs (Zheng et al., 2004). ACh also drives spontaneous waves of action potential firing in neighboring RGCs through nAChRs (Feller et al., 1996) until the second week of development (Bansal et al., 2000;

Wong et al., 2000; Zhou and Zhao, 2000). Initially, SACs propagate cholinergic excitation through gap junctions before transitioning to nAChR-mediated signaling, which is abolished in late development. Ultimately, mature cholinergic synapses remain and

GABAergic synapses transition from being excitatory to inhibitory. These cholinergic synapses represent the first mature, functional circuits in the retina.

Broad nicotinic and muscarinic receptor expression at birth suggests that ACh is capable of modulating various retinal neurons though different signaling pathways (Ford and Feller, 2012). In the CNS, paracrine cholinergic signaling mediates development prior to synapse maturation. Similar volumetric transmission may account for cholinergic regulation during retinal development. In support, diffuse ACh release from SACs is sufficient to drive robust RGC firing prior to synapse maturation. Moreover in adulthood,

RGCs with dendrites outside the proximity of SACs have been shown to respond to ACh release (Schmidt et al., 1987). Thus, synapses between SACs and RGCs may not be necessary to drive RGC excitability as volumetric transmission may account for the far reaching, extra-synaptic ACh-mediated activity seen in the mature retina.

Thesis Objectives

The overall objective of this thesis is to determine whether the unique temporal dynamics of ipRGC photoresponses are subject to modification by endogenous retinal neuromodulators. In Chapter 2, I demonstrate that ipRGC photoresponses are modulated through cAMP/PKA-mediated signaling. Moreover, in Chapter 3, I illustrate that adenosine is an endogenous neuromodulator capable of modulating ipRGC photoresponse through an adenosine A1 receptor-mediated, cAMP/PKA-dependent mechanism. In addition, I show that adenosine A1-mediated suppression of ipRGC photoresponses occurs endogenously after isolated retinas are dark-adapted. In

Chapter 4, I determine, albeit surprisingly, that ipRGCs can be directly stimulated through a melanopsin-independent pathway by cholinergic activation of muscarinic receptors. An outline of the intraceullar mechanisms responsible for the modulatory pathways explored in this thesis are depicted in Figure 1.2.

Figure 1.1. Differences in vertebrate and invertebrate phototransduction.

A schematic representation of the distinct vertebrate phototransduction (a) and invertebrate phototransduction (b) signaling cascades are depicted, as previously published (Hatori and Panda, 2010). The vertebrate phototransduction cascades utilize

Gt (transducin) protein, which triggers phosphodiesterase (PDE) and results in closure of cyclic nucleotide-gated (CNG) ion channels and hyperpolarization. However, the invertebrate cascade relies on Gq-PLCβ signaling that ultimately results in transient receptor potential (TRP) channel activation and subsequent depolarization. 35

Blue Light Adenosine ACh Muller Starburst glia amacrine ATP VGCC TRPC 6/7 A1 Melanopsin M1/3/5 ACh Adenosine

Adenylate PLCβ4 Cyclase Gi Gq Gq AC inhibition ↓ light-evoked: PIP2 • VGCC opening • ipRGC firing duration IP3 DAG

ipRGC Cyclic AMP PKA production ?

Figure 1.2. Proposed model for modulation of ipRGC photoresponses.

The Gq/11-PLCβ4 ipRGC phototransduction cascade is depicted with each proposed neuromodulatory pathway. On left, the cAMP/PKA signaling pathway for Chapter 2 is shown along with the Gi-mediated modulation of ipRGC light responses through activation of A1 receptors by adenosine. A1-mediated suppression of the adenylate cyclase pathway could decrease light-evoked calcium influx through voltage-gated calcium channels (VGCCs). On right, Chapter 4 is outlined with the muscarinic acetylcholine receptor (mAChR)-mediated stimulation of ipRGCs. The intracellular ipRGC diagram corresponds to the ipRGC dendrite boxed region in the schematic representation of the proposed retinal sources of adenosine and acetylcholine.

CHAPTER 2

MODULATION OF ipRGC PHOTORESPONSES THROUGH A CYCLIC AMP/PKA-

MEDIATED PATHWAY

As outlined in Chapter 1, ipRGCs are retinal output neurons and transmit signals to the brain via their axons, which exit the eye in bundles contained in the optic nerve.

The brain regions innervated by ipRGCs are diverse (Hattar et al., 2006; Brown et al.,

2010), but a common theme of known ipRGC functions is that these photoreceptors primarily act as irradiance detectors, signaling information regarding the amount of light present in the environment. Similar to other retinal ganglion cells (RGCs), but unlike rods and cones, ipRGCs are post-synaptic neurons that have extensive dendritic arbors

(Berson et al., 2010) along which synaptic connections with retinal bipolar and amacrine cells occur (Belenky et al., 2003; Wong et al., 2007). This property affords ipRGCs the ability to integrate network signals within the retina and supports the premise that interactions between ipRGCs and other retinal neurons can modify the light responses of these ganglion cell photoreceptors. Modulation of information regarding ambient light levels that is transmitted by ipRGCs could have profound inﬂuences on the variety of functions that are regulated by these cells, which include circadian rhythm photoentrainment, pupil constriction, sleep/wakefulness and even perceptual brightness discrimination (Do and Yau, 2010; Lucas, 2013).

In this work, I tested the hypothesis that the unique temporal characteristics of ipRGC light responses are subject to modiﬁcation by changes in internal levels of cyclic

AMP (cAMP), an important secondary messenger in many physiological signaling pathways (Missale et al., 1998). Changes in internal cAMP levels modulate the light responses of Drosophila and Limulus photoreceptors (Chyb et al., 1999; Battelle, 2002), suggesting that this may be a common feature of invertebrate photoreception. As phylogenetic analyses of the melanopsin protein indicate that it is more homologous to these invertebrate opsins than to vertebrate rod/cone opsins (Koyanagi et al., 2005;

Peirson and Foster, 2006), I postulated similar cAMP-dependent modulation of ipRGCs.

This cyclic nucleotide has already been shown to play a role in network adaptation at the ganglion cell level in vertebrates, as alterations in non-photosensitive RGC spiking have been linked to the changes in internal cAMP levels during light exposure (Vaquero et al.,

2001). Thus, my goal here was to characterize the effects of intracellular changes in cAMP on the inherent light responses of ganglion cell photoreceptors in the rat retina.

Materials and Methods

Multielectrode array (MEA) recordings from rat retinas

The Ohio State University Institutional Animal Care and Use Committee approved all procedures. Neonatal and adult Long Evans rats (Charles River,

Wilmington, MA), housed under a 12/12 h light/dark cycle, were killed by decapitation after isoﬂurane overexposure (by inhalation in a sealed container) and the eyes were enucleated. The adult animals were dark adapted for 1 h prior to enucleation and dissections were performed under dim red light (625 nm; DiCon LED, Richmond, CA) to minimize rod/cone photobleaching. Enucleation of the neonatal animals occurred under standard room lighting conditions, and these retinal dissections were performed under a white microscope light. For both the neonatal and the adult animals, the eyes were

38 hemisected and the posterior eyecup was pinned down in a Sylgard-coated (Dow

Corning, Midland, MI) dish containing Ames medium (Sigma-Aldrich, St Louis, MO) supplemented with10 mM Hepes (pH 7.4; Sigma-Aldrich). The vitreous was removed with a pair of curved forceps, by placing the forceps underneath the vitreous and pulling up from the optic nerve head. Using a paintbrush, each retina was transferred to a glass coverslip and trimmed to obtain square retinal pieces, which were placed ganglion cell- side down in a chamber with a 60-electrode array (30 μm diameter, 200 μm inter- electrode spacing; Multi-Channel Systems, Reutlingen, Germany) and anchored with a weighted, circular nylon mesh (ALA Scientiﬁc Instruments, Farmingdale, NY). The array- mounted retina from one eye was then placed onto a heated stage that was continuously

−1 superfused (1 ml min ﬂow rate), via a peristaltic pump (Gilson, Middleton, WI), with

Ames medium (10 mM Hepes, pH 7.4) that was bubbled with 100% oxygen and pre- heated through a perfusion cannula (PH-01, Multi-Channel Systems). The temperature of the Ames medium in the chamber was 32–34°C. The retinal piece from the second eye was stored on the array in Hibernate-A medium (Brain Bits, Springfield, IL) supplemented with 2% B-27 (Life Technologies, Grand Island, NY) and maintained in the dark until the recordings from the first retina were completed. After transfer to the stage, which occurred under dim red light (GBX-2 Safelight filters; Kodak, Pittsburgh,

PA), the retinas were perfused with the heated, oxygenated Ames medium for 1 h in the dark prior to the ﬁrst light exposure.

The light stimuli (all 20 s in duration) were generated using a blue LED source

(470 nm module, Colibri system; Zeiss, Oberkochen, Germany) that was delivered from above the microscope stage. The Colibri system was attached to an upright microscope

(Axio Examiner; Zeiss) and the blue light was ﬁltered through a bandpass interference

filter (BP 474/28; Zeiss). Using a triple-pass beamsplitter (filter set 62 HE; Zeiss), the light was then directed through a 40× objective (Achroplan, NA 0.75) onto the array- mounted retina, passing through the weighted nylon mesh and hitting the top rod/cone layer first. The blue light stimulus was chosen based on previous work showing that rat ipRGCs are most sensitive to 480 nm light (Berson et al., 2002).

MEA voltage recordings were ampliﬁed and digitized at 25 kHz using a PC-based

A/D interface card and MCRack software (Multi-Channel Systems). The signals were bandpass ﬁltered with cutoffs at 200 Hz and 3 kHz. A detection threshold of –5 standard deviations (SD) from baseline electrical activity was used to identify spike waveforms that were then separated from the continuous data. Clustered spike data were sorted into individual units with Ofﬂine Sorter software (Plexon Inc., Dallas, TX). Specifically, spikes that appeared simultaneously on 80% of the channels were removed as noise artifacts, and remaining waveforms were aligned to the global minimum. The waveforms were represented in a 3D feature space and the optimum number of spike clusters was determined using a T-distribution Expectation-Maximization algorithm (degrees of freedom multiplier = 10). The waveforms were then further sorted using an iterative K- means algorithm, with a threshold of 2 SD used to remove outlier waveforms. Using

Neuro-Explorer software (Nex Technologies, Madison, AL), the number of spikes occurring in 1 s bins was counted, and these data were used to generate spike frequency graphs and total counts of the light-evoked spikes. To identify ipRGCs, spike counts were determined for: (1) the 10 s (–10 to 0 s) pre-stimulus period (‘x’); 2) the ﬁrst

10 s (0–10 s) of the light stimulus period (‘y’); and 3) the second 10 s (10–20 s) of the light stimulus period (‘z’). In the postnatal retinas, identiﬁcation of ‘ipRGC units’ was based on all of the following criteria being met: y 50; y 2x; z 50; and z 2x. Based on

40 initial visual inspections of the data, these criteria identified units exhibiting robust and prolonged light responses characteristic of ipRGC responses. In the adult retina recordings, the same ipRGC identification criteria were applied to the data obtained in the presence of a synaptic blocker cocktail that silenced rod/cone-driven signaling. Due to the criteria employed, the identified neonatal or adult ipRGC units may have been biased towards selection of M1-type ipRGCs, as the M1 subtype exhibits the most robust light-evoked responses (Schmidt and Kofuji, 2009; Ecker et al., 2010). However, while these ipRGC subtypes have been described for mice, it is not currently clear if the same subtypes exist in the rat.

Pharmacology and solution preparation

Stock solutions were prepared, aliquoted and stored at -80°C, with non-water soluble compounds being dissolved in dimethyl sulfoxide (DMSO). The final working concentration of DMSO in the Ames medium or HBSS was  0.1%. Unless stated otherwise, each compound was perfused onto retinas for 5 min prior to light stimulation.

To investigate cAMP/PKA signaling pathways, forskolin was obtained from

Sigma-Aldrich; 8-Br-cAMP (8-bromoadenosine-3',5'-cyclic monophosphate sodium salt),

8-Br-cGMP (8-bromoguanosine cyclic 3',5'-monophosphate sodium salt), MDL 12330A

((±)-N-[(1R*,2R*)-2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine hydrochloride) and

PKI 14-22 amide (protein kinase inhibitor-(14-22)-amide, myristoylated) were obtained from Tocris Bioscience (Minneapolis MN); Sp-5,6-DCl-cBIMPS (5, 6-dichloro-1-ß-D- ribofuranosylbenzimidazole-3’, 5’-cyclic monophosphorothioate, Sp-isomer) and 8- pCPT-cGMP (8-(4-chlorophenylthio)guanosine-3’, 5’-cyclic monophosphate) were obtained from Biolog (Bremen, Germany).

Melanopsin-based light responses were isolated in the intact adult retinas by superfusing a cocktail of synaptic blockers onto the retinas to inhibit rod/cone-driven signaling (Wong et al., 2007). Glutamatergic signaling was inhibited using 100 μM metabotropic glutamate receptor agonist L-AP4 (L-(+)-2-Amino-4-phosphonobutyric acid;

Tocris) and 25 μM AMPA receptor antagonist NBQX (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydr obenzo[f]quinoxaline-7-sulfonamide; Tocris). Cholinergic signaling was inhibited using

10 μM muscarinic receptor antagonist atropine (Sigma-Aldrich) and 100 μM nicotinic receptor antagonist tubocurarine (Tocris). GABAA-,GABAB-, and GABAC-receptors were blocked with 50 μM picrotoxin (Tocris), 5 μM CGP 54626 (3-[[(3,4- dichlorophenyl)methyl]amino]propyl] diethoxymethyl)phosphinic acid; Tocris), and 100

μM TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid; Sigma-Aldrich), respectively. Glycinergic signaling was inhibited by 10 μM strychnine (Tocris). Light responses recorded in the adult retinas in the presence of Ames’ medium containing this blocker cocktail were deemed to be the melanopsin-dependent responses of ipRGCs.

Data analysis

Preparation of ﬁnal graphs and statistical comparisons were performed using

Sigmaplot 12 software (Systat Software, San Jose, CA).The overall light-evoked spike counts were quantiﬁed by adding the number of spikes that occurred during the 20 s light pulses with those that occurred during the initial 80 s post-stimulus for each unit. All data are expressed as the mean ± standard error of the mean (SEM). Due to the higher number of light-sensitive units in the neonatal retinas, multiple units were often recorded on a single electrode. The number of units identiﬁed by the spike sorting software in each postnatal retina was not always identical across different treatment conditions (i.e.

42 before, during and after drug treatment). The number of units (n) reported in the Results therefore represented the total number of units identiﬁed in the ‘drug present’ condition.

Statistical differences in spike counts obtained from postnatal retinal recordings were determined using one-way analysis of variance (ANOVA) and Holm–Sidak post hoc testing. In the adult retinas, there were fewer (usually < 4) light-sensitive units recorded from each retina, allowing responses from individual cells to be matched across the different conditions (before, during and after drug treatment). Statistical differences in spike counts obtained from adult retina recordings were determined using one-way repeated-measures ANOVA and Holm–Sidak post hoc testing.

Results

Modulation of ipRGC light responses through a cAMP-mediated pathway

While melanopsin-dependent light responses in rodent ipRGCs are present from birth, rod-and cone-driven light responses in ganglion cells do not occur until later in development (Hannibal and Fahrenkrug, 2004; Sekaran et al., 2005; Tu et al., 2005).

This enables the neonatal rodent retina to serve as a useful preparation for studying ipRGC photoresponses in the absence of rod/cone-inﬂuenced signaling pathways. Using

MEA recordings from the ganglion cell layer, I recorded light-evoked action potential

ﬁring from postnatal (P) retinas of Long-Evans rats (example from P8 retina in Figure

2.1a). These responses persisted in the presence of a cocktail of glutamate, acetylcholine, GABA and glycine receptor antagonists (see Methods for details), thereby conﬁrming that the light responses reﬂected the inherent melanopsin-related responses of ipRGCs (Figure 2.1b). In the absence of synaptic blocker cocktail, I observed the emergence of rod/cone-driven responses (characteristics discussed in next section; see

Figure 2.4) to coincide closely with eye opening, which usually occurred at P15 in the

Long-Evans rats. In this study, all MEA retinal recordings denoted as ‘neonatal ipRGCs’ were obtained from rats during their second week of life (age P8–P14).

Prior to eye opening, spontaneous waves of correlated activity sweep across the retina that are essential for proper development and reﬁnement of retinal circuitry.

During the second week of life in rodents, the neurotransmitter that drives retinal waves switches from acetylcholine (Stage II) to glutamate (Stage III) (Ford and Feller, 2012). In addition to this excitatory input, ganglion cells also receive GABA-and glycine-mediated inhibitory input during the waves (Blankenship et al., 2009). Thus, the synaptic blocker cocktail was designed to block all excitatory and inhibitory drive to RGCs and, as expected, the waves of activity were silenced in postnatal retinas during superfusion of the cocktail. Although the peak ﬁring frequency of ipRGCs was not signiﬁcantly affected, the mean duration of ipRGC responses to the 20 s bright (7.1 × 1015 photons s−1 cm−2) blue light stimulus was shortened after exposure to the cocktail for 5 min (compare

Figure 2.1b to 2.1a). In quantifying this effect, the recovery of mean spike frequency to a near baseline rate of 2 spike s−1 occurred 25 s earlier (37 s versus 62 s post-light onset) with the blocker cocktail present compared to when it was absent (Figure 2.1c).

As the membrane depolarization associated with the spontaneous waves of activity are known to induce elevations in intracellular cAMP in retinal neurons (Meyer-

Franke et al., 1995; Dunn et al., 2006), I hypothesized that the shortened ipRGC responses were due, at least in part, to reductions in intracellular cAMP caused by the silencing of spontaneous activity. To test this hypothesis, I investigated the effect of forskolin on neonatal ipRGC responses to 20 s blue light exposure in the presence

(Figure 2.2a) and absence (Figure 2.2b) of the synaptic blocker cocktail. Forskolin

44 induces cAMP elevations in retinal neurons (Dunn et al., 2006). The light irradiance (3.9

× 1015 photons s−1 cm−2) was reduced from that used in Figure 2.1 to prevent response saturation or depolarization block from potentially masking forskolin’s effects, and this light stimulus was used for all subsequent experiments. With the cocktail absent, waves of activity persisted and light pulses were timed to occur after a wave passed through the recorded retinal region, as the average inter-wave interval is longer than 60 s at later

(stage III) postnatal ages (Blankenship et al., 2009). In this blocker-free condition, the recovery of mean spike frequency to a rate of 2 spikes s−1 took 24 s longer (based on 1 s binning of spike counts) after exposure to 10 μM forskolin for 5 min, relative to the pre- treatment response (Figure 2.2c). Total counts of light-evoked spiking were quantified by adding together the number of spikes that occurred during 20 s light exposure plus 60 s post-stimulus period. With lengthening of the response duration, the mean light-evoked spike count was also significantly higher during forskolin treatment, as compared to both pre-(‘control’) and post-treatment (‘washout’) conditions (Figure 2.2d). Similarly, forskolin significantly increased (P < 0.001; t test) mean ipRGC spike counts with synaptic blockers present (N = 3 retinas; n = 60 cells; data not shown graphically but example in

Figure 2.2a), indicating that forskolin’s effect was not due to increased release of the major excitatory and inhibitory neurotransmitters.

Forskolin directly stimulates the cAMP-generating enzyme adenylate cyclase without interacting with cell-surface receptors (Seamon et al., 1981). Changes in internal cAMP can regulate a number of intracellular signaling pathways, including the actions of the phosphorylating enzyme PKA, which requires cAMP for its activation. However, forskolin can also affect cells through cAMP-independent mechanisms (Laurenza et al.,

1989). To demonstrate that the effect of forskolin on ipRGC light responses involved a

45 cAMP-dependent signaling pathway, I assessed whether blockade of either adenylate cyclase or PKA altered the effect of forskolin. In the presence of 50 μM MDL 12330A, a widely used speciﬁc inhibitor of adenylate cyclase (Seifert et al., 2012), the response lengthening effect of the 10 μM forskolin treatment was abolished (Figure 2.2e,g). The actions of forskolin were similarly blocked when retinas were treated with the cell- permeable version of PKI-(14–22)-amide (10 μM; Figure 2.2f,g), a compound that renders PKA inactive by speciﬁcally binding to this enzyme’s endogenous catalytic sites

(Murray, 2008), thereby demonstrating that PKA is a downstream component of the forskolin-initiated signaling cascade.

I next tested whether cyclic nucleotide analogues and protein kinase agonists could mimic the effects of forskolin. Superfusion of neonatal retinas with the cell- permeable cAMP analogue 8-Br-cAMP (1 mM) for 5 min significantly increased the total number of light-evoked spikes fired by ipRGCs (Figure 2.3a,c). To control for any non- specific effects of the brominated compound, retinas were also treated with the cGMP analogue 8-Br-cGMP (1 mM) and no discernible effects on ipRGC responses were observed (Figure 2.3b,c). Similar to 8-Br-cAMP, bath application of the potent PKA activator Sp-5,6-DCl-cBIMPS (cBIMPS; 100 μM) increased ipRGC spiking elicited by light stimulation (Figure 2.3d, f). The continued lengthening of ipRGC light responses after drug washout can probably be attributed to the fact that this compound, unlike 8-Br- cAMP, is resistant to degradation by phosphodiesterases (Murray, 2008). Conversely, stimulation of the cGMP-dependent kinase, protein kinase G (PKG), with 8-pCPT-cGMP

(100 μM) did not signiﬁcantly affect light-evoked ﬁring (Figure 2.3e,f), either during drug application or 20 min after washout. Taken together, the results shown in Figures 2.2 and 2.3 provide strong evidence that stimulation of the signaling cascade involving

46 adenylate cyclase, cAMP and PKA results in an increase in the number and duration of light-evoked spikes ﬁred by neonatal ipRGCs.

The wave-dependent elevations of cAMP that occur in postnatal RGCs play a critical role in the reﬁnement of the connections between these retinal output neurons and their appropriate brain targets (Stellwagen and Shatz, 2002). We next sought to determine whether the cAMP-mediated effect on ipRGC light responses was solely a developmental phenomenon or if it remained after retinal circuit wiring was completed.

After eye opening in the Long-Evans rats, which occurred at approximately P15, faster and more transient light responses could be recorded from the ganglion cell layer, consistent with expected rod/cone-driven signaling. The inherent responses of ipRGCs were identified based on the persistence of light-evoked spiking in the presence of synaptic blocker cocktail (see Methods for details). During superfusion of the synaptic blocker cocktail, the units that only exhibited transient (<5 s) responses to the 20 s light pulses prior to cocktail application (presumed non-photosensitive RGCs receiving rod/cone-driven signals) were eliminated from the recordings. In addition to their melanopsin-based photosensitivity, ipRGCs also receive synaptic input from bipolar and amacrine cells (Belenky et al., 2003; Wong et al., 2007), and the rod/cone-driven component was evident by comparing the difference in spiking during the first couple of seconds of the light responses recorded from the older (age > 2 months) rat retinas before and after application of the synaptic blocker cocktail (Figure 2.4). Light responses from subsequently identified ipRGCs, recorded pre-cocktail application, exhibited an abrupt burst in spiking with short latency from light onset (Figure 2.4c). This is consistent with the initial component of adult ipRGC light responses, recorded in the absence of synaptic blockers, being rod/cone-driven. Although ipRGC light responses persisted for

47 many seconds after light onset with cocktail present, the responses from these same cells were even more prolonged in the pre-treatment recordings (compare Figures 2.4a and 2.4b; data summary in Figure 2.4c). When the synaptic connections between these cells and other retinal neurons were not inhibited, more background spiking activity was evident in the dark (Figure 2.4a,c), suggesting that the spontaneous activity present in the older retinas promotes lengthening of ipRGC light responses in a similar fashion to how wave-mediated activity inﬂuenced the neonatal ipRGC light responses (Figure 2.1).

In the presence of synaptic blocker cocktail (to identify light-responding ipRGCs),

I treated the older retinas for 5 min to either 10 μM forskolin (Figure 2.5a, b) or 1 mM 8-

Br-cAMP (Figure 2.5c,d). Similar to the neonatal retinal experiments (Figures 2.2 and

2.3), both compounds signiﬁcantly increased the total number of spikes ﬁred during and after (20 s light exposure plus 60 s post-illumination period) light stimulation. Thus, the effect of cAMP on ipRGC light responses is not restricted to early development, and is instead a fundamental feature of rat melanopsin-based ipRGC photoresponses.

Discussion

A hallmark of ipRGC physiology is that these cells exhibit remarkably sustained spiking responses during light exposures that can persist for many seconds after the light is turned off. In this work, I demonstrate that this unique characteristic of ipRGCs is modifiable. Specifically, I identified a cAMP-dependent signaling pathway that regulates response duration, including total number of spikes fired, in light-stimulated rat ipRGCs.

Neuromodulation of ipRGCs through a cAMP-dependent pathway

The bath application of forskolin, which stimulates increased cAMP levels within retinal neurons (Dunn et al., 2006), signiﬁcantly increased light-evoked spiking in ipRGCs recorded from either neonatal or adult rat retinas. The effect of forskolin could be blocked by pretreating retinas with inhibitors of either adenylate cyclase or PKA

(using MDL 12330A and PKI-[14–22]-amide, respectively), implicating both enzymes in the pathway that mediates forskolin’s actions. Consistent with the effect being cAMP- dependent, light-evoked ipRGC spiking was signiﬁcantly increased following exposure to a synthetic brominated analogue of cAMP, but not to a comparable cGMP analogue.

Moreover, treatment of retinas with a PKA agonist (Sp-5,6-DCl-cBIMPS) affected ipRGC action potential ﬁring in a similar fashion to forskolin, while a PKG agonist (8-pCPT- cGMP) had negligible effect. Taken together, the results indicate that an intracellular signaling cascade, which involves stimulation of PKA by adenylate cyclase-generated cAMP, inﬂuences the overall duration of light-evoked ipRGC spiking. In this regard, mammalian ipRGCs are similar to certain invertebrate photoreceptors, for which cAMP has also been shown to modulate photoresponses (Chyb et al., 1999; Battelle, 2002).

Potential downstream targets mediating cAMP’s effect on ipRGC light responses

In this study, I did not examine the downstream targets of PKA that facilitate alterations in the duration of ipRGC spiking responses to light. Three possibilities that warrant future investigation include PKA-mediated phosphorylation of: 1) voltage-gated calcium channels (VGCCs), 2) canonical transient receptor potential channels (TRPCs) and 3) hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

During light exposure, calcium flows into ipRGCs through L-type VGCCs that are opened after the strong depolarization associated with action potential firing (Hartwick et al., 2007). Phosphorylation of L-type VGCCs by cAMP-dependent PKA is well established in the mammalian heart and skeletal muscle (McDonald et al., 1994; Hosey et al., 1996; Gao et al., 1997; Catterall, 2000). VGCC-mediated calcium influx can modulate neuronal output by regulating activation of K+ channels (Sah and Faber, 2002) and it has been previously demonstrated that the retinal neuromodulator adenosine, acting through the cAMP/PKA pathway, induces an outward current in K+ non- photosensitive rat RGCs (Newman, 2003). Thus, an adenosine-driven reduction in the phosphorylation of VGCCs by PKA could produce a decrease in calcium influx along with a concomitant increase in K+ efflux. Membrane hyperpolarization would be the result of these two outcomes, leading to a reduction in spikes fired by the ipRGCs. In addition, reduced VGCC-mediated calcium influx may directly affect the primary light-gated current, as there is evidence that peak amplitude of the ipRGC photocurrent is reduced when intracellular calcium levels are lowered (Warren et al., 2006).

A second possibility is that PKA inhibition may directly or indirectly affect TRPCs to modulate ipRGC light responses. There is anatomical and physiological evidence that a TRPC is the light-gated channel in ipRGCs (Warren et al., 2006; Hartwick et al., 2007;

Sekaran et al., 2007). More recent evidence using conditional single-and double-gene knockout mice indicate that the initial ipRGC photocurrent is mediated through a

TRPC6/7 heterodimeric channel (Xue et al., 2011). Consensus phosphorylation sites have been shown on TRPC proteins (Kwan et al., 2004), raising the possibility that stimulation of the cAMP/PKA signaling pathway could directly activate the light-gated channel. However, there is currently conﬂicting evidence as to whether phosphorylation

50 of TRPC6 by PKA activates (Shen et al., 2011), inhibits (Horinouchi et al., 2012) or has no effect (Hassock et al., 2002) on the current carried by this channel. Further characterization of the physiological and biochemical features of TRPC6/7 heterodimeric channels (see Itsuki et al., 2014) is necessary to evaluate the potential effects of PKA- driven phosphorylation on their carried current.

HCN (also known as Ih) channels, which have been previously identiﬁed on ipRGCs (Van Hook and Berson, 2010), represent a third potential target for mediating the effect of altered cAMP levels on ipRGC light responses. HCN channels mediate an inward cation current that is activated at hyperpolarized membrane potentials, and typically contribute to the overall resting membrane potential and post-spiking rebound depolarization that occurs in many neurons, including other RGCs (Lee and Ishida,

2007). These channels possess cAMP-speciﬁc binding domains that modify their activation (Ulens and Siegelbaum, 2003), and dopamine-mediated increases in intracellular cAMP have been shown to shift the activation curve of HCN channels in non-photosensitive RGCs (Chen and Yang, 2007). Thus, forskolin-stimulated increases in internal cAMP levels could facilitate the opening of HCN channels, causing depolarization of ipRGC resting membrane potentials. However, blockade of HCN channels has yet to be shown to substantially alter ipRGC light responses and it appears that a substantial hyperpolarized environment is required for ipRGC HCN channel activation (Van Hook and Berson, 2010). It remains to be demonstrated that cAMP can shift the activation curves of these channels into a physiologically relevant range.

Functional implications of cAMP-mediated neuromodulation of ipRGCs

Dopamine is a potential endogenous candidate for lengthening ipRGC light responses in a similar fashion to that observed in our experiments with forskolin treatment. However, it has recently been reported that activation of dopamine D1 receptors, known to stimulate cAMP/PKA-dependent pathways (Neve et al., 2004), inhibited photocurrents in isolated rat ipRGCs as measured through patch voltage-clamp recordings (Van Hook et al., 2012). A similar inhibitory effect was noted by these authors when forskolin plus 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor that also acts as a non-specific adenosine receptor antagonist) was applied to voltage- clamped ipRGCs. While these findings appear to conflict with the action of forskolin described in the present study, it should be noted that this prior study reported that either forskolin or D1 receptor agonist application had additional effects on ipRGCs that were distinct from the reduction in photocurrent. Exposure of ipRGCs to either compound caused an increase in the holding current (and decrease in input resistance) measured during voltage-clamp recordings, indicating an inward current was being activated by the treatments. In accordance with this, forskolin and the D1 agonist induced membrane depolarization and increased background spiking when current-clamp or cell-attached

(extracellular) recording techniques were employed on the cultured ipRGCs (Van Hook et al., 2012). This effect of forskolin on ipRGC membrane properties is consistent with the increased duration in light-evoked spiking associated with forskolin treatment found in the present study, as forskolin-induced depolarization could prolong the recovery of ipRGC membrane potential to a level below the spiking threshold for these neurons. It is therefore possible that the two disparate actions of dopamine and forskolin described by

Van Hook and colleagues are due to separate consequences of PKA activation: 1) the

52 reduction in photocurrent is caused by phosphorylation of the melanopsin photopigment itself, as there is evidence that melanopsin can be inhibited through PKA-mediated phosphorylation when it is heterologously expressed in HEK cells (Blasic et al., 2012); while 2) the ipRGC depolarization is mediated by the action of PKA on ion channels such as VGCCs, TRPCs or HCN channels, as discussed in the preceding section.

Although the two mechanisms appear to counteract each other, my experiments with extracellular MEA recordings indicate that the latter mechanism has a greater role in shaping the light-evoked spiking response of ipRGCs. Using neonatal and adult rat retinal ﬂatmount preparations, my results demonstrate that an increase in light-evoked spiking is the net result associated with an elevation of internal cAMP levels within ipRGCs. As action potentials are the signals that are transmitted to the brain targets innervated by these neurons, the alteration in light-evoked spiking is the output response that would ultimately inﬂuence the functions regulated by these photoreceptors.

In summary, this work establishes an intracellular cAMP/PKA-mediated pathway that regulates light-evoked spiking in rat ipRGCs. Upon activation of this pathway, an increase in ipRGC spike firing is observed. Overall, the results of this study suggest that the light sensitivity of ipRGCs is not a static characteristic, but may instead be regulated by light-and circadian-driven changes in extracellular retinal levels of cAMP-related neuromodulators such as adenosine and dopamine.

Figure 2.1. Effect of spontaneous activity blockade on neonatal ipRGC light responses.

Extracellular MEA recordings of light-evoked spikes from 12 ganglion cells from a P8 rat retina A) before and B) during treatment with a cocktail of glutamate, ACh, GABA and glycine antagonists. Different shading of spike trains facilitates matching of individual cells in the two conditions. C) Mean spike frequency (spikes per 1 s bins) recorded before, during and after light stimulation (N = 3 retinas; n = 70 cells). The ipRGC responses to 20 s blue light (470 nm; 7.1 × 1015 photons s−1 cm−2) shortened in duration following 5 min exposure to synaptic blocker cocktail. 54

Figure 2.2. Effect of forskolin, an adenylate cyclase stimulator, on neonatal ipRGC light responses.

A) Example light response recordings from a cell before and during 5 min treatment with

10 μM forskolin, in the presence of the synaptic blocker cocktail; B) from another cell

continued 55

Figure 2.2. continued with the blocker cocktail absent. ‘Control’ responses were recorded from retinas superfused with Ames medium alone. Summary of mean spike frequency (C, spikes s−1) and D) mean overall spike counts from light-stimulated ipRGCs (N = 3, n = 67), recorded in the absence of the blocker cocktail. ∗P < 0.05, one-way ANOVA, post hoc Holm–

Sidak test. Example light responses recorded from ipRGCs before and during the 5 min exposure to 10 μM forskolin plus E) adenylate cyclase inhibitor MDL 12330A (MDL; 50

μM) or F) PKA inhibitor PKI-(14–22)-amide (10 μM). G) Mean spike counts show that both MDL (N = 3; n = 64) and PKI (N = 2; n = 19) blocked the effect of forskolin on ipRGC light responses. NS, not significant (P > 0.05, one-way ANOVA).

Figure 2.3. Role for cAMP and PKA in modifying neonatal ipRGC light responses.

Example spike rasters from a light-stimulated ipRGC before, during and after 5 min exposure to the cyclic nucleotide analogues A) 8-Br-cAMP (1 mM) or B) 8-Br-cGMP (1 mM). C) Mean spike counts showing that the cAMP analogue (N = 3; n = 35), but not the cGMP analogue (N = 4; n = 54), significantly increased ipRGC spiking. Similarly, the non-hydrolysable PKA agonist Sp-5,6-DCl-cBIMPS (D; N = 3; n = 38) significantly affected ipRGC light responses, while the PKG agonist 8-pCPT-cGMP (E; N = 3; n = 47) did not. F) Mean spike count data illustrating effects of the 5 min treatments with the protein kinase agonists. ∗∗P < 0.01, ∗P < 0.05, one-way ANOVA, Holm–Sidak. 57

Figure 2.4. Effect of synaptic activity blockade on adult ipRGC light responses.

Spike rasters from six ipRGCs recorded from three adult (>2 months old) rat retinas A) before and B) during treatment with cocktail of glutamate, ACh, GABA and glycine antagonists that blocked synaptically driven activity. C) Mean spike frequency (1 s binning) recorded over time in the light-stimulated retinas (N = 10; n = 35). In addition to eliminating the rod/cone-driven spiking, evident by the absence of the short-latency initial burst in spiking, perfusion with synaptic blocker cocktail shortened the duration of ipRGC responses to 20 s blue light (470 nm; 3.9 × 1015 photons s−1 cm−2). 58

Figure 2.5. Role for cAMP in modifying adult ipRGC light responses.

A) Example spike rasters and B) mean spike count data for light-stimulated ipRGCs (N =

3; n = 14) recorded from adult (>2 months old) rat retinas before, during and after 5 min treatment with 10 μM forskolin. Similarly, C) example rasters and D) mean spike count data for adult ipRGCs (N = 3; n = 9) treated with 1 mM 8-Br-cAMP for 5 min. ∗∗ P < 0.01,

∗ P < 0.05, one-way repeated-measures ANOVA, Holm–Sidak. All light responses

(control, drug treatment, washout) in these data were recorded in the presence of synaptic blocker cocktail to confirm recordings were from ipRGCs. Stimulus was blue light (470 nm; 3.9 × 1015 photons s−1 cm−2).

CHAPTER 3

ADENOSINE MODULATES ipRGC PHOTORESPONSES THROUGH A cAMP/PKA-

MEDIATED PATHWAY

Akin to other retinal ganglion cells (RGCS), ipRGCs are retinal output neurons and transmit signals to the brain via their axons in in the optic nerve. Therefore, unlike rods and cones, ipRGCs are post-synaptic photoreceptors that have extensive dendritic arbors (Berson et al., 2010) along which synaptic connections with retinal bipolar and amacrine cells occur (Belenky et al., 2003; Wong et al., 2007). This property affords ipRGCs the ability to integrate network signals within the retina and supports the premise that interactions between ipRGCs and other retinal neurons can modify the light responses of these ganglion cell photoreceptors. Indeed, in Chapter 2, I provide evidence that ipRGC light responses are subject to modulation through an intracellular cAMP/PKA signaling pathway. Intriguingly, modulation of invertebrate photoreceptor light responses has been shown to occur through the cAMP/PKA pathway, indicating that modulation of ipRGCs shares common features with invertebrate phototransduction beyond homology between their photopigments and a rhombic signaling cascade.

Ultimately, modulation of information regarding ambient light levels transmitted by these ganglion cell photoreceptors could profoundly inﬂuence ipRGC-regulated non-image forming functions such as photoentrainment of circadian rhythms, pupil constriction, sleep/wakefulness and even perceptual brightness discrimination (Do and Yau, 2010;

Lucas, 2013).

In this study, I sought to identify an endogenous retinal neuromodulator capable of modifying ipRGC responses through a cAMP-mediated pathway. One candidate, dopamine, is known to alter neuronal cAMP levels through G protein-coupled pathways and recent evidence indicates that ipRGC light responses are indeed affected by this retinal neuromodulator (Van Hook et al., 2012). In this work, however, I instead focused on adenosine, another known G protein-coupled neuromodulator of the CNS. In contrast to dopamine, which is released after light stimulation (Iuvone et al., 1978; Nir et al.,

2000), retinal adenosine levels are thought to rise at night and during prolonged darkness (Ribelayga and Mangel, 2005). Adenosine can be directly released into the extracellular retinal milieu through nucleoside transporters, or formed after enzymatic conversion of ATP by extracellular ecto-nucleotidases (Housley et al., 2009). In the inner retina, Mϋller glial cells (Newman, 2001, 2003, 2004), cholinergic (‘starburst’) amacrine cells (Neal and Cunningham, 1994), and possibly some ganglion cells (Newman, 2005) are thought to be the major sources of ATP that is subsequently converted to adenosine.

Activation of A1 and A3 adenosine receptors are typically linked to inhibition of the enzyme adenylate cyclase, which causes subsequent lowering of intracellular cAMP levels, while A2A and A2B receptor activation leads to increased cAMP generation

(Fredholm et al., 2001). Adenosine receptors are found throughout the mammalian retina, with the A1 receptor especially prevalent in the ganglion cell layer (Blazynski and

Perez, 1991). Stimulation of A1 adenosine receptors on non-photosensitive RGCs results in the inhibition of voltage-gated calcium channels and glutamate-induced calcium influx

(Hartwick et al., 2004). This previous work indicates that adenosine suppresses excitatory neurotransmission at the ganglion cell level, but the effect of this neuromodulator on melanopsin-based irradiance detection by ipRGCs is currently

61 unknown. My goal here was to characterize the effects of extracellular adenosine, and intracellular changes in cAMP, on the inherent light responses of ganglion cell photoreceptors in the rat retina.

Methods

Multielectrode array (MEA) recordings from rat retinas

All procedures were approved by The Ohio State University Institutional Animal

Care and Use Committee and conducted as previously described (see Chapter 2). In brief, postnatal and adult Long Evans rats, maintained under a 12 h light/dark cycle, were decapitated following isoflurane overexposure. Rat eyes were enucleated, hemisected and the posterior eyecup was placed in a dish containing Ames medium (10 mM HEPES, pH 7.4; Sigma-Aldrich). Adult animals were dark adapted for at least 1 h prior to enucleation and dissections were performed under dim red light (625 nm; DiCon

LED) to minimize rod/cone photobleaching. Each retina was trimmed and placed ganglion cell-side down in a 60-electrode array chamber (30 m diameter, 200 m inter- electrode spacing; Multi-Channel Systems) and anchored with a weighted, nylon mesh.

The array was then placed onto a heated stage that was continuously superfused (1 ml/min; 32-34C) via peristaltic pump with oxygenated Ames medium. Retinas were maintained under these conditions for 1 h in the dark prior to first light exposure.

The light stimuli were generated using a blue LED source (470 nm module,

Colibri system; Zeiss) attached to an upright microscope (Axio Examiner; Zeiss). The blue light stimulus, chosen due to rat ipRGC peak sensitivity at 480 nm light (Berson et al., 2002), was filtered through a bandpass interference filter (BP 474/28; Zeiss). A triple-

62 pass beamsplitter (62 HE; Zeiss) directed light stimuli through a 40x objective

(Achroplan, NA 0.75 W) onto the array-mounted retina.

Multielectrode array (MEA) voltage recordings were amplified and digitized and the number of spikes occurring in 1 s bins were counted and used to generated spike frequency graphs and total counts of neuronal spiking (NeuroExplorer software). To identify ipRGCs, spike counts were determined for: 1) the 10 s (-10 to 0 s) pre-stimulus period (‘x’); 2) the first 10 s (0 to 10 s) of the light stimulus period (‘y’); and 3) the second

10 s (10 to 20 s) of the light stimulus period (‘z’). Identification of ‘ipRGC units’ were based on all of the following criteria being met: y  50; y  2x; z  50; and z  2x.

Statistical differences in the spike counts were determined using one-way, or one-way repeated measures, ANOVA and Holm-Sidak post-hoc testing (Sigmaplot 12; Systat).

Calcium imaging of ipRGCs isolated by immunopanning

All culture, imaging and immunohistochemistry supplies and solutions were obtained from Sigma-Aldrich unless noted otherwise. For the immunopanning procedure, I used a crude serum containing antibodies raised against rat melanopsin.

This serum was collected from rabbits that had been immunized with a peptide (by Open

Biosystems, Huntsville, AL) matching the N terminus of rat melanopsin

(KMNSPSESRVPSSLTQDPSF, conjugated to KLH carrier protein; sequence from

Hattar et al., 2002). A portion of the serum was purified against peptide–bovine serum albumin (BSA) affinity columns and titred by indirect enzyme-linked immunosorbent assay (purification by Open Biosystems). The purified antibodies were stored at –80°C in

50% glycerol aliquots and used for immunohistochemical staining of rat retinal sections to verify antibody effectiveness. Posterior eyecups were dissected from adult Long

Evans rats, rinsed with 0.1 M PBS, and ﬁxed in 4% paraformaldehyde/PBS for 1 h. After rinsing with 0.1 M PBS three times, the tissue was embedded in a 20% sucrose/PBS cryoprotection solution overnight at 4°C. The following day, the eyecups were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Torrance, CA) and cut into

20 μm slices using a cryostat. Sections were slide-mounted and stored at –80°C until use. Prior to use, slides were defrosted and washed with 0.1 M PBS before being blocked for 1 h with a PBS solution containing 10% normal goat serum (Life

Technologies), 2% BSA and 0.3% Triton X-100. The primary anti-melanopsin antibodies were diluted (1:2000) in an incubation PBS-based buffer containing 1% BSA, 1% normal goat serum, 0.3% Triton X and 0.01% sodium azide, and the retinal sections were covered in this solution overnight at 4°C. Sections were washed three times with 0.1 M

PBS for 15 min before being incubated for 1 h at room temperature in buffer diluted

(1:1000) with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 dye

(Life Technologies). After incubation, the sections were rinsed in 0.1 M PBS three times for 15 min and coverslipped in Flourmount (SouthernBiotech, Birmingham, AL). Immuno- labelled retinal sections were visualized using an inverted light microscope (Axio

Examiner; Zeiss) equipped with a mercury-based light source.

Puriﬁed ipRGC cultures were prepared from litters of 5 to 6 day-old Long Evans rats (6–8 per session), killed by isoﬂurane overexposure and decapitation, using an anti- melanopsin plate adhesion immunopanning technique that has been previously described (Hartwick et al., 2007). In brief, retinas were dissected in Hibernate-A medium

(Brain Bits) supplemented with B-27 (Life Technologies) and gentamicin. The retinas were incubated for 30 min in Ca2+/Mg2+-free Dulbecco’s phosphate buffered solution

(DPBS; Life Technologies) containing papain (Worthington Biochemicals, Lakewood,

NJ), L-cysteine and DNase. The retinas were then mechanically triturated and washed in

Ca2+/Mg2+-containing DPBS enzyme inhibitor solutions containing DNase and sequentially increasing concentrations of ovomucoid (Roche Diagnostics, Indianapolis,

IN) and BSA. The cells were resuspended in DPBS with added insulin and BSA, and incubated on dishes that had been pre-coated with lectin (Griffonia [Bandeiraea] simplicifolia lectin I; Vector Laboratories, Burlingame, CA) to aid in elimination of non- speciﬁc binding of contaminating cell types, such as macrophages and microglia.

The remaining cell suspension was incubated on a Petri dish that had been coated ﬁrst with goat anti-rabbit IgG antibodies (Jackson ImmunoResearch, West Grove,

PA), and then with rabbit anti-rat melanopsin antibodies (in crude serum; described above). After this incubation, non-adherent cells were discarded by rinsing the dish with

DPBS. Adherent cells were exposed to a 0.125% trypsin solution (8 min at 37°C) and then released by pipetting an enzyme-inhibiting solution (30% fetal bovine serum in

NbActiv4 medium; BrainBits) along the dish. The isolated immunopanned cells were plated on 12 mm poly-D-lysine (PDL)/laminin-coated coverslips in NbActiv4 supplemented with 40 ng ml−1 brain-derived neurotrophic factor (BDNF), 10 ng ml−1 ciliary neurotrophic factor (CNTF), 10 μM forskolin and 10 mg ml−1 gentamicin, and they were maintained in an incubator at 37°C in the dark, under a 5% CO2–air environment.

Calcium imaging methodology was modiﬁed from previous work (Hartwick et al.,

2007). After 1–2 days in culture, coverslip-plated ipRGCs were loaded with 10 μM fura-2

AM calcium indicator dye (Life Technologies) that had been solubilized in Hank’s balanced salt solution (HBSS; with 10 mM Hepes, pH 7.4) containing 0.1% pluronic F-

127 (Life Technologies). After 30 min in the dark at 37°C, the fura-loaded cells were placed in a chamber (Bioptechs, Butler, PA) on the stage of the Axio Examiner

65 microscope (Zeiss) that was constantly superfused with heated (32–34°C), oxygenated

HBSS (10 mM Hepes, pH 7.4). Individual cells were located using a dim white microscope light, which aided in the identiﬁcation of light-responding cells (i.e. cells that exhibited elevated calcium levels due to this light that then recovered in the dark). These cells were then maintained in the dark for at least 15 min before being stimulated with 20 s pulses of the same blue LED light (470 nm; Colibri, Zeiss) described above for the

MEA experiments.

Images of ﬂuorescence (510 nm emission) were captured using a 20× objective

(Plan-Apochromat, NA 1.0; Zeiss) and a cooled charged-coupled device camera

(Sensicam; Cooke, Romulus, MI, USA) attached to the microscope. The two excitation lights were generated by 365 and 380 nm LED modules in the Colibri illumination system, and both lights were further ﬁltered through 360 and 380 nm (FF01-380/14,

FF01-360/12; Semrock, Lake Forest, IL) narrow-band ﬁlters, respectively. A custom dichroic beam-splitter (365DCLP; Chroma Technology, Rockingham, VT) separated the two LED modules within the Colibri casing, and the two excitation lights then passed through a triple-pass beamsplitter (ﬁlter set 62 HE; Zeiss) in the microscope before being directed through the objective. Using Imaging Workbench 6.0 software (Indec

Biosystems, Santa Clara, CA), the ﬂuorescence intensity in individual ipRGC somata was captured and quantiﬁed for ratiometric calcium imaging. The narrow-band 360 nm light (currently the lowest wavelength available for the Colibri system) is close to the isosbestic point of fura-2. Thus, as the intracellular free Ca2+ increased, the fura-2

(360/380 nm) ratio increased due to: 1) a decrease in ﬂuorescence with 380 nm excitation; and 2) unchanged ﬂuorescence with 360 nm excitation (or a smaller decrease relative to 380 nm).

Fura-2 ratios were obtained every 5 s during light stimuli and every 10 to 40 s during the intervening dark periods. Light responsive cells were defined as those exhibiting a greater than 15% rise in fura-2 ratio from baseline. The ∆fura-2 ratio was calculated as peak minus baseline fura-2 ratio. Baseline was determined by averaging the fura-2 ratio of the three ratios preceding each 20 slight pulse while the peak was defined as the maximum fura-2 ratio observed within the 60 s following light onset.

Immunohistochemistry

Posterior eyecups were dissected from adult Long Evans rats, rinsed with 0.1 M

PBS, and fixed in 4% paraformaldehyde/PBS for 1 h. After rinsing with 0.1 M PBS three times, tissue was embedded in a 20% sucrose/PBS cryoprotection solution overnight at

4°C. The following day, eyecups were embedded in optimal cutting temperature (OCT) solution (Tissue-Tek, Torrance, CA) and cut into 20 μm slices using a cryostat. Sections were slide-mounted and stored at -80°C until use. Prior to use, slides were defrosted and washed with 0.1 M PBS before being blocked for 1 h with a PBS solution containing

10% normal goat serum (NGS; Life Technologies), 2% BSA, and 0.3% Triton X-100.

Sequential staining was used to double-label the A1 adenosine receptor and melanopsin.

Each primary antibody was diluted in an incubation PBS-based buffer containing1% BSA, 1% NGS, 0.3% Triton X, and 0.01% sodium azide. The primary anti-

A1 adenosine receptor rabbit antibodies (A268; Sigma-Aldrich) were diluted 1:2000 in the incubation buffer and the retinal sections were covered in this solution overnight at 4°C.

Sections were washed three times with 0.1 M PBS for 15 min before the sections were incubated for 1 h at room temperature in buffer diluted (1:1000) with goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 dye (Life Technologies). Sections

67 were again washed three times in 0.1 M PBS for 15 min, and incubated with 1:500 anti-

α-actin rabbit antibodies (Millipore, Billerica, MA) to mask any unbound secondary antibodies from interfering with the double-labeling procedure. The sections were then incubated for 2 h at room temperature with fluorescently tagged antibodies directed at the N-terminus of rat melanopsin that had been stored in 50% glycerol aliquots (final buffer dilution: 1:1000). To obtain these antibodies, the crude serum used for the immunopanning procedure was purified against peptide-BSA affinity columns and titered by indirect ELISA (purification by Open Biosystems). Since the anti-melanopsin antibodies were rabbit-raised, same as the anti-A1 adenosine receptor antibodies, we first conjugated them to an Alexa 594 fluorescent tag with a Zenon antibody conjugation kit (Life Technologies). After incubation, the sections were rinsed in 0.1 M PBS three times for 15 min and coverslipped in Flourmount (SouthernBiotech, Birmingham, AL).

Some of the retinal sections were single-labeled with either anti-A1 adenosine receptor or anti-melanopsin antibodies.

Immuno-labeled retinal sections were visualized using an inverted light microscope (Axio Examiner; Zeiss) equipped with a mercury-based light source (X-cite

200DC; Lumen Dynamics, Mississauga, Canada) or a spectral confocal microscope

(FV1000; Olympus, Center Valley, PA) equipped with HeNe and Argon lasers. Images were obtained as either single optical sections or z-stacks, and analyzed using commercial software (Zeiss Zen or Olympus FV10 Viewer 4). Contrast and brightness was adjusted uniformly in comparative sections using Photoshop (Adobe, San Jose,

CA). Images were rotated and cropped to properly orient retinal layers for figures.

Pharmacology and solution preparation

Stock solutions were prepared, aliquoted and stored at -80°C, with non-water soluble compounds being dissolved in dimethyl sulfoxide (DMSO). The final working concentration of DMSO in Ames medium or HBSS was  0.1%. Unless stated otherwise in the Results, each compound was perfused onto the retinas for 5 min prior to light stimulation. To investigate adenosine-mediated signaling pathways, adenosine, CPA

(N6-cyclopentyladenosine), Cl-MECA ( 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H- purin-9-yl]-1-deoxy-N-methyl-β-D-ribofuranuronamide), NBMPR (6-S-[(4- nitrophenyl)methyl]-6-thioinosine), and DPCPX (8-cyclopentyl-1,3-dipropylxanthine) were all obtained from Tocris Bioscience.

Melanopsin-based photoresponses were isolated in adult retinas by superfusing a synaptic blocker cocktail to inhibit rod/cone-driven signaling (Wong et al., 2007; Sodhi and Hartwick, 2014). The cocktail contained inhibitors of glutamatergic signaling (100 μM

L-AP4, 25 μM NBQX, and 10 μM MK-801), glycinergic signaling (10 μM strychnine) and

GABAA-, GABAB-, and GABAC-receptors (50 μM picrotoxin, 5 μM CGP 54626, and 100

μM TPMPA) from Tocris. In adult retinas, light responses recorded in the presence of

Ames’ medium containing the blocker cocktail were considered ipRGC responses.

Results

Modulation of ipRGC light responses through A1 adenosine receptor activation

Adenosine is a known neuromodulator present in the inner retina (Hartwick et al.,

2004) that inﬂuences intracellular cAMP levels by either stimulating or inhibiting adenylate cyclase. I therefore tested the effect of adenosine on MEA-recorded ipRGC spiking, seeking to determine whether this endogenously present compound modiﬁes

69 ipRGC light responses through the cAMP-mediated pathway outlined above. Adenosine

(500 μM) was bath applied to neonatal (P8–14) rat retinas in the presence of adenosine transport inhibitor NMBPR (20 μM) (Figure 3.1a). After 5 min exposure to these two compounds, the duration of the ipRGC responses was considerably shortened (Figure

3.1b), with significantly fewer light-evoked spikes fired (Figure 3.1c). As the effect of the adenosine treatment was most consistent with a reduction in cAMP levels (i.e. opposite effect as occurred with forskolin treatment), I investigated whether adenosine’s effect is mediated through A1 receptors. These adenosine receptors are linked to Gi-mediated inhibition of adenylate cyclase (Fredholm et al., 2001). After treatment with adenosine plus NMBPR (20 min later), the retinas were exposed to these compounds again with the A1 receptor antagonist DPCPX (20 μM) present in the bath. This antagonist blocked the effect of adenosine, with responses recorded during the bath application of all three agents together (adenosine, NMBPR and DPCPX) being no different from the initial control light responses recorded in Ames medium alone (Figure 3.1a-c). Confirming the involvement of A1 receptors, exposure of neonatal retinas to the specific agonist CPA

(10 μM) produced a similar effect as adenosine + NMBPR in reducing the duration of light-evoked ipRGC spiking (Figure 3.1d-f). A3 receptors have also been linked to Gi protein-mediated inhibition of adenylate cyclase and have been found on mammalian ganglion cells (Zhang et al., 2006), but the selective A3 agonist 2-Cl-IB-MECA (10 μM) did not signiﬁcantly (P > 0.05, one-way repeated-measures ANOVA; compared to control and washout conditions) affect the number of light-evoked spikes ﬁred by the ipRGCs in the neonatal retinas (N=3 retinas, n=107 cells; data not shown).

To investigate whether the effect of adenosine on neonatal ipRGC light responses was mediated through a decrease in internal cAMP levels, I next assessed

70 whether the pharmacological elevation of cAMP could reverse the shortening effect CPA had on the duration of light-evoked ipRGC spiking. Exposure for 5 min to CPA (10 μM) alone signiﬁcantly decreased the duration of ipRGC light responses (Figure 3.2a-c), consistent with previous experiments (Figure 3.1d-f). Before the next light exposure,

CPA was washed from the bath and the retinas were constantly superfused with 1 mM

8-Br-cAMP for 20 min, with 10 μM CPA being added to the superfusing medium for the last 5 min. The duration of the responses and total number of light-evoked spikes ﬁred by the ipRGCs was signiﬁcantly increased after this CPA plus 8-Br-cAMP pre-treatment as compared to application of CPA alone (Figure 3.2a-c).

In determining whether the effects of adenosine were a unique feature of developing retinas, I tested whether A1 receptor activation could alter the light responses recorded from ipRGCs in adult (>2 month old) rats. As before, these experiments were done in the presence of bath-applied synaptic blocker cocktail to identify light-responding ipRGCs. Similar to the results obtained using neonatal retinas, 500 μM adenosine (with

20 μM NMBPR) decreased the total number of spikes fired by the ipRGCs, and the inhibitory effect of adenosine was blocked by pre-treatment with the A1 receptor antagonist DPCPX (Figure 3.3a, b). Treating the retinas with CPA (10 μM) for 5 min also signiﬁcantly reduced light-driven ipRGC spiking, and this inhibitory effect persisted after the 20 min washout period (Figure 3.3c, d). These results provide physiological evidence that the effect of adenosine on adult ipRGCs is mediated through A1 receptor activation.

As the MEA experiments reported above involved bathing the ﬂat-mounted retinas with the tested compounds, I considered the possibility that adenosine may be acting on other retinal cells, and adenosine’s effect on ipRGCs was an indirect one due to the release of different transmitters/modulators from these adenosine-stimulated cells.

The experiments on the adult retinas were done in the presence of synaptic blockers

(glutamate, GABA, glycine, acetylcholine receptor antagonists), thereby reducing, but not eliminating, the likelihood of this possibility. To conﬁrm that the observed effects are due to the direct effect of adenosine on A1 receptors present on ipRGCs, I used fura-2 calcium imaging on ipRGCs from near-puriﬁed cultures that had been generated through an immunopanning technique that we have used previously (Hartwick et al., 2007). For this technique, I used antibodies targeted against the extracellular N terminus of the melanopsin photopigment (Figure 3.4a). The cells maintain their light sensitivity for the

first 2–3 days in culture, which was measured as light-evoked calcium influx using fura-2 calcium imaging techniques (Figure 3.4b). The calcium signal directly correlates to ipRGC spiking, as the influx is predominantly due to the opening of voltage-gated calcium channels that occurs after action potential firing (Hartwick et al., 2007).

Adenosine (100 μM) significantly reduced the light-evoked calcium signals, which were measured as an increase in the fura-2 ratio (Figure 3.4c,d). The effect of adenosine was abolished when the isolated cells were pre-and co-treated with the A1 adenosine receptor antagonist DPCPX (10 μM; Figure 3.4c,e). As the effects of adenosine on ipRGC light responses were preserved in these purified cultures, these results confirm that adenosine acts directly on A1 receptors expressed by the rat ipRGCs themselves.

To lend further support to the hypothesis that adenosine modulates ipRGC light responses though an A1 receptor, immunohistochemistry was performed on vertical sections to visualize A1 receptor expression on ipRGCs. A Zenon Alexa Fluor 594 rabbit

IgG labeling kit (Molecular Probes, Invitrogen) was used to eliminate species cross reactivity when detecting melanopsin expression due to the fact that most commercially available A1 primary antibodies, and our generated melanopsin antibody, were rabbit

72 raised. Specifically, an Alexa 594 fluorescent tag was directly conjugated to the melanopsin primary antibody to visualize ipRGC staining. The adenosine A1 receptor primary antibody and an Alexa 488 secondary anti-rabbit antibody was used to isolate A1 expression (see Methods for further details). Staining for anti-A1 revealed a crescent-like expression pattern that was predominately localized to ganglion cell somata in the RGC layer (Figure 3.5a), consistent with previous literature outlining A1 receptor expression in the RGC layer (Braas 1986, 1987; Blazynski, 1989; Blazynski and Perez, 1991). Double labeling for the A1 receptor and melanopsin revealed co-localization of A1 transcript on ipRGC somata (Figure 3.5b,c). Taken together with the calcium imaging experiments, A1 expression is present on neonatal and adult ipRGC somata and thereby confirms a means for adenosine-mediated suppression of ipRGC photoresponses. While A1 receptor labeling was present in most melanopsin-immunopositive cells, a subset of ipRGCs did not contain detectable levels of A1 expression. This may be due to the fact that all ipRGC subtypes may not express A1 receptors, as our MEA recording and calcium imaging methodologies are biased towards M1 ipRGCs.

Adenosine A1 receptor-mediated activity does not alter ipRGC sensitivity

As shown thus far, adenosine A1 receptor activation results in clear suppression of light-evoked ipRGC firing duration. However, whether this suppression is due to a change in ipRGC light sensitivity is unknown. To determine whether adenosine alters neonatal rat ipRGC sensitivity through A1 receptor activation, I recorded ipRGC photoresponses, in the presence and absence of CPA, to varying intensities of light. The intensity of consecutive 20 s blue light pulses was adjusted through the use of neutral

13 density (ND) filters. The dimmest light (denoted as 2 ND) corresponded to 7.1 × 10

−1 −2 14 −1 photons s cm at 470 nm, the intermediate light (1 ND) was 7.1 × 10 photons s

−2 15 −1 −2 cm , and the brightest light (0 ND) was 7.1 × 10 photons s cm . The brightest light

15 stimuli was brighter than the stimulus used in previous experiments (3.9 × 10 photons

−1 −2 s cm ) in order to assure detection of ipRGC light responses during the dimmest light exposure. Stimuli were tested in order either from dimmest to brightest or vice versa, to compensate for any non-specific effects due to presentation order. As expected, light- evoked peak firing frequency and duration in neonatal ipRGCs decreased as light intensity decreased, regardless of whether CPA (10 μM) was absent (Ames’ medium alone; Figure 3.6a) or present (Figure 3.6b). A significant reduction in the total light- evoked spikes fired was observed in the presence of CPA as compared to Ames medium alone (Figure 3.6c). However, latency to peak firing was not significantly affected (Figure 3.6d), despite the reduction in overall firing seen with CPA exposure

(Figure 3.6c). Latency increased when comparing the responses of the dimmest versus brightest light, indicating that the mean latency serves as an indicator of ipRGC sensitivity to these light stimuli. Thus, as latency was not altered to a given light intensity, these results suggest that the effect of CPA was not due a reduction in ipRGC light sensitivity. Instead, adenosine A1-mediated suppression appears to be due a modulation of either ipRGC photoresponse offset or spike frequency during the light exposures, instead of a change in response onset.

I hypothesized that the reduction in ipRGC light response offset and total number of spikes fired, in the presence of CPA, would be most apparent if the light stimuli was presented from dimmest to brightest, as the repeated light exposure could elevate cAMP levels (Vaquero et al., 2001) and counter some of the effects of the A1 adenosine receptor activation. Indeed, suppression of light-evoked ipRGC firing and response 74 duration was the greatest in the 2 ND condition when light was presented from dim to bright (Figure 3.7d) as compared to bright to dim (Figure 3.7c). Without CPA present, ipRGC responses were also more robust when the light stimuli order went from dim to bright (Figure 3.7b) as compared to bright to dim (Figure 3.7a). This may be due to the brighter light stimuli inducing a greater increase in internal cAMP levels, which then affects the subsequent stimuli. Thus, there were significant order effects, on ipRGCs maintained in either regular Ames (Figure 3.7e) or Ames supplemented with CPA

(Figure 3.7f), for their responses to the dimmest light stimulus.

Endogenous adenosine accumulation during dark adaptation leads to suppression of ipRGC photoresponses through A1 receptor activation

In the retina, the concentration of extracellular adenosine is differentially regulated through the circadian clock and environmental lighting conditions (Ribelayga and Mangel, 2005). Environmental lighting conditions modulate extracellular adenosine levels with peak adenosine accumulation present during dark adaptation. Overall, the circadian clock and environmental lighting conditions work in congruence to maximize retinal adenosine levels at night and during prolonged darkness (Ribelayga and Mangel,

2005). In order to determine whether endogenous adenosine inhibits ipRGC light responses through an A1 receptor, I examined light-evoked ipRGC firing following dark adaptation. Given that MEA recordings are done ex vivo, environmental light can be more accurately manipulated than circadian time of day. Specifically, retinas were placed in an MEA chamber for 2.5 hr in the dark in Hibernate-A medium supplemented with either adenosine transport inhibitor NMBPR (20 μM) alone or with NMBPR and adenosine A1 antagonist DPCPX (20 μM). NMBPR was added to both conditions to

75 reduce adenosine turnover. After dark adaptation, retinas were perfused with Ames medium for 20 min prior to the presentation of the first light pulse. A series of 5 successive light pulses was presented, separated by 5 min in the dark between each pulse. In the presence of NMBPR alone, dark adaptation led to a suppression of mean peak firing frequency and total spikes fired in light-evoked ipRGCs (Figure 3.8a,c).

Suppression was most pronounced during the initial light pulse and gradually peaked by the final light exposure. This suppression of ipRGC peak firing frequency and spikes fired in response to the initial stimuli did not occur in retinas treated with DPCPX during dark adaptation (Figure 3.8b,d), as no significant difference was observed from initial to final 20 s light exposure. Overall, the reduction in firing frequency and total spikes observed following 2.5 hr dark adaptation was apparent even during the final light pulse, as compared to dark adapting conditions in which DPCPX was present. These results indicate endogenous adenosine accumulation following dark adaptation induces global suppression of ipRGC light responses through activation of the A1 receptor.

Discussion

My previous work demonstrates that the characteristic ipRGC light response are subject to modulation through a cAMP/PKA-dependent pathway. In this work, I specifically showed that the duration of ipRGC action potential firing evoked by bright light stimulation is significantly shortened following the activation of A1 adenosine receptors present on ipRGCs. This study therefore provides evidence that adenosine, a retinal neuromodulator known to influence intracellular cAMP levels, can modify the temporal properties of ipRGC light responses and alter the irradiance information that is conveyed by these photoreceptors to the brain.

Neuromodulation of ipRGCs by adenosine through a cAMP-dependent pathway

I sought to identify an endogenous neuromodulator that could modify ipRGC light responses through the cAMP/PKA pathway identified in Chapter 2. I assessed adenosine, known to be released in the retina at night or after dark adaptation

(Ribelayga and Mangel, 2005), as such a candidate. In my experiments, bath application of adenosine (in the presence of adenosine transporter inhibitor NMBPR) resulted in a reduction in the number of light-evoked spikes ﬁred by ipRGCs. As adenosine had the opposite effect on ipRGC light responses to forskolin, I investigated whether the actions of adenosine were mediated through activation of A1 receptors, which are heavily expressed in the RGC layer (Braas et al., 1987; Blazynski and Perez, 1991). Stimulation of A1 receptors induces a decrease in internal cAMP levels through Gi protein-related inhibition of adenylate cyclase activity (Fredholm et al., 2001). In support of an A1 receptor-based mechanism: 1) the effects of adenosine could be blocked through the use of the selective A1 antagonist DPCPX; and 2) the A1 agonist CPA (but not the A3 agonist 2-Cl-IB-MECA) mirrored the effects of adenosine by reducing the duration of light-stimulated ipRGC spiking after treatment. Increasing intracellular cyclic nucleotide levels by pre-exposing the retinas to 8-Br-cAMP reversed the suppression of ipRGC photo-responses by CPA, thereby supporting cAMP-dependent signaling pathways as underlying adenosine’s effect. Treatment of CPA did not, however, alter the latency of the ipRGC responses with light onset. Therefore, rather than causing an overall decrease in ipRGC light sensitivity, adenosine appears to inhibit spiking during light exposures and shortens the period of persistent firing that occurs in these photoreceptors with light offset.

Adenosine significantly affected the light responses of ipRGCs from either neonatal (8–14 day old) or adult (>2 month old) rat retinas. As compounds were bath applied to the intact retina preparations, I considered the possibility that adenosine was affecting ipRGCs indirectly through its action on other retinal cells, resulting in the release of different neurotransmitters or neuromodulators onto ipRGC dendrites.

However, adenosine reduced the light-induced calcium signal, which directly correlates with the number of spikes ﬁred by these photoreceptors (Hartwick et al., 2007), in cultured ipRGCs that were isolated from synaptic contacts with other retinal cells. As the effect of adenosine on the immunopanned ipRGCs was blocked through co-application of the A1 antagonist DPCPX, these experiments conclusively established that A1 adenosine receptors are located on ipRGCs and that adenosine can directly affect these photoreceptive neurons. This conclusion was further verified by the immunolocalization of the A1 receptor on melanopsin-expressing ipRGCs in fixed retinal tissue.

Functional implications of adenosine-mediated neuromodulation of ipRGCs

The reduction in light-evoked spiking exhibited by neonatal and adult ipRGCs with adenosine present indicates that this endogenous neuromodulator alters the response of these photoreceptors to environmental light cues in vivo. Retinal adenosine levels exhibit circadian variation and peak at night (Ribelayga and Mangel, 2005), a time period that has also been associated with low cAMP levels in the ganglion cell layer

(Vaquero et al., 2001). The source of extracellular adenosine may be from degraded

ATP that is released either from glia (Newman, 2003, 2004) or from starburst amacrine cells (Neal and Cunningham, 1994; Santos et al., 1999). In support of the latter source, the effects of adenosine on the inner retina can be blocked by tetrodotoxin (Newman,

2005), which would be expected to affect spiking starburst amacrine cells more than non-spiking glial cells. Synaptic release of adenosine by amacrine cells may account for the absence of significant circadian changes in the inherent light sensitivity of ipRGCs as described by Weng et al. (2009). In their study, ipRGC sensitivity across 24 h periods was assessed in the presence of synaptic blockers (Weng et al., 2009), which may have blunted the night-time rise in retinal adenosine levels and masked a circadian variation in ipRGC sensitivity that was regulated by this extrinsic neuromodulator.

Extracellular adenosine and intracellular cAMP levels are also influenced by light exposure. Prolonged dark adaptation reduces cAMP immunoreactivity in the ganglion cell layer during the subjective day while the opposite is true for light-adapted retinas

(Vaquero et al., 2001). Similarly, dark adaptation leads to increased extracellular adenosine in the retina (Ribelayga and Mangel, 2005). While synaptically isolated ipRGCs show increases in their inherent light sensitivity following dark adaptation (Wong et al., 2005), similar to other photoreceptors, my data suggest that the extrinsic effects of rising adenosine levels in the dark inhibits these cells and leads to global suppression of ipRGC responses to light after dark adaptation in vivo. Thus, dark adaptation of ganglion cell photoreceptors varies greatly from that of rod and cone photoreceptors. Such opposing affects can be reconciled with the fact that all three photoreceptors regulate vastly dissimilar functions and as such adenosine accumulation in the dark may lead to modulation through distinct mechanisms in the outer versus the inner retina.

The inhibition of light-evoked ipRGCs by adenosine at night, or after dark adaptation, could significantly affect the photic functions that are affected by these photoreceptors, such as circadian photoentrainment, pupil constriction and alertness.

Notably, adenosine has been found to act pre-synaptically at A1 receptors to attenuate

79 light-induced phase shifts in the suprachiasmatic nucleus, the site of the central circadian clock (Hallworth et al., 2002). As ipRGCs are the pre-synaptic neurons that innervate the suprachiasmatic nucleus (Hattar et al., 2006), the inhibition of ipRGCs by adenosine may contribute to some of the known effects for this neuromodulator on the circadian clock (Watanabe et al., 1996; Hallworth et al., 2002; Sigworth and Rea, 2003).

Light signaling by ipRGCs is also thought to increase alertness (Lockley et al., 2006), and therefore the inhibition of ipRGC spiking at night by adenosine would be expected to promote sleepiness. With respect to pupil constriction, the contribution of ipRGCs to the pupillary light reﬂex is well established (Hattar et al., 2003; Lucas et al., 2003) and can be assessed in humans by measuring the prolonged pupil constriction that persists after a bright light stimulus is extinguished (Gamlin et al., 2007). This post-illumination pupil response (PIPR) exhibits a circadian rhythm, becoming less pronounced during the subjective night (Zele et al., 2011). This is consistent with my hypothesis that ipRGC are suppressed at night due to adenosinergic modulation.

ipRGCs have also been implicated in the exacerbation of photophobia that is associated with migraines, as they project to thalamic pain centers that synapse with dura-sensitive neurons (Noseda et al., 2010). Orange-tinted glasses that ﬁlter out blue light have been shown to reduce the frequency of migraine attacks with short periods of usage (Good et al., 1991). There has also been interest in developing pharmacological treatments for photophobia that target light signaling by ipRGCs. A small-molecule antagonist of melanopsin-based phototransduction has been recently described, and this compound reduces light aversive behavior in rodents (Jones et al., 2013). As I found that adenosine modiﬁes ipRGC light responses without fully inhibiting them, compounds that target A1 receptors represent another therapeutic avenue to be explored for this

80 condition. Alternatively, repeated adaptation periods in the dark may help to maximize retinal adenosine levels and reduce ipRGC signaling to the thalamic pain centers.

In summary, this work demonstrates that endogenous retinal adenosine can reduce ipRGC spiking via A1 receptor stimulation through an intracellular cAMP/PKA- mediated pathway previously shown to regulate light-evoked spiking in rat ipRGCs

(Sodhi and Hartwick, 2014). This work suggests that the gain of ipRGC light response is not a static characteristic, but instead is regulated by light-and circadian-driven changes in extracellular retinal levels of adenosine and other cAMP-related neuromodulators.

Figure 3.1. Adenosine A1 receptor-mediated modulation of neonatal ipRGC light responses.

A) Example rasters from an ipRGC treated for 5 min with 500 μM adenosine (ADO) + 20

μM NMBPR (ADO transport inhibitor) in the absence and presence of 20 μM DPCPX (A1 receptor antagonist). B) – C) Summary graphs of mean spike frequency (B; 1 s binning) and mean spike counts for these experiments (C; N = 3; n = 97). D–F) Similar to ADO treatment, a 5 min treatment with A1-receptor agonist CPA (D; 10 μM) shortened light responses E) and reduced the total number of light-evoked spiking in neonatal ipRGCs

(F; N = 3; n = 39). ∗∗P < 0.01, ∗P < 0.05, one-way ANOVA, Holm–Sidak. Stimulus was blue light (470 nm; 3.9 × 1015 photons s−1 cm−2) 82

Figure 3.2. Effect of adenosine on neonatal ipRGC light responses can be reversed by cAMP application.

A) Example raster, B) mean spike frequency and C) mean spike counts for light- stimulated ipRGCs (N = 3; n = 83) treated for 5 min with 10 μM CPA alone and in co- treatment with 1 mM 8-Br-cAMP. For the last named, the cells were perfused with 8-Br- cAMP for 15 min before the 5 min co-treatment. ‘Control’ responses were the initial pretreatment responses recorded with only Ames medium in the bath. ∗∗ P < 0.01

ANOVA, Holm–Sidak. 83

Figure 3.3. Adenosine A1 receptor-mediated modulation on adult ipRGC light responses.

A) Example rasters and B) mean spike counts for light-stimulated ipRGCs (N = 2; n =

12) treated for 5 min with 500 μM adenosine (ADO) + 20 μM NMBPR in the presence and absence of 20 μM DPCPX. Similar to ADO treatment, C) 5 min treatment with the A1 receptor agonist CPA (10 μM) reduced the D) total number of light-evoked spiking in ipRGCs (N = 4; n = 8) recorded from adult (>2 months old) rat retinas. All of these recordings were performed with synaptic blocker cocktail present. ∗∗P < 0.01, ∗P < 0.05, one-way repeated-measures ANOVA, Holm–Sidak.

Figure 3.4. Adenosine inhibits light-evoked calcium responses from isolated ipRGCs in purified cultures.

A) Immunolocalization of melanopsin in three ipRGCs located in the ganglion cell layer

(GCL) of a retinal slice from an adult rat. The primary N-terminal melanopsin antibodies used here were employed in the immunopanning procedure to generate ipRGC cultures.

Scale bar = 20 μm. B) Light evoked a rise in internal calcium levels, measured as an increase in the fura-2 ratio, in isolated ipRGCs. Example pseudocolored images of fura-2 fluorescence ratios in (i) a cultured ipRGC before (ii), immediately after (iii) and 50 s after

(iv) exposure to 20 s light pulse. Scale bar = 25 μm. C) Data summary for the

continued

Figure 3.4. continued experiments involving adenosine alone (n = 11) and adenosine plus DPCPX (n = 8). ∗∗P

< 0.01, one-way repeated-measures ANOVA, Holm–Sidak. D) and E), example traces showing that adenosine (D; ADO; 100 μM) reversibly inhibited the light-evoked calcium signals, and ADO’s effect could be blocked by adding the A1 receptor antagonist DPCPX

(E; 10 μM) to the bath. Stimulus was blue light (470 nm; 3.9 × 1015 photons s−1 cm−2).

Figure 3.5. Adenosine A1 receptor expression on ipRGC somata in adult rat retinas.

Immunolocalization of melanopsin and the adenosine A1 receptor in the ganglion cell layer (GCL) of 20 μm retinal slices from an adult rat imaged by scanning confocal microscope. A) Anti-A1 immunoreactivity was localized to RGC somata in the GCL in crescent-like expression pattern. B) – C) Melanopsin immunoreactivity is depicted in a single ipRGC on left, A1 receptor expression in the center, and the merged image showing co-localization on the right. The primary N-terminal melanopsin antibodies used here were employed in the immunopanning procedure to generate ipRGC cultures. The two representative melanopsin-positive ipRGCs shown in the pictomicrographs demonstrate that A1 receptor expression is localized to ipRGC somata, as depicted in the overlay images. Scale bar = 20 μm (A), 10 μm (B and C). 87

Figure 3.6. Adenosine A1 receptor activation alters ipRGC photoresponses to light of varying intensities.

Mean spike frequency for light-stimulated neonatal ipRGCs in A) regular Ames medium

(N=6, n= 93) and B) following 5 min treatment of Ames supplemented with A1 agonist

CPA (10 μM) (N = 6; n = 94). Overall, CPA treatment reduced C) total light-evoked spiking in ipRGCs at all light intensities (2 ND—0 ND) but did not affect ipRGC sensitivity as D) response latency was not altered. Stimulus was blue light (470 nm) presented through neutral density filters. 7.1 × 1015 photons s−1 cm−2= 0 ND, 7.1 × 1014 photons s−1 cm−2= 1 ND, 7.1 × 1013 photons s−1 cm−2= 2 ND. ∗∗P < 0.01, one-way repeated- measures ANOVA, Holm–Sidak. 88

Figure 3.7. Effect of presentation order, involving light stimuli of different irradiances, on ipRGC light-evoked spiking.

Mean spike frequency, after stimulation with light stimuli presented in brightest-to- dimmest order, for neonatal ipRGCs A) maintained in regular Ames medium and C) following 5 min treatment with A1 agonist CPA (10 μM). Similarly, results using stimuli presented in dimmest-to-brightest order is shown for ipRGCs treated with B) Ames medium alone or D) CPA. Total spikes fired during the dimmest light exposure was reduced when it was presented first (grey) versus last (black) in either the E) regular

Ames (N=3, n= 61) or F) CPA treatment (N= 3, n= 57) conditions, with a greater suppression with CPA present. ∗ P < 0.05, ∗∗P < 0.01, one-way ANOVA, Holm–Sidak 89

Figure 3.8. Extracellular endogenous adenosine induces global suppression of ipRGC light responses that can be reversed through A1 receptor antagonism.

A) Mean spike counts C) and firing frequency for neonatal ipRGCs (N = 3; n = 15) stimulated with 5 successive exposures to 20 s light pulses (470 nm, 7.1 ×1015 photons s−1 cm−2), with 5 min dark between each pulse. Following 2.5 hr dark exposure, significant suppression of light-evoked firing was observed during the initial light pulse with gradual increases in spiking occuring with each successive light pulse. B) and D)

Addition of A1 antagonist DPCPX (20 μM) to the bath during 2.5 hr dark exposure reversed the suppression of light-evoked spiking in ipRGCs (N = 3; n = 66) as initial and final light pulses showed no significant difference in total spikes fired. NMBPR (20 μM) was present in the bath during both conditions. ∗∗P < 0.01, one-way repeated-measures

ANOVA, Holm–Sidak. 90

CHAPTER 4

MUSCARINIC ACETYLCHOLINE RECEPTOR-MEDIATED STIMULATION OF

RETINAL GANGLION CELL PHOTORECEPTORS

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are photoreceptors found in the mammalian inner retina that primarily serve as irradiance detectors, signaling information about ambient light levels to the brain through their axons in the optic nerve (Do and Yau, 2010; Lucas, 2013). Evidence indicates that ipRGC phototransduction, initiated by melanopsin photopigment, involves Gq protein-coupled phospholipase C (PLC) activation (Graham et al., 2008; Xue et al., 2011). Unlike rods and cones, ipRGCs are post-synaptic neurons that possess dense dendritic arbors

(Berson et al., 2010), along which synapses with bipolar and amacrine cells occur

(Belenky et al., 2003; Wong et al., 2007). This property affords ipRGCs the ability to integrate retinal network signals and indicates that light responses of these photoreceptors may be modified by neuromodulators present in the inner retina. Indeed, recent evidence demonstrates that ipRGC light responses are affected by dopamine and adenosine (Van Hook et al., 2012; Sodhi and Hartwick, 2014), two compounds that accumulate in the retina after prolonged exposure to either light or darkness, respectively (Nir et al., 2000; Ribelayga and Mangel, 2005).

I postulated that another candidate neuromodulator of ipRGCs is acetylcholine

(ACh), which is released exclusively from ‘starburst’ amacrine cells (SACs) in the inner mammalian retina. There are two SAC subtypes, differentiated based upon their

91 dendritic stratification in the inner plexiform layer (IPL) of the retina: one stratifies in the outer ‘OFF’ sublamina and releases ACh in response to light offset, while the other localizes to the inner ‘ON’ sublamina and releases ACh during light onset (Masland and

Mills, 1979; Famiglietti, 1983; Vaney, 1984). The post-synaptic actions of acetylcholine are mediated through either nicotinic receptors (nAChRs) or muscarinic receptors

(mAChRs) present on other retinal neurons. While nAChRs are ligand-gated ion channels, mAChRs are coupled to either Gs/i protein-adenylate cyclase (M2 and M4 receptors) or Gq protein-PLC (M1, M3 and M5 receptors) signaling pathways (see review by Brown, 2010). Thus, metabotropic mAChRs are generally associated with slower neuromodulatory actions, as opposed to the more rapid neurotransmission that occurs with nAChR activation. mAChR expression and activity has been characterized in the ganglion cell layer, and acetylcholine is known to modulate the synaptically-driven light responses of non-photoreceptive RGCs (Schmidt et al., 1987; Baldridge, 1996; Kittila and Massey, 1997; Strang et al., 2010). mAChRs have not been specifically identified in ipRGCs, but akin to other RGCs, the dendrites of these photoreceptors reside in the IPL.

I recently demonstrated that ipRGC light responses are affected by adenosine through its ability to alter intracellular levels of the cyclic nucleotide cAMP (Sodhi and Hartwick,

2014). In this work, I sought to determine whether acetylcholine had a similar neuromodulatory action on rat ipRGCs. Surprisingly, I instead found that activation of ipRGC mAChRs causes robust spiking in these neurons and that a relatively dim, flickering light stimulus can be used to stimulate ipRGCs through a melanopsin- independent mechanism involving endogenous release of acetylcholine in the retina.

Materials and Methods

Multielectrode array (MEA) recordings on rat retinas

All procedures were approved by The Ohio State University Institutional Animal

Care and Use Committee. Long Evans rats (Charles River), used at ages either before

(8-14 days old) or after (3-15 weeks old) eye opening, were decapitated following isoflurane overexposure. The latter older animals were dark adapted for at least 1 h prior to enucleation and dissected under dim red light (625 nm) to minimize rod/cone photobleaching. As described in previous work (Sodhi and Hartwick, 2014), retinal pieces were dissected, placed RGC-side down in a 60-electrode array chamber (Multi-

Channel Systems, Reutlingen, Germany), and moved to the microscope stage where it was continuously superfused (1 ml/min) with heated (32-34C), oxygenated Ames medium (10 mM HEPES, pH 7.4; Sigma-Aldrich). Retinas were maintained under these conditions for 1 h in the dark prior to first light exposure.

Blue or yellow light stimuli were generated using an LED source (Colibri system;

Zeiss) attached to an upright microscope (Axio Examiner; Zeiss). The blue light stimulus, chosen due to peak melanopsin sensitivity at 480 nm light (Berson et al., 2002), has been previously used (Sodhi and Hartwick, 2014). For the yellow stimulus, a ‘white’ LED

(module 423052-9120) was first filtered through a bandpass interference filter (BP

585/35; Zeiss) and then a triple-pass beamsplitter (set 62 HE; Zeiss). In some experiments, the yellow light was flickered at 6 Hz using Imaging Workbench 6.0 software (IndecBiosystems).

Details on the MEA recordings have been previously described (Sodhi and

Hartwick, 2014). Briefly, voltage recordings were amplified and digitized, and clustered spike data were sorted into individual units. Spikes occurring in 1 s bins were counted to

93 generate spike frequency graphs and total counts of neuronal spiking. To identify ipRGCs, spike counts were determined for: 1) the 10 s (-10 to 0 s) pre-stimulus period

(‘x’); 2) the first 10 s (0 to 10 s) of the light stimulus period (‘y’); and 3) the second 10 s

(10 to 20 s) of the light stimulus period (‘z’). Identification of ‘ipRGC units’ were based on all of the following criteria being met: y  50; y  2x; z  50; and z  2x. ‘ON’ RGCs were defined as units that exhibited >10 spikes during the first 1 s of light onset, but didn’t meet the ipRGC spiking criteria. ‘OFF’ RGCs were defined as cells that showed a >50% decrease in first 10 s of light exposure, relative to 10 s pre-stimulus period. Statistical differences in the spike counts were determined using one-way, or one-way repeated measures, ANOVA and Holm-Sidak post-hoc testing (Sigmaplot 12; Systat Software).

Calcium imaging of ipRGCs isolated by immunopanning

Purified ipRGC cultures were prepared from 5 to 6 day-old Long Evans rats (6-8 per session) using an anti-melanopsin plate adhesion immunopanning technique

(Hartwick et al., 2007). After incubation of mixed retinal cell suspensions on lectin-coated dishes, ipRGCs were isolated from the remaining cells using a rabbit anti-rat melanopsin antibodies (crude serum; described in Sodhi and Hartwick, 2014) coated dish. The ipRGCs were released and plated on PDL/laminin-coated coverslips in neurotrophin- supplemented NbActiv4 culture medium as before (Sodhi and Hartwick, 2014).

After 1-2 days in culture, coverslip-plated ipRGCs were loaded for 30 min with 10

μM Fura-2 AM calcium indicator dye (Life Technologies), solubilized in Hank’s Balanced

Salt Solution (HBSS; with 10 mM HEPES, pH 7.4) containing 0.1% pluronic F-127. The fura-loaded cells were placed in the microscope chamber and superfused with heated

(32-34°C), oxygenated HBSS. After dark adaptation of >15 min, fluorescence (510 nm

94 emission) images of individual cells were captured using a 20x objective (Plan-

Apochromat, NA 1.0 W) and a microscope-attached cooled charged-coupled device camera (Sensicam; Cooke). Using Imaging Workbench 6.0 (Indec) software, changes in the fura-2 ratio (360/380 nm excitation) were quantified and used as a marker for changes in intracellular free Ca2+ (for details, see Sodhi and Hartwick, 2014). Fura-2 ratios were obtained every 5 s during light stimuli or drug exposure and every 10-40 s during the dark periods. Light responsive cells were defined as those exhibiting a greater than 15% rise in fura-2 ratio from baseline. The ∆fura-2 ratio was calculated as peak

(maximum ratio during 60s following light onset) minus baseline (average of 3 ratios preceding each light pulse) ratios.

Pharmacology and solution preparation

Aliquoted stock solutions were stored at -80°C, with non-water soluble compounds being dissolved in dimethyl sulfoxide (DMSO). The final working concentration of DMSO was  0.1%. Carbachol, tubocurarine, AF-DX 384, 4-DAMP and physostigmine were obtained from Tocris Bioscience; atropine was from Sigma-Aldrich.

Melanopsin-based light responses were confirmed in the older retinas by superfusing a cocktail of synaptic blockers to inhibit rod/cone-driven signaling (Wong et al., 2007). The cocktail contained inhibitors (all from Tocris) of glutamatergic signaling (100 μM L-AP4,

25 μM NBQX, 10 μM MK-801) and, in some experiments, inhibitors of glycine (10 μM strychnine) and GABA receptors (50 μM picrotoxin, 5 μM CGP 54626, 100 μM TPMPA).

Pupillary light reflex (PLR) in human subjects

A brief case history from each subject was obtained to rule out all exclusion criteria. The subject’s visual acuity was measured, and a grading of the anterior chamber angle of the left eye was made by a licensed optometrist (Phil Yuhas) using the Van

Herrik technique. Exclusion criteria for all subjects included: (1) diagnosis of any form

(i.e. secondary, closed-angle, primary open-angle) of glaucoma or a history of optic nerve disease other than glaucoma (e.g. optic neuritis), (2) history of having surgery

(e.g. trabeculectomy) or taking medications (e.g. cholinergic agonists/antagonists) known to profoundly affect pupillary function or shape, (3) history of traumatic brain injury requiring hospitalization, (4) pregnancy, (5) history of unreactive pupil responses,

(6) history of retinopathy (e.g. diabetic retinopathy, retinal detachment), (7) presence of significant cataract (any posterior sub-capsular opacification, or nuclear sclerosis graded

> 1+) at last eye examination, (8) refractive error greater than a spherical equivalent of

+/- 6 diopters. At the time of this report, preliminary data has been collected from two

(one male, one female) subjects.

One drop of 1.0% tropicamide was instilled in the subject’s left eye, followed by a

30 minute dark adaptation period. The 30 minute dark adaptation prior to the first stimulus was to allow time for the left pupil to dilate. At the end of the dark adaptation period, the subject was placed in front of a custom pupillometer, consisting of a computer-controlled LED light source (DiCon LED; Richmond, CA) and an infrared video camera (Sony HDR-XR500V; Tokyo, Japan). The camera provides close-up images of the iris and pupil under infrared illumination, which are recorded with a sampling frequency of 60 frames per second. The session is as follows (red light stimulus = 1012 photons/s/cm2): 30 min dark adaptation – 5 min continuous red light – 15 min dark – 6

Hz red light – 15 min dark – 30 Hz red light. The pupil videos were downloaded onto a password-protected computer for analysis with ImageJ software (pupil size, rate of re- dilation, amplitude of pupil fluctuation) and Excel (Microsoft) software to produce normalized pupil measurements. Pupil size was measured by fitting a circle to the pupil in a given frame and the area of that pupil was calculated. Finalized graphs were completed on Sigmaplot software.

Results

Using MEA recordings from retinas dissected from 5-12 week old rats, I first investigated whether carbachol modifies ipRGC light responses in a comparable manner to that reported for adenosine (see Sodhi and Hartwick, 2014), as certain mAChRs similarly influence adenylate cyclase-related signaling pathways. ipRGCs were identified based on spiking criteria and the persistence of their light responses (Figure 4.1a; top, bottom) in the presence of glutamate antagonists to block synaptic activity (see

Methods). However, rather than a neuromodulatory action, I was surprised to observe that the application of carbachol (100 μM) evoked prolonged action potential firing in the ipRGCs prior to light exposure (Figure 4.1a; middle). With carbachol present, ipRGC responses to the concurrent application of light were significantly reduced in terms of either peak firing frequency (Figure 4.1b) or total spikes fired (Figure 4.1c). Suppression of the ipRGC light response appeared to be due to a dose-dependent effect of carbachol as marginal stimulation of ipRGCs with 5 μM carbachol did not significantly affect ipRGC peak firing frequency (Figure 4.1d) or total spikes fired (Figure 4.1e) during concurrent light exposure. However, in the presence of 10 μM carbachol, ipRGC light-evoked firing peak firing frequency was attenuated ~25% (Figure 4.1d) and a significant reduction in

97 total spikes fired was observed, as compared to baseline control levels (Figure 4.1e).

Thus, the stimulatory actions of light and carbachol were not additive, as the ipRGC responses to carbachol partially occluded those evoked by light.

I next applied carbachol for 1 min to array-mounted retinas superfused with

Ames’ medium alone (no synaptic blockers) in order to assess whether this effect of carbachol was specific to ipRGCs. ‘OFF’ RGCs exhibit elevated baseline spiking in the dark that decreases during light exposure (Figure 4.2a(i); top), and their light responses were abolished with synaptic blockers present (Figure 4.2a(iii); top). Units identified as ipRGCs had sustained spiking during the 20 s light pulses (Figure 4.2a(i); middle) that persisted with synaptic blockers present (Figure 4.2a(iii); middle). For comparison, the more transient light-evoked increases in spiking that occurred in units classified as ‘ON’

RGCs (Figure 4.2a(i); bottom) were eliminated by the blocker cocktail (Figure 4.2a(iii); bottom). Of these 3 broad cell types, the stimulatory effect of carbachol was most pronounced on ipRGCs (Figure 4.2a(ii)), as these cells exhibited sustained spiking during the bath application of carbachol (Figure 4.2b,c). In contrast, carbachol caused a smaller increase in spiking in ON RGCs, and it instead inhibited spiking in the OFF

RGCs (Figure 4.2b,c), consistent with previous work examining cholinergic effects on these two cell types (Masland and Ames, 1976; Strang et al., 2010). In the recordings obtained from 6 retinas, 17 units fired >150 spikes during the 60 s carbachol exposure.

Only 1 of the 17 units did not meet the ipRGC identification criteria; this unit did not exhibit any definitive light response and so could not be classified into one of the 3 broad cell types.

The pharmacology of the receptor mediating carbachol’s effect on ipRGCs was examined. Carbachol (100 μM) was applied to retinas (from rats aged 5-12 weeks) for 1

98 min to distinguish units that exhibited robust (>150 spikes over 60 s) spiking responses to this cholinergic agonist (Figure 4.3ai,bi). At the end of the experiments, and consistent with the data shown in Figure 4.1, cells with strong carbachol responses were confirmed to be ipRGCs based on the persistence of light responses during superfusion of a cocktail of glutamatergic, glycinergic and GABAergic antagonists (see Methods). Pre- treating the retinas with the ionotropic nAChR antagonist tubocurarine (200 μM) for 5 min partially suppressed postnatal ipRGC firing (Figure 4.3c) but did not significantly alter the adult ipRGC responses to a second carbachol exposure (Figure 4.3aii,d), consistent with a previous report indicating adult ipRGCs lack nAChR expression (Wong et al., 2007). Conversely, pre-treatment with the metabotropic mAChR antagonist atropine (20 μM) for 5 min resulted in the response to the third carbachol treatment being almost completely suppressed in both postnatal (Figure 4.3c) and adult ipRGCs

(Figure 4.3aiii,d). In further parsing candidate mAChR subtypes, I tested whether AF-DX

384 (M2/M4 antagonist) or 4-DAMP (M1/M3 antagonist) attenuated carbachol’s actions in adult ipRGCs to avoid any developmental differences in receptor expression. The latter compound, but not the former, nearly abolished the ipRGC responses to carbachol, indicating that the effects were mediated through Gq-coupled mAChRs (M1 or M3) rather than Gi-coupled ones (Figure 4.3d). To rule out the possibility that the effects of atropine or 4-DAMP were due to response ‘run-down’ to the three carbachol exposures, atropine or 4-DAMP were each tested alone on separate groups of cells (n = 5; N = 2 retinas for both groups) with the effects being similar (with atropine present, carbachol response was 5.7% ± 4.1 of that with atropine absent; with 4-DAMP present, carbachol response was 9.7% ± 4.8 of that with 4-DAMP absent) to those shown in Figure 4.3.

In the first two weeks after a rat’s birth, prior to the opening of eyelids, rod and cone-driven retinal circuitry is immature and light-evoked spiking in the RGC layer is entirely melanopsin-dependent (Tu et al., 2005). This enables the neonatal rodent retina to serve as a useful preparation for studying ipRGC photoresponses in the absence of rod/cone-influenced signaling pathways. I obtained MEA recordings from 8-14 day old

(pre-eye opening) rat retinas and found, analogous to the data from the older retinas, carbachol stimulated a strong spiking response in units designated as ipRGCs due to their light sensitivity (Figure 4.4a). Also similar to the adult findings illustrated in Figure

4.3, atropine (20 μM) and 4-DAMP (20 μM) essentially eliminated carbachol-evoked spiking in the neonatal ipRGCs (with antagonist present, responses were 5.8% ± 3.0 [N

= 3; n = 47] and 5.0% ± 1.4 [N = 3; n = 77], respectively), of those recorded during initial carbachol treatments (Figure 4.3c). Thus, the pharmacology of the carbachol-evoked ipRGC responses is consistent with a Gq-coupled muscarinic receptor pathway regardless of developmental age.

The data obtained using MEA recordings did not exclude the possibility that the effect of carbachol on ipRGCs occurred through an indirect mechanism. For example, carbachol may stimulate neurotransmitter release from amacrine cells synaptically connected to ipRGCs. To conclusively confirm that activation of muscarinic receptors located on ipRGCs themselves can produce spiking responses in these photoreceptors,

I monitored the intracellular calcium levels in isolated neonatal ipRGCs that had been immunopanned into near-purified cultures (Hartwick et al., 2007). Light-induced calcium influx in ipRGCs, which is predominately due to voltage-gated calcium channels opening following action potential firing, directly correlates with ipRGC spiking (Hartwick et al.,

2007). Application of carbachol (100 μM) to the isolated photosensitive neurons for 1 min

100 produced a rise in the fura-2 fluorescence ratio, indicative of an increase in calcium levels. The cells were also stimulated with light to verify their ipRGC identity, and the carbachol responses were comparable to those evoked by 20 s pulses of bright blue light (Figure 4.4b,c). Comparable to the MEA experiments on intact retinas, atropine pre- treatment blocked the carbachol-induced calcium responses in the isolated cells (Figure

4.4d,e). Thus, these results provide physiological evidence that mAChRs are expressed directly on ipRGCs, and that the activation of the receptors can directly stimulate action potential firing in these photoreceptors.

While the preceding data indicated that the cholinergic agonist carbachol could stimulate ipRGC spiking, I next investigated whether this mAChR-related mechanism could also be activated through the endogenous release of acetylcholine within the retina. First, I applied the cholinesterase inhibitor physostigmine (20 μM), which causes extracellular ACh levels to rise by preventing its hydrolysis, to retinas obtained from rats either before (8 to 14 day old) or after (4 to 7 weeks old) eye opening. Upon application of this compound, there was an increase in spiking measured in identified ipRGC units

(Figure 4.5a). The spiking responses could be prevented by pre-treating the retinas with atropine (Figure 4.5b), thereby demonstrating that endogenous ACh can stimulate ipRGCs through a mAChR-mediated pathway.

Through cone-driven retinal circuits, the release of acetylcholine from SACs can be evoked by certain light stimuli, with flickering light being an especially effective stimulus. It has previously been reported that light that flashes on and off at a rate of 3 to

10 Hz maximizes the ACh release from SACs (Masland and Livingstone, 1976; Massey and Neal, 1979; Masland et al., 1984; Massey and Redburn, 1985; O’Malley and

Masland, 1993; Firth et al., 2003), likely because this optimally stimulates both (ON and

101

OFF) SAC types. I applied flashing (6 Hz) yellow light for 5 min to MEA-mounted retinas obtained from rats aged 4 to 12 weeks. The stimulus was designed to be below threshold for melanopsin activation (2x1012 photons/s/cm2, measured at 585 nm), but at a sufficient intensity to stimulate SACs through cone-driven circuitry. During the stimulus, there was usually a delayed spiking response in units that were identified as ipRGCs at the end of the experiment (persistent light responses with synaptic blocker cocktail present). Specifically, ipRGCs showed a delayed, but robust, spiking response (see

Figure 4.5c for example) during the flickering light exposure. The ipRGCs that didn’t respond to the flickering light were recorded from two retinas that lacked the typical rod- and cone-driven responses in surrounding channels, suggesting that the outer retinal photoreceptors may have been damaged or inadvertently bleached during dissection.

The data from these two retinas were not included in the mean data summaries (Figure

4.5f).

It is well established that ipRGCs receive glutamatergic, GABAergic and glycinergic input from other retinal neurons, but this input is generally more transient in nature (Wong et al., 2007). The delayed spiking that occurred in response to the flashing yellow light was sustained and prolonged, similar to the ipRGC responses to carbachol described above. To determine whether the responses to the flickering yellow stimulus involved mAChR activation, I pre- and co-treated the retinas with atropine during yellow light exposure. Atropine significantly reduced the spiking response when the flashing light stimulation was repeated (Figure 4.5d,f). In addition, the responses were specific to the use of a flickering light stimulus, known to be an effective stimulus for endogenous

ACh release, as the sustained spiking responses were not elicited from the ipRGCs when the same light was left continuously on for 5 min (Figure 4.5e,f). This result also

102 further confirmed that the ipRGC responses to the yellow light was not due to a summation in photon capture over time by ipRGC photopigment, indicating again that the yellow light was used at an intensity that was below threshold for melanopsin activation.

Bolstering the role for cone-driven circuitry in mediating the delayed ipRGC responses to the flickering yellow light, I demonstrated that these responses were eliminated following the application of the cocktail containing glutamatergic antagonists

(Figure 4.5g, representative of 6 ipRGCs tested). Furthermore, I found that the same flickering light stimulus did not stimulate a spiking response in neonatal (8 to 14 day old) ipRGCs, which were characterized based on their response to bright blue light (Figure

4.5h). These control experiments verified that the yellow light stimulus was at an irradiance that was below threshold for stimulating intrinsic melanopsin-related light responses in these ganglion cell photoreceptors.

To explore, indirectly, whether mAChR-mediated stimulation of ganglion cell photoreceptors potentially affects ipRGC-driven behaviors in human subjects, I assessed the effects of 6 Hz flickering light stimuli on the pupillary light reflex (PLR). The

PLR is a non-invasive, objective measure that can be used to extrapolate the preceding findings to the clinic in human subjects. Human subjects were presented with three light stimuli— continuous, 6 Hz flickering, 30 Hz flickering. As in the animal studies, each stimulus was presented for 5 min. However, a red light stimulus (625 nm; 2x1012 photons/s/cm2) was used to sufficiently stimulate human cone-driven circuitry, but remained below the threshold for melanopsin activation. The PLR recorded in human subjects revealed that pupil constriction was most pronounced during the 6 Hz flickering light stimuli (Figure 4.6a,b). These effects were specific to 6 Hz flickering light exposure,

103 as continuous light and 30 Hz flickering light stimuli elicited a similar but reduced percentage of pupil constriction as compared to 6 Hz flicker (Figure 4.6a,b). These preliminary PLR results bolster the hypothesis that, through the use of flickering light, the

ACh-driven ipRGC stimulation observed can influence ipRGC-regulated behaviors.

Discussion

A defining feature of photoreceptors is their ability to endogenously absorb photons of light and convert them into an electrical signal, independent of input from the surrounding retinal milieu. ipRGCs exhibit remarkably sustained spiking responses during light exposures that can persist for many seconds following light offset. My current work demonstrates that ipRGCs can also be directly stimulated through activation of the mAChR-mediated Gq-coupled PLC signaling pathway, independent of melanopsin-based phototransduction, to produce prolonged, high frequency firing characteristically observed with ipRGC light responses. This effect appears to be specific to ipRGCs, as such robust firing was not observed in non-photosensitive RGCs.

I have demonstrated that activation of mAChRs, specifically Gq-coupled mAChRs, induces robust ipRGC firing, which can be reversed through mAChR antagonism. This contrasts with a previous study, where ipRGCs reportedly did not respond to cholinergic stimulation during an initial screen of neurotransmitters (Wong et al., 2007). Herein I present substantial evidence to refute this claim, including calcium imaging on isolated ipRGCs that clearly demonstrates their intrinsic response to muscarinic stimuli. A notable difference is that Wong et al. performed their experiment using ACh, while we chose to use carbachol due to its increased longevity in bath applications given its resistance to breakdown by acetylcholinesterase. Moreover, Wong

104 et al. may not have detected stimulation of ipRGCs to puff-applied ACh with the inclusion of cesium in their Ames bath medium, due to blockade of hyperpolarization-activated non-selective cation (Ih) channels that have been linked to muscarinic activity in the CNS

(Zhu and Uhlrich, 1998; Zhu and Heggleund, 2001; Fisahn et al., 2002). In this study, treatment with acetylcholinesterase inhibitor produced similar effects to carbachol, providing evidence that endogenous ACh released in the retina is capable of driving the responses we observed. In agreement with Wong et al, I also show that adult ipRGCs lack nAChRs. Although my data suggest that mAChR stimulation is responsible for the observed phenomenon in adult retinas, a small degree of nAChR-driven activity was noted during neonate experiments. In the developing retina, RGCs express nAChRs as nAChR-driven spontaneous wave activity in postnatal RGCs is crucial for proper refinement of retinal circuitry (Blankenship et al., 2009; Ford and Feller, 2012). Thus, ipRGCs may express nAChRs and mAChRs during development, but nAChR expression is apparently lost in mature ipRGCs.

The calcium imaging experiments demonstrate that ipRGCs directly respond to mAChR stimulation. However, whether these effects are due to volumetric or synaptic release of acetylcholine was not explored. In the CNS and in the retina, volumetric transmission is well documented during development (see Picciotto et al., 2012, Ford and Feller, 2012). In the developing retina, volumetric release allows acetylcholine to accumulate and act in a diffuse manner to affect extrasynaptic cholinergic receptors present on RGCs, including ipRGCs (Syed et al., 2004; Ford and Feller, 2012). Similar extrasynaptic effects have been observed in the CNS (see Fuxe et al., 2012 for review).

Typically, ACh is thought to be synaptically released in the mature CNS and retina, as the occurrence of volumetric release during adulthood is a controversial topic. However,

105 synapses between ipRGCs and SACs have not been reported in the retina. Given that

SACs are found in the vicinity of ipRGCs (in the IPL and RGC layer), I postulate that diffuse ACh release from these cells is sufficient to drive ipRGC stimulation not only in development, but in the mature retina. Nevertheless, further investigation is needed to truly rule out synaptic ACh release, as recent evidence indicates that ipRGC dendrites partially co-stratify with ChAT positive processes in the macaque retina (Chang et al.,

2014 Society for Neuroscience meeting, 433.08/EE26).

Identification of a novel, melanopsin-independent pathway for activating sustained ipRGC firing fundamentally challenges the notion that ipRGCs are only activated by bright, blue light, which is the light that the melanopsin protein has the highest probability of absorbing (Berson et al., 2002; Dacey et al., 2005). My work shows that ACh released from SACs, which can be driven by a dim flickering yellow light, is sufficient to directly activate ipRGCs and elicit robust spiking in these photoreceptors.

Moreover, data from two human subjects demonstrates that this flickering light may potentially drive greater pupil constriction as compared to continuous light. Flickering light is known to activate motion detectors in the retina, including SACs which are subsequently driven to release ACh (Masland and Mills, 1979; Masland et al., 1984;

Pourcho and Osman, 1986; Voigt, 1986; Brandon, 1987; Brecha et al., 1988; Kosaka et al,. 1988) and GABA (Brecha et al., 1998; O’Malley and Masland, 1989). In turn, direction selective RGCs respond to this pre-synaptic GABA and ACh release to convey motion in our environment (see Taylor and Vaney, 2003 for review). In contrast, ipRGCs are not directionally selective (Estevez et al., 2012) and therefore, their activation by

SACs would not contribute to motion detection. The flickering light experiments in this study demonstrate that while ipRGCs are not motion detectors, these cells respond to

106 changes in motion or object presentation in our environment in a unique manner. Thus, this suggests SACs have a novel, ACh-dependent role in stimulating RGCs that goes beyond solely conveying motion in our environment in a directionally selective manner, as seen in these ganglion cell photoreceptors. Flickering light exposure is often perceived as being brighter than continuous light and has been implicated in exacerbating migraine-induced photophobia (Digre and Brennan, 2012). A recent study showed that ipRGC signaling to the ventro-posterior thalamus, elicits migraine-induced photophobia (Noseda et al., 2010). Thus, perhaps SACs play a role in migraine-related photophobia, as ACh released from SACs in response to flickering is capable of driving ipRGC signaling to thalamic pain centers in the brain.

Ultimately, the melanopsin-independent pathway we describe has the potential to be exploited clinically by pharmacologic drugs or even non-pharmacological approaches

(e.g. flickering light) to influence non-visual functions regulated by ipRGCs. Alteration of ipRGC function is a feasible strategy, as demonstrated by the recent use of a small- molecule melanopsin antagonist (Jones et al., 2013). Simply “turning on” ipRGCs with a drug compound offers versatile use for correcting conditions in which ipRGC stimulation is abnormal, as in seasonal affective disorder, and to enhance processes such as alertness and cognition (Altimus et al., 2008; Roecklein et al., 2009; LeGates et al.,

2012).

107

A ipRGC Light Responses (Synaptic Blockers Present) B C Pre-Carbachol Treatment

100 mM Carbachol Present

Post-Carbachol Treatment

D Light response: Synaptic blockers absent Carbachol response: Synaptic blockers absent Light response: Synaptic blockers present OFF RGC

ipRGC

ON RGC

E F

Figure 4.1. Carbachol-evoked spiking in rat ipRGCs.

A) Light responses from example ipRGC, recorded before, during and after exposure to

100 µM carbachol. Recordings were done in the presence of cocktail containing glutamatergic antagonists to identify responding units as ipRGCs. B) Graphs of mean spike frequency (spikes/s) and C) mean counts of spikes fired during the 20 s light pulse show that the carbachol application stimulated ipRGC spiking, which partially occluded ipRGC responses to subsequent light exposure. Occlusion is due to a concentration dependent effect as 10 µM carbachol, but not 5 µM carbachol, partially occludes ipRGC

D) light-evoked firing frequency and E) total spikes fired during 20 s light exposure. ** P

< 0.01, T-test. All light stimuli were blue LED-generated (470 nm; 3.9x1015 phots/s/cm2).

108

Figure 4.2. Robust carbachol-evoked spiking is specific to rat ipRGCs versus non- photosensitive RGCs.

A) Example responses to (i) light and (ii) carbachol recorded from an OFF-type RGC, ipRGC, and ON-type RGC in the same retina, while superfused with Ames medium alone. (iii) Light responses were also obtained with synaptic blocker cocktail present to confirm cell identification. B) Mean spike frequency (spikes/s) and C) mean overall spike counts (during first minute of exposure) for carbachol recorded from 5 OFF-type RGCs,

16 ipRGCs and 14 ON-type RGCs during MEA experiments on 6 retinas. ** P < 0.01, one-way ANOVA, Holm-Sidak posthoc. All light stimuli were blue LED-generated (470 nm; 3.9x1015 phots/s/cm2). 109

Figure 4.3. Carbachol’s effect on ipRGCs is mediated by Gq-coupled mAChR activation.

A) Example spike rasters for an adult ipRGC exposed to (i) carbachol (100 µM), first alone, and then in the presence of (ii) the nAChR antagonist tubocurarine (200 µM) and

(iii) the mAChR antagonist atropine (20 µM). B) Example spike rasters for another ipRGC exposed to (i) carbachol (100 µM) alone and in the presence of (ii) the M2/M4 mAChR antagonist AF-DX 384 (20 µM) or (iii) the M1/M3 mAChR antagonist 4-DAMP (20

µM). Summary of data, normalized within-cell to initial carbachol response, showing effect of cholinergic antagonists on C) postnatal and D) adult ipRGCs. ** P < 0.01,

Friedman RM ANOVA, Dunnett’s post hoc; compared to initial carbachol response. 110

Figure 4.4. Effect of carbachol on neonatal ipRGCs, using MEA retinal recordings and calcium imaging on isolated ipRGCs in purified cultures.

A) Example spike raster from MEA recording of ipRGC in 10-day old retina (pre-eye opening). The ipRGC responds to both carbachol (100 µM) and blue light (470 nm;

3.9x1015phots/s/cm2). B) Representative trace of fura-2 fluorescence ratio showing that carbachol (100 μM) stimulated an increase in internal calcium levels in isolated light- responding ipRGCs in culture. C) Data summary showing magnitude of the responses, with the response to a bright blue light (470 nm; 3.9x1015phots/s/cm2; 20 s pulse) used for comparative reference (n = 15). D) Example trace and E) data summary graph illustrating that carbachol’s effect was blocked with application of the mAChR antagonist atropine (20 μM; n = 9). ** P< 0.01, paired T-test. 111

Figure 4.5. Effect of endogenous acetylcholine, stimulated by cholinesterase inhibition or use of flickering light stimuli, on ipRGCs ex vivo.

A) Graphs of mean spike frequency (spikes/s) and B) mean counts of spikes fired during

500 s recording show that application of cholinesterase inhibitor physostigmine produced a delayed increase in ipRGC spiking, which was blocked by atropine pre-treatment

continued 112

Figure 4.5. continued

(20 μM; N = 3, n = 9). ** P < 0.01, T-test. C) Spike rasters from an example ipRGC with characteristic sluggish response to blue light (top), that exhibited delayed, but repeatable

(middle, bottom), spiking responses to dim yellow light stimulus flickering at 6 Hz. D)

Spike rasters from other ipRGCs showing flickering yellow light –induced response was blocked by atropine (20 μM), and E) was not elicited during continuous yellow light presentation. Note, atropine did not block the ipRGC responses to blue light. F)

Summary of data for spike counts during the 300 s flickering yellow light exposures that was repeated in the absence (N = 4, n = 14) or presence (N = 10, n = 22) of atropine, or given following 300 s of continuous yellow light exposure (N = 7, n = 16).** P < 0.01, paired T-test. G) Spike rasters from representative ipRGC showing that a cocktail of glutamatergic antagonists blocked the response to 6 Hz flickering yellow light (compare top to bottom), although the characteristic responses to blue light persisted with the cocktail present (middle). H) Representative raster from a neonatal ipRGC depicting a lack of response (spike was due to spontaneous wave activity) to 6 Hz dim flickering yellow light exposure for 5 min. ** P < 0.01, one-way ANOVA, Holm-Sidak posthoc.

Lights were yellow (585 nm; 2.0x1012phots/s/cm2) and blue (470 nm;

3.9x1015phots/s/cm2) LED-generated.

113

Figure 4.6. Effect of continuous versus flickering light exposure on pupillary constriction in human subjects in vivo.

A) Traces depicting mean pupillary constriction frequency recorded during 5 min exposure to continuous red light, 6 Hz flickering red light, and 30 Hz flickering red light.

B) Corresponding total percentage of pupil constriction (during 5 min of exposure) recorded during continuous light, 6 Hz flickering light, and 30 Hz flickering light exposure on 2 subjects. Pupil constriction was signiﬁcantly more pronounced during the 6 Hz flicker condition, as compared to both continuous light and 30 Hz light exposure. ** P <

0.01, one-way ANOVA, Holm-Sidak posthoc. All light stimuli were red LED-generated

(625 nm; 2x1012 phots/s/cm2).

114

CHAPTER 5

CONCLUSIONS

Summary of Key Findings

In the presence of light, ipRGCs exhibit sustained depolarization, in contrast to the transient hyperpolarizing responses of rods and cones. The persistence of this response with light offset underlies the reduced temporal resolution exhibited by these ganglion cell photoreceptors. The overall aim of this thesis was to determine whether the unique temporal dynamics of ipRGC photoresponses are subject to modification by endogenous retinal neuromodulators. As post-synaptic photoreceptors, ipRGCs are capable of integrating photic information transmitted from pre-synaptic neurons regulated by rod- and cone-driven signaling. In fact, the light responses of ipRGCs are composed of both an extrinsic (rod- and cone-driven) and an intrinsic (melanopsin- related) component. Given that ipRGCs possess dense dendritic nets that span the entire retina, I hypothesized that these ganglion cell photoreceptors were capable of being modulated by extrinsic input from the retinal network.

In Chapter 2, I demonstrate that ipRGC photoresponses are indeed subject to modulation through an intracellular cAMP/PKA-mediated signaling pathway. Specifically, stimulation of the cAMP/PKA pathway leads to prolonged light-evoked ipRGC firing. To expand upon these findings, I sought to identify an endogenous retinal neuromodulator capable of modulating ipRGC photoresponse through the cAMP/PKA pathway (Chapter

3). I illustrated that adenosine is one such endogenous neuromodulator that suppresses

115 light-evoked ipRGC firing following activation of the Gi-coupled A1 receptor that is present on ipRGC somata, as confirmed through calcium imaging experiments on dissociated ipRGCs. Notably, I show that endogenous adenosine A1-mediated suppression of ipRGC photoresponses occurs after maintaining the isolated retinas in the dark for a prolonged period, consistent with previous work finding that retinal adenosine levels elevate after dark adaptation. Dark adaptation serves to increase sensitivity and contrast detection in conventional photoreceptors. However, in ganglion cell photoreceptors, dark adaptation leads to a global suppression of light-evoked responses that does not appear to be due to a reduction in sensitivity.

In Chapter 4, I determine that ipRGCs can be directly stimulated through a melanopsin-independent pathway by cholinergic activation of muscarinic receptors present on ipRGCs. The prolonged action potential firing evoked by cholinergic compounds appeared to specifically occur in ipRGCs. In addition, I was able to reproduce the effects of cholinergic compounds on ipRGC firing with yellow 6 Hz flickering light stimuli that was below the threshold for melanopsin activation. Flickering light stimuli are known to stimulate ACh release in the retina, and the robust ipRGC responses were blocked with mAChR antagonists. Moreover, preliminary evidence suggests that these results could be translatable to humans as examination of pupillary constriction in two subjects revealed that 6 Hz flickering red light produces a greater mean pupil constriction as compared to continuous red light exposure. Overall, these results suggest that extrinsic cone-driven signaling is capable of producing sustained melanopsin-independent action potential firing in ipRGCs through the stimulation of mAChRs by ACh that is released by SACs.

116

Significance

Taken together, these studies indicate that ipRGC photoresponses are much more complex than previously appreciated. The temporal characteristics of these ganglion cell photoreceptors are not a static feature. Instead, ipRGCs are highly dynamic, capable of altering their responses to light after exposure to retinal neuromodulators such as adenosine and dopamine. Remarkably, these ganglion cell photoreceptors can also exhibit sustained spiking after stimulation by acetylcholine released through cone-driven signaling. Extrinsic input serves to extend the dynamic range of ipRGCs, allowing them to respond to dim and rapid light stimuli beyond the range of detection required of melanopsin-dependent phototransduction. However, extrinsic input-generated ipRGC photoresponses in dim light are typically transient in nature. Thus, the extrinsic ACh-mediated stimulation of ipRGCs uncovered in this work is truly novel in that it results in robust, prolonged firing that is unlike anything previously observed.

In some regards, the fact that the presumed M1 ipRGCs examined in this work are subject to such profound modulation is intriguing when considering the intricacies of this subtype. The prototypic M1 cells express the greatest levels of melanopsin photopigment and thus rely heavily on intrinsic melanopsin-dependent signaling to drive their photoresponses (Schmidt and Kofuji, 2010, 2011). On the other hand, M2 cells rely less on intrinsic melanopsin-dependent signaling to drive their photoresponses. Instead, extrinsic rod- and cone-driven signaling has a greater contribution in shaping M2 photoresponses (Schmidt and Kofuji, 2010, 2011) and as such, it would not be as unexpected for these cells to be subject to modulation. However, I speculate that the neuromodulators that govern modulation of M1 ipRGCs are representative of the more

117 diffuse spread of these compounds that results in broad effects upon neurons in multiple retinal layers. Dopamine and adenosine are prime candidates for such modulators, as their effects are usually due to global accumulation that occurs over large regions of the retina. Although M1 ipRGCs may receive less synaptic input that other ipRGC subtypes, my results demonstrate that their characteristic sluggish light responses can be modified through the extracellular accumulation of these types of neuromodulators. In addition, ipRGC responses may be affected by volumetric ACh release by SACs. Thus, global changes in the retinal milieu would influence M1 ipRGC-driven non-image forming functions, as opposed to the auxiliary role ipRGCs play in image-forming vision.

Neuromodulators are defined by the diffuse nature of their release and long- lasting effects at multiple post-synaptic neurons. As such, fluctuations in the global concentration of retinal neuromodulators could modulate neuronal activity at multiple levels. The highlighted work suggests that retinal adenosine accumulation at night and during dark-adapted conditions would affect photoreceptors in the inner and outer retina in a distinct manner. In the outer retina, adenosine modulates photoreceptor coupling to enhance sensitivity and contrast detection. However, my data reveal that adenosine inhibits the responses of inner retinal photoreceptors. Thus, adenosinergic modulation varies due to the separate functions regulated by this neuromodulator in the dark. For example, the action of adenosinergic modulation on rod/cone photoreceptors increases the sensitivity of vision at night while its inhibitory actions on ipRGCs during nighttime may aid in limiting stimulation to the brain centers that regulate wakefulness. Therefore, it is not surprising that modulation of ipRGCs by compounds such as adenosine may not be similar to its actions on classical photoreceptors, given the vastly separate functions regulated by the different photoreceptors.

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In addition to the effect of ACh on ipRGCs shown in my work, ACh is known to stimulate DSGCs in the retina. DSGCs play a role in the ability of the visual system to detect moving objects. While the purpose of cholinergic stimulation of ipRGCs is not clear, ipRGCs are not considered motion detectors. Therefore, ACh-driven stimulation of these ganglion cell photoreceptors represents a novel, distinct role for SACs that is separate from the regulation of DSGCs to convey motion in our environment.

Adenosinergic modulation in the RGC layer has already been documented as adenosine inhibits glutamate-driven responses in non-photosensitive RGCs through A1 receptor activation (Hartwick et al., 2007). Given my findings depicting A1 receptor-mediated suppression of ipRGC light responses, it appears that ipRGCs share some commonalties with other RGCs with respect to their neuromodulation. This can be partly attributed to the fact that ipRGCs and non-photosensitive RGCs are post-synaptic output neurons that integrate network effects within the retina.

The neuromodulation of ipRGCs showcased in this body of work further inform the complexity of ipRGC light responses and melanopsin-driven functions. While these ganglion cell photoreceptors appear to be unlike classical photoreceptors, they share some commonality with non-photosensitive RGCs. However, if this work is any indication, much is still unknown about ipRGCs since their discovery over a decade ago.

To date, ipRGCs have been shown to be modulated (Van Hook and Berson, 2012;

Sodhi and Hartwick, 2014) and to drive modulation themselves of retinal interneurons and cones (Zhang et al., 2008, 2012; Muller et al., 2010). Thus, unlike other retinal neurons, ipRGCs convey photic information in a bi-directional manner within the retina.

Recently, there has been a great deal of work focused on quantifying luminance or

‘melanopic’ lux (Enazi et al., 2011; Brown et al., 2013; Lucas et al., 2014) by essentially

119 filtering the overall irradiance of a given light through the melanopsin spectral sensitivity curve, which has a peak sensitivity of 480 nm. However, my findings suggest that this method of calculating melanopic lux may be too simplistic to determine light-evoked stimulation of ipRGCs. The calculation of scotopic and photopic lux utilizes the spectral sensitivity curve for rods and cones, respectively. Similarly, the calculation of melanopic lux assumes that the light responses of ganglion cell photoreceptors are solely due to the capture of photons by the melanopsin photopigment. However, unlike rods and cones, ipRGCs are post-synaptic retinal neurons. My work demonstrates that the characteristic prolonged spiking associated with melanopsin-based photoresponses can be altered by modulators like adenosine, and mimicked through stimulation of these cells by cone-driven ACh release. Thus, calculating melanopic lux based solely on the irradiance and spectral distribution of a given light is unlikely to be an accurate correlate of the magnitude of the subsequent evoked signal carried by ipRGCs to their brain targets. As a field, we have yet to form a complete understanding of how other retinal circuits modify ipRGC light responses, as well as the role these ganglion cells photoreceptors play in modulating other retinal neurons.

Future Directions

Adenosinergic modulation of ipRGC-driven pupil constriction in humans

In Chapter 3, I demonstrated that adenosine A1-mediated activity inhibits light- evoked ipRGC firing and that this inhibition can be observed through elevation of endogenous adenosine following dark adaptation ex vivo. However, whether adenosinergic modulation affects ipRGC-driven NIF functions in vivo was not examined.

Given that ipRGCs contribute to the PLR, examination of the post illumination pupil

120 response (PIPR) can be used to determine whether a physiological correlate for increased adenosine in the dark exists in human subjects. Due to the sluggish nature of ipRGC phototransduction, ipRGC firing persists after light onset as opposed to rod and cone responses. This results in prolonged pupil constriction that occurs for seconds after light stimulation. Therefore, the PIPR provides a measure of the ipRGC contribution to the neural circuitry responsible for the PLR (Gamlin et al., 2007). Using PLR methodology outlined in Chapter 4, I could examine the consensual PIPR in response to the following protocols using blue light (470 nm; 2x1013 photons/s/cm2): 1) 30 min dark adaptation – 20 s blue light – 5 min dark – 20 s blue light – 5 min dark – 20 s blue light;

2): 30 min photopic light adaptation (10 cd/m2) – 20 s blue light – 5 min dark – 20 s blue light – 5 min dark – 20 s blue light. I anticipate that a reduction in the PIPR response (i.e. the pupil will re-dilate more rapidly with light offset) after dark adaptation, as compared to after light adaptation. Furthermore, I predict the PIPR will gradually increase (i.e. pupil will remain more constricted with light offset) with each additional light pulse occurring after the prolonged dark adaptation, given the results obtained in Figure 3.8a,c. These results would support the hypothesis that light exposure reduces extracellular adenosine levels, increasing cAMP levels in ipRGCs, resulting in more prolonged firing by these photoreceptors as shown in my work (Chapter 2 and 3). However, as dark adaptation has been shown to increase rod/cone photoreceptor sensitivity, while light adaptation reduces their sensitivity, it would be of interest to determine whether the initial rod/cone- driven pupil constriction that occurs in response to the light pulses would be reduced after light adaptation in this experimental protocol.

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Mechanisms underlying acetylcholine-mediated stimulation of ipRGCs

In Chapter 4, I demonstrated that ipRGCs are capable of being directly stimulated by acetylcholine, resulting in prolonged spiking in these photoreceptors that resembles their melanopsin-dependent light responses. Given these unexpected results,

I then focused on determining whether the effect was mediated through activation of mAChRs located on ipRGCs and examining whether flickering light could evoke these responses in ipRGCs by stimulating the endogenous retinal release of ACh. However, the mechanisms underlying the depolarizing responses of ipRGCs to carbachol were not elucidated. My results indicate that a Gq-coupled mAChR (M1 or M3) mediated the effects of ACh. To confirm the involvement of a Gq-PLC pathway, future experiments could include MEA retinal recordings to monitor carbachol-evoked ipRGC spiking in the presence of PLC inhibitor U 73122 (20 μM), to determine whether PLC inhibition terminates the mAChR-induced ipRGC excitability. Recordings should be repeated with inactive analog U 73343 as a negative control to confirm that the effects observed were not due to the non-specific effects of U 73122.

Gq-protein coupled muscarinic receptors have been shown to initiate PLC signaling cascades (Brown, 2010) and there is evidence that a Gq-PLC cascade is involved in the melanopsin phototransduction pathway (Do and Yau, 2010). Thus, it is possible that after activation of ipRGC mAChRs, the stimulation of PLC leads to opening of the same TRPC6/7 channels that mediate the depolarizing responses associated with the melanopsin phototransduction cascade (hypothesis outlined in Figure 1.2). In support of a shared downstream pathway between mAChR signaling and melanopsin phototransduction, I found that concurrent light and carbachol exposure partially occludes ipRGC photoresponses, rather than producing an additive effect (Figure 4.1).

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As the concentration of carbachol is lowered (see Figure 4.1), the occlusion of ipRGC photoresponses is minimized.

However, another possibility is that mAChR activation causes ipRGC depolarization through a distinct mechanism involving channels other than TRPC channels. Activation of mAChRs can lead to the opening of non-selective cation (Ih) channels (e.g. Zhu and Uhlrich, 1998; Zhu and Heggleund, 2001; Fisahn et al., 2002), which are activated by membrane hyperpolarization to produce stimulatory effects in the brain (see Kase and Imoto, 2012 for review). In the retina, Ih currents have been observed in non-photosensitive RGCs and ipRGCs and serve to boost excitability, as increased spiking is observed following hyperpolarization (Eng et al., 1990; Skaliora et al., 1993; Tabata and Ishida, 1996; O’Brien et al., 2002). While Ih currents exist in ipRGCs (VanHook and Berson, 2010), the physiological conditions during which these channels would be activated in ganglion cell photoreceptors has yet to be determined.

Thus, a second possibility that accounts for the effect of carbachol and ACh on ipRGCs is the direct stimulation of Ih currents through a metabotropic pathway involving mAChRs.

To explore whether the carbachol-evoked responses involve TRPC 6/7 and

PLCβ4 that mediate melanopsin-dependent responses, ipRGC responses to carbachol could be examined using whole cell voltage-clamp (-70mV) and current-clamp recordings from TRPC 6/7 and PLCβ4 knockout mice. Transgenic TRPC 6/7-/- and

PLCβ4-/- mice, each crossed into Opn4:tdTomato for ipRGC identification (Xue et al.,

2011), should display the lack of an ipRGC response upon exposure to bright blue light stimulation (470 nm; 7.1x1015 phots/s/cm2). In accord with the shared pathway hypothesis, mAChR-induced ipRGC firing should also be absent in these mice due to the lack of expression of TRPC 6/7 and PLCβ4.

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If negative results are obtained through these experiments on knockout mice, I would then explore whether the Gq-mAChR mechanism is directly linked to Ih channels.

Direct coupling to Ih channels would provide one explanation for why Wong and colleagues (2007) did not observe a response in ipRGCs exposed to brief pulses of

ACh, as their use of cesium in the bath would block Ih channels. To assess whether ipRGCs are directly activating Ih channels, ion substitution experiments can be done with whole cell patch clamp in which 100 μM carbachol is bath applied with Ames medium in the absence and presence of cesium. Blockade of robust ipRGC firing would support the involvement of Ih, but alternative mechanisms would have to be ruled out. For example, post synaptic activation of Gq-coupled mAChRs on ipRGCs may lead to neuronal excitation through inhibition of Ca+2-activated K+ channels (Brown, 2010) or sub- threshold K+ channels as observed in the brain (Brown and Adams, 1980; Halliwell and

Adams, 1982; McCormick and Price, 1985; McCormick and Williamson, 1989; Douglas et al., 2002). The carbachol-induced calcium influx observed in my experiments (Figure

4.4) would provide a potential source for activation of Ca+2-activated K+ channels. Thus, ion substitution experiments using patch clamp may address the channels involved through regulation of the concentration of cesium, calcium, and potassium in the bath medium.

Potential endogenous neuromodulators of ipRGCs

Given that the temporal dynamics of ipRGC photoresponses have been shown to be modifiable by dopamine (Van Hook and Berson, 2012) and adenosine (Chapter 3), additional endogenous retinal neuromodulators warrant consideration for their potential effects on ipRGC responses. Dopaminergic and adenosinergic modulation of ipRGCs

124 both rely on cAMP/PKA-mediated signaling. Moreover, dopamine and adenosine are implicated in the regulation of sleep/wake cycles and circadian rhythms, which are regulated by the M1 ipRGCs that were likely the predominant ipRGC recorded in my data presented in this thesis. Thus, turning to additional cAMP/PKA-associated modulators of sleep/wake cycles and circadian rhythms in the CNS may provide promising candidate modulators of ipRGCs.

Histamine

One such endogenous neuromodulator candidate is histamine. Histaminergic projections in the retina originate from the tuberomammilary nucleus of the posterior hypothalamus. These retinopetal axons, which originate from the tuberomammilary nucleus and terminate in the retina, comprise the sole source of retinal histamine as histamine-positive cells have not been identified in the retina (Airaksinen and Panula,

1988; Panula et al., 1989; Labandeira-Garcia et al., 1990; Gastinger et al., 2001). In dark-adapted conditions, histamine has been shown to inhibit the light-evoked firing of

ON RGCs and reduce their sensitivity to light (Akimov et al., 2010). Histaminergic inhibition of dopaminergic amacrine cells has also been observed (Weber and Schlicker,

2001).

Preliminary data I have gathered indicates that histamine may indeed modulate ipRGC light responses, as histamine prolonged light-evoked firing and total spikes fired in neonatal ipRGCs (Figure 5.1a,b). However, it should be noted that a 20 min exposure to histamine was required to elicit a significant effect, as opposed to the 5 min perfusion of the compounds presented in this thesis. This discrepancy may be due to the fact that significant accumulation of histamine, which is highly diffusible, is needed. Experiments

125 in which endogenous adenosine was bath applied (Chapter 3) utilized a transport inhibitor (NMBPR) to prevent adenosine turnover. Similarly, a histamine N- methyltransferase inhibitor (SKF 91488 dihydrochloride) may be needed to prevent histamine breakdown in order for shorter histamine exposures to produce sufficient effects on ipRGC light-evoked firing.

Histaminergic activity is perhaps mediated through Gs-coupled H2 receptors as the effects of histamine on ipRGC firing appear to be analogous to those obtained when stimulating the cAMP/PKA signaling pathway with compounds such as forskolin

(Chapter 2). On the other hand, Gq-coupled H1 receptors have been localized to dopaminergic amacrine cells (Gastinger et al., 2006; Greferath et al., 2009) and to bundles in the RGC layer in the rodent retina (Greferath et al., 2009). H1 receptor activation leads to inhibition of dopaminergic amacrine cells in guinea pig and rat retinas

(Weber and Schlicker, 2001; Marshak et al., 2008 ARVO e-abstract 5792). Given that ipRGCs utilize a Gq-coupled phototransduction mechanism, histamine acting at H1 receptors may affect ipRGC photoresponses. In the brain, histamine is involved in sleep/wake cycles with peak levels observed during arousal states (Steininger et al.,

1999; Vanni-Mercier et al., 2003). Therefore, histamine is high during the night in nocturnal animals (Prast et al., 1992), such as rodents, and high in humans during the day. These findings are in line with night-time suppression of dopamine observed in rodents. However, histamine accumulation at night may serve to modulate rodent ipRGCs through a mechanism separate from the cAMP/PKA pathway associated with

A1-mediated suppression of ipRGC light responses, as adenosine and histamine would counteract each other during the night when accumulation of both modulators peaks.

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Pituitary adenylate cyclase-activating peptide (PACAP)

Pituitary adenylate cyclase-activating peptide (PACAP) is a protein that has been localized to the axons of SCN-projecting ipRGCs (Hannibal et al., 2002, 2004). In the brain, PACAP has been shown to act as a neuromodulator and activation of PAC1 receptors results in stimulation of the cAMP/PKA signaling pathway through a Gs- coupled mechanism (see Vaundry et al., 2009 for review). PACAP is involved in several functions. Of note, muscarinic receptors and PACAP receptors have been shown to regulate REM sleep in the pontine reticular formation (Ahnaou et al., 1999, 2000). Given that ipRGCs 1) express PACAP, 2) regulate sleep/wake cycles, 3) and have been shown to be subject to cAMP/PKA-mediated modulation, PACAP is a promising candidate for

Gs-coupled ipRGC-mediated modulation.

In fact, PACAP may enhance ipRGC spiking responses as a counter-balance to the adenosine-mediated inhibition observed in my work. Dopamine has been shown to be one such opposing regulator of ipRGCs. Dopamine modulates ipRGCs through a Gs- coupled mechanism and its endogenous levels peak during light exposure (Van Hook and Berson, 2012). However, Gs-coupled modulation of ipRGCs through PACAP may be particularly noteworthy given that ipRGCs themselves express PACAP and evidence indicates that some ipRGC axons terminate within the retina instead of projecting to the brain (Barnard et al., 2006; Zhang et al., 2008, 2012; Joo et al., 2013). Thus, it stands to reason that PACAP release in the retina from ipRGCs would occur during ipRGC stimulation, as seen during light exposure. If the intra-retinal ipRGC axon collaterals terminate near other ipRGCs, the released PACAP would act on ipRGCs themselves, leading to an increase in cAMP and enhancement of ipRGC photoresponses. Such a modulatory mechanism would suggest that the adaptation that occurs in ipRGCs

127 following light exposure would be the opposite of what occurs in light-adapted rods and cones; the increased excitability of these neurons could be associated with an increase in sensitivity rather than a decrease. Notably, histamine and PACAP may display a truly unique modulation of ipRGCs that has not been uncovered. In my work, the outlined modulatory mechanisms rely on integration of top-down, one-way transmission of photic information that originates from rods and cones. However, ipRGC participation in bidirectional photic information flow may allow ipRGCs to not only be modulated by these neuromodulators, but to also drive release of modulators like histamine and PACAP within the retina.

Opioids

Endogenous opioids are known to directly bind to three G-protein-coupled opioid receptors— μ, δ, and κ (see Williams et al., 2001). Of these, μ-opioid receptor expression has been localized to rabbit retinas (Cunningham et al., 1992) and appears to be expressed on ipRGC dendrites in the mouse retina (Gallagher and Vigh, unpublished findings). Morphine was the first characterized ligand for μ-opioid receptors and as such, is typically associated with mediating its effects through these receptors.

However, further characterization has revealed additional ligands of μ-opioid receptors, such as DAMGO. DAMGO is considered the most selective μ-opioid receptor agonist

(see McDonald and Lambert, 2005). Preliminary data I have gathered indicates that

DAMGO may modulate ipRGC light responses in a similar manner to adenosine, as

DAMGO application resulted in an inhibition of light-evoked firing in adult ipRGCs

(Figure 5.1c,d). Given that μ-opioid receptors are Gi-coupled, these results are in line with the A1-mediated effects observed in my work. However, the physiological conditions

128 associated with the accumulation of endogenous retinal opioids are not known. Thus, the light conditions that would promote opioid-mediated modulation of ipRGCs are unclear.

In the brain, opioids have been shown to act as neuromodulators that inhibit ACh release (e.g. Ruzicka and Jhamandas, 1991; Mulder et al., 1991; Arenas et al., 1991;

Heijna et al., 1992). Perhaps similar modulatory actions are observed in the retina, in order to inhibit ipRGC firing and suppress the pain associated with photophobia.

Specifically, ipRGCs may mediate aversion to bright light exposure (Matynia et al., 2012) and are implicated in migraine-related photophobia (Noseda et al., 2010). Given that these light aversive mechanisms may be due to ACh-mediated ipRGC stimulation (as discussed in the subsequent section), endogenous opioids may serve as suppressors of neuronal excitability in ipRGC during aversive events as is seen in the brain. Thus, ipRGC modulation through Gi-coupled μ-opioid receptors could perform a distinct function than the Gi -coupled A1 receptor modulation described in my work.

Clinical Implications

Regulation of sleep/wake cycles

The studies described in this body of work focus on elucidating the underlying mechanisms through which retinal neuromodulators alter ipRGC light responses.

Subsequently, it may be possible to manipulate the neuromodulation of ipRGCs to aid in regulation of sleep/wake cycles in patients with sleep disorders or for shift workers. For example, my work indicates that dark-adapting conditions suppress ipRGC firing through adenosinergic modulation. The converse should be true in light-adapting conditions, as cAMP is elevated and would instead lead to the enhancement of ipRGC photoresponses

129 as shown in Chapter 2. Therefore, blue light exposure during the morning may aid in regulating proper sleep cycles. Such non-invasive therapy is already being utilized in the form of blue light boxes, to treat seasonal affective disorder (SAD) and regulate sleep.

However, such devices recommend at least 30 min a day of usage (see Parry, 2003).

Instead of using a continuous light, my data suggest that very brief exposures (5 min) to flickering light may be sufficient to “jump start” the ipRGC system. Though extensive testing is needed to confirm whether this would occur, exposure to light for such a short duration may be more manageable for people suffering from sleep disturbances.

Migraine-related photophobia

Numerous studies indicate that flickering light increases retinal ACh levels through release from starburst amacrine cells (SACs), the only source of ACh in the retina (Masland and Mills, 1979; Masland et al., 1984; Pourcho and Osman, 1986; Voigt,

1986; Brandon, 1987; Brecha et al., 1988; Kosaka et al., 1988). These cells have been shown regulate non-photosensitive RGCs such as DSGCs (see Taylor and Vaney,

2003) and my data suggest that they are capable of driving ipRGC excitability through a muscarinic signaling pathway (Chapter 4). Thus, I hypothesize that ACh-mediated ipRGC signaling contributes to the photophobia associated with flickering light exposure.

Flickering light may amplify migraine-related pain by maximizing stimulation of the ventro-posterior thalamus, a region involved in pain that has been shown to be innervated by ipRGCs (Noseda et al., 2010). Furthermore, flickering light-generated headaches have also been reported in healthy patients (Brundrett, 1974; Wilkins et al.,

1989; Kennedy et al., 1998). Thus, elucidating the mechanism underlying sensitivity to flickering light would be beneficial to the general population.

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One method of assessing whether ACh-mediated ipRGC signaling may account for hypersensitivity to flickering light is to utilize the pupillary light reflex (PLR).

Assessment of light-evoked pupil constriction through the PLR provides an objective, clinical measure that serves as a functional readout of ipRGC-driven behavior. While the sample size is limited, the PLR experiments described in Chapter 4 demonstrate that 6

Hz flickering light induces greater pupilliary constriction as compared to both continuous and 30 Hz flickering light, in healthy subjects. Thus, the PLR in human subjects appears to be more sensitive to flickering light in the range that is reported to elicit maximal ACh release from SACs (Masland and Livingstone, 1976; Massey and Neal, 1979; Masland et al., 1984; O’Malley and Masland, 1993; Firth et al., 2003). To determine whether flickering light plays a role in migraine-related photophobia, comparison of the PLR in healthy subjects versus subjects with migraine could be assessed using the paradigm outlined in Chapter 4. Patients with migraine are photophobic even when not experiencing migraine symptoms and are more sensitive to bright light intensity than normal patients (Digre and Brennan, 2012). Thus, I would anticipate subjects with migraine to have a substantially greater PLR, due to greater sensitivity to all three red light stimuli (continuous, 6 Hz, 30 Hz) than healthy subjects. However, pupillary constriction should be most pronounced in the 6 Hz condition and is expected to be far greater than that observed in healthy subjects. These experiments would provide insight as to why flickering light is perceived as a more aversive stimulus than constant light and subsequently, a potential mechanism through which the sensation of photophobia is enhanced. Ultimately, this work may provide a means for evaluating photophobia in patients with migraine and establish a rationale for the development of novel therapies to treat photophobia by targeting neuromodulators that alter ipRGC responses.

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Figure 5.1. Histamine and opioids as potential endogenous neuromodulators of ipRGCs.

A) Mean spike frequency (spikes/s) and B) mean overall spike counts for histamine recorded from neonatal ipRGCs (N = 1, n = 15) during MEA experiments. Histamine (10

μM; 20 min) application increased firing frequency and total spikes fired in light-evoked ipRGCs. Conversely, application of μ-opioid agonist DAMGO (10 μM) revealed a reduction in light-evoked C) mean spike frequency and D) mean overall spikes fired in adult ipRGCs (N = 5, n = 6). Adult recordings were performed with synaptic blocker cocktail present in each condition. * P < 0.05, ** P < 0.01, one-way ANOVA RM, Holm-

Sidak posthoc. All light stimuli were blue LED-generated (470 nm; 3.9x1015 phots/s/cm2).

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