Elsevier

Neuroscience

Volume 303, 10 September 2015, Pages 178-188
Neuroscience

Melatonin modulates M4-type ganglion-cell photoreceptors

https://doi.org/10.1016/j.neuroscience.2015.06.046Get rights and content

Highlights

  • Intrinsically photosensitive retinal ganglion cells (ipRGCs) are novel photoreceptors.

  • We tested if M4-type ipRGCs are modulated by melatonin, a hormone secreted at night.

  • Both exogenous and endogenous melatonin altered M4 cells’ light-evoked responses.

  • M4 cells express the MT1 receptor, suggesting melatonin might act directly on them.

  • We also detected circadian variations in M4 cells’ electrophysiology.

Abstract

In the retina, melatonin is secreted at night by rod/cone photoreceptors and serves as a dark-adaptive signal. Melatonin receptors have been found in many retinal neurons including melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), suggesting it could modulate the physiology of these inner retinal photoreceptors. Here, we investigated whether melatonin modulates the alpha-like M4-type ipRGCs, which are believed to mediate image-forming vision as well as non-image-forming photoresponses. Applying melatonin during daytime (when endogenous melatonin secretion is low) caused whole-cell-recorded M4 cells’ rod/cone-driven depolarizing photoresponses to become broader and larger, whereas the associated elevation in spike rate was reduced. Melanopsin-based light responses were not affected significantly. Nighttime application of the melatonin receptor antagonist luzindole also altered M4 cells’ rod/cone-driven light responses but in the opposite ways: the duration and amplitude of the graded depolarization were reduced, whereas the accompanying spiking increase was enhanced. These luzindole-induced changes confirmed that M4 cells are modulated by endogenous melatonin. Melatonin could induce the above effects by acting directly on M4 cells because immunohistochemistry detected MT1 receptors in these cells, although it could also act presynaptically. Interestingly, the daytime and nighttime recordings showed significant differences in resting membrane potential, spontaneous spike rate and rod/cone-driven light responses, suggesting that M4 cells are under circadian control. This is the first report of a circadian variation in ipRGCs’ resting properties and synaptic input, and of melatoninergic modulation of ipRGCs.

Introduction

Melatonin is a hormone synthesized and secreted by the pineal gland and other tissues including the retina (Lerner et al., 1958, Cardinali and Rosner, 1971, Bubenik et al., 1974, Tosini and Menaker, 1996, Stefulj et al., 2001). Although the biochemistry and physiological actions of melatonin have been investigated extensively, its roles in the retina remain poorly understood. Melatonin is secreted at night by rod/cone photoreceptors and the melatonin receptors MT1 and MT2 have been detected in all five classes of retinal neurons including rod/cone photoreceptors, horizontal cells, bipolar cells, amacrine cells and ganglion cells, suggesting this hormone performs diverse neuromodulatory functions (Huang et al., 2013, Wiechmann and Sherry, 2013).

Two recent studies reported the expression of melatonin receptors in intrinsically photosensitive retinal ganglion cells (ipRGCs) in mice and rats (Sengupta et al., 2011, Sheng et al., 2015). IpRGCs are inner retinal photoreceptors that contain the photopigment melanopsin and mediate not only pattern vision but also non-image-forming visual responses such as pupillary constriction and circadian photoentrainment (Do and Yau, 2010, Ecker et al., 2010, Schmidt et al., 2014). Though ipRGCs are directly light-sensitive, they nevertheless receive rod/cone-driven synaptic input through bipolar and amacrine cells (Wong et al., 2007, Zhao et al., 2014). Thus, melatonin could modulate ipRGCs by acting either directly on them or on their presynaptic circuits, or both. Here, we tested this hypothesis by focusing on the alpha-like M4-type ipRGCs, whose unusually large somas enable them to be identified for whole-cell recordings with relative ease (Estevez et al., 2012, Schmidt et al., 2014, Reifler et al., 2015), thereby obviating the need for fluorescent labeling (Berson et al., 2002, Ecker et al., 2010). We studied the effects of exogenous and endogenous melatonin on M4 cells’ resting membrane potential, spontaneous spike rate, rod/cone-driven (“extrinsic”) light responses, and melanopsin-mediated (“intrinsic”) light responses. We also looked for the expression of melatonin receptors in these neurons.

Section snippets

Ethical approval

All experimental procedures were approved by the University Committee on Use and Care of Animals at the University of Michigan, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23). All efforts were made to minimize the number of animals used and their suffering.

Animals

A total of ∼150 male and female Sprague–Dawley rats aged 2–4 months were used. The animals were maintained in a 12-h light, 12-h dark

Daytime melatonin application modulates M4 cells’ extrinsic light responses

We examined the effects of exogenous melatonin during the animals’ subjective day, when endogenous melatonin secretion is low (Cahill and Besharse, 1992, Tosini and Menaker, 1996). In normal Ames’ medium, M4 cells had a resting membrane potential of −63.1 ± 1.2 mV and spiked spontaneously at 36.7 ± 6.2 Hz. Bath application of 10 nM melatonin had no significant effects on either of these resting properties (Fig. 1B, C). By contrast, extrinsic photoresponses were significantly altered. In response to a 1

Discussion

To date, only two studies have investigated neuromodulation of ipRGC physiology: dopaminergic modulation of M1 cells (Van Hook et al., 2012), and adenosinergic modulation of unclassified ipRGCs (Sodhi and Hartwick, 2014). The present work focused on the M4 type because it is relatively easy to identify for whole-cell recordings. Considering that M4 cells contribute to image-forming as well as non-image-forming vision (Estevez et al., 2012, Schmidt et al., 2014), our results suggest that

Author contributions

D.D.H. conducted pilot experiments and made the recordings shown in Fig. 5A; W.P. generated and analyzed all other electrophysiological data; K.Y.W. conceived the project, designed the research, performed the immunohistochemistry and confocal imaging, and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

Acknowledgments

This work was funded by National Eye Institute (NEI) Grant R00 EY018863 to K.Y.W., a Research to Prevent Blindness Scientific Career Development Award to K.Y.W., National Science Foundation Graduate Student Research Fellowship DGE 1256260 to D.D.H., and NEI Grant P30 EY007003 to the Kellogg Eye Center.

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