Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

Ribelayga CP (2012) Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina. PLoS ONE 7(11): e50602. doi:10.1371/journal.pone.0050602 Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina Xiaoqin Liu 0 Zhijing Zhang 0 Christophe P. Ribelayga 0 Gianluca Tosini, Morehouse School of Medicine, United States of America 0 1 Department of Ophthalmology and Visual Science, The University of Texas Health Science Center at Houston, Medical School , Houston , Texas, United States of America, 2 The University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences , Houston, Texas , United States of America Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue. - Funding: The work was funded by the National Eye Institute (R01 EY018640 and P30 EY010608), www.nei.nih.gov/, a Challenge Grant from Research to Prevent Blindness, http://www.rpbusa.org/rpb/, and the Hermann Eye Fund, http://www.cizikeye.org/hermann_eye_fund.htm/. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. Competing Interests: The authors have declared that no competing interests exist. Circadian clocks orchestrate metabolism, physiology, and behavior with respect to the 24-h rotations of the Earth and the associated variations in the external world. These internal timekeeping mechanics provide living beings with the adaptive advantage of anticipating and preparing for the daily geophysical fluctuations of their environment [1]. The core machinery of circadian clocks is a well-conserved cellular mechanism based on a set of genes-the clock genes- and their protein products-the clock proteins- interlocked in transcriptional-translational feedback loops that self-regenerate with a period close to 24-h [1]. In mammals, fundamental elements of the clock mechanism have been identified. These include the transcription activators CLOCK, NPAS2, and BMAL1 and their inhibitors PERIOD (PER) and CRYPTOCHROME (CRY) [2]. Many, if not all, aspects of the physiology and function of the vertebrate retina vary on a daily basis. These include photoreceptor disk shedding, gene expression, the synthesis and release of neurohormones and neurotransmitters (such as melatonin and dopamine), neuronal light responses, and components of the electroretinogram [3,4,5,6]. Importantly, most of these rhythms persist in constant conditions (i.e. constant darkness) with a period of approximately 24 h, reflecting their control by endogenous circadian clocks [3,4,5,6]. The formal demonstration that the vertebrate retina contains a circadian clock came from the classic in vitro work on retinal and photoreceptor melatonin by Cahill and Besharse [7,8]. Those works on Xenopus were then followed by essentially similar papers on mouse retina [9,10,11]. In mammals however, notwithstanding intense research, our knowledge of the origin of retinal circadian rhythms remains incomplete. In particular, despite widespread clock gene expression in the retinal tissue [4,5] and functional evidence supporting the presence of a clock in the photoreceptor layer [9,10,11] and in the inner retina [12,13,14], it is still unknown whether the clock components are expressed in most or in specific retinal cells. To date, the only retinal cell type in which concurrent expression of the core clock components has been consistently observed is the dopaminergic amacrine cell [12,15,16,17]. In addition, it is still largely unknown whether rhythms of clock gene transcript expression translate into rhythms of clock protein accumulation in retinal cells. In an attempt to identify the circadian clock neurons in mouse retina, we used a semi-quantitative immunocytochemical approach to investigate the expression of six key circadian clock proteins in a number of retinal neurons labeled with specific markers. Our data indicate that the core clock elements are expressed in most neurons in the mouse retina and reveal a large degree of homogeneity within a same cell type and of heterogeneity between cell types not only in the amount but also in the rhythmic occurrence of clock protein expression. This important disparity in clock protein expression among cell types suggests that circadian rhythms in the retina are built upon distinct subpopulations of neuronal cellular clocks. Our observations raise the possibility that the strong heterogeneity we observed in the retina, and that others have observed in the suprachiasmatic nucleus of the hypothalamus (SCN) [18], is a general feature of circadian clock organization in mammalian tissues. Materials and Methods Animals and Lighting Conditions This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol was approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston (Protocol Number: HSC-AWC-09-095, renewed 12043). Most of the experiments were conducted on adult (16 months) C57Bl/6J mice of ei (...truncated)