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All six types of ipRGCs were tracer-coupled to amacrine cells. (A) Neurobiotin staining patterns of six representative Opn4 Cre/+ ; Z/EG ipRGCs. Arrowheads highlight Neurobiotin-filled somas near each ipRGC. (B) None of the tracer-coupled somas were immunopositive for the ganglion cell marker RBPMS, indicating they were amacrine cells. Asterisks in the right panel mark the locations of the ipRGC-coupled somas shown in the left panel. Note that the asterisks do not colocalize with RBPMS + somas. (C) Population-averaged numbers of amacrine cells tracer-coupled to each M1-M6 ipRGC, including ipRGCs lacking coupled somas. The number above each column is the number of ipRGCs analyzed for that ipRGC type. Error bars are SEM.
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Purpose:
Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with...
Contexts in source publication
Context 1
... somas were observed for all six ipRGC types, including 9 of 19 M1 cells (47%), 31 of 39 M2 cells (79%), 32 of 45 M3 cells (71%), 24 of 30 M4 cells (80%), 19 of 26 M5 cells (73%), and 20 of 24 M6 cells (83%). As shown in the exemplary data in Figure 1A, some of the coupled somas appeared to be in contact with the injected ipRGCs' dendrites, potentially suggesting dendrosomatic gap junctions, whereas others were outside the ipRGCs' dendritic fields, which probably connected with the ipRGCs via dendrodendritic gap junctions or an intermediary cell. To learn whether any of the somas were ganglion cells, 110 somas (98 in the GCL and 12 in the INL) coupled to 19 ipRGCs were tested with the RBPMS antibody, which labels all and only ganglion cells, 30 and none were stained (Fig. 1B). ...
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... dendritic fields, which probably connected with the ipRGCs via dendrodendritic gap junctions or an intermediary cell. To learn whether any of the somas were ganglion cells, 110 somas (98 in the GCL and 12 in the INL) coupled to 19 ipRGCs were tested with the RBPMS antibody, which labels all and only ganglion cells, 30 and none were stained (Fig. 1B). Since Neurobiotin FIGURE 2. ipRGCs were tracer-coupled with a wide range of soma sizes. (A) Frequency distribution of the soma diameters of all the in ganglion cells does not diffuse into glia, 22,31 we inferred that the ipRGC-coupled somas were amacrine cells. Figure 1C shows the population-averaged number of amacrine cells coupled ...
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... Frequency distribution of the soma diameters of all the in ganglion cells does not diffuse into glia, 22,31 we inferred that the ipRGC-coupled somas were amacrine cells. Figure 1C shows the population-averaged number of amacrine cells coupled to each M1-M6 ipRGC, including ipRGCs lacking coupled somas. ...
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... both mouse lines would presumably be affected more or less equally, so all observed control versus knockout differences should remain valid. Another caveat is that the low percentage of coupled M1 cells could have been due to the difficulty of injecting their relatively small somas, 18,25,32 and M1 cells indeed seemed less well filled than M2-M6 (Figs. 1A, 5B). Nevertheless, our finding that M1 cells couple with about half as many somas as M2 and M3 agrees with Müller et al. 11 While Müller et al. 11 saw ipRGC-coupled somas only in the GCL, we found some in the INL. Considering that they injected Neurobiotin via 120-to 145-MΩ microelectrodes for 3 minutes whereas we injected using ...
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... our finding that M1 cells couple with about half as many somas as M2 and M3 agrees with Müller et al. 11 While Müller et al. 11 saw ipRGC-coupled somas only in the GCL, we found some in the INL. Considering that they injected Neurobiotin via 120-to 145-MΩ microelectrodes for 3 minutes whereas we injected using similar electrodes but Figure 1C. ***P < 0.001. ...
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Citations
... The neuronal types of the retina are composed of photoreceptors and horizontal, bipolar, amacrine, and ganglion cells. There is a large array of connexins on these cells in mice and humans, with previous research demonstrating the presence of several Cx subtypes, including Cx35, Cx36, Cx43, Cx45, Cx57, Cx59, among others [52][53][54][55]. Furthermore, studies have largely focused on the role connexins play in material transport and signaling transmission. ...
Fibrosis initially appears as a normal response to damage, where activated fibroblasts produce large amounts of the extracellular matrix (ECM) during the wound healing process to assist in the repair of injured tissue. However, the excessive accumulation of the ECM, unresolved by remodeling mechanisms, leads to organ dysfunction. Connexins, a family of transmembrane channel proteins, are widely recognized for their major roles in fibrosis, the epithelial–mesenchymal transition (EMT), and wound healing. Efforts have been made in recent years to identify novel mediators and targets for this regulation. Connexins form gap junctions and hemichannels, mediating communications between neighboring cells and inside and outside of cells, respectively. Recent evidence suggests that connexins, beyond forming channels, possess channel-independent functions in fibrosis, the EMT, and wound healing. One crucial channel-independent function is their role as the primary functional component for cell adhesion. Other channel-independent functions of connexins involve their roles in mitochondria and exosomes. This review summarizes the latest advances in the channel-dependent and independent roles of connexins in fibrosis, the EMT, and wound healing, with a particular focus on eye diseases, emphasizing their potential as novel, promising therapeutic targets.
... Six ipRGC subtypes, known as M1-M6, have been reported and are mainly classified by morphological characteristics, such as cell size, dendrite size, and complexity of dendrite structure [57]. The M1 type ipRGC was reported to project mainly to the non-imaging visual brain regions, such as the suprachiasmatic nucleus (SCN) and olivary pretectal nucleus (OPN) [58]. ...
Myopia, a common ophthalmic disorder, places a high economic burden on individuals and society. Genetic and environmental factors influence myopia progression; however, the underlying mechanisms remain unelucidated. This paper reviews recent advances in circadian rhythm, intrinsically photosensitive retinal ganglion cells (ipRGCs), and dopamine (DA) signalling in myopia and proposes the hypothesis of a circadian rhythm brain retinal circuit in myopia progression. The search of relevant English articles was conducted in the PubMed databases until June 2023. Based on the search, emerging evidence indicated that circadian rhythm was associated with myopia, including circadian genes Bmal1, Cycle, and Per. In both humans and animals, the ocular morphology and physiology show rhythmic oscillations. Theoretically, such ocular rhythms are regulated locally and indirectly via the suprachiasmatic nucleus, which receives signal from the ipRGCs. Compared with the conventional retinal ganglion cells, ipRGCs can sense the presence of light because of specific expression of melanopsin. Light, together with ipRGCs and DA signalling, plays a crucial role in both circadian rhythm and myopia. In summary, regarding myopia progression, a circadian rhythm brain retinal circuit involving ipRGCs and DA signalling has not been well established. However, based on the relationship between circadian rhythm, ipRGCs, and DA signalling in myopia, we hypothesised a circadian rhythm brain retinal circuit.
... Other reports have also suggested the connections between J-RGCs and bipolar cells by morphology [45] and function [24]. The predominant (possibly the only) subtype of connexins for gap junctions between J-RGCs and amacrine cells have been suggested to be Cx36 [25], and the Cx36 f/f mice have been successfully used to knock out Cx36 in retinal neurons [48,49]. However, after we used it to remove the Cx36-mediated gap junction coupling in J-RGCs, the excitatory inputs were still direction-selective, indicating that the direction selectivity of excitation is not fully dependent on the electrical synapses. ...
Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons’ stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity.
... Nonsynaptic NMDA receptors very closely associated with Cx36 gap junctions have been shown to contribute significantly to the signaling that drives CaMKII phosphorylation of Cx36 in AII amacrine cells (Kothmann et al., 2012). However, as described above, it is possible that Cx36-GCaMP is not expressed in AII amacrine cells, and it is known that Cx36 is expressed by additional cell types in the inner retina, including retinal ganglion cells and other amacrine cells (Hidaka et al., 2002;Schubert et al., 2005;Hoshi and Mills, 2009;Pan et al., 2010;Kirkby and Feller, 2013;Harrison et al., 2021). Furthermore, most ganglion cells and some amacrine cells use NMDA receptors in postsynaptic response to glutamatergic bipolar cells (Massey and Miller, 1990;Marc, 1999;Gustafson et al., 2007). ...
Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.
... (B2) M5 ipRGC electrically coupled to a CRH + AC drives feedforward inhibition of a wide-field amacrine cell (WAC) that is electrically coupled to an M2 ipRGC. Reproduced with permission from Pottackal et al. (2021). ...
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are photoreceptors located in the ganglion cell layer. They project to brain regions involved in predominately non-image-forming functions including entrainment of circadian rhythms, control of the pupil light reflex, and modulation of mood and behavior. In addition to possessing intrinsic photosensitivity via the photopigment melanopsin, these cells receive inputs originating in rods and cones. While most research in the last two decades has focused on the downstream influence of ipRGC signaling, recent studies have shown that ipRGCs also act retrogradely within the retina itself as intraretinal signaling neurons. In this article, we review studies examining intraretinal and, in addition, intraocular signaling pathways of ipRGCs. Through these pathways, ipRGCs regulate inner and outer retinal circuitry through both chemical and electrical synapses, modulate the outputs of ganglion cells (both ipRGCs and non-ipRGCs), and influence arrangement of the correct retinal circuitry and vasculature during development. These data suggest that ipRGC function plays a significant role in the processing of image-forming vision at its earliest stage, positioning these photoreceptors to exert a vital role in perceptual vision. This research will have important implications for lighting design to optimize the best chromatic lighting environments for humans, both in adults and potentially even during fetal and postnatal development. Further studies into these unique ipRGC signaling pathways could also lead to a better understanding of the development of ocular dysfunctions such as myopia.
... To complement our pharmacological approach, we used mice that lack specific connexin proteins to assess the impact of disrupted gap junction coupling among ipRGCs on photoaversion. In adults, ipRGCs express Cx30.2 (Meyer et al., 2016) and Cx36 (Harrison et al., 2021). To identify the connexins expressed in ipRGCs in development, we performed immunohistochemical analyses of retinas of mice from three transgenic lines in which regulatory elements of the Cx30.2 (Cx30.2 ...
... Coupling between ipRGCs has not been observed. Cx36 has been implicated in ipRGC-AC coupling (Harrison et al., 2021) and a subset of ipRGCs express Cx30.2 (Meyer et al., 2016). Cx45 is only thought to be present in bistratified RGCs in the adult (Schubert et al., 2005), and thus could be present in M3s. ...
... M2-M6 ipRGCs are extensively coupled during development, and this strongly contributes to the light response (Arroyo et al., 2016;Caval-Holme et al., 2019). In contrast to the adult (Harrison et al., 2021), we found little evidence for Cx36 expression in ipRGCs during development (Fig. 5), consistent with previous work from our laboratory showing that Cx36 expression / Cre, as in the Cx45 KO). Cx30.2 KO: 7 FOV from 2 mice. ...
Aversive responses to bright light (photoaversion) require signaling from the eye to the brain. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) encode absolute light intensity and are thought to provide the light signals for photoaversion. Consistent with this, neonatal mice exhibit photoaversion prior to the developmental onset of image vision, and melanopsin deletion abolishes photoaversion in neonates. It is not well understood how the population of ipRGCs, which constitutes multiple physiologically distinct types (denoted M1-M6 in mouse), encodes light stimuli to produce an aversive response. Here, we provide several lines of evidence that M1 ipRGCs that lack the Brn3b transcription factor drive photoaversion in neonatal mice. First, neonatal mice lacking TRPC6 and TRPC7 ion channels failed to turn away from bright light, while two photon Ca2+ imaging of their acutely isolated retinas revealed reduced photosensitivity in M1 ipRGCs, but not other ipRGC types. Second, mice in which all ipRGC types except for Brn3b-negative M1 ipRGCs are ablated, exhibited normal photoaversion. Third, pharmacological blockade or genetic knockout of gap junction channels expressed by ipRGCs, which reduces the light sensitivity of M2-M6 ipRGCs in the neonatal retina, had small effects on photoaversion only at the brightest light intensities. Finally, M1s were not strongly depolarized by spontaneous retinal waves, a robust source of activity in the developing retina that depolarizes all other ipRGC types. M1s therefore constitute a separate information channel between the neonatal retina and brain that could ensure behavioral responses to light but not spontaneous retinal waves.SIGNIFICANCE STATEMENT:At an early stage of development, prior to the maturation of photoreceptor input to the retina, neonatal mice exhibit photoaversion-upon exposure to bright lights, they turn away and emit ultrasonic vocalizations-a cue to their parents to return them to the nest. Neonatal photoaversion is mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs)-a small percentage of the retinal ganglion cell population that expresses the photopigment melanopsin and depolarizes directly in response to light. Caval-Holme et al. show that photoaversion is mediated by a subset of ipRGCs, called M1-ipRGCs. Moreover, they find M1-ipRGCs have reduced responses to retinal waves, providing a mechanism by which the mouse distinguishes light stimulation from developmental patterns of spontaneous activity.
... More recently, some non-spiking displaced ACs were also found to receive gap junctional ipRGC input (Pottackal et al., 2021). All six known ipRGC types couple with displaced ACs (Muller et al., 2010;Harrison et al., 2021a), which contain at least four neuromodulators (Harrison et al., 2021a;Pottackal et al., 2021). But many fundamental questions remained: 1) Do ipRGCs couple with additional morphological/physiologic types of ACs? 2) Do the six ipRGC types couple with different AC types? ...
... More recently, some non-spiking displaced ACs were also found to receive gap junctional ipRGC input (Pottackal et al., 2021). All six known ipRGC types couple with displaced ACs (Muller et al., 2010;Harrison et al., 2021a), which contain at least four neuromodulators (Harrison et al., 2021a;Pottackal et al., 2021). But many fundamental questions remained: 1) Do ipRGCs couple with additional morphological/physiologic types of ACs? 2) Do the six ipRGC types couple with different AC types? ...
... To answer them, we used a gap junction-permeable fluorescent tracer, PoPro1 (Hoshi et al., 2006), to identify ipRGC-coupled ACs in living retinas for electrophysiological and morphological analysis. We also examined the functional consequences of disrupting ipRGC-AC coupling by knocking out the gap junction protein connexin36 (Cx36) selectively in ipRGCs (Harrison et al., 2021a). ...
Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only anterogradely to drive behavioral responses, but also retrogradely to some amacrine interneurons to modulate retinal physiology. We previously found that all displaced amacrine cells with spiking, tonic excitatory photoresponses receive gap-junction input from ipRGCs, but the connectivity patterns and functional roles of ipRGC-amacrine coupling remained largely unknown. Here, we injected PoPro1 fluorescent tracer into all six types of mouse ipRGCs to identify coupled amacrine cells, and analyzed the latter’s morphological and electrophysiological properties. We also examined how genetically disrupting ipRGC-amacrine coupling affected ipRGC photoresponses. Results showed that ipRGCs couple with not just ON- and ON/OFF-stratified amacrine cells in the ganglion-cell layer as previously reported, but also OFF-stratified amacrine cells in both ganglion-cell and inner nuclear layers. M1- and M3-type ipRGCs couple mainly with ON/OFF-stratified amacrine cells, whereas the other ipRGC types couple almost exclusively with ON-stratified ones. ipRGCs transmit melanopsin-based light responses to at least 93% of the coupled amacrine cells. Some of the ON-stratifying ipRGC-coupled amacrine cells exhibit transient hyperpolarizing light responses. We detected bidirectional electrical transmission between an ipRGC and a coupled amacrine cell, although transmission was asymmetric for this particular cell pair, favoring the ipRGC-to-amacrine direction. We also observed electrical transmission between two amacrine cells coupled to the same ipRGC. In both scenarios of coupling, the coupled cells often spiked synchronously. While ipRGC-amacrine coupling somewhat reduces the peak firing rates of ipRGCs’ intrinsic melanopsin-based photoresponses, it renders these responses more sustained and longer-lasting. In summary, ipRGCs’ gap junctional network involves more amacrine cell types and plays more roles than previously appreciated.
Background
The mammalian retina contains an autonomous circadian clock that controls various aspects of retinal physiology and function, including dopamine (DA) release by amacrine cells. This neurotransmitter plays a critical role in retina development, visual signalling, and phase resetting of the retinal clock in adulthood. Interestingly, bidirectional regulation between dopaminergic cells and melanopsin-expressing retinal ganglion cells has been demonstrated in the adult and during development. Additionally, the adult melanopsin knockout mouse (Opn4−/−) exhibits a shortening of the endogenous period of the retinal clock. However, whether DA and / or melanopsin influence the retinal clock mechanism during its maturation is still unknown.
Results
Using wild-type Per2Luc and melanopsin knockout (Opn4−/−::Per2Luc) mice at different postnatal stages, we found that the retina generates self-sustained circadian rhythms from postnatal day 5 in both genotypes and that the ability to express these rhythms emerges in the absence of external time cues. Intriguingly, only in wild-type explants, DA supplementation lengthened the endogenous period of the clock during the first week of postnatal development through both D1- and D2-like dopaminergic receptors. Furthermore, the blockade of spontaneous cholinergic retinal waves, which drive DA release in the early developmental stages, shortened the period and reduced the light-induced phase shift of the retinal clock only in wild-type retinas.
Conclusions
These data suggest that DA modulates the molecular core of the clock through melanopsin-dependent regulation of acetylcholine retinal waves, thus offering an unprecedented role of DA and melanopsin in the endogenous functioning and the light response of the retinal clock during development.
Chemical and electrical synapses (gap junctions) are widely prevalent in the nervous system. Gap junctions emerge long before chemical synapses, allowing communication between developing cells, and are thought to be involved in establishing neural circuits. Mounting evidence indicates that these two modalities of synaptic transmission closely interact during retinal development and that such interactions play a critical role in synaptogenesis and circuit formation during the perinatal period. In vertebrates, gap junctions consist of two connexins which in turn are made up of six connexins (Cx). To what extent Cx45 and Cx36, the most abundant connexins in the retina, are involved in synaptogenesis and retinal circuit formation is not known. The here presented immunohistochemical study used stainings of Cx45, Cx36 and Synaptophysin in the outer and inner (IPL) plexiform layers of postnatal day 8–16 mice retinas to shed light on the role of connexins during critical neuronal developmental processes. Cx45 and Cx36 expressions in both plexiform layers of the mouse retina increased till eye opening and dropped afterwards. The percentage of heterotypic Cx45/Cx36 gap junctions is also higher before the critical event of eye opening. Finally, Cx45 is closely located and/or colocalized with Synaptophysin also shortly before eye opening in the IPL of the mouse retina. All findings point towards a pivotal role for Cx45 during postnatal synaptogenesis in the mouse retina. However, a more functional study is needed to determine the role of Cx45 during synaptogenesis and circuit formation. Cx45 expression and colocalization with Synaptophysin, a marker for presynaptic terminals and synaptogenesis, is stronger before eye opening in the mouse retina. Heterotypic Cx45/Cx36 gap junctions are also more frequent before the critical event of eye opening.
The above article from Synapse , published online on March 7, 2021, https://doi.org/10.1002/syn.22200 in Wiley Online Library ( http://onlinelibrary.wiley.com/ ), has been retracted by agreement between the journal Editor in Chief, Dr. Benjamin Hall, and Wiley Periodicals LLC. The retraction has been agreed due to unattributed overlap between this article and the following article, Sondereker KB, Stabio M, Renna J, Crosstalk: The diversity of melanopsin ganglion cell types has begun to challenge the canonical divide between image‐forming and non‐image‐forming vision, 528, 12 ( https://doi.org/10.1002/cne.24873 ) published in Journal of Comparative Neurology .
REFERENCE
Xiao, J., Lin, X., Qu, J., Zhang, J., Morphological and functional diversity of intrinsically photosensitive retinal ganglion cells. Synapse . 2021; 75 :e22200. https://doi.org/10.1002/syn.22200
