The mouse retina at E16–18 exhibits spontaneous correlated transients (Bansal et al., 2000; Syed et al., 2004), termed stage 1 retinal waves, despite the immature state of retinal circuits. At E16–18, several postmitotic cell types are present in the retina, including broad classes of retinal ganglion cells (RGCs) and amacrine cells (ACs), as well as proliferating progenitors which will go on to produce cells such as rods, bipolar cells, and Muller glia postnatally (Figure 1A; Cepko, 2014). By E17, there are no chemical synaptic structures (Hoon et al., 2014), though migrating SACs release ACh (Wong, 1995), and they, along with RGCs, express nicotinic acetylcholine receptors. One potential mode of cell–cell communication is via gap junction coupling between RGCs as well as between progenitor cells (Cook and Becker, 2009), which have been proposed to be the primary substrate mediating stage 1 waves (Catsicas et al., 1998; Syed et al., 2004; Wong et al., 1998).

To begin to understand how cholinergic signaling and gap junction coupling govern stage 1 waves, we first used calcium imaging to characterize their spatiotemporal properties. Retinas were isolated from E16–18 mice that were either bath loaded with the organic calcium dye Cal 520 or from mice expressing the genetically encoded calcium indicator GCaMP6s under the Vglut2 promoter (Vglut2;GCaMP6s). The earliest age at which we could detect reliable waves was E16 (Video 1). Stage 1 retinal waves were recorded on retinal whole mounts using a custom-built epifluorescent macroscope.

To assess the spatiotemporal properties of stage 1 waves, we divided the retinal surface into small square ROIs (roughly 10 µm × 10 µm), about 7 µm apart. ΔF/F traces for each ROI were rasterized based on a detection algorithm that identified the timing of peak changes in fluorescence, which we call transients (Figure 1B). These rasterized transients were used for subsequent analysis. We first computed the time between spontaneous transients by measuring the inter-transient interval (ITI) for each ROI and found that the distribution peaked at around 80 s (Figure 1C). We also described the size of individual waves by computing the sum of transients that occurred simultaneously (i.e., percent of ROIs that participated in each wave). The distribution of wave sizes was broad, ranging from 6 to 90% of the retina, with a mean and standard deviation of 46 ± 21% (Figure 1D).

In addition to propagating waves, we also observed small non-propagating calcium events (defined by transients present in a few neighboring ROIs that never propagated beyond 160 × 160 μm²; Video 2 and Figure 1—figure supplement 1). These events were like those described previously in Bansal et al., 2000. Note that these small non-propagating transients were included in the ITI but not in the analysis of propagating wave sizes.

Finally, we calculated the average propagation speed of stage 1 waves to range from 145 μm/s to 237 μm/s (average ± SD: 181 ± 24 μm/s; Figure 1E, Figure 1—figure supplement 2), similar to stage 2 waves we observed with an average speed of 177 μm/s ± 62 μm/s (see Table 1 for wave speed summary data). We also measured the distribution of stage 1 wave initiation sites in WT retinas and saw no evidence of an initiation site bias (Figure 1—figure supplement 3).

Previous work based on epifluorescent calcium imaging experiments performed in embryonic mice has shown that retinal waves, as defined by correlated changes in fluorescence, are reduced in frequency and size by curare, a competitive antagonist for nAChRs (Bansal et al., 2000). However, in roughly the equivalent developmental period in rabbit, blockade of all fast neurotransmitter receptor, including nAChRs, had no impact on wave frequency (Syed et al., 2004). Rather, waves in rabbit are blocked after the application of 18β-glycyrrhetinic acid, a gap junction antagonist (Syed et al., 2004).

To determine the relative role of gap junctions and nAChRs on the frequency and area of stage 1 waves in mice, we used two-photon calcium imaging and pharmacology in retinas isolated from E16–18 mice bath loaded with Cal 520. To block gap junctions, we bath applied the gap junction antagonist meclofenamic acid (MFA, 50 μM), which reversibly blocks electrical coupling between retinal interneurons (Veruki and Hartveit, 2009) and developing ipRGCs (Caval-Holme et al., 2019). Application of the gap junction blocker MFA (50 μM) nearly abolished stage 1 waves, causing a significant reduction in frequency of waves and cell participation during waves (Figure 2A-C). We also found a small but significant reduction in wave amplitude, quantified as the average maximum response amplitude of all cells participating in individual waves (Figure 2D).

MFA has notable off-target effects including overall cell health after long exposures (Kuo et al., 2016). Previous studies from our lab show that at P6 MFA does not reduce light-induced calcium transients in M1-iRGCs, the ability of M4-ipRGCs to fire action potentials, nor the amplitude of depolarization-induced calcium transients (Caval-Holme et al., 2019). Note this is in contrast to another gap junction antagonist, carbenoxolone, which inhibits light responses in cultured ipRGCs (Bramley et al., 2011). Using whole-cell voltage-clamp recordings, we observed that MFA induced a significant decrease in voltage-activated potassium conductance (Figure 2—figure supplement 1). Note that a reduction in K+ conductance would increase excitability of cells and is therefore unlikely to be the reason for the observed decrease in wave activity in the presence of MFA. Hence, we conclude that MFA’s block on retinal waves is via their impact on gap junctions.

We next assayed the impact on nAChR-antagonists on stage 1 waves. For stage 2 waves, both spontaneous calcium transients and compound excitatory synaptic events are completely blocked by bath application of dihydro-ß-erythroidine hydrobromide (DHβE, 8 μM) (Ford et al., 2012), which preferentially targets nAChRs containing α4 and β2-subunits (Harvey and Luetje, 1996; Harvey et al., 1996). We found that DHβE also dramatically reduced activity associated with stage 1 retinal waves (Figure 2E-H). However, in contrast to stage 2, some waves persisted in the presence of DHβE, but recruited fewer neurons and therefore had smaller areas (Figure 2G). Stage 1 waves were blocked by general nAChR antagonists: both hexamethonium (Figure 2I-J; Hex, 100 μM), a non-selective nAChR antagonist, and epibatidine (Figure 2K-L; EPB, 10 nM), an nAChR agonist that potently desensitizes all nAChRs (Corrie et al., 2020; Spang et al., 2000), blocked stage 1 wave events. Hence, both gap junctions and multiple subunit combinations of nAChRs mediate the initiation and propagation of stage 1 waves .

Our results thus far indicate that both the frequency and area of stage 1 retinal waves are modulated by the activation of different subtypes of nAChRs as well as gap junction coupling. To further differentiate the role of different nAChRs, we characterized mice where the β2 subunit of the nicotinic acetylcholine receptor is genetically ablated (β2-nAChR-KO). β2-nAChR-KO mice have severely disrupted stage 2 retinal waves (Bansal et al., 2000; Rossi et al., 2001) and have served as a model system for assessing the role of stage 2 waves in driving different developmental events (Ackman et al., 2012; Burbridge et al., 2014).

We observed that β2-nAChR-KO retinas exhibited stage 1 waves with different spatiotemporal properties than those of WT retinas (Figure 3A and B, see also Video 3). Specifically, β2-nAChR-KO retinas exhibited longer IEIs (Figure 3C and D), and individual waves propagated over smaller areas β2-nAChR-KO (Figure 3E and Figure 1—figure supplement 1). In sharp contrast to WT retina, waves in the β2-nAChR-KO retina were unaffected by the addition of Hex but showed a significant reduction in both area and frequency with the application of MFA (Figure 3D and E). These results suggest that in the absence of β2-nAChRs, gap junctions are the primary remaining component of the stage 1 wave generation mechanism. Furthermore, we found that stage 1 waves in the β2-nAChR-KO propagated at a significantly slower speed than those observed in WT mice (average ± SD: 99 ± 15 µm/s compared to 181 ± 24 µm/s for WT, n = 10 waves per condition, p=3.9073e^–8) (Figure 3F), consistent with a distinct mechanism of propagation.

Finally, we wanted to test whether the properties of stage 1 waves in WT or β2-nAChR-KO were influenced by light activation of ipRGCs via the 474 nm imaging light used to excite the calcium dye on the macroscope. ipRGCs have been shown to be responsive to 476 nm light during both embryonic and postnatal development (Emanuel and Do, 2015; Verweij et al., 2019). Previous studies have shown a light-dependent increase in the wave frequency of stage 2 waves in the β2-nAChR-KO during the first postnatal week (Kirkby and Feller, 2013). This light modulation of the spatiotemporal properties of β2-nAChR-KO during the first postnatal week depends on ipRGC melanopsin expression, as well as an increase in gap junction conductance between ipRGCs and other RGCs (Arroyo et al., 2016; Kirkby and Feller, 2013). However, we found no difference in the frequency of waves recorded using a two-photon microscope (based on 920 nm illumination) to those recorded on the macroscope and in either the WT or β2 nAChR-KO embryonic retinas (Figure 3 and Figure 3—figure supplement 1). Hence, we conclude that light activation of ipRGCs does not significantly influence the spatiotemporal properties of stage 1 retinal waves in either WT or the β2 nAChR-KO retinas. These results are consistent with the fact that light stimulation of the retina does not modulate the frequency of stage 2 waves and only begins to do so when conventional photoreceptors come online during stage 3 waves (Tiriac et al., 2018, Renna et al., 2011).

RGCs undergo a period of dramatic cell death during the first two postnatal weeks of development, the majority occurring during the first postnatal week (Abed et al., 2022; Braunger et al., 2014). Whether this cell death process is regulated by retinal waves is unknown. We looked specifically at intrinsically photosensitive ganglion cells (ipRGCs) for several reasons. First, ipRGCs have completed proliferation (Lucas and Schmidt, 2019; McNeill et al., 2011) and appear to be fully differentiated by E16 (Shekhar et al., 2022; Whitney et al., 2022), the onset of stage 1 waves. ipRGCs undergo a period of dramatic cell death during the first two postnatal weeks of development, the majority occurring during the first postnatal week. Prevention of cell death profoundly disrupts several important developmental processes in the retina – including spacing of ipRGC somas as well as rod- and cone-mediated circadian entrainment through the activation of ipRGCs (Chen et al., 2013). However, the exact mechanism regulating ipRGC cell death is unknown. Here we assessed the impact of disrupting stage 1 and 2 waves on the density and distribution of ipRGCs.

We first set out to determine whether ipRGCs participate in stage 1 waves. To do this, we conducted two-photon calcium imaging of RGCs in the ganglion cell layer (GCL) of retinas isolated from Opn4^Cre/+;Ai9 E16–18 mice (Ecker et al., 2010), which express tdTomato in ipRGCs (Figure 4A) enabling us to assess the differential participation of RGCs and ipRGCs during stage 1 waves (Figure 4B). On average, both RGCs and ipRGCs participated in most waves with no significant differences between the two groups (Figure 4C; average ± SD: RGCs 78.78 ± 21.48%; ipRGCs 84.64 ± 16.86%). We also found no significant differences in the amplitude of the calcium response that RGCs and ipRGCs exhibit in response to stage 1 waves (Figure 4D). Hence, ipRGCs are depolarized by stage 1 waves similarly to stage 2 waves (Caval-Holme et al., 2019; Caval-Holme et al., 2022; Chew et al., 2017).

Since β2-nAChR-KO retinas exhibited reduced retinal activity during both embryonic and early postnatal development, we used this mouse as a model to determine whether normal stage 1 and 2 wave activity is important for regulating the density and distribution of ipRGCs. To this end, we isolated retinas from Opn4^Cre/+;Ai9 and Opn4^Cre/+;Ai9;(β2-nAChR^-/-) at P1 and P7. These retinas express tdTomato in all melanopsin-expressing cells regardless of subtype. Retinas were imaged using the macroscope (Figure 5A and B).

We observed a dramatic decrease in the density of ipRGCs in WT retinas from P1 to P7 (Figure 5B and C; p=1.17 × 10^–7), consistent with previous studies of ipRGCs (Chen et al., 2013) and which coincides with peak levels of RGC apoptosis, the primary cause of RGC death during development (Braunger et al., 2014; Young, 1984). We found that β2-nAChR-KO retinas exhibited the same density of ipRGCs at P1 as WT retinas, suggesting that the decrease in activity in stage 1 waves does not regulate ipRGC cell density. Like WTs, β2-nAChR-KO retinas also exhibited a decrease in the density of ipRGCs from P1 to P7 (p=4.99 × 10^–11). At P7, we observed a small but significant increase in ipRGC densities at P7 in β2-nAChR-KO mice than in WT mice (568 ± 33 ipRGCs/µm² in WT vs. 654 ± 78 ipRGCs/µm² in β2-nAChR-KO, n = 8 retinas in each genotype). We cannot determine whether this small difference is due to the smaller size of retinas in β2-nAChR-KO retinas (Xu et al., 1999) or reflects a true increase in cell number. Overall, these data indicate that the cell death processes that regulate ipRGC density during the first postnatal week persist despite a significant reduction in wave activity.

To determine the impact of this developmental decrease in cell density on the mosaic organization of ipRGCs, we computed the regularity index, which is equal to the average nearest-neighbor distance divided by the standard deviation. A large regularity index is associated with a non-random distribution of somas. Despite the expected increase in nearest-neighbor distance in WT and β2-nAChR-KOs retina between P1 and P7 (Figure 5D, see Table 2), there was only a small decrease in the regularity index. Interestingly, the measured mean regularity indices of 2.8 ± 0.12 (WT P1), 2.9 ± 0.16 (β2-nAChR-KO P1), 2.3 ± 0.13 (WT P7) and 2.6 ± 0.2 (β2-nAChR-KO P1) fall within the range of what would be predicted by a random distribution of cells with soma diameters between 7–10 μm (Keeley et al., 2020). Hence, the decrease in cell density does not appear make the soma organization more ordered. This might be expected since our analysis does not differentiate between ipRGC subtypes, each of which likely forms an independent retinal mosaic. Together these data indicate that although retinal waves provide a robust source of depolarization for embryonic and early postnatal ipRGCs, reducing wave activity does not significantly influence the processes that regulate their density or organization. Whether elimination of all spontaneous activity affects these processes remains to be determined.

Our findings support a model in which there are both key differences and some similarities between stage 1 and 2 waves. The distribution of ITI is peaked at roughly 80 s for stage 1 waves that is a slightly longer interval than the peak interval reported for stage 2 (compare Figure 1C here to Figure 7D in Ford et al., 2012). Stage 1 and 2 waves also propagate at similar speeds. Key differences are that spontaneous activity between E16 and E18 included small non-propagating transients and propagating waves of a broad range of sizes. In contrast, after P1, there are no small non-propagating transients, and stage 2 waves mostly propagate over large areas of the retina, as observed both in vitro (Feller et al., 1997; Hilgen et al., 2017) and in vivo (Ackman et al., 2012). Note the spatiotemporal properties of stage 2 waves also vary dramatically across the developmental period spanning P1–P10 (Ge et al., 2021; Hilgen et al., 2017; Maccione et al., 2014) and therefore we are making these comparisons to the earlier stage 2 waves.

The similarity in frequency of waves and propagation speed indicates that the mechanisms responsible for initiating and propagating large waves are similar for stage 1 and 2 waves. Indeed, stage 1 and 2 waves share a dependence on nAChR signaling. Starburst amacrine cells (SACs) are the sole source of ACh in the retina. During the period of development concurrent with stage 1 waves, SACs begin to migrate toward the INL and send projections to the IPL via homotypic contacts (Ray et al., 2018). Also during this period, SACs start to express choline acetyltransferase at E17 in the rat retina (Kim et al., 2000), equivalent to E15.5 in mice (Schneider and Norton, 1979), and show a response to nicotine in the fetal rabbit retina between E20 and 27 (Wong, 1995), corresponding to stage 1 and 2 waves in rabbit (Syed et al., 2004). Together these studies support the idea that during embryonic development, SACs are not only releasing ACh but also forming a cholinergic network, similar to that of stage 2 wave propagation (Ford and Feller, 2012). Here, we have shown that stage 1 waves fail to initiate in the presence of the general nAChR antagonists, hexamethonium (Hex) and epibatidine (EPB). This suggests that spontaneous depolarization of SACs is important for wave initiation and that the cholinergic circuits that mediates waves start earlier than expected.

One key difference in ACh signaling between stage 1 and 2 waves is their sensitivity to the specific nAChR antagonist, DHβE. Though DHβE reduced the number of stage 1 waves, many smaller calcium transients persisted. In contrast, DHβE is a potent blocker of all activity during stage 2 waves (Ford et al., 2012). DHβE is an nAChR antagonist with a greater affinity for nAChRs containing α4 and β2 subunits in heterologous systems (Ho et al., 2020; Papke et al., 2010). The fact that all stage 1 wave activity is blocked by Hex and EPB suggests that different subunit combinations of nAChRs, on both SACs and RGCs, are independently contributing to either the initiation or propagation of stage 1 waves.

The different sensitivity to nAChR antagonists between stage 1 and 2 waves is highlighted in the patterns of retinal waves in β2-nAChR-KO mice. β2-nAChR-KO mice have significantly reduced stage 2 cholinergic waves (Bansal et al., 2000; Burbridge et al., 2014; Xu et al., 2015; Xu et al., 2016) and as such have served as the canonical model for studying the role of stage 2 cholinergic waves in eye-specific segregation, retinotopic maps, retinal and collicular direction selectivity, and in the optokinetic reflex (Arroyo and Feller, 2016; Grubb et al., 2003; Thompson et al., 2017; Tiriac et al., 2022; Wang et al., 2009). In contrast, stage 1 waves in the β2-nAChR-KO mice persist, albeit they propagate with slower speed and cover smaller areas of the retina (Figure 3). Despite this difference in spatiotemporal properties, both stage 1 and 2 waves that persist in the β2-nAChR-KO mice are blocked by gap junction receptor antagonist rather than blockers of fast neurotransmitter receptors (Kirkby and Feller, 2013). Recent evidence suggests that embryonic activity influences many aspects of visual system development including eye specific segregation and retinotopic mapping of retinal projections to the brain (Guillamón-Vivancos et al., 2022), as well as cortical innervation of the visual thalamic nucleus (Moreno-Juan et al., 2023). Whether the various visual system phenotypes observed in β2-nAChR-KO mice can be attributed in part to reduced stage 1 waves remains to be determined.

In our hands, global blockade or desensitization of nAChRs completely abolished stage 1 waves. This result appears to conflict with previous studies in mice showing that application of curare, a competitive antagonist for nAChRs, preserves small non-propagating transients (Bansal et al., 2000). One possibility is that in contrast to hexamethonium, curare has mixed affinity for neuronal nAChRs. A second is that curare was acting via other neurotransmitter receptors where it has some cross-reactivity not shared by the receptor antagonists that we used (Spirova et al., 2019; Wotring and Yoon, 1995).

In addition to the ACh signaling, gap junctions also play a role in mediating stage 1 retinal waves. Here, we use the gap junction antagonist meclofenamic acid (MFA), which was previously shown to reversibly block junctional conductance (Veruki and Hartveit, 2009), dye coupling (Pan et al., 2007), and spikelets between developing RGCs (Caval-Holme et al., 2019). Application of MFA led to a significant reduction in both the frequency and size of stage 1 waves. The results we observed in MFA are consistent with the pharmacological studies of stage 1 waves in other species: stage 1 waves in rabbit are insensitive to nAChR antagonists (Syed et al., 2004), as are early waves in developing chick retina (Catsicas et al., 1998). Note that waves in these species were also sensitive to antagonists of various metabotropic receptors, indicating that neurotransmitters are still important for propagating waves in these systems. However, there is the important caveat that MFA can also have some off-target effects that might impact wave propagation (Kuo et al., 2016). Previously we showed that in P6-7 retina MFA did not alter depolarization-induced calcium transients and did not reduce the excitability of RGCs. Here, we show that short applications (10 min) of MFA increased input resistance of RGCs and did not appear to block compound EPSCs associated with waves or voltage-gated sodium and potassium channels. Future experiments with independent measures of gap junction coupling – such as tracer coupling and potentially knockout of connexin proteins – are necessary to have a more complete understanding of how electrical synapses contribute to wave propagation properties.

We also observed a difference between stage 1 and 2 waves in β2-nAChR-KO retinas. Embryonic β2-nAChR-KO retinas still exhibited stage 1 waves but at reduced frequency and size when compared to WT retinas. Though the spatiotemporal properties of stage 2 waves recorded in vitro for P1-P8 β2-nAChR-KO retinas are highly dependent on recording conditions with results ranging from sparse activity to high frequency (Bansal et al., 2000; Stafford et al., 2009; Xu et al., 2016), in vivo waves in β2-nAChR-KO are infrequent and weakly depolarizing (Burbridge et al., 2014). The activity that persists in β2-nAChR-KO retinas both embryonically and postnatally is resistant to nAChR antagonists, as was observed previously (Bansal et al., 2000). Rather the remaining activity in β2-nAChR-KO retinas is completely blocked by MFA, thereby suggesting that stage 1 and 2 waves in β2-nAChR-KO retinas rely solely on gap junctions (Kirkby and Feller, 2013).

We propose a model for stage 1 waves consistent with these observations (Figure 6). Stage 1 wave initiation is dependent on the spontaneous depolarization of SACs, but wave propagation is dependent on both gap junction coupling and nAChR activation. Hence, stage 1 wave initiation is similar to stage 2 waves where the spontaneous depolarization of a SAC activates neighboring SACs, leading to the volumetric release of ACh responsible for wave propagation both in the INL and GCL (Ford et al., 2012). The model for stage 1 wave propagation is also similar to stage 2 wave propagation, though a complete description of how gap junction coupling is mediating stage 2 wave propagation is needed. The pharmacological and genetic block/ablation of gap junctions have yet to reveal a phenotype (Blankenship et al., 2011; Caval-Holme et al., 2019; Kirkby and Feller, 2013; Singer et al., 2001; Torborg et al., 2005). Interestingly, pure gap junction-mediated waves, such as those we observed in the β2-nAChR KO mouse, are considerably slower than waves in WT retina. A recent computational model of gap junction-mediated stage 1 waves, where waves are initiated by RGCs undergoing rare and random depolarizations that propagate entirely via electrical synapses, argues that the speed of propagation is limited by the slow rate at which the junctional currents charge up the membrane capacitance of neighboring RGCs (Kähne et al., 2019). Hence, the faster speed of waves mediated by a combination of nAChRs and gap junctions indicates that diffuse release of ACh leads to faster propagation than electrical synapses alone.

Early intrinsic light responses of ipRGCs have been implicated in several developmental events (Aranda and Schmidt, 2021), including retinal vascularization (Rao et al., 2013), maturation of circadian circuits (McNeill et al., 2011), and the maturation of the lens to prevent myopia (Chakraborty et al., 2022). Here, we report that ipRGCs are robustly activated by stage 1 waves, similar to our observations that ipRGCs participate in stage 2 waves (Arroyo et al., 2016; Caval-Holme et al., 2022; Kirkby and Feller, 2013). Thus, it is possible that depolarization via stage 1 waves may contribute to some of these ipRGC-dependent developmental processes.

To begin to explore to role of stage 1 and 2 waves in ipRGC development, we monitored the impact of chronically altered waves on the distribution and density of ipRGCs across the retina. Notably, ipRGCs undergo extensive apoptotic cell death, with the peak of apoptosis occurring between P2–P4 (Chen et al., 2013). Prevention of apoptosis during this developmental period doubles the density of ipRGCs and dramatically increases the clumping of M1-ipRGC somas. In some systems, correlated network activity has been implicated in cell proliferation and cell death. For example, retinal wave activity promotes neurite outgrowth and potentially survival among RGCs (Goldberg et al., 2002). Additionally, in the developing primary somatosensory and motor cortices, higher levels of spontaneous electrical activity were shown to have a neuroprotective effect (Blanquie et al., 2017b). However, here we show in the β2-nAChR-KO mouse, which has significantly diminished stage 1 and 2 retinal waves, the normal developmental loss of ipRGCs between P1 and P7 is maintained. Thus, stage 1 and 2 retinal waves are not required for ipRGC apoptosis; however, it is possible that the residual wave activity in β2-nAChR-KO is sufficient to activate pro-survival pathways. A deeper understanding of how spontaneous activity modulates RGC survival pathways (e.g., Ahmed et al., 2022) is warranted.

All animal procedures were approved by the UC Berkeley Institutional Animal Care and Use Committee and conformed to the NIH Guide for the Care and Use of Laboratory Animals (AUP-2015-10-8080-2), the Public Health Service Policy, and the SFN Policy on the Use of Animals in Neuroscience Research. For our calcium dye-based calcium imaging, Opn4^Cre/+;Ai9 mice were generated by crossing mice Opn4^Cre/+ B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato) Hze}/J mice (Ai9) mice (stock # 007909, Jackson Laboratory, Bar Harbor, ME) to the Opn4^Cre/+ reporter mouse (T. Schmidt, Northwestern University, Evanston, IL). For our GCaMP6s-based calcium imaging, we generated Vglut2;GCaMP6s mice by crossing B6J.129S6(FVB)-Slc17a6^tm2(cre)Lowl/MwarJ mice (stock # 028866) to B6J.Cg-Gt (ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}/MwarJ mice (stock # 028863). ipRGC density measurements were conducted on P1–P7 mice of either sex using Opn4^Cre/+;Ai9.

To obtain mice that were precisely at the correct embryonic age, we set up timed pregnancies and checked vaginal plugs every morning for 4 days after the animals were paired. This approach led to an uncertainty of age of ± 1 day. Hence, we grouped data across E16–18. Since we observed more variance within litters than across litters, we assume age was not a determining factor in our findings. We used the β2-nAChR-KO mouse line in which the β subunit of the nicotinic acetylcholine receptor is knocked out as a genetic model in which cholinergic retinal spontaneous activity is disrupted. For experiments regarding the influence of spontaneous retinal activity on the distribution of ipRGCs across the retina, we used β2^-/-::Opn4^Cre/+;Ai9 (β2-nAChR-KO) mice, generated by crossing β2^-/- (A. Beaudet, Baylor University, Waco, TX) mice to Opn4^Cre/+;Ai9 mice to label all melanopsin-expressing cells. All mouse lines are maintained on a C57BL/6 genetic background. All animals used for two-photon calcium imaging experiments and immunohistochemistry were housed in 12 hr day/night cycle rooms.

On the day of the experiment, pregnant dams were deeply anesthetized via isoflurane inhalation and fetuses were harvested via a cesarean section. tdTomato-positive fetuses were identified using miner goggles (Biological Laboratory Equipment Services and Maintenance Ltd., model: GFsP-5). Fetuses were kept alive in 50 ml Falcon tubes filled with oxygenated (95% O₂ 5% CO₂) ACSF (in mM, 119 NaCl, 2.5 KCl, 1.3 MgCl₂, 1 K₂HPO₄, 26.2 NaHCO₃, 11 D-glucose, and 2.5 CaCl₂). Fetuses were then euthanized sequentially by decapitation. Eyes were immediately enucleated and retinas were dissected at room temperature in oxygenated ACSF under a dissecting microscope. Isolated retinas were mounted whole over a 1–2 mm² hole in nitrocellulose filter paper (Millipore) with the photoreceptor layer side down and transferred to the recording chamber of a two-photon microscope for imaging. The whole-mount retinas were continuously perfused (3 ml/min) with oxygenated ACSF warmed to 32–34°C by a regulated inline heater (TC-344B, Warner Instruments) for the duration of the experiment. Additional retina pieces were kept in the dark at room temperature in ACSF bubbled with 95% O2, 5%CO2 until use (maximum 8 hr).

For the calcium imaging experiments, retinas were bath loaded with the calcium indicator Cal 520 AM (AAT Bioquest) for 1–2 hr at 32°C.

Two-photon fluorescence measurements were obtained with a modified movable objective microscope (MOM) (Sutter instruments, Novator, CA) and made using an Olympus ×60, 1.00 NA, LUMPlanFLN objective (Olympus America, Melville, NY) for single-cell resolution imaging (field of view, FOV: 203 × 203 µm) or a Nikon ×16, 0.80 NA, N16XLWD-PF objective (Nikon, Tokyo, Japan) for large FOV (850 × 850 um) imaging. Two-photon excitation was evoked with an ultrafast pulsed laser (Chameleon Ultra II; Coherent) tuned to 920 nm to image Cal520, GCaMP6s, and tdTomato. Laser power was set between 6.5 and 12 mW for imaging of Cal520 and tdTomato expression. The microscope system was controlled by the ScanImage software (https://www.scanimage.org/). Scan parameters were [pixels/line × lines/frame (frame rate in Hz)]: [256 × 256 (1.48 Hz)], at 2 ms/line. This MOM was equipped with a through-the-objective light stimulation and two detection channels for fluorescence imaging.

Epifluorescent calcium imaging were obtained on a custom-built macroscope with an Olympus XLFLUOR4X/340 4× 0.28 NA objective, a Teledyne Kinetix camera. Collectively, this macroscope has 4.7 mm × 4.7 mm FOV, and 1.5 µm/pixel. All movies were taken at a 10 Hz frame rate and pixels were binned 4 × 4 bringing the resolution down to 5.9 µm/pixel, still maintaining single-cell resolution. Cal520 and GCaMP6s excitation was evoked with a 474 nm LED. A full description and building instructions can be found at https://github.com/Llamero/DIY_Epifluorescence_Macroscope, (Smith, 2022).

Macroscope recordings of stage 1 events were used for the manual detection of initiation sites. Small non-propagating events were identified as local regions of correlated calcium activity with fixed areas and no wave fronts. For this analysis only, we separated waves into small and large events to see whether there were differences in initiation sites. Small propagating waves were identified as regions of correlated calcium activity with no fixed areas and with wave fronts covering up to 25% of the retina. Large propagating waves were identified as regions of correlated calcium activity with no fixed areas and with wave fronts covering up to 90% of the retina.

We blocked gap junctions via application of the gap junction blocker MFA (50 µM, Sigma-Aldrich). We blocked the nicotinic acetylcholine signaling pathway via application of the broad nicotinic receptor antagonists hexamethonium (Hex, 100 µM, Sigma-Aldrich) and epibatidine (EPB, 10 nM, Sigma-Aldrich) as well as the specific antagonist dihydro-ß-erythroidine hydrobromide (DHßE, 8 µM, Sigma-Aldrich).

The following procedure was used for all pharmacology experiments: We recorded baseline activity in ACSF for 8 min before pharmacological agents were applied to the perfusion system. We then waited 15–30 min for the agents to take effect before acquiring another 8 min recording session.

To attempt to assay off-target effects of MFA, we used whole-cell voltage-clamp recordings to compare voltage-gated ion channels on RGCs in E16–18 retina but found inconsistent results (Figure 2—figure supplement 1). We associate this high variance with a rapid changing complement of ion channels during development and the quick washout of these conductances during whole-cell recordings.

Movies were preprocessed for motion correction using a MATLAB code from the Flat Iron Institute (https://github.com/flatironinstitute/NoRMCorre; Pnevmatikakis, 2019). The baseline movie frame (F0) was computed by taking the temporal median projection of all the movie frames. Each movie frame (F) was normalized by dividing its difference from the baseline frame (F-F0) by the baseline frame ((F-F0)/F0) to produce a ΔF/F0 movie. For movies taken on the two-photon microscope, circular ROIs were drawn on all cells within the FOV. Additional circular ROIs were drawn for tdTomato+ cells. For movies taken on the macroscope a grid of 10 µm × 10 µm squares, which were spaced 1.5 pixels apart, were drawn over the whole surface of the retina using a custom FIJI macro. The ROIs and the ΔF/F0 movie were then imported into MATLAB for further analysis using custom algorithms. Traces for each FOV and ROI were computed as the mean value of the pixels enclosed by the ROI in each frame of the ΔF/F0 movie.

For transient frequency analysis, event detection was done using the findpeaks function in MATLAB, with the minimum threshold set to greater than at least 10 times the standard deviation of the baseline fluorescence, which corresponds to at least 10% ΔF/F₀. The ITI for each ROI was calculated by finding the difference between the frame of each detected transient. This difference was then converted to seconds by multiplying it to the movie’s frame rate.

The area of waves was calculated for all movies taken on the macroscope. To do this, we first z-scored the percent active ROI traces and used the findpeaks function to detect individual waves, with the threshold set to a z-score of 1. After determining the time of waves, we removed any waves that occurred within the first and last 6 s of the movie due to them being edge cases. We then summed the number of active ROIs within 12 s around the wave times. This number was then divided by the total number of ROIs to get the percentage of active ROIs.

To determine whether neurons participate in waves in the two-photon calcium imaging data, we employed the following bootstrapping strategy: we randomly sampled the activity of individual neurons outside of wave times a thousand times to build a baseline of neural activity. We then set a threshold of 95th percentile to statistically determine whether neurons exhibited a greater calcium response during a wave than at rest. We then calculated the percentage of waves each cell participated in and averaged this value for every FOV. Similarly, mean response amplitudes were calculated for each cell and then averaged for each FOV.

For a description of statistics used, please refer to the figure captions.

To image the density of ipRGCs in fixed retinas, dissected retinas from P1 and P7 mice were fixed in 4% PFA for 30 min. The fixed retinas were subsequently mounted on a slide with vectashield and a cover slip, then imaged within an hour of mounting on the macroscope. For P1 retinas, Z-stacks were acquired by manually turning the focus knob.

We first identified the centroid of each ipRGC. For P7 retinas, where there is more space between cells, we employed the following automatic segmentation. Images were bandpass filtered and somata automatically segmented using the Morpholibj (Legland et al., 2016) classic watershed tool to obtain 8-bit binarized masks. The masks were then processed in MATLAB in order to obtain the centroid locations and nearest-neighbor distances for each soma. For P1 retinas, where there is less space between cells and in fact cells seem to form clusters, automatic segmentation was not possible. Therefore, cells were manually marked using the ImageJ multipoint tool and soma locations exported as a CSV file. For all ages, the centroid data was imported to MATLAB for further analyses.

Density was quantified by dividing the microscope field of view up into 200 ×200 μm squares, manually excluding ones that did not cover the retina or covered partial or damaged parts of the retinas. Out of the resulting squares (~150 per P7 retina, ~100 per P1 retinas), 75 squares (for P7 retinas) and 50 squares (for P1 retinas) were randomly selected and the average density of TdTom+ cells in those squares calculated.

To quantify the nearest-neighbor distances, we used a custom-written MATLAB code that, for each ipRGCs, identified the closest neighbor using the shortest Euclidean distance.

Details of statistical tests, number of replicates, and p values are indicated in the figures and figure captions. p values < 0.05 were considered significant.