Identification of melanopsin-responsive pretectal neurons
We first set out to identify neurons in the mouse pretectum that received input from ipRGCs. To this end, we performed multielectrode (32 channel) recordings from the PON and surrounding pretectum of 26 anaesthetised Opn1mwR mice. Although the pretectum receives input from both ipRGCs and other RGC types [35, 36], a characteristic feature of melanopsin phototransduction is a sluggish and sustained elevation in firing in response to high intensity short-wavelength (‘blue’) light [5]. Accordingly, to screen for cells likely to receive input from ipRGCs, we first evaluated responses to monochromatic 460-nm light steps (10 s duration from darkness) across a range of intensities (Fig. 1a; 14–16 Log melanopsin effective photons/cm2/s; termed here ‘Mel High’) predicted to robustly activate melanopsin-based responses in all known classes of ipRGCs [37,38,39].
Across all primary retinorecipient targets, visual responses driven by rods and cones are characterised by an acute increase in firing rate within a few hundred milliseconds following stimulus onset that typically decays substantially on prolonged stimulation [11, 12, 16, 17, 27, 40]. By contrast, where present, response elements originating with melanopsin build up over several seconds and persist even under very long exposure [7, 11, 16, 17, 40]. We first then quantified early and later components of the response to bright 460-nm light steps (0–500 ms and 5–10 s following stimulus onset respectively) to identify neurons with the sustained changes in firing consistent with melanopsin input (Fig. 1b).
As expected, based on previous data [11], a substantial number of the pretectal neurons we isolated from these multielectrode recordings (n = 72/230 visually responsive cells) exhibited sustained increases in firing in response to such stimuli (Fig. 1b, c). By contrast, the remaining cells exhibited responses incompatible with a significant contribution from ipRGCs (Fig. 1b, c): the majority (n = 121/158) exhibited only very transient increases in firing at the onset and offset of the light step (as reported previously; [11]), while a smaller subset (n = 37) exhibited light-driven decreases in firing rate (OFF responses).
To more specifically establish that the population of cells exhibiting sustained responses to Mel High stimuli did indeed correspond to those receiving input from ipRGCs, we next evaluated their responses to polychromatic light steps, matched to provide identical stimulation of L- and S-cones but a significantly weaker impact on melanopsin (Fig. 1a; ~ 500-fold weaker, termed here ‘Mel Low’). We chose this approach, initially, since we predicted it would allow us to evaluate the impact of much larger variations in melanopsin excitation than are achievable using more selective melanopsin-isolating stimuli, without encountering the rod-intrusion that can occur following light-adaptation [16, 40, 41]. Hence, while the Mel Low/High stimuli used here also differed substantially in their impact on rods, our expectation was that, at the two higher intensities tested, both should be sufficiently bright to produce a saturating rod response when presented as light steps from darkness (≥ 12.9 and 15 Log rod effective photons/cm2/s for Mel Low and High respectively; c.f. [42]).
Consistent with our expectation that Mel High and Low stimuli should produce equivalent rod/cone-mediated responses, light-responsive cells whose properties were incompatible with ipRGC input (those with transient or OFF responses) exhibited equivalent responses to both stimuli (Additional file 1: Figure S1a,b). Similarly, as expected given the very sluggish nature of melanopsin phototransduction, for the majority of the cells with sustained responses (n = 60/72), the initial increase in firing (peak occurring during first 500 ms of light step) was statistically equivalent for Mel Low/High at one or both of the top two intensities tested (within-cell t tests across 10 trials at each stimulus). Further analysis of this group of cells indicated that, in fact, there was no detectable effect of stimulus on early response components (mean firing during first 500 ms) at any intensity (Fig. 1d, e; two-way RM ANOVA with Sidak’s post-tests, all P > 0.05). Importantly, however, Mel High/Low responses diverged at later timescales (Fig. 1d) such that, over the last 5 s of the response, the Mel High-evoked increase in firing was significantly greater at all intensities tested (Fig. 1e; two-way RM ANOVA with Sidak’s post-tests). Based on this pronounced and selective enhancement of late components of the Mel High response (across a range where melanopsin phototransduction is active; [37]), we consider this group of cells melanopsin responsive (MR). These observations align well with the kinetics of PON neuronal responses observed previously in rodless/coneless mice [11]. This conclusion is also further supported by additional data reported below and later in the manuscript (Figs. 5 and 7 and associated additional files), including the anatomical location of such cells which, as expected based on known ipRGC projections [43, 44], were strongly clustered in the region of the PON.
By contrast to the above, we also observed a small number of cells (n = 12) with sustained responses where both early and later components of the responses were significantly enhanced for Mel High stimuli, an effect that was most apparent at the highest intensity tested (Additional file 2: Figure S2a,b). Although we cannot definitively rule out a melanopsin contribution to the responses of this group of cells, this difference even in the earliest portions of the response suggests contributions from a photoreceptor other than melanopsin (presumably an unexpected contribution from rods; see below). Accordingly, for subsequent analysis, we consider this rare group of cells (~ 5% of the light-responsive cells identified here) as non-MR.
To provide further confidence in our classification of cells as melanopsin responsive, we also performed parallel sets of experiments in melanopsin knockout red cone animals (Opn1mwR; Opn4−/−; n = 5 mice) and mice lacking functional cone photoreception (Cnga3−/−; n = 4). Among both groups of mice, a small population of cells (n = 8/41 and n = 3/24 for Opn1mwR; Opn4−/− and Cnga3−/− respectively) exhibited behaviour equivalent to that described above: a global enhancement in responses to the Mel High vs. Low stimulus that emerged at higher intensities (Additional file 2: Figure S2c-f). Since we observe this behaviour in recordings from animals lacking either cone or melanopsin phototransduction, we conclude this must reflect an unexpected difference in rod-mediated responses that appears in certain cells under high light intensities. This may reflect the emergence of bleaching adaptation [41] or perhaps even the contribution of an atypical phototransduction pathway [45]. In either case, the impact of such a mechanism appears to be sufficiently restricted (being essentially absent from the other classes of visually responsive cells we recorded; Fig. 1f–i; Additional file 1: Figure S1) that it does not significantly interfere with our ability to identify MR cells.
Of particular note here then are the more commonly encountered sustained cells exhibiting statistically identical initial increases in firing to Mel High/Low steps at high stimulus intensities (n = 9 and n = 5 for Opn1mwR; Opn4−/− and Cnga3−/− respectively). As for the large population of Opn1mwR cells matching these criteria (i.e. MR cells), early components of the responses of both Opn1mwR; Opn4−/− (Fig. 1f, g) and Cnga3−/− cells (Fig. 1h, i) to Mel High vs. Low stimuli were in fact equivalent at all intensities (two-way RM ANOVAs with Sidak’s post-tests, all P > 0.05). Cells in Cnga3−/− animals (where melanopsin remains functional) also retained the pronounced enhancement across later components of the Mel High response at all intensities (Fig. 1h, i; two-way RM ANOVA with Sidak’s post-tests). By contrast, responses of Opn1mwR; Opn4−/− cells were qualitatively different (Fig. 1f, g), instead showing statistically equivalent responses at all but the highest intensity where Mel Low responses were very marginally reduced (Sidak’s post-test, P = 0.03; presumably reflecting the threshold appearance of a mechanism equivalent to that illustrated in Additional file 2: Figure S2). Collectively then, these data support our classification of cells as MR and provide further confidence that elements of the Opn1mwR MR-cell responses ascribed to melanopsin do not instead reflect significant stimulus-related difference in rod/cone activation.
Cone influences on melanopsin-responsive pretectal neurons
Having identified pretectal neurons that received input from ipRGCs, we next aimed to comprehensively define how their activity was modulated by cone input and determine whether any exhibited the colour-opponent behaviour observed previously in another major ipRGC target—the SCN [27]. To this end, starting with a polychromatic background stimulus that resembled a wildtype mouse’s experience of natural daylight (Fig. 2a), we adjusted the spectral composition to generate carefully calibrated pairs of stimuli designed to differ in apparent brightness for one or both cone opsins without significantly altering rod or melanopsin excitation (Fig. 2b; see also ‘Methods’ for further details). In particular, we generated four stimulus pairs where transitions between each element selectively modulated excitation of just L- or S-cone opsin in isolation, or both opsins in unison (‘L + S’) or in antiphase (‘L − S’). We then applied 0.25 Hz square-wave cycles of these cone-isolating stimulus pairs at a range of contrasts up to 75% Michelson (sevenfold change in apparent brightness).
The majority of MR units tested with these stimuli (n = 50/60) exhibited robust responses to at least a subset of the cone-isolating conditions tested (Fig. 2c–g). Moreover, a substantial proportion of these (n = 24/50) changed their firing rates in opposite directions when we selectively modulated L- or S-cone excitation in isolation; that is, almost half of these MR cells exhibited colour-opponency. Among this group, S-ON/L-OFF behaviour was substantially more common than the converse (n = 17 vs. n = 7 with L-ON/S-OFF responses); however, both classes of opponent MR cells usually exhibited a significant bias (on average ~ 3-fold larger responses) towards the excitatory (ON) component of the response (Fig. 2c, d, g).
We next inspected contrast response functions for ON and OFF responses among these colour opponent cells, by quantifying the percentage variance in firing rate accounted for by stimuli providing 15–75% contrast for the target cone population (see ‘Data analysis’ section in ‘Methods’ for further details). This analysis indicated, while OFF responses were reliably smaller at all tested contrasts (Fig. 2e; two-way RM ANOVA with Sidak’s post-tests), the sensitivity of ON and OFF responses was similar. Consistent with this behaviour, while these opponent MR cells were capable of responding to stimuli that modulated cone ‘illuminance’ without changing chromaticity (L + S stimulus), their responses were significantly larger when activation of the two cone opsins was modulated in antiphase to produce pronounced changes in colour (Fig. 2e; two-way RM ANOVA with Sidak’s post-tests). This chromatic response enhancement was evident even under the lowest contrast tested (15% Michelson).
Anatomically intermingled with the cell populations described above (Additional file 3: Figure S3), the remaining MR cells that exhibited robust responses to our cone-isolating stimuli lacked any clear evidence of opponent behaviour. In most cases, the responses of this group of cells were strongly biased towards one of the two opsins, with similar numbers of cells primarily driven by L-cone or S-cone opsin (Fig. 2g, n = 12 and n = 14 respectively). At the level of the population average then (Fig. 2d), achromatic (L + S) modulations in cone illuminance drove much more robust changes in firing than antiphasic (L − S) modulations (due to differential modulation of L- and S-biased subpopulations). By contrast, at the level of individual cells, responses to L + S stimuli tended to be only marginally elevated relative to those produced by L − S stimuli (Fig. 2e, two-way RM ANOVA with Sidak’s post-tests), consistent with the typically very weak responses driven by the ‘non-dominant’ opsin.
This diversity in the responses of pretectal MR units and, especially, the prevalence of cells exhibiting S-ON/L-OFF type colour opponent responses recapitulates our previous findings in the SCN [27], where retinal input comes almost exclusively via ipRGCs. By contrast, among the pretectal neurons identified here where we did not find clear evidence of melanopsin-dependent responses (Additional file 4: Figure S4), S-ON/L-OFF responses were significantly less common (Fig. 2g, n = 16/170, vs. 17/60 MR cells; Fisher’s exact test, P = 0.001) while the prevalence of L-ON/S-OFF and non-opponent cone-driven responses was similar (P = 0.136 and P = 0.446 respectively). Instead, across this group, there was a higher proportion of cells lacking detectable responses (n = 81/170 vs. 10/60 MR cells, P < 0.0001), indicating that either such cells primarily receive rod input or, perhaps more likely, that they are specialised to detect specific stimulus feature, e.g. motion or spatial contrast [46]. These differences in the relative proportions of response types held true also when we analysed data separately for non-MR cell populations (Additional file 4: Figure S4 g) designated ‘transient’ or ‘OFF’ but not the very rarely encountered group with sustained responses (where it is harder for us to definitively rule out a melanopsin contribution). Collectively then, alongside the recent identification of a subtype of mouse ipRGC (M5) with colour opponent input [28] and our earlier data [27], the present findings indicate a close association between the presence of melanopsin input and S-ON/L-OFF chromatic input.
To rule out the possibility that our assessment of cone preferences (and especially our inability to detect such responses in subsets of cells) simply reflected the relatively high background light intensity used, in a subset of experiments (n = 10 Opn1mwR mice), prior to collecting the data reported in Fig. 2, we also applied identical cone-isolating stimuli but at a background 10× dimmer (ND1; intensity ~equivalent to sunrise/sunset). In fact, we found all cell groups tested displayed remarkably consistent responses at both background light intensities (Additional file 5: Figure S5a), barring a small number of cells that only responded under one of the two intensities (n = 12 and n = 6 responding only at ND1 and ND0 respectively from 133 cells tested). Response amplitudes were, however, modestly increased at lower irradiance (Additional file 5: Figure S5b, paired t test P < 0.0001). Since, for MR cells, peak firing rates were also at least nominally higher at the lower intensity (Additional file 5: Figure S5c), we suspect the slight reduction in response amplitude at higher irradiance reflects some degree of contrast adaption (since the higher irradiance also followed the lower in these experiments). Most importantly, however, the fundamental nature of the cone-dependent modulations in firing (opponent vs. non-opponent) was retained across all cell types.
Validation of cone-isolating stimuli
Having established the impact of cone-isolating stimuli on the activity of visually responsive neurons in the PON and surrounding pretectum, we next sought to determine the extent to which contributions from photoreceptors other than those specifically targeted by our stimuli could have influenced the results.
We first evaluated the possibility of a contribution from melanopsin. Since the cone-isolating stimuli we employed provided negligible melanopic contrast (< 6%, equivalent to a 0.05log unit change), well below the level required to evoke detectable responses [47], we considered it most unlikely that this inner retinal photoreceptor exerted any significant influence over the responses reported above. Consistent with that expectation, pretectal neurons recorded in Opn1mwR;Opn4−/− mice under the same conditions behaved in an identical manner to those in mice with functional melanopsin: we readily identified both colour opponent and non-opponent neurons in the same proportions as in Opn1mwR mice and those response preferences were stable when we changed irradiance (Additional file 6: Figure S6).
Since our cone-isolating stimuli also provided negligible contrast for rods (1.7 and 3.3% for L- and S-opsin-isolating stimuli respectively), we also considered a significant contribution from rods most unlikely. Indeed, the rod contrast associated with our cone-isolating stimuli was lower than previously reported thresholds for rod-based responses under light adaptation [41, 47,48,49]. Nonetheless, to more directly rule out the possibility that our cone-isolating stimuli drove responses via photoreceptors other than those targeted, we next evaluated the impact of a stimulus pair designed to produce no detectable contrast for either cone opsin (< 0.05% Michelson contrast), while presenting a significant change in rod and melanopsin activation (Fig. 3a; ‘cone-silent’; 45 and 43% contrast for rod and melanopsin respectively).
Importantly, we found that across all classes of visually responsive pretectal neurons (colour-opponent and non-opponent, MR and non-MR) in both Opn1mwR and Opn1mwR;Opn4−/− mice, this cone-silent stimulus produced consistently negligible modulations in the firing rate. Indeed, changes in firing associated with presentation of this stimulus were in all cases significantly lower than those evoked even by the weaker of the two (non-dominant) single-cone stimuli for that cell (Fig. 3b; one-way RM ANOVAs with Dunnett’s post-tests, all P < 0.001). These data thus rule out the possibility that the responses we ascribe to cones in fact originate with rods and/or melanopsin.
To further confirm our interpretation above, we also evaluated the impact of cone-isolating stimuli on pretectal neurons in Cnga3−/− animals. As expected, given the absence of a critical component of the cone-phototransduction cascade, presentation of these cone-isolating stimuli did not produce detectable responses in Cnga3−/− cells (Fig. 3c, n = 24 visually responsive neurons reported in Fig. 1 and Additional files 1 and 2). As such, statistical comparison of the maximal response of each cell to any of the cone-isolating stimuli we applied revealed a pronounced difference relative to those of cells from both Opn1mwR and Opn1mwR;Opn4−/− mice (Fig. 3c, Kruskal-Wallis test with Dunn’s multiple comparisons, both P < 0.0001; analysis based on all visually responsive neurons, regardless of response to cone-isolating stimuli). In addition, equivalent analyses to those reported above (Fig. 3b, c) for subsets of cells tested at 10-fold lower irradiance produced identical results (Additional file 7: Figure S7); in no case did we observe any evidence consistent with any significant rod or melanopsin intrusion.
To complement the important control experiments described above, we also considered whether variations in cone opsin sensitivity or prereceptoral filtering, relative to those assumed when generating our stimuli, might result in off-target cone-driven responses under conditions designed to silence one or the other class. In particular we were keen to rule out the possibility that the colour-opponent responses we detected might instead have reflected some ‘overshoot’, such that stimuli expected to be silent in fact induced detectable negative contrast responses in that cone population.
To this end, we first modelled the impact of very substantial variations in the cone opsin peak sensitivity (λmax) and/or in the short-wavelength cut-off imposed by the mouse lens (Fig. 3d–f; in each case ± 25 nm—far beyond any reasonable estimate of errors associated with the published values). In fact, we found that even unfeasibly large deviations from the assumptions inherent in our stimulus design had negligible effects, particularly in the case of predicted S-opsin responses which were effectively insensitive to these manipulations. Moreover, while the potential impact on L-opsin-driven responses was slightly larger, stimuli expected to be L-cone silent continued to present negligible contrast even following the most extreme deviations in both lens transmission and opsin sensitivity (Fig. 3c; <±2% contrast).
We also considered other sorts of prereceptoral filtering. In humans, under certain circumstances, stimuli expected to be cone silent generate a percept of the retinal vasculature [50], due to differential stimulation of cones that lie in the shadow of blood vessels (penumbral cones). Given the very large size of mouse RGCs (especially ipRGCs; [43]), it is most unlikely that any of the pretectal units we sampled from could receive input exclusively from penumbral cones. Nonetheless, we assessed the possible impact of such a phenomenon, by modelling the effect of varying contributions of penumbral cones alongside deviations in L- and S-opsin λmax (Fig. 3g–i). Again, we found that neither mechanism (singly, in combination or even with the addition of variations in lens cut-off) significantly altered predicted S-opsin-driven responses. Moreover, while including penumbral cones could nominally increase off-target contrast for stimuli expected to be L-cone silent (Fig. 3h), even in the implausible case that a cell received input exclusively from penumbral cones, the expected magnitude of this effect (< 8% negative L-opsin contrast) was far too small to account for the colour opponent responses we observed (where responses driven by the weaker of the two cone opsins were at least as large as those driven by 15% contrast stimuli applied to the stronger; see Fig. 3b). Indeed, our further analysis revealed that for any noticeable interference due to off-target effects to occur, not only would input have to come from an unfeasibly high proportion of penumbral cones but also that L-opsin would have to have a far more short-wavelength λmax than any reasonable expectation (Fig. 3i). Our experimental data rule out this most unlikely possibility. Hence, additional modelling indicated that any deviation in cone-opsin λmax sufficient to result in detectable off-target effects would result in an even larger unintended contrast for our cone-silent stimulus (Additional file 7: Figure S7c). As reported above (Fig. 3b), this stimulus did not produce detectable responses.
In sum then, we can exclude the possibility that responses originating with rods, melanopsin or unintended stimulation of cones interfere with our conclusions regarding cone-specific influences on pretectal neurons. Additional analysis reported below (Figs. 4, 5 and 7) provides further confirmation that our stimuli work as expected under a variety of different conditions, such that stimuli with very different spectral compositions but similar predicted photoreceptor contributions consistently produce equivalent responses.
Influence of cones on mouse pupil responses
Insofar as our data above reveal that cone inputs provide high-amplitude chromatic or illuminance signals to subsets of pretectal MR units, we next investigated the extent to which these distinct sources of visual information were important for regulation of the primary physiological output known to be under PON control—the pupil. For these experiments, we used the same 75% contrast cone-isolating stimuli described above and, based on the previously reported dynamic range for mouse pupil responses [24, 25, 51] and our data showing moderately enhanced cone-based responses (Additional file 5: Figures S5 and Additional file 6: Figure S6), presented these at the lower of the two backgrounds tested (10-fold dimmer than that in Fig. 2b, corresponding to typical irradiance at sunrise/sunset). Since previous data reveal important contributions of melanopsin and rods to mouse pupil responses [25, 51, 52], we also aimed to understand how any influence of cones compared with that originating with those other photoreceptor classes under our conditions. As such we included an additional stimulus (‘all opsin’) that provided a spectrally neutral modulation in light intensity providing 75% contrast for every class of mouse photoreceptor. Finally, to account for the more sluggish nature of pupil responses relative to PON neuronal activity, we extended our stimulus presentation here to 5 s at each phase and presented these as single cycle ‘bright-dim’ and ‘dim-bright’ modulations (stimuli interleaved and order randomised between animals; 2–4 trials/stimulus/animal). This allowed us to test all stimuli multiple times in a single animal while avoiding any confound due to contrast adaptation. We then went on to record consensual pupil responses to these stimuli from awake gently restrained mice (Fig. 4a; n = 10 Opn1mwR mice).
Across both stimulus polarities tested, we reliably observed significantly larger pupil diameters during ‘dim’ vs. ‘bright’ phases when the stimuli selectively modulated either L- or S-cone opsin in isolation (Fig. 4b; paired t tests all P < 0.05). The same was also true when we modulated just L + S opsin or all opsins in unison to produce achromatic changes in illuminance. By contrast, stimuli that modulated L and S-opsin in antiphase (and thereby produced large changes in colour without changing illuminance) failed to evoked significant changes in pupil diameter (Fig. 4b; paired t tests; P = 0.23 and 0.32 for positive-negative and negative-positive modulations respectively).
As expected, baseline pupil diameter did not vary significantly between any of the stimulus conditions (one-way RM ANOVA, P = 0.11) nor did the difference in pupil diameter between dim and bright phases vary as a function of stimulus polarity (two-way RM ANOVA, P > 0.05 for polarity and interaction). As such, we averaged ‘bright’-‘dim’ change in pupil diameter across both stimulus polarities for comparisons between stimuli (Fig. 4c). This analysis confirmed that modulations in pupil size evoked by achromatic L + S opsin modulations were significantly larger than those produced by chromatic, antiphasic modulations in the two cone opsin classes (Fig. 4c; one-way RM ANOVA with Sidak’s post-test, P < 0.001). Moreover, the change in pupil size evoked by L + S cone opsin modulations was not significantly different from that predicted by a simple linear sum of the responses to stimuli modulating just L- or S-opsin in isolation (Fig. 4c, paired t test, P = 0.49). Collectively then, these data indicate that, despite the presence of large numbers of chromatic MR units in the mouse PON, cone-derived chromatic signals do not noticeably contribute to regulation of the mouse pupil. Instead signals from L- and S-opsins combine in an additive manner to drive pupil constriction.
To rule out the possibility that this lack of chromatic modulation of the mouse pupil simply reflected differences in the temporal waveform of the stimuli between those used for electrophysiology and pupillography, we also evaluated neurophysiological responses of a subset of pretectal MR units under the latter stimulus paradigm. As expected, these recordings confirmed that both colour opponent and non-opponent MR neuronal responses remained readily identifiable under the conditions used for pupillography (Fig. 5a, b; n = 7 S-ON/L-OFF and 9 non-opponent neurons from 5 Opn1mwR mice). A similar finding was true also for pretectal neurons in Opn1mwR;Opn4−/− mice tested under these conditions (Additional file 8: Figure S8a,b).
In addition to demonstrating that cones exert an additive rather than chromatic influence on mouse pupil regulation, the analysis above also highlighted another important feature of pupil control under our experimental conditions; when comparing the average change in pupil size between ‘bright’ and ‘dim’ across the full 5-s stimulus duration, there was no detectable difference between stimuli that modulated just L- and S-opsin vs. those that which modulated all mouse opsins equally (Fig. 4c, one-way RM ANOVA with Sidak’s post-test, P = 0.85). This indicates that, under the photopic conditions studied here, pupil responses to relatively small/rapid changes in illumination are dominated by cone rather than rod and/or melanopsin-derived signals.
To explore this conclusion in more detail, we next analysed the difference in pupil size between dim and bright stimulus phases of L + S vs. all-opsin stimuli as a function of time-post stimulus transition (Fig. 4d). This analysis in fact revealed a progressive divergence between pupil responses to the two stimuli, with both types of stimuli producing equivalent responses for the first 3 s and all-opsin modulations producing larger changes in pupil size across the later components of the stimuli (Fig. 4d; two-way RM ANOVA with Sidak’s post-tests). Equivalent effects were also observed in the electrophysiological responses of MR neurons (Fig. 5c, d). This gradual appearance of a cone-independent influence on pupil responses and PON neurophysiological activity closely matches that expected from melanopsin, based on pupil data reported for melanopsin only and melanopsin knockout animals [25, 51, 52] and electrophysiological recordings from the PON and other ipRGC target regions [11, 16, 27, 40, 44, 47, 53, 54]. By contrast, rod-driven pupil and electrophysiological responses are much faster [25, 41, 47] (see also data from Cnga3−/− mice presented in Fig. 1 and Additional file 1: Figure S1 and Additional file 2: Figure S2). Importantly then, the close similarity between initial components of L + S and all-opsin responses observed here (Fig. 4d and Fig. 5c, d) rules out any overt contribution from rods under the conditions we employed. Also consistent with our interpretation that the divergence in response to these two stimuli at later timepoints originates with melanopsin, this feature was lacking from the neurophysiological responses of Opn1mwR;Opn4−/− cells tested under these conditions (Additional file 8: Figure S8c,d). Further data reported below confirm and extend this conclusion under a variety of different conditions (Fig. 7 and associated additional files).
These data therefore confirm additive contributions from both cone types and melanopsin to pupillary control, with cones dominating early components of the responses and melanopsin becoming a progressively more important contributor under extended exposure.
Temporal properties of cone influences on melanopsin-responsive neurons
Given our data above, we next sought to better understand the temporal properties of cone inputs to MR units in the PON. In particular, a number of previous studies have provided evidence that S-cone contributions to ipRGC-dependent physiological responses (including PON neuronal activity) may exhibit temporal properties that are distinct from those originating with longer-wavelength-sensitive cones [11, 30, 55]. Here then, we examined in more detail the temporal tuning properties of cone inputs to MR cells by applying cone-isolating stimuli as sinusoidal oscillations across a range of temporal frequencies (0.025 to 5 Hz, 75% contrast, ND0).
We first examined the responses of MR cells to their ‘optimal’ stimulus type (L − S and L + S stimuli for colour-opponent and non-opponent cells respectively) and found that the majority of MR neurons that responded to square-wave modulations also exhibited significant responses across a wide range of sinusoidal stimulus frequencies (Fig. 6a, b). Indeed, substantial numbers of MR cells continued to track modulations in cone-derived illuminance or chromatic signals even at the lowest temporal frequency tested (Fig. 6b, n = 32/50). This observation applied similarly to both colour-opponent and non-opponent MR neurons (n = 18/24 and 14/26 respectively; Fisher’s exact test, P = 0.15). By contrast, non-MR cells (Additional file 9: Figure S9) generally showed significant modulations in the firing rate over a narrower range of temporal frequencies, with a much lower proportion of cells capable of tracking the lowest temporal frequency we tested (Fig. 6b; n = 32/89 cells that responded to square-wave stimuli, Fisher’s exact test, P = 0.002 vs. MR units). Similarly, while a substantial proportion of MR cells in fact exhibited the most robust modulations in firing rate at the lowest frequency tested (n = 18/50; quantified as percent variance in firing rate explained by a 0.025-Hz stimulus), this was less commonly observed across non-MR cells (n = 15/89; Fisher’s exact test P = 0.013). Collectively then, these data support previous suggestions that cone inputs to ipRGCs are unusually sustained [14, 15, 53] and indicate that MR neurons in the pretectum can use cone inputs to track even quite gradual changes in illuminance or colour.
We next examined whether the L- and S-opsin-driven responses of MR units differed in their ability to track low-frequency modulations. In line with the data reported above, we found that low-frequency sinusoidal modulations of the cone opsin class to which that cell was biased (as determined from response to square-wave stimuli, Fig. 2) reliably evoked robust responses in a significantly larger population of MR vs. non-MR neurons (Fig. 6c; Kolmogorov-Smirnov test, P < 0.0001). Further analysis classes confirmed that, as expected based on data presented above (Fig. 2), there were near equivalent proportions of both MR and non-MR neurons that preferentially responded to low-frequency stimuli targeting L- or S-opsin (Fig. 6d; MR: n = 22 L-biased vs. 28 S-biased, non-MR: 38 vs. 51; Fisher’s exact test vs. equal proportions—both P > 0.05). Hence, for low temporal frequency stimuli, colour opponent cells almost always exhibited more robust variations in the firing rate when we modulated the opsin providing the excitatory/ON component of their response (Fig. 6e), whereas subsets of achromatic cells preferentially responded to low-frequency L- or S-opsin-isolating stimuli (Fig. 6d, e).
Relationship between cone and melanopsin inputs
The data described above indicate that cone inputs exert a powerful influence over MR-cell firing activity, even for relatively modest and gradual changes in illumination (sevenfold variation in intensity over 40 s). Nonetheless, data presented earlier in the manuscript (Figs. 1, 4 and 5) provide clear evidence for a progressive transition between cone- and melanopsin-derived influences on pretectal MR cell activity and pupil responses following extended exposure. We next then sought to more comprehensively define the relative influence of cones and melanopsin on MR-cell firing in response to the smaller, dynamic changes in light intensity a mouse might encounter during exploratory activity close to dawn and dusk.
For this analysis, we compared responses to square-wave or sinusoidal stimuli that selectively modulated L- and S- opsin in unison (data in Figs. 2 and 6), with equivalent stimuli that modulated all photoreceptor classes equally (all-opsin). As discussed above, in principle, any difference in the response to these stimuli could reflect a contribution of either rods or melanopsin. However, the relatively high background light intensity employed for the experiments should substantially impair any rod-mediated responses (Fig. 2b; ND0; 15.2 log rod effective photons). Indeed, the related analyses reported earlier in the manuscript at 10-fold lower irradiance (Figs. 4 and 5) certainly did not reveal any evidence of significant rod contributions.
As expected then, given the relatively sluggish nature of melanopsin responses, across all MR units that reliably responded to cone-isolating stimuli (n = 50), changes in the firing rate evoked by rapid L + S opsin-targeted stimuli (0.25 Hz square wave) were in fact remarkably similar to those produced by equivalent stimuli targeting all opsin classes (Fig. 7a). Nonetheless, when comparing the difference in firing rates between bright and dim stimulus phases, we found that by the last 400 ms at each phase there was a progressive divergence in the contrast response functions for the two stimuli such that, at the highest contrast, responses were greater for the all opsin stimuli (Fig. 7b; two-way RM ANOVA with Sidak’s post-test, P < 0.05). This divergence in responses between stimuli that modulate both cone opsin types with or without also engaging melanopsin became even more pronounced when we examined responses to low temporal frequency modulations (Fig. 7a). Indeed, comparison of the temporal frequency tuning relationship for L + S and all opsin stimuli across MR units revealed that, whereas responses were identical for frequencies of 0.5 Hz and greater, there was a substantial enhancement in responses at the two lowest frequencies tested (Fig. 7b, two-way RM ANOVA with Sidak’s post-tests, both P > 0.001).
Although the selective response enhancement for all opsin stimuli within a specific temporal domain matched well what one would predict for melanopsin, we next sought to confirm this was indeed the case. As such, we first performed the same analysis described above for the other classes of pretectal neurons encountered in Opn1mwR mice that lacked conclusive evidence of melanopsin input (non-MR). As expected, we did not observe any significant response enhancement for all opsin stimuli across any tested contrast, temporal frequency or cell class (Additional file 10: Figure S10; two-way RM ANOVA with Sidak’s post-tests, all P > 0.05). We also compared the responses of pretectal neurons in melanopsin knockout animals (n = 23 Opn1mwR; Opn4−/− cells responding at ND0) under the same conditions. Again, we observed no significant differences between L + S and all opsin stimuli at any contrast or temporal frequency (Fig. 7c, d; two-way RM ANOVA with Sidak’s post-tests, all P > 0.05). Finally, we performed a similar analysis of contrast response functions for units tested with square-wave stimuli at ND1 (Additional file 11: Figure S11) which again confirmed that Opn1mwR; Opn4−/− cells exhibited identical responses to L + S and all-opsin stimuli while Opn1mwR MR unit responses for these stimuli diverged in a manner equivalent to that shown at higher irradiance.
In sum, the analyses described above support our view that the appearance of a cone-independent component of the MR cell responses at high-contrast/low temporal frequency conditions reflects the contribution of melanopsin photoreception. Collectively then, these data indicate that, in line with previous suggestions [26], for the relatively small and rapid variations in light intensity/spectral composition most commonly encountered during exploratory activity during the day [53], MR neuronal responses are primarily driven by cones. By contrast, melanopsin signals become increasingly important for tracking slower/larger changes in irradiance, such as those encountered across twilight or when moving between areas with substantial differences in access to natural light.