The Second Arden Shakespeare Series: A theoretical discussion and analysis

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the non-aversive, spontaneous recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless–coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance

Examples of movements of a mouse required to activate PIR sensors at different heights from the cage floor

<p>Near-infrared LED outside and below the cage shows the activation of the sensor.</p

Electronic Supplementary Material 4: Data In Figures 1, 2, 3, S2, S3, S4, And S5 from Modulation of recognition memory performance by light requires both melanopsin and classical photoreceptors

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the non-aversive, spontaneous recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless–coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance

Electronic Supplementary Material 1: Figures S1–S6, Supplemental Methods, And Supplemental Results And Discussion from Modulation of recognition memory performance by light requires both melanopsin and classical photoreceptors

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the non-aversive, spontaneous recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless–coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance

Primer Sequences.

<p>Primer Sequences.</p

Summary of Opn4L and Opn4S expression profiles and timeline for maturation of M1 and M2 type pRGCs.

<p>A) Graph showing the mean number of cells in which Opn4S and Opn4L or only Opn4L were detected per retinal section at each development time point. Note the increasing numbers of Opn4S positive cells (also expressing Opn4L after P0) detected between P0 and P3 and also the increase in number of Opn4L only cells detected at P14 compared to earlier time points. B) Graph showing the mean number of cells from each retinal section that could be reliably classified as either M1, M2 or displaced M1 type pRGCs (dM1) based on morphology and stratification of dendrites. In all cases, cells classified as M1 and M2 type pRGCs were positive for Opn4S and Opn4L or Opn4L only respectively. Changes in immuno-positive cells were assessed against the previous time point in a stepwise manner. Significant differences are indicated, where the cell type is indicated by markers of the respective colours. This data demonstrates significant increases in Opn4S expression at P0–P5 and Opn4L at P5–14. Corresponding increases in the number of cells identified as M1 and M2 cells were observed at P0–P5, and P5–P14 respectively. * = p<0.05, ** = p<0.001, *** = p<1.0E-6. The overall correlation between the number of cells expressing Opn4S and Opn4L and those that could be classified as M1 type pRGCs based on morphology was 0.91. All cells expressing only Opn4L were classified as M2 type pRGCs. C) Summary of the classifications assigned to cells at each time point shown as a percentage of the total number of cells identified. As shown, cells resembling M1 type pRGCs were the dominant cell type identified in the early postnatal retina, with M2 type cells not detected reliably until P14. Number of sections examined was typically 25–30 per retina, from n = 3–4 retina per time point. Cells with dendrites located in multiple layers of the IPL or were clearly bistratified were classified as Multi / M3. Cells for which dendrites could not be clearly identified were classified as undefined. Prior to P10, all cells classified as undefined expressed both Opn4S and Opn4L (except at P0 where Opn4L could not be detected) and most likely represent early M1 type cells. In order to estimate the total number of cells that went undetected at each time point using the Opn4L and Opn4S antibodies (levels of under-reporting), the mean number of cells identified per retinal section using the isoform specific antibodies was compared to values observed from similar analysis of sections stained using the highly sensitive UF006 antibody that recognises both Opn4L and Opn4S.</p

Differential expression of Opn4S is observed early in postnatal development in cells resembling M1 and M2 type pRGCs.

<p>A) Double labelling with UF006 (red) and Opn4S (green) antibodies showing the differential expression of Opn4S in M1 and M2 type pRGC subtypes as early as P3. B) Double labelling with Opn4L (red) and Opn4S (green) antibodies at P30 showing the morphology and pattern of melanopsin expression in M1 and M2 type pRGCs of the adult retina. Note the similarities in morphology and levels of staining for M1 type pRGCs observed at P3 and P30, compared to the changes in morphology and staining that are observed for M2 type cells between these time points. For all images DAPI nuclear counterstain is show in blue. Merged images are show in right hand panels. Scale bar for all images = 50 µm.</p

Differential expression and of Opn4L and Opn4S during development correlates with the maturation of M1 and M2 type pRGCs.

<p>Immunohistochemistry images showing the expression of Opn4L (red) and Opn4S (green) throughout postnatal retinal development, highlighting the variety of cell types detected at each time point. Note that Opn4L and Opn4S positive cells resembling M1 type pRGCs can be preliminarily identified as early as P3 and clearly be P5, whereas cells resembling M2 type pRGCs are detected only very weakly at P10 and not reliably detected until P14, following a large increase in levels of Opn4L staining. For all images DAPI nuclear counterstain is show in blue. ON and OFF correspond to the ON and OFF sublamina of the inner plexiform layer. M1 = M1 type pRGCs, M2 = M2 type pRGCs, dM1 = displaced M1 type pRGC, M3 = M3 type pRGC, but is also used to label cells that are multi stratified during early development, ND = not defined, for cells that could not be classified based on morphology alone. Scale bar for all images = 50 µm.</p

Expression of <i>Opn4L</i> and <i>Opn4S</i> mRNA during postnatal development.

<p>qPCR analysis showing the differential expression of Opn4S (A) and Opn4L (B) mRNA during postnatal development of the retina, highlighted by the changing ratio of Opn4L/Opn4S mRNA (C). Developmental profiles for rhodopsin (D), middle wave sensitive (MWS) cone opsin (E) and tyrosine hydroxylase (TH) are shown to indicate the timeline for the development of the classical visual system. All expression data is shown normalised to the expression of three housekeeping genes (Gapdh, Pmsb2, Arbp). Statistical results are shown for comparisons to data at P5. * = p<0.01, ** = p<0.001, *** = p<0.0001. For Opn4L, Opn4S and TH no significant differences were observed between P0 and P5, but significant increases were detected for rhodopsin (p = 0.001) and MWS (p = 0.02) when comparing these time points.</p

Representative actograms of <i>Grm2/3</i>^+/+ and <i>Grm2/3</i>^-/- mice during a 12:12 h light/dark (12:12 LD) cycle.

<p>Each row depicts a single 24 h period. The light and dark grey shading corresponds to periods of (100 lux) light and dark, respectively. (A) Representative wheel-running actograms. The black bars denote periods of wheel-running activity, binned in 6 min epochs. The height of the bars corresponds to the number of wheel rotations within each epoch. (B) Representative passive-infrared (PIR) actograms. The black bars denote periods of home-cage activity, binned in 6 min epochs. The height of the bars corresponds to % time active within each epoch. ZT = zeitgeber time.</p