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. 2020 Jan 15;40(3):585-604.
doi: 10.1523/JNEUROSCI.1626-19.2019. Epub 2019 Nov 25.

Long-term Monocular Deprivation during Juvenile Critical Period Disrupts Binocular Integration in Mouse Visual Thalamus

Carey Y L Huh  1 , Karim Abdelaal  2 , Kirstie J Salinas  3 , Diyue Gu  4 , Jack Zeitoun  3 , Dario X Figueroa Velez  3 , John P Peach  5 , Charless C Fowlkes  6 , Sunil P Gandhi  1   7
Affiliations

Long-term Monocular Deprivation during Juvenile Critical Period Disrupts Binocular Integration in Mouse Visual Thalamus

Carey Y L Huh et al. J Neurosci. .

Abstract

Study of the neural deficits caused by mismatched binocular vision in early childhood has predominantly focused on circuits in the primary visual cortex (V1). Recent evidence has revealed that neurons in mouse dorsolateral geniculate nucleus (dLGN) can undergo rapid ocular dominance plasticity following monocular deprivation (MD). It remains unclear, however, whether the long-lasting deficits attributed to MD during the critical period originate in the thalamus. Using in vivo two-photon Ca2+ imaging of dLGN afferents in superficial layers of V1 in female and male mice, we demonstrate that 14 d MD during the critical period leads to a chronic loss of binocular dLGN inputs while sparing response strength and spatial acuity. Importantly, MD leads to profoundly mismatched visual tuning properties in remaining binocular dLGN afferents. Furthermore, MD impairs binocular modulation, reducing facilitation of responses of both binocular and monocular dLGN inputs during binocular viewing. As predicted by our findings in thalamic inputs, Ca2+ imaging from V1 neurons revealed spared spatial acuity but impaired binocularity in L4 neurons. V1 L2/3 neurons in contrast displayed deficits in both binocularity and spatial acuity. Our data demonstrate that critical-period MD produces long-lasting disruptions in binocular integration beginning in early binocular circuits in dLGN, whereas spatial acuity deficits first arise from circuits further downstream in V1. Our findings indicate that the development of normal binocular vision and spatial acuity depend upon experience-dependent refinement of distinct stages in the mammalian visual system.SIGNIFICANCE STATEMENT Abnormal binocular vision and reduced acuity are hallmarks of amblyopia, a disorder that affects 2%-5% of the population. It is widely thought that the neural deficits underlying amblyopia begin in the circuits of primary visual cortex. Using in vivo two-photon calcium imaging of thalamocortical axons in mice, we show that depriving one eye of input during a critical period in development chronically impairs binocular integration in thalamic inputs to primary visual cortex. In contrast, visual acuity is spared in thalamic inputs. These findings shed new light on the role for developmental mechanisms in the thalamus in establishing binocular vision and may have critical implications for amblyopia.

Keywords: amblyopia; binocular vision; critical period; dorsolateral geniculate nucleus; thalamus; visual cortex.

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Figures

Figure 1.
Figure 1.
Long-term critical-period MD leads to a loss of binocular thalamocortical inputs. A, Schematic of dLGN virus injection, GCaMP6s expression in thalamocortical axons in V1. B, Experimental timeline. C, In vivo two-photon Ca2+ imaging was performed in awake, head-fixed mice. D, An example cranial window with binocular zone mapped using widefield intrinsic signal imaging. Scale bar, 1 mm. E, An example FOV (summed projection) of dLGN boutons imaged in bV1 L1–2/3 of a control mouse. Boutons color-coded according to peak SF during contra-eye (left) and ipsi-eye (right) viewing. Scale bar, 10 μm. F, Same field as in E, but color-coded for OD. Scale bar, 10 μm. G, Ca2+ signals in a binocular (top) and two monocular (middle, contra-only; bottom, ipsi-only) boutons in response to drifting gratings presented to contra- or ipsi-eye. Gray represents individual traces. Black represents mean trace. Purple and orange bars represent time of stimulus presentation. Scale bar, 2 μm. Responses to 8 orientations at peak SF are shown. H, Number of visually responsive dLGN boutons that are ipsi-only, binocular, and contra-only per FOV in control versus MD mice (mean ± SEM per field). Control, ipsi-only: 63 ± 6; binocular: 10 ± 2; contra-only: 95 ± 8 boutons per field. MD, ipsi-only: 57 ± 4; binocular: 3 ± 1; contra-only: 73 ± 6 boutons per field. Linear mixed-effects model, effect of MD for ipsi-only: F = 0.35, p = 0.57; binocular: F = 6.74, p = 0.01; contra-only: F = 4.40, p = 0.04; n = 17 fields in 5 control mice, 17 fields in 5 MD mice. I, Percentage of visually responsive boutons that are ipsi-only, binocular, or contra-only per field in control versus MD mice (mean ± SEM per field). Control, ipsi-only: 38.1 ± 2.6%; binocular: 5.6 ± 1.3%; contra-only: 56.3 ± 2.6%. MD, ipsi-only: 41.2 ± 2.1%; binocular: 2.3 ± 0.4%; contra-only: 56.5 ± 2.1%. Linear mixed-effects model, effect of MD for ipsi-only: F = 0.23, p = 0.64; binocular: F = 6.29, p = 0.03; contra-only: F = 0.02, p = 0.90; n = 17 fields in 5 control mice, 20 fields in 6 MD mice. J, Ipsi-only, binocular, and contra-only fractions of visually responsive dLGN boutons in control versus MD mice (χ2(2) = 46.96, p = 6 × 10−11). K, Violin and overlaid box plots of response amplitude Rpref of binocular and monocular boutons in control versus MD mice. Linear mixed-effects model, effect of MD: F = 0.29, p = 0.59; binocular versus monocular: F = 229.22, p = 2 × 10−16. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. J, K, n = 2866 boutons in 5 control mice, 3503 boutons in 6 MD mice. ns, Not significant at p > 0.05. *p < 0.05, ****p < 0.0001.
Figure 2.
Figure 2.
Loss of binocular dLGN boutons without a reduction in response strength. A, ODI histogram of dLGN boutons in control versus MD mice (mean ± SEM per field, n = 17 fields in 5 control mice, 20 fields in 6 MD mice). B, Violin and overlaid box plots of mean response amplitude Rpref of dLGN boutons in control versus MD mice. Linear mixed-effects model, effect of MD: F = 0.32, p = 0.58; binocular versus monocular: F = 259.47, p = 2 × 10−16; contra versus ipsi: F = 44.48, p = 2 × 10−11; n = 2866 boutons in 5 control mice, 3503 boutons in 6 MD mice. C, D, The binocular bouton loss following critical-period MD (Fig. 1H,J) is shown using two additional statistical criteria in determining visual responsiveness: more liberal (C: p < 0.05) or more conservative (D: p < 0.005) than the typical criterion used in this study (p < 0.01; see Materials and Methods). Linear mixed-effects models, effect of MD for ipsi-only: p = 0.75 (C), p = 0.55 (D); for binocular: p = 0.01 (C), p = 0.01 (D); for contra-only: p = 0.93 (C), p = 0.05 (D). χ2 tests: for C, χ2(2) = 37.50, p = 7 × 10−9, n = 8099 versus 11,028 visually responsive boutons in total, 10% versus 7% binocular (control vs MD); for D, χ2(2) = 54.25, p = 1 × 10−12, n = 1887 versus 2211 visually responsive boutons in total, 6% versus 2% binocular (control vs MD). The binocular bouton loss in MD mice remains statistically significant under different data inclusion criteria. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. ns, Not significant at p > 0.1. †p < 0.1, *p < 0.05, ****p < 0.0001.
Figure 3.
Figure 3.
GCaMP6s labeling in dLGN is comparable between control and MD mice used for functional thalamocortical axon imaging. A, Example fluorescence sections of dLGN neurons labeled following GCaMP6s virus injection in control (left) and MD (right) mice that were used for in vivo two-photon Ca2+ thalamocortical axon imaging. Sections were immunostained for GFP. Scale bar, 100 μm. Example sections in A are from the same mice shown in Figure 12B. B, Heatmaps showing spatial distribution of labeled dLGN neurons in control and MD mice. Heatmaps are based on summed cell counts across all sections and mice. C, Numbers of dLGN neurons labeled were similar between functionally imaged control versus MD mice (mean ± SEM by animal values; t test, p = 0.56). D, Quadrants used in quantifying spatial distribution of labeled dLGN neurons. Scale bar, 100 μm. E, Numbers of labeled dLGN neurons were similar between control and MD mice across all quadrants (mean ± SEM by animal values; two-way ANOVA, control vs MD: F = 0.65, p = 0.43, effect of quadrant: F = 3.31, p = 0.03, interaction: F = 0.15, p = 0.93). B–E, n = 4 control and 5 MD mice, cells counted from three sections per animal.
Figure 4.
Figure 4.
Intact SF processing in thalamocortical boutons following long-term critical-period MD. A, Example FOVs of dLGN boutons imaged in bV1, color-coded according to peak SF of bouton during contralateral- and ipsilateral-eye presentation in control versus MD mice. Scale bar, 10 μm. B, Example SF tuning curves of monocular (top row, contra-only; bottom row, ipsi-only) boutons in control mice. Purple represents contralateral-eye trials. Orange represents ipsilateral-eye trials. Solid lines indicate mean response amplitudes. Dotted lines indicate fitted curves based on mean values. Fits omitted if curve-fitting failed to merge. C, Example SF tuning curves of monocular boutons in MD mice. Same convention as in B. D, Mean probability distribution of peak SF in binocular (top) and monocular (bottom) dLGN boutons' contralateral- (left) and ipsilateral-eye (right) responses (mean ± SEM by animal values). Mean values were fitted with a local regression smoothing function. E, Mean peak SF of boutons in control versus MD mice (mean ± SEM by animal; three-way ANOVA, control vs MD: F = 0.54, p = 0.47, binocular vs monocular: F = 4.32, p = 0.04, contra vs ipsi: F = 2.42, p = 0.13). F, Percentage of boutons with peak SF of 0.48–0.96 cpd (“High-SF boutons”; mean ± SEM by animal values; three-way ANOVA, effect of MD: F = 0.57, p = 0.46, binocular vs monocular: F = 15.05, p = 0.004, contra vs ipsi: F = 0.03, p = 0.87). D–F, n = 2866 boutons in 5 control mice, 3503 boutons in 6 MD mice.
Figure 5.
Figure 5.
Comparison of SF tuning properties of dLGN boutons, V1 L4 and L2/3 neurons. A–C, Control data only. A, Raincloud plots represent distributions of peak SF in binocular (left) and monocular (right) (purple represents contra-only; orange represents ipsi-only) dLGN boutons during contralateral- (top) and ipsilateral-eye (bottom) visual stimulation. Individual data points are plotted jittered to avoid overplotting. Black filled circles and lines represent mean ± SD. B, Distribution of peak SF in V1 L4 neurons. C, Distribution of peak SF in V1 L2/3 neurons. D, Mean peak SF (mean ± SEM of all sample) of binocular, contra-only, and ipsi-only dLGN boutons (left), V1 L4 neurons (middle), and V1 L2/3 neurons (right) in control mice. Linear mixed-effects models. Boutons versus L4 neurons: F = 2.37, p = 0.16; boutons versus L2/3 neurons: F = 10.96, p = 0.004; L4 versus L2/3 neurons: F = 0.06, p = 0.80; binocular versus contra-only boutons: F = 7.94, p = 0.004; binocular versus ipsi-only boutons: F = 1.98, p = 0.15; contra-only versus ipsi-only boutons: F = 2.29, p = 0.12; binocular versus contra-only L4 neurons: F = 11.83, p = 0.0006; binocular versus ipsi-only L4 neurons: F = 0.001, p = 0.96; contra-only versus ipsi-only L4 neurons: F = 13.46, p = 0.0002; binocular versus contra-only L2/3 neurons: F = 42.71, p = 9 × 10−11; binocular versus ipsi-only L2/3 neurons: F = 1.54, p = 0.21; contra-only versus ipsi-only L2/3 neurons: F = 38.33, p = 9 × 10−10. E, Mean peak SF (± SEM) of binocular, contra-only, and ipsi-only dLGN boutons and V1 L4, L2/3 neurons in MD mice (same convention as D). Linear mixed-effects models. Boutons versus L4 neurons: F = 4.26, p = 0.05; boutons versus L2/3 neurons: F = 130.63, p = 3 × 10−10; L4 versus L2/3 neurons: F = 58.09, p = 1 × 10−6; binocular versus contra-only boutons: F = 24.37, p = 8 × 10−7; binocular versus ipsi-only boutons: F = 8.61, p = 0.003; contra-only versus ipsi-only boutons: F = 4.90, p = 0.02; binocular versus contra-only L4 neurons: F = 3.91, p = 0.04; binocular versus ipsi-only L4 neurons: F = 0.50, p = 0.47; contra-only versus ipsi-only L4 neurons: F = 24.67, p = 9 × 10−7; binocular versus contra-only L2/3 neurons: F = 40.21, p = 3 × 10−10; binocular versus ipsi-only L2/3 neurons: F = 3.37, p = 0.06; contra-only versus ipsi-only L2/3 neurons: F = 52.40, p = 8 × 10−13. F, Violin and overlaid box plots of SF tuning bandwidth in binocular, contra-only, and ipsi-only dLGN boutons (left), V1 L4 neurons (middle), and V1 L2/3 neurons (right) in control versus MD mice. Linear mixed-effects models. Boutons: effect of MD: F = 2.65, p = 0.12; effect of eye group: F = 4.42, p = 0.01. Post hoc tests, binocular versus contra-only: F = 4.09, p = 0.04; binocular versus ipsi-only: F = 7.49, p = 0.006; contra-only versus ipsi-only: F = 4.02, p = 0.04. V1 L4 neurons: effect of MD: F = 1.04, p = 0.30; effect of eye group: F = 0.72, p = 0.48. V1 L2/3 neurons: effect of MD: F = 0.54, p = 0.47; effect of eye group: F = 0.42, p = 0.65. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. All panels: n = 2866 boutons in 5 control mice, 3503 boutons in 6 MD mice; 572 L4 neurons in 3 control mice, 565 L4 neurons in 2 MD mice; 1051 L2/3 neurons in 9 control mice, and 1355 L2/3 neurons in 6 MD mice. ns, Not significant at p > 0.1. †p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 6.
Figure 6.
In V1 L2/3 neurons, long-term critical-period MD leads to reductions in binocularity and spatial acuity. A, ODI histogram of V1 L2/3 neurons in control versus MD mice (mean ± SEM per field, n = 10 fields in 9 control mice, 15 fields in 6 MD mice). B, Percentage of visually responsive V1 L2/3 excitatory neurons that are ipsi-only, binocular, and contra-only per field in control versus MD mice (mean ± SEM per field). Linear mixed-effects model, effect of MD for ipsi-only: F = 0.78, p = 0.38; binocular: F = 8.09, p = 0.008; contra-only: F = 1.09, p = 0.30. C, Ipsi-only, binocular, and contra-only fractions of visually responsive V1 L2/3 neurons in control versus MD mice (χ2(2) = 17.42, p = 0.0001). D, Cumulative distribution of ODI values from V1 L2/3 neurons in control versus MD mice (−1 ODI: ipsi-only; 1: contra-only; Kolmogorov–Smirnov test, p = 0.01). E, Violin and overlaid box plots of response amplitude Rpref of binocular and monocular V1 L2/3 neurons in control versus MD mice. Linear mixed-effects model, effect of MD: F = 1.30, p = 0.27; binocular versus monocular: F = 59.43, p = 1 × 10−14. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. F, Mean probability distribution of peak SF in binocular (top) and monocular (bottom) V1 L2/3 neurons' contralateral- (left) and ipsilateral-eye (right) responses (mean ± SEM by animal values). There is a leftward shift of SF distribution curves in MD mice compared with controls. Mean values were fitted with a local regression smoothing function. G, Mean peak SF of V1 L2/3 neurons in control versus MD mice (mean ± SEM by animal values). Three-way ANOVA, effect of MD: F = 10.03, p = 0.002; binocular versus monocular: F = 6.53, p = 0.01; contra versus ipsi: F = 39.82, p = 6 × 10−8. Post hoc tests, effect of MD in binocular-contra: p = 0.01; binocular-ipsi: p = 0.45; monocular-contra: p = 0.01; monocular-ipsi: p = 0.14. H, Percentage of V1 L2/3 neurons with peak SF of 0.48–0.96 cpd (high-SF neurons; mean ± SEM by animal values). Three-way ANOVA, effect of MD: F = 20.79, p = 3 × 10−5; binocular versus monocular: F = 7.21, p = 0.009; contra versus ipsi: F = 57.40, p = 5 × 10−10. Post hoc tests, effect of MD in binocular-contra: p = 0.005; binocular-ipsi: p = 0.15; monocular-contra: p = 0.009; monocular-ipsi: p = 0.01. A–H, n = 1051 neurons in 9 control mice, 1355 neurons in 6 MD mice. I–K, The decrease in binocular fraction and acuity deficits following critical-period MD (C,G,H) is shown using a more liberal statistical criterion (p < 0.05) in determining visual responsiveness than the typical criterion used in this study (p < 0.01; see Materials and Methods). I, Same convention as in C. χ2(2) = 10.95, p = 0.004; 37% versus 31% binocular (control vs MD). J, Same convention as in G. Three-way ANOVA, effect of MD: F = 7.48, p = 0.008; binocular versus monocular: F = 8.03, p = 0.006; contra versus ipsi: F = 21.03, p = 2 × 10−5. Post hoc tests, effect of MD in binocular-contra: p = 0.02; binocular-ipsi: p = 0.08; monocular-contra: p = 0.11; monocular-ipsi: p = 0.55. K, Same convention as in H. Three-way ANOVA, effect of MD: F = 10.50, p = 0.002; binocular versus monocular: F = 7.87, p = 0.007; contra versus ipsi: F = 25.75, p = 5 × 10−6. Post hoc tests, effect of MD in binocular-contra: p = 0.04; binocular-ipsi: p = 0.01; monocular-contra: p = 0.05; monocular-ipsi: p = 0.44. I–K, n = 1332 neurons in 9 control mice, 1881 neurons in 6 MD mice. ns, Not significant at p > 0.1. †p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 7.
Figure 7.
In V1 L4 neurons, long-term critical-period MD leads to reduced binocularity but preserved spatial acuity. A, Example FOVs (summed projection) of bV1 L4 neurons, color-coded according to peak SF of neuron during contralateral- and ipsilateral-eye visual stimulation in control versus MD mice. Scale bar, 100 μm. B, ODI histogram of V1 L4 neurons in control versus MD mice (mean ± SEM per field, n = 10 fields in 3 control mice, 10 fields in 2 MD mice). C, Percentage of visually responsive V1 L4 excitatory neurons that are ipsi-only, binocular, and contra-only per field in control versus MD mice (mean ± SEM per field). Control, ipsi-only: 32.5 ± 3.3%; binocular: 7.1 ± 1.7%; contra-only: 60.4 ± 3.4%. MD, ipsi-only: 36.1 ± 2.9%; binocular: 3.6 ± 0.8%; contra-only: 60.2 ± 2.4%. Linear mixed-effects model, effect of MD for ipsi-only: F = 0.75, p = 0.39; binocular: F = 3.67, p = 0.06; contra-only: F = 0.002, p = 0.96. D, Ipsi-only, binocular, and contra-only fractions of visually responsive V1 L4 neurons in control versus MD mice (χ2(2) = 14.57, p = 0.0006). E, Violin and overlaid box plots of response amplitude Rpref of binocular and monocular V1 L4 neurons in control versus MD mice. Linear mixed-effects model, effect of MD: F = 0.08, p = 0.78; binocular versus monocular: F = 2.41, p = 0.12. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. F, Mean probability distribution of peak SF in binocular (top) and monocular (bottom) V1 L4 neurons' contralateral- (left) and ipsilateral-eye (right) responses (mean ± SEM by field values). Mean values were fitted with a local regression smoothing function. G, Mean peak SF of V1 L4 neurons in control versus MD mice (mean ± SEM by field values). Linear mixed-effects model, effect of MD: F = 0.70, p = 0.53; binocular versus monocular: F = 2.30, p = 0.13; contra versus ipsi: F = 16.41, p = 0.0001. H, Percentage of V1 L4 neurons with peak SF of 0.48–0.96 cpd (high-SF neurons; mean ± SEM by field values). Linear mixed-effect model, effect of MD: F = 1.48, p = 0.22; binocular versus monocular: F = 9.19, p = 0.003; contra versus ipsi: F = 14.60, p = 0.0002. B–H, n = 572 neurons in 3 control mice, 565 neurons in 2 MD mice. I–K, The decrease in binocular fraction and the lack of acuity deficits following critical-period MD (D,G,H) are shown using a more liberal statistical criterion (p < 0.05) in determining visual responsiveness than the typical criterion used in this study (p < 0.01; see Materials and Methods). I, Same convention as in D. χ2(2) = 9.91, p = 0.007; 13% versus 9% binocular (control vs MD). J, Same convention as G. Linear mixed-effects model, effect of MD: F = 1.42, p = 0.31; binocular versus monocular: F = 1.33, p = 0.25; contra versus ipsi: F = 15.67, p = 0.0001. K, Same convention as in H. Linear mixed-effect model, effect of MD: F = 5.43, p = 0.02; binocular versus monocular: F = 0.75, p = 0.38; contra versus ipsi: F = 10.84, p = 0.001. I–K, n = 1080 neurons in 3 control mice, 1163 neurons in 2 MD mice. ns, Not significant at p > 0.05. **p < 0.01, ***p < 0.001.
Figure 8.
Figure 8.
Binocular mismatch in thalamocortical boutons following long-term critical-period MD. A, Example tuning curves of binocular boutons in control (top row) and MD (bottom row) mice (3 examples each). Each pair of plots show SF (left) and orientation tuning (right) at SF indicated with arrowheads of a binocular bouton shown in inset. Scale bar, 2 μm. Purple represents contralateral-eye trials. Orange represents ipsilateral-eye trials. Solid lines indicate mean response amplitudes. Dotted lines indicate fitted curves based on mean values. Fits omitted if curve-fitting failed to merge. B, Scatter plot of response amplitudes of binocular dLGN boutons to preferred stimuli (Rpref) during contra- (y axis) versus ipsi-eye (x axis) viewing. Black crosses represent mean values. C, OD distribution of binocular boutons (mean ± SEM per field, n = 17 fields in 5 control mice, 20 fields in 6 MD mice). D, Violin and overlaid box plots of ODI values of binocular boutons in control versus MD mice (t test: p = 0.01). In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. E, Proportion plots of contra- versus ipsi-eye peak SF of binocular boutons in control versus MD mice. Unity (dotted line) represents perfect match. F, Violin and overlaid box plots of interocular difference in peak SF (contra-ipsi, in octaves) for binocular boutons (Wilcoxon rank sum test: p = 0.0008). G, Fractions of SF-matched, Contra-Acute (peak SF is greater in contra-eye response) or Ipsi-Acute (peak SF is greater in ipsi-eye response) binocular boutons in control versus MD mice (χ2(2) = 20.75, p = 3 × 10−5). H, Rain cloud plots represent distributions of preferred direction in orientation- or direction-selective binocular boutons in control versus MD mice. I, Scatter plots of preferred orientation of binocular boutons during contra- versus ipsi-eye viewing in control versus MD mice. J, Violin and overlaid box plots of interocular difference in preferred orientation for binocular boutons in control versus MD mice (Wilcoxon rank sum test: p = 0.0003). B–G, n = 171 binocular boutons in 5 control mice, 90 binocular boutons in 6 MD mice. H–J, n = 74 (control) and 48 (MD) OS/DS binocular boutons. *p < 0.05, ***p < 0.001, ****p < 0.0001.
Figure 9.
Figure 9.
Comparison of orientation/direction tuning properties of dLGN boutons versus V1 L2/3 neurons. A, B, Control data only. A, Raincloud plots represent distributions of global orientation selectivity index (gOSI) values in binocular, contra-only, and ipsi-only boutons (left) and V1 L2/3 neurons (right). V1 L2/3 neurons are more orientation-selective than dLGN boutons. Linear mixed-effects models. Boutons versus L2/3 neurons: F = 10.85, p = 2 × 10−5; boutons: binocular versus contra-only: F = 2.98, p = 0.08; binocular versus ipsi-only: F = 44.28, p = 7 × 10−11; contra-only versus ipsi-only: F = 15.25, p = 9 × 10−5. V1 L2/3 neurons: binocular versus contra-only: F = 23.98, p = 1 × 10−6; binocular versus ipsi-only: F = 9.91, p = 0.001; contra-only versus ipsi-only: F = 1.41, p = 0.23. B, Distributions of global direction selectivity index (gDSI) values in binocular, contra-only, and ipsi-only boutons and V1 L2/3 neurons (same convention as A). dLGN boutons are more direction-selective than V1 L2/3 neurons. Linear mixed-effects models: Boutons versus L2/3 neurons: F = 15.90, p = 1 × 10−7; boutons: binocular versus contra-only: F = 0.12, p = 0.72; binocular versus ipsi-only: F = 8.23, p = 0.004; contra-only versus ipsi-only: F = 34.46, p = 5 × 10−9. V1 L2/3 neurons: binocular versus contra-only: F = 23.53, p = 1 × 10−6; binocular versus ipsi-only: F = 6.42, p = 0.01; contra-only versus ipsi-only: F = 1.31, p = 0.25. C, Violin and overlaid box plots of gOSI in dLGN boutons (left) and V1 neurons (right) in control versus MD mice. Linear mixed-effects models. Boutons: effect of MD: F = 1.01, p = 0.33; binocular versus contra-only: F = 1.96, p = 0.16; binocular versus ipsi-only: F = 41.71, p = 1 × 10−10; contra-only versus ipsi-only: F = 43.59, p = 4 × 10−11. V1 L2/3 neurons: effect of MD: F = 0.95, p = 0.34; binocular versus contra-only: F = 32.54, p = 1 × 10−8; binocular versus ipsi-only: F = 11.44, p = 0.0007; contra-only versus ipsi-only: F = 2.81, p = 0.09. D, Violin and overlaid box plots of gDSI in dLGN boutons (left) and V1 neurons (right) in control versus MD mice. Linear mixed-effects models. Boutons: effect of MD: F = 1.37, p = 0.26; binocular versus contra-only: F = 0.0009, p = 0.97; binocular versus ipsi-only: F = 11.38, p = 0.0007; contra-only versus ipsi-only: F = 44.76, p = 2 × 10−11. V1 L2/3 neurons: effect of MD: F = 1.03, p = 0.32; binocular versus contra-only: F = 50.41, p = 1 × 10−12; binocular versus ipsi-only: F = 3.38, p = 0.06; contra-only versus ipsi-only: F = 14.25, p = 0.0001. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. All panels: n = 2866 boutons in 5 control mice, 3503 boutons in 6 MD mice; 1051 V1 L2/3 neurons in 9 control mice, 1355 L2/3 neurons in 6 MD mice. ns, Not significant at p > 0.1. †p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 10.
Figure 10.
Long-term critical-period MD impairs binocular facilitation of thalamocortical boutons. A, Illustration of three modes of binocular modulation of visual responses: both only, remaining responsive, and suppressed. Broken lines indicate noise floor. B, Example FOVs (summed projection) of dLGN boutons imaged in bV1, color-coded according to eye group, including both-only in control versus MD mice. Scale bar, 10 μm. C, Example Ca2+ signals in a binocular (top), contra-only and ipsi-only (middle), and both-only (bottom) boutons in response to drifting gratings presented to contra-eye, ipsi-eye, and both eyes. Black represents mean trace. Gray represents mean ± SEM of 8 repeats. Colored bars represent time of stimulus presentation. Scale bar, 2 μm. Responses to 8 orientations at peak SF are shown. D, Fractions of dLGN boutons according to mode of binocular modulation in control versus MD mice (χ2(2) = 119.40, p = 2 × 10−16). E, Percentage of boutons that are both-only, remaining responsive, suppressed per field in control versus MD mice (mean ± SEM per field). Control, both-only: 29.6 ± 1.6%; remaining responsive: 15.8 ± 3.4%; suppressed: 54.5 ± 4.1%. MD, both-only: 33.9 ± 1.8%; remaining responsive: 5.6 ± 1.5%; suppressed: 60.4 ± 2.3%. t tests, effect of MD for both-only: p = 0.10; remaining responsive: p = 0.01; suppressed: p = 0.22; n = 12 fields in 5 control mice, 9 fields in 5 MD mice. F, Fractions of visually responsive dLGN boutons according to eye group in control versus MD mice (χ2(3) = 57.23, p = 2 × 10−12). G, Percentages of boutons in each eye group (ipsi-only, binocular, and contra-only, both-only) in control versus MD mice (mean ± SEM per field). t tests, effect of MD for ipsi-only: p = 0.32; binocular: p = 0.02; contra-only: p = 0.10; both-only: p = 0.10. H, Response amplitude Rpref of boutons under different viewing conditions (mean ± SEM of all sample). Linear mixed-effects models. Control: contra-eye versus both-eye viewing in binocular boutons: F = 17.07, p = 5 × 10−5; monocular versus both-eye viewing in contra-only boutons: F = 79.16, p = 2 × 10−16; in ipsi-only boutons: F = 53.39, p = 7 × 10−13; MD: contra-eye versus both-eye viewing in binocular boutons: F = 0.003, p = 0.96; monocular versus both-eye viewing in contra-only boutons: F = 77.93, p = 2 × 10−16; in ipsi-only boutons: F = 75.43, p = 2 × 10−16. Numbers inside bars indicate numbers of boutons. I, Violin and overlaid box plots of response amplitude Rpref of boutons during contra-eye, ipsi-eye, and both-eye viewing conditions, normalized to contra-eye viewing median values (medians indicated by numbers below violin plots). Linear mixed-effects models, contra-eye versus both-eye viewing in controls: F = 4.59, p = 0.03; in MD mice: F = 1.06, p = 0.30. In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. D–I, n = 2784 boutons in 5 control mice, 2443 boutons in 5 MD mice. ns, Not significant at p > 0.05. *p < 0.05, ***p < 0.001, ****p < 0.0001.
Figure 11.
Figure 11.
Long-term MD-induced impairment of binocular modulation affects binocular and monocular thalamocortical boutons. A, B, Normalized Ca2+ signals from two example dLGN boutons from control (A) and MD (B) mice. Binocular dLGN boutons (left) and contra-only monocular boutons (right), as well as their respective bouton images and orientation tuning curves. Traces are in response to drifting gratings presented to contra-eye, ipsi-eye, and both eyes. Black represents mean trace. Gray represents mean ± SEM of 8 repeats. Colored bars represent time of stimulus presentation. Scale bar, 2 μm. Numbers above tuning curves indicate binocular modulation values (binocular Rpref/dominant-eye monocular Rpref). C, Binocular, contra-only, and ipsi-only dLGN boutons are shown rank-ordered according to binocular modulation. Both binocular facilitation and suppression exist in control (left) and MD (right) mice. D, Binocular modulation of visual responses for binocular, contra-only, and ipsi-only boutons in control versus MD mice (mean ± SEM of all sample; numbers inside bars indicate number of boutons; t tests against 1 for binocular control: p = 0.08; contra-only control: p = 2 × 10−8; ipsi-only control: p = 0.002; binocular MD: p = 0.18; contra-only MD: p = 0.34; ipsi-only MD: p = 0.07. t tests between control versus MD for binocular: p = 0.03; contra-only: p = 0.002; ipsi-only: p = 0.16. E, Scatter plots and linear regression of binocular modulation as a function of dominant-eye monocular Rpref for binocular, contra-only, and ipsi-only boutons in control (gray) and MD (red) mice. F, Scatter plots and linear regression of binocular modulation as a function of absolute OD for binocular boutons in control (gray) and MD (red) mice. C–F, Broken lines indicate no binocular modulation. E, F, Numbers on top right indicate estimated slope (symbols indicate p values). Solid colored lines indicate linear regression fits. Gray represents control. Red represents MD. C–F, n = 496 boutons in 5 control mice, 186 boutons in 4 MD mice. ns, Not significant at p > 0.1. †p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 12.
Figure 12.
No evident structural loss of thalamocortical connectivity following long-term critical-period MD. A, Heatmaps showing spatial distribution of dLGN neurons labeled following GCaMP6s virus injection in control (left) and MD (right) mice. Of 10 mice included in this dataset, 6 were part of functional dataset obtained using in vivo two-photon calcium imaging. Heatmaps are based on summed cell counts across all sections and mice. B, Left, Example fluorescence images (maximal projection of confocal z stacks) of V1 coronal sections showing dLGN axon labeling in control and MD mice. Scale bar, 100 μm. Sections were immunostained for GFP. Right, Semiautomatically traced axons in L1–2/3 in control and MD mice. Scale bar, 50 μm. Images in B are from the same mice shown in Figure 3A. C, Number of dLGN neurons labeled in control versus MD mice (mean ± SEM by animal; t test: p = 0.19). D, Mean fluorescence intensity of labeling in V1 sections from control versus MD mice, shown for all layers, L2/3 only, and L4 only (mean ± SEM by animal; two-way ANOVA, effect of MD: F = 1.95, p = 0.18, effect of layer: F = 31.14, p = 2 × 10−7). E, Traced axon length per volume (μm per μm3) across binned cortical depths in V1 L1–2/3 in control versus MD mice (mean ± SEM by section; linear mixed-effects model, effect of MD: F = 0.04, p = 0.84, effect of cortical depth: F = 65.64, p = 8 × 10−13). F, Violin and overlaid box plots represent distribution of traced axon radius in V1 L1–2/3 in control versus MD mice (linear mixed-effects model, effect of MD: F = 0.35, p = 0.57). In box plots, middle mark indicates the median, and bottom and top edges indicate 25th and 75th percentiles, respectively. All panels, n = 5 control and 5 MD mice, 3 sections per animal.
Figure 13.
Figure 13.
Schematic model of abnormal binocular integration in mouse dLGN following long-term critical-period MD. Summary of main findings. In normal mice, binocular dLGN neurons relay binocularly matched visual inputs to V1. Monocular inputs sum to give rise to larger responses during binocular viewing (binocular facilitation, indicated by “+b” and binocular modulation values of > 1). Monocular dLGN neurons also display binocular facilitation because the input from the nondominant eye, albeit subthreshold, acts in synergy with the dominant-eye input. In MD mice, the percentage of binocular dLGN neurons is reduced, and surviving binocular neurons relay mismatched visual information to V1. MD mice lack binocular facilitation of binocular and monocular dLGN neurons, potentially due to the mismatch in suprathreshold and subthreshold visual inputs. In the case of binocular neurons, binocular viewing leads to even lower activity levels compared with monocular viewing through the dominant eye (binocular suppression, indicated by “−b” and binocular modulation values < 1). For simplicity, the model does not depict other modes of binocular modulation, such as binocular activation and complete suppression (“both only” and “suppressed” in Fig. 10).
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