1. Neuroscience
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Homeostatic regulation of perisynaptic matrix metalloproteinase 9 (MMP9) activity in the amblyopic visual cortex

  1. Sachiko Murase  Is a corresponding author
  2. Dan Winkowski
  3. Ji Liu
  4. Patrick O Kanold
  5. Elizabeth M Quinlan  Is a corresponding author
  1. University of Maryland, United States
Research Advance
Cite this article as: eLife 2019;8:e52503 doi: 10.7554/eLife.52503
5 figures and 1 additional file

Figures

Parallel increase in MMP2/9 activity following LRx at thalamo-cortical synapses in deprived and non-deprived V1b.

(A) Top: Experimental timeline. Subjects received cMD from eye opening (postnatal day 14, (P14) until adulthood (>P90). 10 days of DE was followed by 4 hr of LRx. MMP2/9 biomarker (4 μl of 2 mg/ml Dye-quenched gelatin) was delivered i.c. 24 hr prior to 4 hr of LRx. Middle left: Coronal section with DAPI nuclear staining. Layer 4 of binocular region of V1 indicated by red box. Middle right: representative images of MMP2/9 biomarker fluorescence in deprived (dep) and non-deprived (non) V1b in cMD (left) and cMD+LRx subjects (LRx, right). Bottom: Quantification of biomarker puncta reveals no change in puncta size (left), but a parallel and significant increase in puncta density (middle) and fluorescent intensity (right) in dep and non V1b following LRx. One-way ANOVAs, size F(3, 19)=0.52, p=0.67; density F(3, 19)=6.7, p=0.003; intensity F(3, 19)=8.4, p=0.0009; n = 6, 5, 6, 6 subjects for cMD dep, cMD non, LRx dep, LRx non, respectively; *p<0.05, Tukey-Kramer post hoc test. (B) Representative images of MMP2/9 biomarker fluorescence (MMP; green) and marker for thalamic axons (VG2; magenta) in deprived visual cortex in cMD and cMD+LRx subjects. A parallel and significant increase in colocalization of biomarker puncta with VGluT2 following LRx in dep and non V1b. One-way ANOVA, F(3, 19)=9.5, p=0.0005; n = 6, 5, 6, 6 subjects for cMD dep, cMD non, LRx dep, LRx non, respectively; *p<0.05, Tukey-Kramer post hoc test. Co-localization with VGluT2 is lost following 2 μm shift of biomarker image (shift). (C) Representative images of MMP2/9 biomarker (green), VGluT2 (magenta) and parvalbumin fluorescence (PV; blue) in deprived and non-deprived cMD and cMD+LRx subjects. Significant increase in co-localization of biomarker puncta with VGluT2 at PV+ and PV-locations of dep and non V1b following LRx. One-way ANOVAs, PV+ F(3, 12)=17.33, p=0.00012; PV- F(3, 12)=56.17, p<0.0001; n = 4, 4, 4, 4 subjects for cMD dep, cMD non, LRx dep, LRx non, respectively; *p<0.05, Tukey-Kramer post hoc test. (D) Left: Representative immunoblots for active MMP9 (95 kDa), and β-actin from dep and non V1b. MMP9 level is normalized to β-actin and reported as % of cMD non. Right: Quantification of immunoblots reveals a parallel and significant increase in active MMP9 in dep and non V1b following 2 hr of LRx. One-way ANOVA, F(3, 26)=5.6, p=0.004; n = 7, 7, 8, 8 subjects for cMD dep, cMD non, LRx dep, LRx non, respectively: *p<0.05, Tukey-Kramer post hoc test.

Figure 2 with 1 supplement
Parallel decrease in ECM integrity following LRx in deprived and non-deprived V1b.

(A) Representative triple labeled fluorescent micrographs of Wisteria floribunda agglutinin (WFA)-FITC staining (cyan), immunostaining for aggrecan (Agg; yellow) and parvalbumin (PV; magenta) in deprived (dep) and non-deprived (non) V1b in cMD (left) and cMD+LRx subjects (LRx, right). Roman numerals indicate cortical layer. WM = white matter. Inset: High magnification images of triple labeled PV+ interneurons (100X). (B) Fluorescence intensity profiles (mean ± SEM) along vertical depth of V1b. cMD dep (dark blue), cMD non (light blue), LRx dep (dark red), LRx non (red). (C) A parallel and significant decrease in WFA and Agg mean fluorescence intensity 250–400 μm from surface in dep and non V1b following LRx. One-way ANOVAs, WFA F(3, 26)=32, p=0.0001; Agg F(3, 26)=10, p=0.0001; PV F(3, 26)=0.4, p=0.75; n = 7, 7, 8, eight subjects for cMD dep, cMD non, LRx dep, LRx non, respectively; *p<0.05, Tukey-Kramer post hoc test. (D) LRx induces a significant decrease in WFA and Agg fluorescence intensity at PV+ and PV-locations in dep and non V1b. One-way ANOVAs, WFA PV+, F(3, 309)=40.3, p<0.0001; WFA PV-, F(3, 309)=30.1, p<0.0001; Agg PV+, F(3, 309)=29.4, p<0.0001; Agg PV-, F(3, 309)=18.7, p<0.0001; n (subjects, ROIs)=(5, 77 , 5, 73 , 5, 81 , 5, 82), for cMD dep, cMD non, LRx dep, LRx non, respectively; *p<0.05, Tukey-Kramer post hoc test.

Figure 2—figure supplement 1
Parallel decrease in ECM integrity following LRx in deprived and non-deprived V1.

Representative double labeled fluorescent micrographs with Wisteria floribunda agglutinin (WFA)-FITC staining (cyan) and immunostaining for parvalbumin (PV; magenta) in deprived (dep) and non-deprived (non) V1b in cMD (cMD, left) and cMD+LRx subjects (LRx, right). Roman numerals indicate cortical layer. WM = white matter. Inset: High magnification images of double labeled PV+ interneurons (100X).

LRx limited to deprived eye is sufficient to induce perisynaptic MMP2/9 activity at thalamo-cortical synapses in deprived V1b.

(A) Top: Experimental timeline. A light-occluding eye patch was attached to the non-deprived eye before DE. Bottom left: Representative images of MMP2/9 biomarker fluorescence in layer 4 of chronically deprived (dep, contralateral to cMD, ipsilateral to eye patch) and non-deprived (non, ipsilateral to cMD, contralateral to eye patch) V1b of LRx subject. Quantification of biomarker puncta reveals a significant increase in density and intensity in dep vs non V1b following LRx to amblyopic eye; n = 6, six subjects for LRx dep, LRx non, respectively; *p<0.05, Student’s T-test. (B) Representative images of MMP2/9 biomarker fluorescence (MMP, green) and VGluT2 immunoreactivity (VG2, magenta) in dep and non V1b of LRx subject. A significant increase in biomarker colocalization with VGluT2 in dep vs non V1b following LRx to amblyopic eye; n = 6, six subjects for LRx dep, LRx non, respectively; *p<0.05, Student’s T-test. Co-localization with VGluT2 is reduced following 2 μm shift of biomarker image (shift). (C) Representative images of MMP2/9 biomarker (green), VGluT2 (magenta) and parvalbumin fluorescence (PV, blue) in dep and non V1b following LRx. A significant increase in co-localization of MMP2/9 biomarker puncta with VGluT2 at PV+ and PV- immunoreactive locations of dep vs non V1b; n = 4; *p<0.05, Student’s T-test.

Figure 4 with 1 supplement
LRx limited to deprived eye is sufficient to decrease ECM integrity in deprived V1b.

(A) Representative triple labeled fluorescent micrographs of WFA-FITC staining (cyan), immunostaining for aggrecan (Agg; yellow) and parvalbumin (PV; magenta) of deprived (dep, contralateral to cMD, ipsilateral to eye patch) and non-deprived (non, ipsilateral to cMD, contralateral to eye patch) V1b after LRx to amblyopic eye. Roman numerals indicate cortical layer. WM = white matter. Inset: High magnification images of triple labeled PV+ interneurons (100X). (B) Fluorescence intensity profiles (mean ± SEM) along vertical depth of V1b. Dep LRx (dark red), non LRx (blue). (C) A significant decrease in WFA and Agg mean fluorescence intensity 250–400 μm from surface in dep V1b; n = 5; *p<0.05, Student’s T-test. (D) LRx-induced a significant decrease in WFA and Agg fluorescence intensity at PV+ and PV-locations in dep V1b; n (subjects, ROIs)=(5, 73 , 5 78), for LRx dep, LRx non, respectively; *p<0.05, Student’s T-test.

Figure 4—figure supplement 1
LRx limited to deprived eye is sufficient to decrease ECM integrity in deprived V1b.

Representative double labeled fluorescent micrographs with WFA-FITC staining (cyan) and parvalbumin (PV; magenta) of deprived (dep, contralateral to cMD, ipsilateral to eye patch) and non-deprived (non, ipsilateral to cMD, contralateral to eye patch) V1b, after LRx to amblyopic eye. Roman numerals indicate cortical layer. WM = white matter. Inset: High magnification images of triple labeled PV+ interneurons (100X).

DE lowers the threshold for light-induced activation of MMP2/9.

(A) Dark chamber with an imaging window allows maintenance of visual deprivation during two photon live imaging of MMP2/9 biomarker. Left drawing: Top view of a subject wearing a custom aluminum headpost (1 cm diameter) magnetically held to an o-ring in the blackout ceiling of the dark camber (inset; 3 mm diameter magnets APEX magnets; magnetic field generation around V1,<20 gauss). The headpost is secured to a stereotax. The cannula for biomarker delivery is adjacent to theimaging window margin. Right drawing: Side view of a subject in the dark chamber. The headpost is magnetically attached to the o-ring opening of the blackout ceiling (magnet locations, yellow arrows). (B) In vitro emission spectrum of MMP2/9 biomarker A580 (2 ng/ml) incubated with activated rat recombinant MMP9 (rrMMP9, 100 ng). (C) Inset: Experimental timeline. Adult (>P90) WT mice received AAV-CaMKII-GFP~2 weeks before 10 d of DE. Biomarker was delivered 24 hr before imaging. Subjects received 40 s of light stimulation (1 Hz flash of 470 nm LED at 0 or 300 cd/m2). Left: Representative images of GFP (green) and biomarker (MMP, magenta) signals in V1b 10 s prior or 40 s after light stimulation at 0 or 300 cd/m2 in a DE subject. Right: Time course of raw fluorescent intensities (pixel) and ΔF/F of MMP biomarker (top) and co-localized GFP (bottom) within the single ROI denoted by yellow circle, from 10 s before (−10) to 40 s after (+40) light stimulation of 0 or 300 cd/m2 in a DE subject. (D) Summary data: Time course of ΔF/F of MMP biomarker from −10 s to +40 s of light stimulation of 0 or 300 cd/m2 in DE subjects. ΔF/F of MMP biomarker was stable in absence of visual stimulation (0 cd/m2) and increased over time in response to 300 cd/m2 light stimulation (mean ± SEM; Repeated measure ANOVA, F(1, 22), *p<0.001; n = 12 puncta from three subjects each). (E) Biomarker ΔF/F +40 s relative to 0 s as a function of DE (0, hr, 18 hr or 10 d) and light intensity (0, 300, or 150,000 cd/m2). Moderate intensity light did not induce a change in biomarker fluorescence in the absence of DE (blue line, p=0.49, Student’s T-test; n = 11, 10 puncta for 0 and 300 cd/m2, respectively). Following 18 hr of dark adaptation, a significant increase in biomarker fluorescence was observed in response to high, but not moderate intensity light (black line, One-way ANOVA, F(2, 45)=11.5, p<0.0001; n = 12, 12, 24 puncta for 0, 300, 150,000 cd/m2, respectively, *p<0.05, Tukey-Kramer post hoc test). Following 10 d DE, moderate and high intensity light significantly increased biomarker fluorescence (solid red line, One-way ANOVA, F(2, 40)=6.3, p=0.0042; n = 12, 12, 19 puncta for 0, 300, 150,000 cd/m2, respectively, *p<0.05, Tukey-Kramer post hoc test). The increase in biomarker fluorescence by 150,000 cd/m2 stimulation to 10 d DE subjects was inhibited by an MMP9 inhibitor delivered 24 hr before visual stimulation (MMP9i; 5 nM delivered i.c. 24 hr prior to LRx, dashed red line, p=0.13 Student’s T-test; n = 13 puncta for 0 and 150,000 cd/m2).

Data availability

All data generated/analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5.

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