Journal: International Journal of Molecular Sciences

Article Title: Chronic Monocular Deprivation Reveals MMP9-Dependent and -Independent Aspects of Murine Visual System Plasticity

doi: 10.3390/ijms23052438

Figure Lengend Snippet: cMD decreases the strength of the deprived eye pathway in WT but not MMP9 −/− mice. ( A ) Left: Experimental timeline. MD was initiated at eye opening (postnatal day 14 (P14)) and maintained until adulthood (>P90). Right: GCaMP6s expression was targeted to layer 2/3 neurons of WT V1b (AAV1.hSyn1.mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40, Addgene, titer: 1.3 × 10 13 U/mL, 30 nl, AP: 1.0 mm, MD: −3.0 mm, DV: 0.3 mm; at least 3 weeks prior to imaging) to monitor visual evoked calcium transients. ( B ) Representative ΔF/F of the GCaMP6s signal evoked in layer 2/3 of V1b in normal reared (NR) and cMD WTs in response to the presentation of drifting square wave gratings (0.05 cycle/degree, 100% contrast, 28 cd/m 2 at 12 orientations) to contra- and ipsilateral eyes. Blue bar = stimulus onset. Individual trials in grey, average of 5 repeats in black. Ocular dominance score (ODS) = (C − I)/(C + I) was calculated at the preferred orientation for each neuron. ( C ) Examples of two-photon field views of GCaMP6s expression. ( D ) Cumulative distribution of ODS. * p = 1.0 × 10 −4 , KS test. ( E ) Mean CBI of layer 2/3 neurons is significantly lower in cMD than NR WT mice. * p = 0.012, Mann−Whitney test. n = 5 subjects. ( F ) Cumulative distribution of contra and ipsi eye neuronal responses (ΔF/F; * p = 8.3 × 10 −29 for contra, p = 0.91 for ipsi, Student’s t -test, 365 and 201 neurons for NR and cMD, respectively).

Article Snippet: Image acquisition was restricted to 150 to 250 μm from the brain surface to focus on neuronal cell bodies in layer 2/3 in the binocular region of the primary visual cortex (V1b) following targeted delivery of GCaMP6s (AAV1.hSyn1. mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40, Addgene, titer: 1.3 × 10 13 U/mL, 30 nl, A).

Techniques: Expressing, Imaging, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Chronic Monocular Deprivation Reveals MMP9-Dependent and -Independent Aspects of Murine Visual System Plasticity

doi: 10.3390/ijms23052438

Figure Lengend Snippet: Ocular dominance in MMP9 −/− mice is normal and resistant to cMD. ( A ) Left: Experimental timeline. Right: GCaMP6s expression was targeted to layer 2/3 neurons of MMP9 −/− V1b. ( B ) Representative ΔF/F of GCaMP6 signal evoked in layer 2/3 of V1b in normal reared (NR) and cMD MMP9 −/− mice in response to presentation of drifting square wave gratings (0.05 cycle/degree, 1 Hz, 100% contrast, 28 cd/m 2 at 12 orientations) to contra- and ipsilateral eyes. Red bar = stimulus onset. Individual trials in grey, average of 5 repeats in black. Ocular dominance score (OD score) = (C − I)/(C + I) was calculated at the preferred orientation for each neuron. ( C ) Examples of two-photon field views of GCaMP6s expression. ( D ) Cumulative distribution of ODS ( p = 0.42, KS test). ( E ) Mean CBI of layer 2/3 neurons is similar in cMD and NR MMP9 −/− mice. p = 0.38, Mann−Whitney test. n = 6 subjects. ( F ) Cumulative distribution of contra and ipsi eye visual responses (ΔF/F; p = 0.75 for contra, p = 0.63 for ipsi, Student’s t -test, 328 and 595 neurons for NR and cMD, respectively).

Article Snippet: Image acquisition was restricted to 150 to 250 μm from the brain surface to focus on neuronal cell bodies in layer 2/3 in the binocular region of the primary visual cortex (V1b) following targeted delivery of GCaMP6s (AAV1.hSyn1. mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40, Addgene, titer: 1.3 × 10 13 U/mL, 30 nl, A).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Chronic Monocular Deprivation Reveals MMP9-Dependent and -Independent Aspects of Murine Visual System Plasticity

doi: 10.3390/ijms23052438

Figure Lengend Snippet: Stability of GCaMP6s expression and visually responsive neurons over experimental conditions. ( A ) Left: representative neuron from layer 2/3 of V1b expressing GCaMP6s. Right: Quantification of fluorescence along dashed white line reveals a high signal in the cytoplasm relative to the nucleus. ( B ) Top: No difference in the total number of GCaMP6s expressing neurons between WT and MMP9 −/− , normal reared (NR) and cMD subjects (one-way ANOVA, F = 3.0, p = 0.06, n = 5 and 6 subjects for WT and MMP9 −/− mice, respectively). Bottom: Percent of GCaMP6s expressing neurons that are visually-responsive (one-way ANOVA, F = 0.96, p = 0.43). A neuron is defined as visually responsive if the mean ΔF value in response to a visual stimulus of any direction exceeds 3 × STD of baseline (F 0 ) for > 40% of trials during either contra or ipsi eye stimulation. ( C ) Coefficient of variance (CV) of calcium transients (standard deviation over mean ΔF/F (STD/mean) is comparable across all experimental conditions, and is unaffected by the deletion of MMP9 or cMD. Box plots represent the median as a bar, 25th to 75th percentile as box, and max and min as whiskers (WT: one-way ANOVA, F = 7.1, p = 0.10, n = 359, 364, 201, and 194 neurons for NR contra, NR ipsi, cMD deprived, and cMD non-deprived, respectively; MMP9 −/− : one-way ANOVA, F = 4.3, p = 0.11, n = 315, 321, 595, and 595 neurons for NR contra, NR ipsi, cMD deprived, and cMD non-deprived, respectively).

Article Snippet: Image acquisition was restricted to 150 to 250 μm from the brain surface to focus on neuronal cell bodies in layer 2/3 in the binocular region of the primary visual cortex (V1b) following targeted delivery of GCaMP6s (AAV1.hSyn1. mRuby2.GSG.P2A.GCaMP6s.WPRE.SV40, Addgene, titer: 1.3 × 10 13 U/mL, 30 nl, A).

Techniques: Expressing, Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, examples of averaged baseline & stimulus-related raw iOS images, evoked by one train of whisker deflections. Right, example barrel map overlayed over a brightfield image of the blood vessels. Green dots represent the location of GCaMP6s-expressing cells in the C2 barrel column. (B) Average 2PLSM image of GCaMP6s-expressing neurons. (C) Experimental design: the PW, which corresponds to the barrel column containing the GCaMP6s-expressing cells, is always used to read out the sensory stimulus-evoked response. The PW for PRWS (blue) or a control far-away whisker (CW) for CRWS (orange) is stimulated during rhythmic whisker stimulation (RWS, 8Hz, 10min). (D) Experimental protocol: the PW is stimulated at 0.1 Hz for 10 min pre- and post-RWS. RWS (8 Hz, 10 min) is performed on either the PW (PRWS, blue) or a far-away CW (CRWS, orange). Whisker movement index during the stimulus protocol can be found in - . (E & F) Left, example trace of the GCaMP6s fluorescence, in response to PW stimulation (0.1 Hz, 10 min) pre- and post-PRWS ( E ) or CRWS (F) . The signals in E are from the cell circled in B . Right, the PW-evoked response strength (RS, amplitude X whisker-evoked signal probability, (ΔF/F 0 )/Nstim) pre- and post-PRWS ( E , n=1099 cells, **P=0.002, N=11 mice, P=0.5, full descriptive statistics can be found in Table 1-1) or CRWS ( F , n=829 cells, P=0.4; N=11 mice, P=0.6). Grey lines, paired responses. Violin plots depict median (solid) and quartiles (dotted) bars. Squares, the mean over cells (±SEM). Circles, the mean over mice (±SEM). (G) Pre- versus post-RWS RS ((ΔF/F 0 )/Nstim) with the simple linear regression for PRWS (blue, n=1099 cells) and CRWS (orange, n=829 cells). Comparing slopes (PRWS=0.62±0.014, CRWS=0.85±0.019, F=92.9, DFn=1, DFd=1924, ****P<0.0001). (H) Frequency distribution of the pre-RWS RS for PRWS & CRWS, bin size 0.01(ΔF/F 0 )/Nstim. High responders (resp) were identified as outliers (dots above, Iterative Grubb’s outlier test, α=0.0001). (I & J) Violin plot of the RS pre- and post-PRWS and CRWS, for low & moderate responders (I , PRWS, n=1058 cells, ****P<0.0001; N=11 mice, P=0.006; CRWS, n=792 cells, P=0.3; N=11 mice, P=0.7), and for high responders ( J , PRWS, paired t-test, n=41 cells, ***P=0.0008; N=11 mice, P=0.04; CRWS, n=37 cells, ****P<0.0001; N=11 mice, P=0.0001). (K) The pre-RWS/post-RWS ratio (in %) for PRWS and CRWS low & moderate, and high responders (mixed effects model, N=11 mice, P=0.0004; multiple comparisons: PRWS low & moderate vs high, ***P=0.0004; CRWS low & moderate vs high, P=0.1; Low & moderate PRWS vs CRWS, *P=0.018; High PRWS vs CRWS, P=0.7).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Whisker Assay, Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, example trace of GCaMP6s fluorescence from a neuron showing persistent PW-evoked responses (0.1 Hz, 10 min) pre- and post-PRWS. Right, violin and pairwise representation of pre- and post-PRWS (n=465 cells, ****P<0.0001; N=11 mice, P=0.003) or CRWS (Paired t test, n=279 cells, P=0.06; N=11 mice, P=0.2). (B&C) Left, example trace of GCaMP6s fluorescence from neurons of which responses were recruited (B) or suppressed (C) post-PRWS. (B) Right, the mean PW-evoked response strength (RS) of recruited neurons, post-PRWS (n=307 cells) & CRWS (Unpaired t test, n=131 cells, P=0.1; N=11 mice, P=0.5). (C) Right, the mean response strength of suppressed neurons pre-PRWS (n=205 cells) & CRWS (Unpaired t test, n=258 cells, ****P<0.0001; N=11 mice P=0.037). (D) Pie charts with the percentages (%) of persistent (grey), recruited (red), suppressed (blue), no response (white), and high (pink) responders for PRWS (n=1099 cells) & CRWS (n=829 cells, Chi-square=19.8, DF=4, ***P<0.0001).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Raster plot of GCaMP6s fluorescence intensity (ΔF/F 0 ) for PRWS at each acquisition for pre-PRWS (-10 min) and post-PRWS (10, 60, 120, & 180 min). Neurons sorted from top to bottom by decreasing response strength pre- vs post-PRWS (n=410 cells). Arrowheads, examples of the five subpopulations: persistent (persist.), recruited (recruit.), suppressed (suppr.), no response (no resp.), and high (hi resp.) responders. (B) Top, example 2PLSM images of neurons expressing AAV1-hSyn-mRubyGSG-P2A-GCaMP6s across the longitudinal experimental protocol -10 min pre-PRWS, & 10, 60, 120, & 180 min post-PRWS. mRuby (red) serves as an activity-independent marker, whereas GCaMP6s (green) reports Ca 2+ signals upon PW-stimulation. The lower images represent high magnifications of the cells in the square inset on top. (B) Bottom, PW-evoked RS pre-PRWS (-10 min) or CRWS and post-PRWS or CRWS (10, 60, 120, & 180 min) (PRWS n=382 cells, or CRWS n=304 cells; Two-way RM ANOVA, ***P=0.0006; N=6 mice, P=0.026). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10 ****P<0.0001; or 60 ***P=0.0001). (C) PW-evoked Ca 2+ signal probability (P S [#events/Nstim]) (PRWS n=382 cells, CRWS n=304 cells; Two-way RM ANOVA, ****P<0.0001; N=6 mice, P=0.026). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10****P<0.0001; or 60 **P=0.001; or 120 P=0.7; or 180 P=0.99) & CRWS (-10 min vs, 10 P=0.99; or 60, P=0.3; or 120 P=0.2; or 180 P=0.3). (D) PW-evoked Ca 2+ signal amplitudes (Ā S [ΔF/F 0 ]) (PRWS n=382 cells, CRWS n=304 cells; Two-way RM ANOVA, ****P<0.0001; N=6, P=0.25). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10 *P=0.01; or 60 **P=0.002; or 120 P=0.8; or 180 P=0.4) & CRWS (-10min vs, 10 P=0.2; or 60 P=0.6; or 120 P=0.1; or 180 P=0.5). (E) PW-evoked response strength, pre- and 24 hrs post-PRWS or CRWS (PRWS n=162 cells, CRWS n=134 cells; Two-way RM ANOVA, P=0.36; PRWS 3 mice, CRWS 2 mice, P=0.054).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence, Expressing, Activity Assay, Marker

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A, B, Left) Example traces of GCaMP6s (black) or mRuby fluorescence (red) for a 20-sec baseline before and during PRWS or CRWS. Violin and pairwise representation of fluorescence intensities (integrated over 20s) (violin plot median: white bar, quartiles: dotted bars) baseline vs PRWS (n=290 cells, Paired t test, ****P<0.0001; N=3 mice, P=0.027) or CRWS (n=115 cells, Paired t test, P=0.6; N=3 mice, P=0.8). (C) Fluorescence intensity (integrated over 20s) during PRWS vs PW-evoked response strength change (post/pre), (n=115 cells, Pearson r correlation, r=0.0002, P=1.0). Inset, pre- and post-PRWS response strength (n=115 cells, pre=0.027±0.002, post=0.031±0.002, Paired t test, P=0.027). (D) Fluorescence intensity during PRWS (integrated over 20s) vs pre-PRWS. Pink line simple linear regression, black dotted lines 95% confidence intervals. (n=115 cells, Pearson r correlation, r=-0.35, ***P=0.0001; simple linear regression, slope=-0.008, non-zero? P=0.0001).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, Example 2PLSM image of flex.mRuby.GCaMP6s-expressing VIP interneurons in the VIP-Cre mouse line. Right, representative confocal image after post-hoc anti-VIP immunocytochemistry on slices of barrel cortex from 2PLSM imaged VIP-Cre mice (green, anti-VIP; red, AAV1.CAG.Flex.mRuby.P2A.GCaMP6s; blue, Hoechst staining). (B) Pre-RWS PW-evoked response strength (RS) of VIP interneurons (n=341 cells, N=7 mice), and low & moderate (n=1058 cells, N=11 mice) and high (n=41, N=11 mice, one-way ANOVA, ****P<0.0001) responding L2/3 neurons. Squares and circles represent the means ± SEM over cells and mice, respectively. (C) Left, example trace of GCaMP6s (black) or mRuby fluorescence from a VIP interneuron, pre- and post-PRWS. Right, pre- and post-PRWS PW-evoked RS of VIP neurons (Paired t test, n=341 cells, P=0.2; N=7 mice, P=0.45). Grey lines, paired responses. Violin plots depict median (solid) and quartiles (dotted) bars. (D, E), Left, example trace of GCaMP6s fluorescence (black) or mRuby (integrated over 20s) from a VIP interneuron before and during PRWS (D) or CRWS (E) . Right , paired response and violin plots of normalized fluorescence intensity (norm.) during baseline, PRWS (Paired t-test, n=341 cells, ****P<0.0001; N=7 mice, P=0.047) or CRWS (Paired t-test, n=231 cells, ****P<0.0001; N=5 mice, P=0.08). (F) VIP interneurons were imaged at two planes in upper layers of S1, plane (P) 1 is closest to the pia and P2 is 100µm below. (G, H) Left, average VIP interneuron GCaMP6s fluorescence for P1 (darker green) and P2 (light green) for baseline (integrated over 20s) and during PRWS (G) or CRWS (H) . Right, normalized integrated fluorescence intensities duirng PRWS (G , P1 vs P2, Paired t test, **P=0.006) or CRWS (H, P1 vs P2, Paired t test, P=0.18). (I) Circuit diagram summarizing the RWS-evoked plasticity model. PRWS (blue) activates first-order thalamocortical (TC; red) as well as higher-order TC and feedback inputs (green), which activate disinhibitory VIP interneurons (grey). These combined inputs drive a potentiation of PW-evoked responses and a recruitment of neuronal responsivity (28%). CRWS (orange) may only activate higher-order TC and feedback inputs, also activating disinhibitory VIP interneurons, but this is not sufficient to drive potentiation and favors suppression of neurons (30%).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Expressing, Immunocytochemistry, Staining, Fluorescence