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. 2015 Sep 16;35(37):12693-702.
doi: 10.1523/JNEUROSCI.4315-14.2015.

Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1

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Free PMC article

Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1

Noreen Bukhari et al. J Neurosci. .
Free PMC article

Abstract

Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders.

Significance statement: Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction and ocular dominance plasticity in adult visual cortex. This is the first demonstration of adult brain to retain the intrinsic capacity to elevate tPA in an experience-dependent manner but is effectively limited by Lynx1. tPA-Lynx1 may potentially be a new candidate mechanism for interventions that were shown to activate plasticity in adult brain.

Keywords: Lynx1; ocular dominance; plasticity; spine; tPA; visual cortex.

Figures

Figure 1.
Figure 1.
MD-dependent elevation of tPA proteolytic activity in adult V1 of Lynx1KO mice. A, Measurement of tPA activity using amidolytic assay in right V1 homogenates from adult WT and Lynx1 KO mice with (dark gray) and without (white) 4 d of monocular deprivation of left eye (MD). n = 12–18 mice. *p < 0.05 (Kruskal–Wallis test with post hoc Dunn's test, and Mann–Whitney U test). B, tPA serpin, a known molecular inhibitor of tPA, was applied in ex vivo V1 homogenates from adult Lynx1 KO mice after 4 d (4D) of MD and tPA activity measured using amidolytic assay as above. n = 4 mice *p < 0.05 (paired Student's t test). The range and the average age of mice for each subgroup were as follows: WT no MD: 2.6–6.8 months, average 6.1 months, WT MD: 2.6–6.8 months, average 4.7 months, Lynx1 KO no MD: 2.4–13.1 months, average 8.1 months, and Lynx1 KO MD: 2.3–11.0 months, average 5.9 months. As a recent study reported, no significant OD plasticity in WT mice ∼P72 (age range P57–P80) (Stodieck et al., 2014), we also compared the tPA activity only with the mice older than P72 (excluding 2 mice each from KO noMD group and KO MD group who were younger than P72). In these sets of mice over P72, Lynx1KO MD groups (14 mice) maintained a statistically significant increase in tPA activity compared with age-matched controls (p = 0.031 vs WT MD group, 12 mice; p = 0.020 vs Lynx1 KO no MD group, 16 mice, t test). When only older adults (>P110) were compared, Lynx1KO MD groups (7 mice) also maintained statistically significant increase in tPA activity compared with age-matched controls (p = 0.046 vs WT MD group, 6 mice; p = 0.003 vs Lynx1 KO no MD group, 12 mice, t test). Only male mice were used in WT groups. In the Lynx1 KO groups, a comparison between the two genders revealed no statistical difference in tPA activity within the Lynx1 KO no MD group (9 male, 6 female, excluding 3 mice with unidentified gender, p = 0.098, t test) or MD group (7 male and 9 female, p = 0.180, t test).
Figure 2.
Figure 2.
MD-dependent reduction of thin and stubby spine density in adult V1 of Lynx1KO mice is dependent on tPA. A, Adult mice (>postnatal day 60) underwent HSV-GFP injection 1 d after MD. Four days after MD, adult V1 was isolated and processed for spine analysis. B, Representative images of GFP-labeled dendritic spines (green) and corresponding gray schema below labeled with stubby (pink), thin (yellow), mushroom (nonlabeled white) spines in WT, Lynx1 KO, and Lynx1-tPA DKO adult mice V1 after MD. C, Graphs showing MD-dependent changes of total spine density and densities of three subsets (stubby, thin, and mushroom) spines in adult V1 of WT, Lynx1 KO, and Lynx1-tPA DKO mice. Each genotype was initially normalized to its no MD control of the same genotype, and all spine density graphs are represented as percentage change ((MD − no MD)/no MD × 100); n = 4–6. *p < 0.05 (one-way ANOVA and post hoc Tukey test). The age range and the average of the mice for each subgroup were as follows: WT no MD: 3.8–4.3 months, average 4.2 months; WT MD: 3.6–4.1 months, average 3.9 months; Lynx1 KO no MD: 2.8–5.0 months, average 3.3 months; Lynx1 KO MD: 2.5–3.1 months, average 2.6 months; DKO no MD 2.1–4.3 months, average 3.3 months; and DKO MD 2.5–4.9 months, average 3.6 months. There was no statistically significant correlation between age and total spine density within each subset groups (Pearson correlation coefficient analysis: R2 = 0.426, p = 0.160 for WT no MD, R2 = 0.036, p = 0.719 for WT no MD, R2 = 0.815, p = 0.097 for KO no MD, R2 = 0.0572, p = 0.761 for KO MD, R2 = 0.985, p = 0.078 for DKO no MD, R2 = 0.110, p = 0.586 for DKO MD). Although the gender of mice was not controlled in this study (5 male for WT no MD, 3 male and 3 female for WT MD, 3 male and 2 female for KO no MD, 1 male and 5 female for KO MD, 3 male and 1 female for DKO no MD, 3 male and 3 female for DKO MD), the groups that had necessary number of mice for quantification (>3 mice for each gender) did not show gender differences (p = 0.719 WT MD male vs female mice, p = 0.736 for DKO MD male vs female group, t test).
Figure 3.
Figure 3.
tPA-dependent OD shift after MD in adult Lynx1KO mice. A, Adult mice underwent 4 d of MD and in vivo extracellular recordings of visually evoked single unit responses in V1 contralateral to the deprived eye. B, Lynx1-tPA-DKO adult mice (blue histogram represents 5 mice, 83 cells) showed significantly reduced shift in the OD distribution after 4 d MD compared with adult Lynx1KO mice with MD (red histogram represents 5 mice, 87cells). ***p < 0.0001 (χ2 test). C, Cumulative percentage of OD index confirms loss of OD shift by genetic removal of tPA in Lynx1 KO adult mice with MD compared with Lynx1KO adult mice with MD. ***p < 0.001 (K-S test). D, Quantification of adult OD plasticity by CBI that reflects the extent of OD shift after 4 d of MD per animal. Low CBI indicates higher plasticity. Gray area represents CBI range in a nonplastic mouse. CBI = 0.47 for Lynx1KO + MD (n = 5), 0.62 for Lynx1-tPADKO + MD (n = 5). *p < 0.05 (Student's t test). E, Quantification of the changes in magnitude of contralateral and ipsilateral responses after MD, represented as percentage change ((MD − no MD)/no MD × 100). The deprived contralateral eye response demonstrated significant reduction in Lynx1 KO mice (red histogram represents n = 87 cells from 5 mice) compared with that of Lynx1-tPA DKO mice (blue histogram represents n = 83 cells from 5 mice). ****p < 0.0001 (Student's t test). The changes in the nondeprived ipsilateral eye response showed no significant difference between genotypes (n.s., Not significant; p = 0.1036, Student's t test). The range and average age of the mice for each subgroup were as follows: WT no MD: age range: 1.9–3.5 months, average 2.8 months, WT MD: age range 1.7–10.5 months, average 4.8 months, Lynx1 KO no MD: 4–7.5 months, average 6.4 months, Lynx1 KO MD: 2.8–7.4 months, average: 5.3 months, DKO no MD: 2.5–4.2 months, average: 3.2 months, and DKO MD: 2.5–4 months, average 3.6 months. All mice but one WT no MD mouse and two WT MD mice were older than P72, when no OD plasticity is detected after 4 d of MD (Stodieck et al., 2014). Among WT mice with MD, there was no statistically significant difference in OD plasticity (CBI) between adult mice group younger than P72 (2 mice) versus mice group older than P72 (5 mice) (p = 0.599, t test), or between adult mice younger than P110 (3 mice) versus fully adult mice older than P110 (4 mice) subgroups (p = 0.253, t test). When we compared only among fully adult mice older than P110, Lynx1KO MD groups (4 mice) still showed significantly enhanced plasticity compared with control groups (vs WT MD, p = 0.020, 4 mice; vs Lynx1 KO no MD, p = 0.008, 6 mice, t test). Although gender of mice was not controlled in this study (male only for WT no MD and DKO no MD, female only for Lynx1KO no MD, 5 male and 2 female for WT MD, 2 male and 3 female for Lynx1KO MD, 3 male and 2 female for DKO MD), we did not detect significant gender effect on CBI for any MD subgroups (p = 0.970 for WT MD males vs females, p = 0.838 for Lynx1KO MD males vs females, p = 0.602 for DKO MD males vs females, t test). When we compared CBI of only male mice, we still detected a statistically significant increase of plasticity in the Lynx1KO MD group compared with the WT MD group (p = 0.034, t test) or DKO MD group (p = 0.014, t test).
Figure 4.
Figure 4.
Adult specific inhibition of tPA ablates MD-dependent OD shift in Lynx1KO mice. A, AAV-GFP or AAV-tPA serpin was injected in Lynx1 KO adult mice. MD was performed 21 d after brain injection, and in vivo electrophysiology 4 d after MD. B, Representative images of the binocular zone of V1 from three different distances from bregma in anterior–posterior axis (−3.1 mm, −3.5 mm, −4.0 mm) 21 d after AAV-tPA serpin injection showing bicistronically expressed GFP fluorophore. Binocular zone of V1 is outlined by dashed white-line based on Paxinos and Franklin (1997). Scale bar, 250 μm. C, Quantification of tPA serpin expression in AAV-tPA serpin and AAV-GFP-injected mice using ISH, data are represented as mean intensity of tPA serpin mRNA signal/GFP cell (n = 30 cells for AAV-tPA seprin overexpression and n = 28 cells for AAV-GFP overexpression). ***p < 0.001 (Student's t test). D, Quantification of tPA activity after AAV-tPA Serpin or AAV-GFP injection in V1 using amidolytic assay n = 3–5. *p < 0.05 (Student's t test). E, Lynx1 KO + AAV-tPA serpin mice (blue histogram represents n = 6 mice, 97 cells) showed no rightward shift in the OD distribution after 4 d MD in contrast to Lynx1KO + AAV-GFP mice with MD (red histogram represents n = 5 mice, 89 cells). ***p = 0.0005 (χ2 test). F, Cumulative percentage of OD index confirms loss of OD shift after injection of tPA serpin compared with control AAV-GFP injection in Lynx1 KO mice with MD. ***p < 0.001 (K-S test). G, Quantification of adult OD plasticity by CBI. CBI = 0.43 in Lynx1 KO + AAV-GFP and CBI = 0.57 in Lynx1 KO + tPA serpin. *p < 0.05 (Student's t test). Gray area represents CBI range in a nonplastic mouse. All mice were older than P110. The average age and age range of the mice for each subgroup are as follows: Lynx1 KO AAV-GFP MD: 4.9–5.1 months, average 5.0 months, Lynx1 KO AAV-NS MD: 5.3–7.9 months, average 6.8 months. Only females were used for recordings with AAV injections.

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