Optimal level activity of matrix metalloproteinases is critical for adult visual plasticity in the healthy and stroke-affected brain
Neuroplasticity is the ability of the brain to adapt both structurally and functionally to changing patterns of activity induced by the environment or intrinsic factors . In the clinical setting , plasticity is important for tissue repair and neural network rewiring , necessary for recovery and optimal post-injury brain function . The primary visual cortex ( V1 ) is a widely used model region for studying sensory plasticity in young vs . adult brains ( Hofer et al . , 2006; Espinosa and Stryker , 2012; Levelt and Hübener , 2012 ) . In mammals , V1 consists of a monocular and a binocular zone; neurons in the binocular zone respond to stimulation of both eyes but are dominated by the contralateral eye in rodents ( Dräger , 1975; 1978 ) . This ocular dominance ( OD ) can be modified in an experience-dependent manner , by depriving one eye of pattern vision for several days ( known as monocular deprivation or MD ) , as originally observed by Wiesel and Hubel more than 50 years ago ( Wiesel and Hubel , 1965 ) . OD-plasticity has become one of the major paradigms for studying cortical plasticity . In standard-cage raised mice , OD-plasticity in binocular V1 is most pronounced in 4-week-old animals; reduced , yet present , in early adulthood; and absent in animals older than 110 days of age ( Lehmann and Löwel , 2008 ) . In 4-week-old mice , 3-4 days of MD are sufficient to induce a significant OD-shift towards the open eye ( juvenile OD-plasticity ) , while 7 days of MD are needed in 3-month-old animals ( adult OD-plasticity ) ( Gordon and Stryker , 1996; Sawtell et al . , 2003; Frenkel and Bear , 2004; Mrsic-Flogel et al . , 2007; Sato and Stryker , 2008 ) . Although the experimental paradigm is rather similar , molecular mechanisms underlying 'juvenile' and 'adult' OD-plasticity are different: in juvenile mice , OD-shifts are mostly mediated by reductions in deprived eye responses while adult plasticity is predominantly mediated by an increase in open eye responses in V1 ( Hofer et al . , 2006; Espinosa and Stryker , 2012; Levelt and Hübener , 2012 ) . Activity driven modifications in neuronal circuits can be facilitated by degradation of the extracellular matrix ( ECM ) ( Pizzorusso et al . , 2002; de Vivo et al . , 2013 ) , which provides structural and biochemical support for the cells ( Frischknecht and Gundelfinger , 2012 ) . Structural and molecular composition of the ECM changes during development , becoming denser and more rigid in the mature brain ( Frischknecht and Gundelfinger , 2012; de Vivo et al . , 2013 ) . This structural composition is partially regulated by a family of zinc dependent endopeptidases , the matrix metalloproteinases ( MMPs ) , and their enzymatic activity is crucial for proper development , function and maintenance of neuronal networks ( Milward et al . , 2007; Huntley , 2012 ) . A recent study in juvenile rats showed that pharmacological inhibition of MMPs with a broad spectrum inhibitor during the MD-period did not influence the reduction of the closed-eye responses induced by 3 days of MD , yet it prevented the potentiation of the nondeprived eye responses in V1 after 7 days of MD ( Spolidoro et al . , 2012 ) . Whether MMPs are involved in adult OD-plasticity , for which mechanisms are believed to be different ( Hofer et al . , 2006; Sato and Stryker , 2008; Ranson et al . , 2012 ) , is not yet known , and clarifying this point was the first goal of this study . In addition to MMP involvement in healthy brain function , their excessive activity can also be detrimental ( Agrawal et al . , 2008; Huntley , 2012 ) . Both human and animal studies found upregulated activity of MMPs following inflammation , infectious diseases or brain trauma ( Agrawal et al . , 2008; Rosell and Lo , 2008; Morancho et al . , 2010; Vandenbroucke and Libert , 2014 ) , and pharmacological inhibition of MMPs shortly after brain injuries reduced infarct sizes and prompted better recovery ( Romanic et al . , 1998; Lo et al . , 2002; Gu et al . , 2005; Wang and Tsirka , 2005; Yong , 2005; Morancho et al . , 2010; Chang et al . , 2014; Vandenbroucke and Libert , 2014 ) . Stroke can influence synaptic activities within the area directly affected by it , and also in a broader area surrounding the lesion ( Witte et al . , 2000; Murphy and Corbett , 2009 ) . Likewise , impairments in experience-dependent plasticity after a cortical stroke also have been observed in distant brain regions ( Jablonka et al . , 2007; Greifzu et al . , 2011 ) : in 3-month-old mice , a focal stroke in the primary somatosensory cortex ( S1 ) prevented both V1-plasticity and improvements of the spatial frequency and contrast thresholds of the optomotor reflex of the open eye ( Greifzu et al . , 2011 ) . Interestingly , some MMPs were shown to be upregulated within the first 24 hours after focal stroke ( Cybulska-Klosowicz et al . , 2011; Liguz-Lecznar et al . , 2012 ) . Thus , the second goal of our study was to test whether balancing the upregulated MMPs resulting from a focal stroke in S1 would rescue visual plasticity . Using a combination of in vivo optical imaging of intrinsic signals and behavioral vision tests in adult mice , we observed that application of the broad spectrum MMP-inhibitor GM6001 during the 7-day MD-period prevented both OD-plasticity and enhancements of the optomotor response of the open eye . In addition , a single treatment after the S1-stroke rescued plasticity in both paradigms , whereas treatment with the inhibitor two times diminished plasticity-promoting effect . Together , these data reveal a crucial role of MMPs in adult visual plasticity and suggest that MMP-activity has to be within a narrow window for experience-induced plasticity to occur .
MMPs were shown to be critical for open eye potentiation after 7 days of MD in juvenile rats ( Spolidoro et al . , 2012 ) . Since it is believed that the mechanisms underlying juvenile and adult OD-plasticity are different ( Hofer et al . , 2006; Sato and Stryker , 2008; Ranson et al . , 2012 ) , we aimed to test whether MMPs also play a significant role in adult V1-plasticity , in which open-eye potentiation is a major component mediating these changes . First , we induced 7-day-MD in 3-month-old mice and examined whether treatment with GM6001 ( 50mg/kg/day , for 7days ) , a broad-spectrum MMP-inhibitor , can prevent ocular dominance ( OD ) plasticity , measured by in vivo intrinsic signal optical imaging in V1 . Figure 1 shows representative examples of optically recorded activity and polar maps after visual stimulation of the right ( deprived , contralateral ) and left ( open , ipsilateral ) eye in the binocular region of the left V1 in vehicle-treated ( control , Figure 1A , C ) and GM6001-treated adult mice ( GM6001 , Figure 1B , D ) . In both , control and GM6001-treated mice without MD ( Figure 1A , B ) the activity patch induced by stimulation of the contralateral eye was darker than that of the ipsilateral eye; the OD-index ( ODI ) was positive and warm colors dominated the OD-map , indicating typical contralateral eye dominance in V1 . 7 days of MD induced an OD-shift towards the open eye in control ( Figure 1C ) but not in GM6001-treated mice ( Figure 1D ) : in control mice after MD visual stimulation of the contralateral and ipsilateral eye activated V1 rather equally strong; the OD-histogram was shifted to the left and cooler colors appeared in the 2-dimensional OD-map . In contrast , V1-activation of GM6001-treated mice after MD was similar to values without MD and the deprived eye still dominated V1-activity ( compare Figure 1B and D ) . 10 . 7554/eLife . 11290 . 003Figure 1 . Treatment with the MMP-inhibitor GM6001 during MD period prevented OD-plasticity in adult mouse V1 . Representative examples of optically recorded activity and polar maps in V1 of both vehicle-treated mice ( control , A , C ) and animals treated with the MMP-inhibitor GM6001 ( GM6001 , B , D ) , before ( no MD , A , B ) and after monocular deprivation ( 7 days MD , C , D ) . V1-maps after visual stimulation of the contra- ( contra ) and ipsilateral ( ipsi ) eye in the binocular region of V1 . Top panels ( A–D ) display grayscale-coded response magnitude maps ( V1-activation ) and their quantification on the right: histogram of ( C-I ) / ( C+I ) . V1-activation is illustrated as fractional change in reflection ×10-4 . Average V1-activation is illustrated as a number at the lower right corner of each magnitude map; the average OD-index ( ODI ) as a number in the upper right corner of the histograms . Bottom panels represent color-coded polar maps of retinotopy , and the color-coded OD-map . In both control and GM6001-treated mice without MD , V1-activity was dominated by input from the contralateral eye: activity patches after contralateral eye stimulation were darker than after ipsilateral eye stimulation , the average ODI was positive , and warm colors prevailed in the OD-map , illustrating contralateral dominance . After 7 days of MD , OD-plasticity occurred only in control ( C ) but not in GM6001-treated mice ( D ) : in control mice , both eyes activated V1 rather equally strong , the OD-histogram was shifted to the left ( blue arrow in C ) , and colder colors appeared in the OD-map . In contrast , in GM6001-treated mice , the deprived ( contralateral ) eye continued to dominate V1 , the ODI was not reduced and warm colors still dominated the OD-map . Scale bar: 1mm . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 003 Quantification of all imaging data is illustrated in Figure 2 . In control mice OD-indices were significantly reduced after MD ( control , n=6 , 0 . 27 ± 0 . 03 vs . control+MD , n=5 , 0 . 02 ± 0 . 01 , t-test , p<0 . 001 , Figure 2A ) , and the OD-shift was mediated by an increase in open eye responses in V1 ( control , 1 . 3 ± 0 . 2 vs . control+MD , 1 . 8 ± 0 . 1 , t-test , p<0 . 05 , Figure 2B ) , with no concomitant changes in visually driven activity after deprived eye stimulation ( control , 1 . 9 ± 0 . 2 vs . control+MD , n=5 , 1 . 8 ± 0 . 1 , t-test , p>0 . 05 ) , indicating a typical adult type of OD-plasticity . Thus , the vehicle treatment did not interfere with the induction of experience-dependent cortical plasticity in adult mouse V1 . In contrast , in GM6001-treated mice OD-indices were indistinguishable before and after MD ( GM6001 , n=5 , 0 . 23 ± 0 . 02 vs . GM6001+MD , n=5 , 0 . 24 ± 0 . 02 , t-test , p>0 . 05 Figure 2A ) . Likewise , V1-activations via the ipsilateral ( GM6001 , 1 . 2 ± 0 . 2 vs . GM6001+MD , 1 . 2 ± 0 . 04 ) or contralateral eye ( GM6001 , 1 . 9 ± 0 . 2 vs . GM6001+MD , 1 . 9 ± 0 . 1 ) were similar before and after MD ( for all , t-test , p>0 . 05 , Figure 2B ) , indicating that the deprived , contralateral eye still dominated V1 . This absence of any detectable OD-shift during MMP-inhibition strongly suggests a crucial role of MMPs for mediating visual cortical plasticity in the adult brain . 10 . 7554/eLife . 11290 . 004Figure 2 . Inhibition of MMPs during the MD-period prevented adult OD-plasticity . ( A ) Optically imaged OD-indices in animals without MD ( solid symbols ) and after 7 days of MD ( right half of symbols is black ) in control ( vehicle-treated , black ) and in GM6001-treated mice ( grey ) . Symbols illustrate ODI-values of individual cases; means are marked by horizontal lines . ( B ) V1-activation elicited by stimulation of the contralateral ( C ) or ipsilateral ( I ) eye in animals before ( open circles ) and after MD ( closed eye marked as a black filled circle , and striped bar ) . The OD-shift in control mice was mediated by an increase of open eye responses in V1 , while no OD-shift and no changes in V1-actiavtion were observed in GM6001-treated mice after MD . Mean ± SEM , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . ANOVA followed by multiple comparisons with Bonferroni correction was used in A; two-tailed t-test was used in B . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 00410 . 7554/eLife . 11290 . 005Figure 2—source data 1 . Ocular dominance index and V1-activation individual values for Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 005 To test whether injections of GM6001 or vehicle influenced basic properties of V1 , we analyzed V1-activation and the quality of the retinotopic maps in all experimental groups without MD . Essentially , there were no significant differences between control ( vehicle-treated ) and GM6001-treated mice in both map quality and V1-activation ( control/GM6001 , elevation , map scatter: 0 . 9 ± 0 . 2/1 . 1 ± 0 . 1; V1-activation: 3 . 8 ± 0 . 5/3 . 2 ± 0 . 4; azimuth , map scatter: 12 . 1 ± 2 . 9/4 . 8 ± 1 . 9 , V1-activation: 3 . 1 ± 0 . 5/2 . 8 ± 0 . 3; t-test , p>0 . 05 for all comparisons ) , indicating that GM6001/vehicle-treatment did not impact basic map quality nor magnitude of V1-activation . In addition to our optical imaging experiments , we also performed behavioral assessments of visual abilities in the same groups of mice . While the experience-induced improvements in the spatial frequency and contrast thresholds of the optomotor reflex of the open eye after MD are cortex-dependent ( Prusky et al . , 2006 ) , the mechanism underlying these enhancements is not yet fully understood . Using the virtual-reality optomotor setup ( Prusky et al . , 2004 ) and daily testing , we examined the role of MMPs in this kind of interocular plasticity . Spatial frequency and contrast sensitivity thresholds of the optomotor reflex were measured in both vehicle-treated ( control ) and GM6001-treated mice before and during the 7-day MD-period . Mice without MD did not show any experience-induced changes in the spatial frequency ( n=6 , day 0: 0 . 37 ± 0 . 002 cyc/deg vs . day 7: 0 . 37 ± 0 . 001 cyc/deg , t-test , p>0 . 05 , Figure 3A ) or contrast sensitivity thresholds of the optomotor reflex ( day 0 vs . day 7 , for all spatial frequencies , ANOVA , p>0 . 05 , Figure 3B and Table 1 ) . In contrast , vehicle-treated ( control ) mice with 7 days of MD showed the typical enhancements of both the spatial frequency and contrast thresholds of the optomotor reflex: the spatial frequency threshold of the open eye increased on average by 22 ± 1% from day 0 to day 7 ( day 0/7: 0 . 37 ± 0 . 002/0 . 46 ± 0 . 0001 cyc/deg , n=5 , t-test , p<0 . 001 , Figure 3A ) and was thus significantly different from control mice without MD ( n=6 ) that displayed no experience-driven changes ( ANOVA , p<0 . 001 , Figure 3A ) . Likewise , contrast sensitivity of the open eye of control mice increased after MD ( for all spatial frequencies , ANOVA , p<0 . 001 , Table 1 ) , and was different on day 7 compared to control mice without MD ( control , n=5 vs . control+MD , n=5 , t-test , p<0 . 001 , Figure 3B ) . In contrast , in GM6001-treated mice MD did neither induce an improvement of the spatial frequency ( day 0/day 7: 0 . 37 ± 0 . 002/0 . 38 ± 0 . 002 cyc/deg , n=5 , t-test , p>0 . 05 Figure 3A ) , nor of contrast sensitivity thresholds of the optomotor reflex of the open eye ( at all spatial frequencies , ANOVA , p>0 . 05 , Figure 3B ) . Treatment in the no MD groups ( control , GM6001 ) had no effect on baseline spatial frequency and contrast thresholds ( for all measurements , ANOVA , p>0 . 05 , Table 1 , Figure 3 ) , demonstrating that MMP-inhibition did not affect the ability to exhibit the optomotor reflex but prevented the experience-enabled changes induced by MD . Together , our behavioral vision tests indicate an essential role for MMPs in this cortex-dependent interocular plasticity paradigm: MMP-inhibition completely abolished the experience-enabled improvements of the optomotor reflex of the open eye . 10 . 7554/eLife . 11290 . 006Figure 3 . Inhibition of MMPs prevented experience-enabled enhancements of both the spatial frequency ( A ) and contrast sensitivity ( B ) thresholds of the optomotor reflex of the open eye in adult mice after MD . Values of vehicle-treated ( control ) mice are displayed in black ( triangles ) , values of GM6001-treated mice in grey ( circles ) ; values of animals with monocular deprivation ( +MD ) are marked by displaying the right half of the symbol in black . ( A ) Spatial frequency threshold values in cycles/degree ( cyc/deg ) , measured in the optomotor setup plotted against days . ( B ) Contrast sensitivity values on day 7 at 6 different spatial frequencies . Mean ± SEM; ***p<0 . 001 , ns p>0 . 05 . ANOVA followed by multiple comparisons with Bonferroni correction was used; two-tailed t-test was used within the group analysis in A . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 00610 . 7554/eLife . 11290 . 007Figure 3—source data 1 . Optomotry measured spatial frequency and contrast sensitivity thresholds individual values for Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 00710 . 7554/eLife . 11290 . 008Table 1 . Optomotry-measured contrast sensitivity improvements after MDDOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 008Contrast sensitivity Day 0Spatial frequency ( cyc/deg ) ControlGM6001Control+MDGM6001+MD0 . 0313 . 6 ± 0 . 043 . 5 ± 0 . 023 . 5 ± 0 . 013 . 5 ± 0 . 010 . 06412 . 7 ± 0 . 5211 . 8 ± 0 . 1611 . 6 ± 0 . 2312 . 0 ± 0 . 150 . 09211 . 7 ± 0 . 5110 . 9 ± 0 . 1810 . 7 ± 0 . 2211 . 3 ± 0 . 170 . 10310 . 3 ± 0 . 5410 . 2 ± 0 . 1010 . 0 ± 0 . 1610 . 6 ± 0 . 120 . 1926 . 9 ± 0 . 076 . 9 ± 0 . 127 . 0 ± 0 . 116 . 8 ± 0 . 050 . 2723 . 5 ± 0 . 043 . 5 ± 0 . 023 . 5 ± 0 . 013 . 5 ± 0 . 01Day 70 . 0313 . 6 ± 0 . 043 . 5 ± 0 . 025 . 1 ± 0 . 173 . 6 ± 0 . 020 . 06412 . 7 ± 0 . 4711 . 8 ± 0 . 1826 . 9 ± 2 . 3512 . 6 ± 0 . 290 . 09211 . 7 ± 0 . 4411 . 0 ± 0 . 1523 . 3 ± 2 . 1712 . 0 ± 0 . 220 . 10310 . 5 ± 0 . 4910 . 3 ± 0 . 1021 . 8 ± 1 . 8311 . 3 ± 0 . 170 . 1927 . 0 ± 0 . 087 . 0 ± 0 . 1013 . 5 ± 1 . 087 . 5 ± 0 . 210 . 2723 . 5 ± 0 . 043 . 5 ± 0 . 014 . 9 ± 0 . 173 . 8 ± 0 . 25 Upregulated activity of MMPs can negatively contribute to the pathology of stroke as well as other neurodegenerative diseases ( Rosenberg et al . , 1996; Rosenberg , 2002; Agrawal et al . , 2008 ) . Furthermore , increased MMP-activity was observed within 24 hours of a photothrombotically ( PT ) induced stroke , and application of a broad spectrum MMP-inhibitor at the time of the stroke partially rescued impaired barrel cortex plasticity ( Cybulska-Klosowicz et al . , 2011; Liguz-Lecznar et al . , 2012 ) . Here we tested if the stroke-induced impairment of visual cortical plasticity ( Greifzu et al . , 2011 ) can be rescued by inhibiting MMPs after stroke . We examined a total of six groups of mice: all mice received a PT-stroke in their left S1 cortex , about 1 mm anterior to the anterior border of V1 . Groups 1 and 2 received vehicle injections 1 h after PT ( PT vehicle , PT+MD vehicle ) , groups 3 and 4 a single injection of the broad spectrum MMP-inhibitor GM6001 1h after stroke ( PT 1xGM6001 and PT+MD 1xGM6001 1 h ) , group 5 received a single injection of GM6001 24 h after stroke ( PT+MD+1xGM6001 24 h ) and group 6 received 2 injections of GM6001 1 h and 24 h after PT ( PT+MD 2xGM6001 1 h+24 h ) . Groups 2 , 4 , 5 and 6 were additionally subjected to MD . Histological analysis showed no difference in the volume of the lesion between vehicle- and GM6001-treated mice ( vehicle/1xGM6001 1 h/1xGM6001 24 h/2xGM6001 1 h+24 h: 0 . 6 ± 0 . 2/1 . 1 ± 0 . 3/0 . 6 ± 0 . 3/1 . 2 ± 0 . 2 mm3 , ANOVA , p>0 . 05 ) . Since there was no difference in the volume of the lesion between mice that received GM6001 once or twice ( ANOVA , p>0 . 05 ) , we pooled these data for further analysis of lesion size and position . The PT-lesion of vehicle-treated mice measured on average 1 . 1 ± 0 . 01 mm in medio-lateral and 1 . 0 ± 0 . 02 mm in anterior-posterior direction . The lesion center was located 1 . 2 ± 0 . 4 mm anterior to the anterior border of V1 , 1 . 7 ± 0 . 2 mm lateral to the midline , and 1 . 0 ± 0 . 3 mm posterior to the Bregma . For GM6001-treated mice , PT-lesion measured on average 1 . 2 ± 0 . 09 mm in medio-lateral and 1 . 0 ± 0 . 01 mm in anterior-posterior direction . Center of the lesion was situated 0 . 9 ± 0 . 3 mm anterior to the anterior border of V1 , 1 . 9 ± 0 . 1 mm lateral to the midline , and 1 . 2 ± 0 . 2 mm posterior to the Bregma . There was no significant difference in the location nor the size of the lesion between vehicle- and GM6001-treated mice ( ANOVA , p>0 . 05 for every measurement ) . Figure 4 shows representative examples of optically recorded activity and polar maps in V1 of adult S1-lesioned mice before and after MD . In both vehicle- and GM6001-treated mice without MD ( Figure 4A , B ) , the activity patch induced by stimulation of the contralateral eye appeared darker than that of the ipsilateral eye , the ODI was positive and warm colors dominated in the OD-map , indicating that treatment with the MMP-inhibitor or vehicle after the PT-lesion did not influence baseline ocular dominance and sensory-induced activity in binocular V1 ( Figure 4A , B ) . As previously observed in untreated mice , MD after small S1-lesions was also not able to induce an OD-shift towards the open eye in vehicle-treated mice ( Figure 4C ) : V1-activity of PT-lesioned mice after MD was almost indistinguishable from animals without MD ( compare Figure 4A and C ) , and V1 remained dominated by the deprived , contralateral eye . In contrast , GM6001-treatment rescued the OD-shift , since after MD V1 was activated equally strongly by both eyes , the ODI-histogram shifted to the left and colder colors appeared in the OD-map ( Figure 4D ) . 10 . 7554/eLife . 11290 . 009Figure 4 . A single GM6001-treatment rescued OD-plasticity in V1 after a stroke in S1 . Representative examples of optically recorded activity and polar maps in V1 of both vehicle- ( PT+vehicle , A , C ) and single GM6001-treated mice ( PT+1xGM6001 , B , D ) , before ( no MD , A , B ) and after 7 days of MD ( 7days MD , C , D ) . V1-maps after visual stimulation of the contra- ( contra ) and ipsilateral ( ipsi ) eye in the binocular region in V1 . Figure layout and data display as in Figure 1 . In both vehicle- ( A ) and GM6001-treated PT-lesioned mice without MD ( B ) , V1 activation was dominated by the contralateral eye: activity patches induced by contralateral eye stimulation were darker than after ipsilateral eye stimulation , the average ODI was positive , and warm colors dominated the OD-map . ( C ) While 7 days of MD did not induce an OD-shift in vehicle-treated PT mice , and deprived ( contra ) eye patches still dominated V1 , a single treatment with GM6001 rescued OD-plasticity in PT mice ( D ) : both eyes activated V1 about equally strong , the histogram of ODIs shifted to the left ( blue arrow ) and colder colors appeared in the OD-map . Scale bar: 1mmDOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 009 Quantification of all individual cases is presented in Figure 5 . In vehicle-treated mice , OD-plasticity was abolished after PT ( PT vehicle , n=4 , 0 . 25 ± 0 . 02 vs . PT+MD vehicle , n=4 , 0 . 22 ± 0 . 04 , t-test , p>0 . 05 , Figure 5A ) and there was no change in visually driven activity in V1 from either the deprived ( C: PT vehicle , 1 . 9 ± 0 . 1 vs . PT+MD vehicle , 2 . 2 ± 0 . 3 , t-test p>0 . 05 ) or the open eye ( I: PT , 1 . 2 ± 0 . 1 vs . PT+MD , 1 . 5 ± 0 . 3 , t-test , p>0 . 05 ) ( Figure 5B ) . In contrast , treatment once , but not twice , with GM6001 rescued OD-plasticity after stroke . Specifically , mice which received a single injection of GM6001 1 h or 24 h after PT-induction showed a significant OD-shift , while mice receiving two injections ( 1 h+24 h ) after the injury did not display OD-plasticity ( PT+GM6001 , n=5 , ODI=0 . 26 ± 0 . 04 vs . PT+MD+1xGM6001 1 h , n=5 , 0 . 05 ± 0 . 04 , p<0 . 01; vs PT+MD+1xGM6001 24 h , n=4 , 0 . 05 ± 0 . 01 , p<0 . 01 and vs . PT+MD+2xGM6001 1 h+24 h , n=4 , 0 . 17 ± 0 . 04 , p>0 . 05 , ANOVA Figure 5A ) . Interestingly , in the three MD groups treated with GM6001 , deprived eye responses were reduced compared to GM6001-treated mice without MD ( PT+GM6001 , 2 . 3 . ± 0 . 1 vs . PT+MD+1xGM6001 1 h , 1 . 7 ± 0 . 1 , ANOVA , p<0 . 01; vs PT+MD+1xGM6001 24 h , 1 . 4 ± 0 . 1 , p<0 . 001; and vs . PT+MD+2xGM6001 1 h+24 h , 1 . 8 ± 0 . 1 , p<0 . 05 , ANOVA ) and there was no change in open eye responses in V1 ( PT+GM6001 , 1 . 5 ± 0 . 1 vs . PT+MD+1xGM6001 1 h , 1 . 5 ± 0 . 1; vs PT+MD+1xGM6001 24 h , 1 . 3 ± 0 . 1; and vs . PT+MD+2xGM6001 1 h+24 h , 1 . 3 ± 0 . 1 , for all p>0 . 05 , ANOVA ) , indicating strongly reduced but not entirely absent plasticity-promoting effect in the two-days treatment group ( Figure 5B ) . 10 . 7554/eLife . 11290 . 010Figure 5 . Brief MMP-inhibition after a stroke in S1 rescued experience-dependent plasticity in V1 . Quantification of the imaging data , layout and data display as in Figure 2 . ( A ) Optically imaged OD-indices in mice without MD and after 7 days of MD in vehicle-treated 1h after PT ( black ) mice and single GM6001-treated ( PT 1xGM6001; PT+MD 1xGM6001 1 hr and PT+MD 1xGM6001 24 h , light grey ) or two-times GM6001-treated ( PT+MD 2xGM6001 1 h+24 h , dark grey ) after PT mice . ( B ) V1-activation elicited by stimulation of the contralateral ( C ) or ipsilateral ( I ) eye in animals before ( open circles ) and after MD . V1-activation did not change after MD in vehicle-treated PT mice . In contrast , 1xGM6001 ( either 1 h or 24 h ) but not 2xGM6001 ( 1 h+24 h ) treatment rescued OD-plasticity after the PT-lesion ( A ) . There was a significant reduction in deprived eye responses in V1 in the GM6001- but not vehicle-treated mice after MD ( B ) . Mean ± SEM , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . ANOVA followed by multiple comparisons with Bonferroni correction was used; two-tailed t-test was used within the group analysis in B . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 01010 . 7554/eLife . 11290 . 011Figure 5—source data 1 . Ocular dominance indexes and V1-activation individual values for Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 011 We have previously shown that even a small PT-lesion in S1 , i . e . close to but not within V1 , can prevent the experience-induced enhancement of the optomotor reflex of the open eye after MD ( Greifzu et al . , 2011 ) : the S1-lesion abolished improvements in both the spatial frequency and contrast sensitivity thresholds of the optomotor reflex . Since upregulated MMPs can have a detrimental effect on experience-dependent plasticity ( Cybulska-Klosowicz et al . , 2011 ) , and thus on recovery from brain injuries ( Rosell and Lo , 2008; Yang et al . , 2013; Chaturvedi and Kaczmarek , 2014 ) , here we tested whether a brief inhibition of MMPs after a cortical stroke can also rescue lost optomotor enhancement of the open eye after MD . In vehicle-treated mice , MD did not induce any improvements in the spatial frequency thresholds of the optomotor reflex of the open eye ( PT+MD vehicle ) : on day 0 , acuity was 0 . 38 ± 0 . 002 cyc/deg and 0 . 38 ± 0 . 004 cyc/deg on day 7 ( n=4 , t-test , p>0 . 05 , Figure 6A ) ; values were not different from vehicle-treated mice without MD ( PT vehicle , n=4 , vs . PT+MD vehicle , n=4 , p>0 . 05 , Figure 6A ) . Thus , visual improvements after MD in vehicle-treated animals were as compromised as previously described for wild type mice after PT ( Greifzu et al . , 2011 ) . Similarly , we did not observe any improvements in contrast sensitivity thresholds of the optomotor reflex at all spatial frequencies ( n=4 , ANOVA , for all p>0 . 05 , Table 2 ) , and values were similar for mice with or without MD ( PT vehicle , n=4 vs . PT+MD vehicle , n=4 , p>0 . 05 , Figure 6B ) . 10 . 7554/eLife . 11290 . 012Figure 6 . Brief inhibition of MMPs after induction of a photothrombotic lesion in S1 rescued experience-enabled enhancements of both the spatial frequency ( A ) and contrast sensitivity ( B ) thresholds of the optomotor reflex in adult mice . Values of vehicle-treated PT-mice are shown in black and values of GM6001-treated mice in grey . Layout and data presentation as in figure 3 . Mean ± SEM , ***p<0 . 001 , ns p>0 . 05 . ANOVA followed by multiple comparisons with Bonferroni correction and two-tailed t-test was used in A within the group analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 01210 . 7554/eLife . 11290 . 013Figure 6—source data 1 . Optomotry-measured the spatial frequency and contrast sensitivity individual values for Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 01310 . 7554/eLife . 11290 . 014Table 2 . Optomotry-measured contrast sensitivity improvements after MDDOI: http://dx . doi . org/10 . 7554/eLife . 11290 . 014Contrast sensitivityDay 0Spatial frequency ( cyc/deg ) PT+vehiclePT+GM6001PT+MDvehiclePT+MDGM60010 . 0313 . 5 ± 0 . 023 . 5 ± 0 . 043 . 5 ± 0 . 023 . 5 ± 0 . 020 . 06411 . 4 ± 0 . 3511 . 4 ± 0 . 3211 . 8 ± 0 . 5711 . 8 ± 0 . 150 . 09210 . 5 ± 0 . 3610 . 5 ± 0 . 3410 . 7 ± 0 . 3911 . 0 ± 0 . 160 . 1039 . 8 ± 0 . 269 . 9 ± 0 . 2810 . 0 ± 0 . 3710 . 0 ± 0 . 320 . 1926 . 9 ± 0 . 126 . 7 ± 0 . 096 . 7 ± 0 . 136 . 5 ± 0 . 260 . 2723 . 5 ± 0 . 033 . 5 ± 0 . 043 . 4 ± 0 . 013 . 5 ± 0 . 02Day 70 . 0313 . 5 ± 0 . 023 . 5 ± 0 . 043 . 5 ± 0 . 015 . 0 ± 0 . 110 . 06411 . 4 ± 0 . 3911 . 5 ± 0 . 3212 . 1 ± 0 . 4225 . 2 ± 1 . 530 . 09210 . 5 ± 0 . 3610 . 5 ± 0 . 3411 . 4 ± 0 . 2821 . 9 . ± 1 . 140 . 1039 . 9 ± 0 . 249 . 9 ± 0 . 2810 . 6 ± 0 . 2619 . 7 ± 1 . 250 . 1926 . 9 ± 0 . 116 . 8 ± 0 . 077 . 0 ± 0 . 0212 . 8 ± 0 . 910 . 2723 . 5 ± 0 . 023 . 5 ± 0 . 043 . 5 ± 0 . 024 . 9 ± 0 . 12 As there was no significant difference in the spatial frequency ( ANOVA , p>0 . 05 ) or contrast sensitivity thresholds of the optomotor reflex ( ANOVA , p>0 . 05 ) between mice receiving a single or two injections of GM6001 , we pooled data for analysis . In contrast to vehicle-treated , PT-lesioned mice receiving a single or two-times injections of the MMP-inhibitor ( PT+MD+GM6001 ) displayed the typical experience-enabled enhancements in the spatial frequency threshold of the optomotor reflex of the open eye after MD: values increased from 0 . 38 ± 0 . 002 on day 0 to 0 . 45 ± 0 . 003 on day 7 after MD ( n=14 , t-test , p<0 . 001 ) . GM6001-treated PT-lesioned mice also improved contrast sensitivity thresholds of the optomotor reflex of the open eye at all measured spatial frequencies ( n=14 , ANOVA , for all p<0 . 001 , Table 2 ) , and exhibited significant increases in contrast sensitivity on day 7 , when compared to mice without MD ( PT+GM6001 , n=4 , vs . PT+MD+GM6001 , n=14 , ANOVA , p<0 . 001 , Figure 6B ) . Altogether , these data show that brief inhibition of MMPs after the induction of a cortical stroke lesion completely rescued experience-induced enhancements in both the spatial frequency and contrast sensitivity thresholds of the optomotor reflex of the open eye after MD . This finding also suggests that upregulated MMPs in the immediate post-injury period can have a detrimental influence on interocular plasticity in adult mice .
The objective of this study was to examine if MMPs are crucial for adult visual plasticity , and if inhibition of their upregulation following cortical stroke may be beneficial for rescuing lost plasticity . A combination of in vivo optical imaging and behavioral vision tests revealed that an optimal level of MMP-activity is essential for adult visual plasticity to occur in the healthy and stroke-affected brain . In healthy adult mice , MMP-inhibition with GM6001 prevented both ocular dominance plasticity and experience-driven improvements of the optomotor reflex of the open eye after MD , indicating that MMP-activation is required for normal adult plasticity . In addition , blockade of elevated MMP-activity after a cortical stroke rescued compromised plasticity . Specifically , a single but not two-times treatment with GM6001 after a cortical PT-lesion in the neighboring S1 region fully rescued experience-dependent ocular dominance plasticity in adult V1 , which is normally lost under these conditions . These observations suggest that MMP-activity has to be within a narrow window to allow visual plasticity: if MMP-activity is downregulated ( with inhibitors ) or upregulated ( after stroke ) experience-induced plastic changes are compromised . In a healthy brain , the enzymatic activity of MMPs has plasticity promoting effects ( Milward et al . , 2007; Huntley , 2012 ) . Consequently to increased neuronal activity , activated MMPs can lessen physical barriers ( loosen up the ECM ) or via the degradation of certain receptors influence signaling pathways . Such changes within neuronal cells and their synapses thereby alter the structure and activity of neuronal networks ( Milward et al . , 2007; Huntley , 2012 ) . Involvement of MMPs in synaptic circuit remodeling has been mainly studied in the hippocampus , yet , their abundant expression in the central nervous system ( CNS ) indicates a much broader function . For instance , MMP9-deficient mice showed modestly reduced experience-dependent plasticity in the barrel cortex ( Kaliszewska et al . , 2012 ) and MMP3-deficient mice displayed abnormal neuronal morphology in the visual cortex and impaired plasticity induced by long-term monocular enucleation ( Aerts et al . , 2014 ) . Here , we used a different approach to probe the role of MMPs in experience-induced changes in the visual system: we performed the experiments in wild type mice and treated them with the broad-spectrum inhibitor GM6001 or vehicle during 7 days of MD . Consistent with previous findings ( Gordon and Stryker , 1996; Sawtell et al . , 2003; Sato and Stryker , 2008 ) , the OD-shift of vehicle-treated mice was mediated by open-eye potentiation . In contrast , there was no change in the open nor in the closed eye responses and hence no OD-plasticity in V1 after MD in GM6001-treated adult mice . This is in line with recent observations from juvenile rats , in which chronic treatment with GM6001 also prevented open eye potentiation after 7 days of MD ( Spolidoro et al . , 2012 ) . However , treatment in this study only partially prevented the OD-shift , as there was no effect on the reduction of deprived eye responses ( Spolidoro et al . , 2012 ) . A reduction in deprived eye responses in V1 is mostly observed in juvenile rodents after 3-4 days of MD ( Gordon and Stryker , 1996 ) , unless different raising conditions are used such as enriched environment or running wheel ( Greifzu et al . , 2014; Kalogeraki et al . , 2014 ) . In adult , standard-cage raised mice , 6-7 days of MD are necessary for significant OD-shifts and changes are mainly mediated by increases of open eye responses in V1 ( Gordon and Stryker , 1996; Sawtell et al . , 2003; Sato and Stryker , 2008 ) . MMP9 activity was suggested in the potentiation of the open eye responses in juvenile rats , as treatment with GM6001 significantly reduced MMP9-mRNA expression only in the hemisphere where structural changes took place ( Spolidoro et al . , 2012 ) . Since we observed a full blockade of plasticity after GM6001-treatment , it would be of interest to determine whether MMP9 is crucial for open-eye potentiation also in the adult brain . MMP9 has been widely investigated in various plasticity paradigms ( Milward et al . , 2007; Frischknecht and Gundelfinger , 2012; Huntley , 2012; Tsilibary et al . , 2014 ) and one of the molecules shown to stimulate MMP9 secretion and expression in vitro and in vivo is Tumor Necrosis Factor alpha ( TNFalpha ) ( Hanemaaijer et al . , 1993; Candelario-Jalil et al . , 2007 ) . TNFalpha signaling was found to play an important role in the open eye potentiation in juvenile ( Kaneko , et al . , 2008 ) , but not in adult V1 plasticity ( Ranson et al . , 2012 ) , and since our data reveal that MMPs are indispensable for adult V1 plasticity , MMP-activation in the adult brain is most likely not dependent on TNFalpha signaling . Accordingly , this adds to the notion that juvenile and adult V1 plasticity depend on different molecular mechanisms ( Hofer et al . , 2006; Sato and Stryker , 2008; Ranson et al . , 2012 ) . Together , our new data demonstrate a vital role of MMPs for adult visual cortex plasticity , in particular for the increase of open eye responses in V1 after MD , and notably expand the previous studies from juvenile rats . Under normal conditions , MMP-activity supports healthy brain development and function; yet a different outcome of MMP action has been described for diseased brain ( Agrawal et al . , 2008 ) . Under pathophysiological conditions like inflammation , infection or stroke , uncontrolled MMP driven proteolysis can lead to negative consequences ( Rosenberg et al . , 1996; Rosenberg , 2002; Agrawal et al . , 2008 ) . Excessive MMP-activity after stroke caused blood brain barrier disruption , upregulation of inflammatory mediators , excitotoxicity and eventually cell death ( Romanic et al . , 1998; Lo et al . , 2002; Gu et al . , 2005; Wang and Tsirka , 2005; Yong , 2005; Morancho et al . , 2010; Chang et al . , 2014; Vandenbroucke and Libert , 2014 ) . Recent studies reported increased enzymatic MMP9 activity within 24 h after a PT-stroke , and application of a broad spectrum MMP-inhibitor ( FN-439 ) applied at the time of stroke induction , partially rescued impaired barrel cortex plasticity ( Cybulska-Klosowicz et al . , 2011; Liguz-Lecznar et al . , 2012 ) . Here we tested whether inhibition of upregulated MMP-activity ( resulting from PT-stroke ) may rescue cortical plasticity also when the treatment starts after lesion induction . Indeed , a single GM6001-treatment after PT in the neighboring S1 fully rescued plasticity in both of our experimental paradigms , OD- and interocular plasticity . Importantly , successful treatment did not have to start immediately after stroke induction ( 1 h ) because treatment 24 h after had the same beneficial effect , highlighting the therapeutic potential of brief MMP-inhibition for stroke recovery . Beneficial treatment was , however , dependent on the number of injections: a single but not two-times injection of the MMP-inhibitor rescued OD-plasticity . The reduced plasticity-promoting effect in V1 of mice treated twice with GM6001 is likely due to too excessive MMP-downregulation , which in turn interfered with MMP facilitation of MD-induced plasticity . Consistent with this interpretation , it was reported that a particular dosage , timing as well as duration of MMPs-inhibition mattered for reduciton of lesion sizes after intracerebral hemorrhage , blood brain barrier permability or neurovascular remodeling in post-stroke period ( Wang and Tsirka , 2005; Zhao et al . , 2006; Sood et al . , 2008; Chang et al . , 2014 ) . Together with the results from healthy animals , our data suggest that the plasticity-promoting effects of MMPs are dependent on a well-balanced level of activation , and if that balance is disturbed , experience-induced plastic changes are compromised . There are several plausible mechanisms by which MMP-inhibition rescues OD-plasticity after stroke in S1 . Stroke influences brain function in numerous ways e . g . , causing inflammation and apoptosis , or disrupting the tightly regulated balance of neuronal inhibition and excitation ( Neumann-Haefelin et al . , 1995; Schiene et al . , 1996; Witte and Stoll , 1997; Que et al . , 1999a; 1999b; Witte et al . , 2000 ) also in perilesional areas ( Murphy and Corbett , 2009 ) . One of the major consequences of ischemic damage is an elevated level of the neurotransmitter glutamate , leading to excitotoxicity and neuronal death ( Lai et al . , 2014 ) . On the other hand , stroke can lead to increased tonic inhibition in the peri-infarct zone , and reducing this inhibition can promote functional recovery ( Clarkson et al . , 2010 ) . In addition , focal ischemia can induce spreading depression within ipsilateral cortex ( Schroeter et al . , 1995 ) and a recent study showed that this phenomenon upregulated MMPs , leading to a breakdown of the blood brain barrier , edema , and vascular leakage , which was suppressed by GM6001 treatment ( Gursoy-Ozdemir et al . , 2004 ) . Thus , it is likely that treatment with GM6001 shortly after the stroke ( as in the present study ) downregulated MMPs , thus reduced spreading depression , improved disturbed excitation/inhibition balance and allowed plastic changes to occur . Although , we observed clear functional rescue of OD-plasticity after GM6001-treatment , there was no apparent effect on the lesion size: the PT-lesions in GM6001-treated mice were not smaller compared to vehicle-injected mice . This is in line with a recent observation , that a different broad-spectrum MMP-inhibitor ( FN-143 ) did not attenuate brain damage resulting from photothrombosis , but partially rescued barrel cortex plasticity ( Cybulska-Klosowicz et al . , 2011 ) . The present results , together with the above mentioned study , are not easy to reconcile with findings where MMP-inhibitors reduced the volume of a brain injury ( Gu et al . , 2005; Wang and Tsirka , 2005; Vandenbroucke and Libert , 2014 ) . The difference might arise from the method used for stroke induction , dosage of inhibitors , timing and duration of the treatment and severity of the lesion . Behavioral vision tests additionally revealed an involvement of MMPs for interocular plasticity during MD . The optomotor reflex is known to be mediated by subcortical pathways ( Giolli et al . , 2006 ) . While visual capabilities measured by optomotry mainly reflect the properties of the retinal ganglion cells that project to these subcortical structures ( Douglas et al . , 2005 ) , daily testing in the optomotor setup after MD induces a cortex-dependent and experience-enabled enhancement of spatial vision through the open eye ( Prusky et al . , 2006 ) . Although inflammation was shown to interfere with the experience-enabled optomotor changes ( Greifzu et al . , 2011 ) , little is known about the cellular origins or signaling pathways responsible for this plasticity paradigm . Here , we found that daily application of the MMP-inhibitor during MD prevented enhancements in both the spatial frequency and contrast sensitivity thresholds of the optomotor reflex of the open eye , while vehicle-treated control mice displayed the typical experience-enabled optomotor improvements . On the other hand , treatment of mice with the same inhibitor once or twice following cortical stroke rescued the impaired optomotor improvements . Thus , in contrast to the OD-plasticity results , rescue of optomotor improvements was present regardless of the duration of the treatment , adding to the idea that separate mechanisms and different neuronal circuits are responsible for OD- and interocular plasticity ( Greifzu et al . , 2011; 2014; Kang et al . , 2013 ) . Together , our results establish a novel function of MMPs in experience-enabled enhancements of the optomotor reflex of the open eye after MD in adult mice . In conclusion , our present data highlight a critical role of MMPs in adult visual plasticity . They further suggest that upregulation of MMP-activity shortly after a cortical lesion compromises experience-induced visual plasticity , which in turn can be rescued by brief MMP-inhibition . Precise regulation of MMP-activity therefore seems to be essential for facilitating plasticity in the adult brain and offers new opportunities in treatment and recovery after stroke . It remains to be determined which particular MMPs account for the present results .
Male adult ( n=49 , P77-91 ) wild-type C57Bl/6J mice housed in standard cages ( 26×20×14 cm ) on a 12 h light/dark cycle with food and water available ad libitum were used . To assess basic visual capabilities and experience-induced changes of all experimental animals , we measured both the spatial frequency threshold and contrast thresholds of the optomotor reflex using a virtual-reality optomotor system ( Prusky et al . , 2004 ) . Briefly , freely moving mice were positioned on a small platform surrounded by four computer monitors forming a square . A rotating virtual cylinder covered with vertical sine wave gratings ( with a drift speed of 12°/s ) were projected on the monitors . The mice reflexively tracked the gratings by head movements as long as they could see the visual stimuli . Spatial frequency at full contrast and contrast at six different spatial frequencies [0 . 031 , 0 . 064 , 0 . 092 , 0 . 103 , 0 . 192 , 0 . 272 cycles/degree ( cyc/deg ) ] were varied by the experimenter until the threshold of tracking was determined . Contrast sensitivity thresholds measured in percent were converted into Michelson contrasts as described previously ( Goetze et al . , 2010 ) . To stimulate experience-enabled enhancement of visual capabilities behavioral testing was performed daily for 7 days in all mice , starting before MD ( day 0 ) in the MD-groups . To induce plasticity and to study the influence of a stroke on experience-dependent plasticity we used monocular deprivation paradigm ( Gordon and Stryker , 1996 ) . The right eye was closed as described previously ( Greifzu et al . , 2011 ) . Mice were anesthetized with 2% isoflurane in a mixture of O2/N2O ( 75/25% ) , their eye-lids were sutured , and mice returned to their home cages for recovery . For stroke experiments MD was performed directly after surgery for induction of a photothrombotic stroke . Animals were checked daily to ensure that the eye remained closed . Mice in which the eye was not closed completely were excluded from the experiment . The broad spectrum MMP-inhibitor GM6001 ( USBiological , Swampscott , Massachusetts ) was used and prepared as previously described with slight modifications ( Gursoy-Ozdemir et al . , 2004; Wang and Tsirka , 2005; Chen et al . , 2009 ) . Specifically , mice were injected intraperitoneally with either 50 mg/kg GM6001 diluted in 3% DMSO and 2% cyclodextrin in saline to a total volume of 200 µl , or with 200 µl vehicle ( 3% DMSO and 2% cyclodextrin in saline ) . This type of systemic application of GM6001 was used before and shown to successfully inhibit MMPs activity in the brain tissue ( Gursoy-Ozdemir et al . , 2004; Wang and Tsirka , 2005; Chen et al . , 2009 ) . For the healthy brain group , the injections were applied daily for 7 days , starting 1 h after MD/no MD . For the stroke group , treatment started 1 h after stroke induction , with an additional injection 24 h after stroke in one group of animals . The cortical stroke was induced by photothrombosis ( PT ) in the left primary somatosensory cortex ( S1 ) using the Rose Bengal technique ( Watson et al . , 1985 ) as described previously ( Greifzu et al . , 2011 ) . The PT-technique was chosen because it allows small lesions with a reproducible localization , which is particularly relevant when studying post-lesion changes in specific cortical areas in proximity of the lesion . Briefly , mice were box-anesthetized with 2% isoflurane in a mixture of 75/25% of O2/N2O; during surgery , anesthesia was maintained with 0 . 8-1% isoflurane delivered via an inhalation mask . The body temperature was maintained at 37°C via a heating pad . The animals’ head was placed in a stereotaxic frame . The skin above the skull was incised and an optic fiber bundle ( aperture: 1 . 0 mm ) mounted on a cold light source ( Schott KL 1500 , Germany ) was positioned 2 mm lateral to the midline and 1 mm posterior to the bregma . 100 μl Rose Bengal ( 0 . 1% in normal saline ) dye was injected into the tail vein . 5 min after the dye injection , the illumination ( lasting 15 min ) began . The skin above the skull was sutured and animals returned to their home cages for recovery . Lesion size was determined at the end of the behavioral and optical imaging experiments by Nissl staining . Following optical imaging experiments , mice were deeply anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital and perfused transcardially with Phosphate-Buffered Saline ( PBS ) ( pH 7 . 4 , 0 . 1M ) for 2 min followed by 4% paraformaldehyde ( pH 7 . 4 ) for 8 min . The brain was removed and postfixed in 4% paraformaldehyde ( pH 7 . 4 ) for one day , then transferred to a 10% sucrose solution in PBS for one day followed by a 30% sucrose solution in PBS for one or two days . The brains were frozen in methylbutane at -40°C and stored at -80°C . 40 μm thick coronal brain sections were cut on a microtome and Nissl-stained . To determine size and location of the cortical lesions every third section was analyzed under the microscope ( Axioskop , Carl Zeiss ) using AxioVision ( AxioVs 40 4 . 5 . 0 . 0 . ) . Animals in which the lesion extended into the white matter or V1 were excluded from further analyses . We used the Shapiro-Wilk test to check if the data are normally distributed within the group and the F-test to look for equality of variance within the groups . All intra- and intergroup comparisons were analyzed either by a two-tailed t-test or one-way ANOVA followed by multiple comparisons Bonferroni correction . The intergroup comparison of the enhancement of the spatial frequency , and contrast sensitivity thresholds were analyzed by two-way ANOVA with repeated measurements and Bonferroni correction . Significance levels were set as *p<0 . 05 , **p<0 . 01 , and ***p<0 . 001 . Data are represented as means ± SEM .
The ability of the adult brain to undergo plastic changes is of particular interest in medicine , especially regarding recovery from injuries or improving learning and cognition . Matrix metalloproteinases ( MMPs ) have been associated with juvenile experience-dependent primary visual cortex ( V1 ) plasticity , yet little is known about their role in this process in the adult V1 . Activation of MMPs is a crucial step facilitating structural changes in a healthy brain; however , upon brain injury , upregulated MMPs promote the spread of a lesion and impair recovery . To clarify these seemingly opposing outcomes of MMP-activation , we examined the effects of MMP-inhibition on experience-induced plasticity in healthy and stoke-affected adult mice . In healthy animals , 7-day application of MMP-inhibitor prevented visual plasticity . Additionally , treatment with MMP-inhibitor once but not twice following stroke rescued plasticity , normally lost under these conditions . Our data imply that an optimal level of MMP-activity is crucial for adult visual plasticity to occur .
When part of the brain becomes damaged as a result of injury or disease – for example , a stroke – other brain regions can sometimes take over from the damaged part . This is one example of a phenomenon called brain plasticity . The strengthening and weakening of connections between neurons that underlies learning and memory is another , less extreme , example of plasticity . While the brain is most plastic during childhood , it remains malleable to some degree throughout life . The brain’s visual system in particular shows robust and predictable plasticity , and so is often used by neuroscientists to study mechanisms behind plasticity . In young rodents , taping one eye shut for a few days causes inputs from that eye to visual areas of the brain to become weaker . Inputs from the open eye meanwhile become stronger , leading to improved vision in the open eye . Such plasticity also occurs in adult rodents , but the eye must be closed for longer to produce an effect . In young animals , this plasticity depends , in part , on enzymes called matrix metalloproteinases ( MMPs ) . These help to regulate a network of proteins called the extracellular matrix , which provides structural support for cells . Pielecka-Fortuna et al . now provide the first evidence that MMP enzymes also contribute to visual plasticity in adult animals . Blocking the activity of MMPs prevented reorganisation of visual areas of the brains of adult mice in response to eye closure , and prevented vision improvements in the open eye . However , blocking MMP in adult mice whose brains had been damaged by a stroke had the opposite effect . Whereas stroke normally prevents visual system plasticity in response to eye closure , treatment with a single dose of MMP blocker rescued this plasticity . Strikingly , these benefits were lost if the mice were given two doses of MMP blocker , rather than one . These experiments show that MMP levels must be within a narrow range to support plasticity . In the healthy adult brain , blocking MMPs impairs plasticity . After stroke , MMP levels are increased and reducing them rescues plasticity . The next challenge is to identify the specific MMP enzymes responsible , and to determine whether these changes can be exploited to improve recovery from stroke .