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

The deletion of matrix metalloproteinase MMP9 is combined here with chronic monocular deprivation (cMD) to identify the contributions of this proteinase to plasticity in the visual system. Calcium imaging of supragranular neurons of the binocular region of primary visual cortex (V1b) of wild-type mice revealed that cMD initiated at eye opening significantly decreased the strength of deprived-eye visual responses to all stimulus contrasts and spatial frequencies. cMD did not change the selectivity of V1b neurons for the spatial frequency, but orientation selectivity was higher in low spatial frequency-tuned neurons, and orientation and direction selectivity were lower in high spatial frequency-tuned neurons. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, including ocular preference and tuning for spatial frequency, orientation, and direction. However, MMP9−/− mice were completely insensitive to plasticity engaged by cMD, such that the strength of the visual responses evoked by deprived-eye stimulation was maintained across all stimulus contrasts, orientations, directions, and spatial frequencies. Other forms of experience-dependent plasticity, including stimulus selective response potentiation, were normal in MMP9−/− mice. Thus, MMP9 activity is dispensable for many forms of activity-dependent plasticity in the mouse visual system, but is obligatory for the plasticity engaged by cMD.


Introduction
The shift in ocular preference in binocular neurons of the primary visual cortex is the canonical model of receptive field plasticity confined to a postnatal critical period. In mice, the critical period for ocular dominance plasticity peaks during the third postnatal week, after one week of binocular vision. During the critical period, MD induces an early decrease in excitatory drive onto parvalbumin-expressing interneurons, which is permissive for subsequent structural and functional changes in V1b [1]. MD during the critical period decreases the strength of deprived eye responses and the density of spines on the apical dendrites of supragranular neurons [2][3][4]. An extension of the MD duration reveals the potentiation of non-deprived inputs, enabled by deprived eye depression [5] and accompanied by a recovery of dendritic spine density [3]. Changes in spatial acuity induced by brief MD during the critical period can be reversed rapidly [6][7][8]. In contrast, chronic MD (cMD) induced prior to the onset of the critical period and maintained until adulthood induces a significant reduction in the spatial acuity of the chronically-deprived eye, which is highly resistant to recovery [9][10][11].

Extracellular Proteolysis Enables Plasticity in Immature and Mature Circuits
We have previously shown that the visual deficits induced by cMD can be reversed in adults by dark exposure followed by light reintroduction [11]. Activation of extracellular proteolysis has been repeatedly shown to reactivate synaptic plasticity in the adult brain [12][13][14]. Accordingly, an enzyme that degrades the chondroitin sulfate proteoglycans of the extracellular matrix (ECM) reactivates robust ocular dominance plasticity in adult rat V1 [15,16]. Similarly, hyaluronidase, which cleaves the hyaluronic acid backbone of ECM, enhances short-term plasticity in mature cultured neurons and rescues plasticity in adult MMP9 −/− mice [17,18]. Other manipulations that attenuate the perineuronal net specializations of the ECM, such as deletion of cartilage link protein, eliminate the constraint on ocular dominance plasticity with age [19].
MMP9 is of particular interest, as MMP9 activity has been repeatedly shown to couple synaptic activity to structural plasticity in both mature and immature circuits. In the adult hippocampus, theta burst stimulation increases MMP9 activity, and MMP9 inhibition blocks the maintenance of synaptic potentiation and several forms of learning [20][21][22][23]. In cultured hippocampal neurons, the pharmacological induction of synaptic potentiation increases MMP9 activity, specifically at enlarging or emerging spines [24]. In hippocampal slices from young rats, the application of MMP9 is sufficient to potentiate the size and strength of CA3-CA1 excitatory synapses and enable plasticity onto CA2 neurons [25,26]. Many manipulations, including the administration of anti-depressants, reveal an inverse correlation between MMP9 activity and the density of perineuronal net on inhibitory neurons [27]. However, activation of MMP9 has also been implicated in synaptic weakening and may represent an obligatory step for many forms of structural and functional plasticity [28,29]. The reactivation of structural and functional plasticity in adult mouse V1 by light reintroduction following dark exposure requires an increase in activity of endogenous MMP9 [18]. The threshold for visually-evoked activation of perisynaptic MMP9 is regulated by visual experience and lowered by dark exposure at thalamo−cortical, but not cortico−cortical, synapses in adult V1 [30].
Here, we ask if deletion of MMP9 impacts neuronal response properties in binocular and chronically monocularly deprived adults. We find that visual response strength and selectivity is normal in adult MMP9 −/− mice. However, the changes in neuronal response strength and selectivity induced by cMD in wild-type (WT) mice are absent in MMP9 −/− mice.

cMD Decreases the Strength of the Deprived Eye Pathway of WT but Not MMP9 −/− Mice
The critical period for ocular dominance plasticity peaks during the third postnatal week in mice, one week after eye opening. To quantify the impact of a longterm asymmetry in the quality of vision across the two eyes initiated immediately after eye opening, we measured visually-evoked calcium transients in layer 2/3 neurons of awake adult C57BL/6J mice (WT) following chronic monocular deprivation from P14 until adulthood (>P90, up to P170). 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, Figure 1A).
Visually-evoked calcium transients in response to drifting square wave gratings (0.05 cycle/degree (cpd),100% contrast) in 12 directions were analyzed to identify the neuronal response preferences in normal-reared (NR) adult (>P90) wild type (WT) subjects ( Figure 1B). The mean ∆F/F in response to the preferred stimulus was 46 ± 2%, which is predicted to translate to 5-6 action potentials [31] and is consistent with previous reports of visually-evoked firing rates in V1 of adult mice [32][33][34]. The majority of neurons were binocular (ocular dominance score (ODS) = (C − I)/(C + I)), ODS = ±1) and preferred contralateral (ODS > 0.2) eye stimulation ( Figure 1C,D). cMD decreased the preference for 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). Monocular visual responses (ΔF/F) following cMD revealed the strength of response to the stimulation of the deprived, contralateral eye was significantly reduced but the strength of response to the stimulation of the non-deprived, ipsilateral eye was unchanged (mean ± SEM: 46 ± 2% for NR, 22 ± 1% for cMD, Student's t-test, * p = 8.3 × 10 −29 for contra, 26 ± 1% for NR, 26 ± 1% for cMD, p = 0.91 for ipsi, 365 and 201 neurons for NR and cMD, respectively, Figure 1F). Thus, the response to cMD differs from the response to MD initiated during the peak of the critical period, which is biphasic and includes strengthening of the non-deprived input [2,35]. 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 ∆F/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. Monocular visual responses (∆F/F) following cMD revealed the strength of response to the stimulation of the deprived, contralateral eye was significantly reduced but the strength of response to the stimulation of the non-deprived, ipsilateral eye was unchanged (mean ± SEM: 46 ± 2% for NR, 22 ± 1% for cMD, Student's t-test, * p = 8.3 × 10 −29 for contra, 26 ± 1% for NR, 26 ± 1% for cMD, p = 0.91 for ipsi, 365 and 201 neurons for NR and cMD, respectively, Figure 1F). Thus, the response to cMD differs from the response to MD initiated during the peak of the critical period, which is biphasic and includes strengthening of the non-deprived input [2,35].
In MMP9 −/− mice, the expression of GCaMP6s and the neuronal response preferences in normal-reared (NR) adult (>P90) subjects were comparable to WTs (Figure 2A,B).

cMD Decreases the Strength, but not the Selectivity of the Deprived Eye Pathway of WT but not MMP9 −/− Mice
To quantify the impact of cMD on the selectivity of neurons in mouse V1b for visual stimulus spatial frequency and contrasts, we presented low spatial frequency stimuli (0.05 cycle/degree (cpd)) at contrasts ranging from 20-100%, and high contrast stimuli (100%) at spatial frequencies ranging from 0.05 to 0.4 cpd. In NR adult WTs, the magnitudes of evoked calcium transients were positively correlated with visual stimulus contrasts (y = 11 ln(x) − 7, R 2 = 0.92; Figure 5, top left dark blue). cMD significantly reduced the magnitude of the visually-evoked response across the range of contrasts (repeated measures ANOVA, F = 9.7, * p = 0.014, n = 5 subjects; Figure 5B, top left light blue). In NR adult MMP9 −/− mice, response magnitude and stimulus contrast were also positively correlated (y = 16 ln(x) − 29, R 2 = 0.82) and comparable to NR WT, as predicted ( Figure 5B, top right red [18]). However, cMD did not impact the amplitude of the evoked response at any visual stimulus contrast (repeated measures ANOVA, F = 0.20, p = 0.67, n = 5 subjects; Figure 5B, top right orange).
Similarly, in NR WT mice, the response magnitude was negatively correlated with the visual stimulus spatial frequency (y = −8 ln(x) + 22, R 2 = 0.98; Figure 5B, bottom left dark blue). cMD significantly reduced the magnitude of the visually-evoked response across the range of spatial frequencies (repeated measures ANOVA, F = 8.3, * p = 0.02, n = 5 subjects; Figure 5B, bottom left light blue). In NR adult MMP9 −/− mice, response magnitude and visual stimulus spatial frequency were also negatively correlated (y = −9 ln(x) + 18, R 2 = 0.88) and comparable to WTs ( Figure 5B, bottom right red). Again, cMD did not  To quantify the impact of cMD on the selectivity of neurons in mouse V1b for visual stimulus spatial frequency and contrasts, we presented low spatial frequency stimuli (0.05 cycle/degree (cpd)) at contrasts ranging from 20-100%, and high contrast stimuli (100%) at spatial frequencies ranging from 0.05 to 0.4 cpd. In NR adult WTs, the magnitudes of evoked calcium transients were positively correlated with visual stimulus contrasts (y = 11 ln(x) − 7, R 2 = 0.92; Figure 5, top left dark blue). cMD significantly reduced the magnitude of the visually-evoked response across the range of contrasts (repeated measures ANOVA, F = 9.7, * p = 0.014, n = 5 subjects; Figure 5B top left light blue). In NR adult MMP9 −/− mice, response magnitude and stimulus contrast were also positively correlated (y = 16 ln(x) − 29, R 2 = 0.82) and comparable to NR WT, as predicted ( Figure 5B top right red [18]). However, cMD did not impact the amplitude of the evoked response at any visual stimulus contrast (repeated measures ANOVA, F = 0.20, p = 0.67, n = 5 subjects; Figure 5B top right orange).
Similarly, in NR WT mice, the response magnitude was negatively correlated with the visual stimulus spatial frequency (y = −8 ln(x) + 22, R 2 = 0.98; Figure 5B bottom left dark blue). cMD significantly reduced the magnitude of the visually-evoked response across the range of spatial frequencies (repeated measures ANOVA, F = 8.3, * p = 0.02, n = 5 subjects; Figure 5B bottom left light blue). In NR adult MMP9 −/− mice, response magnitude and visual stimulus spatial frequency were also negatively correlated (y = −9 ln(x) + 18, R 2 = 0.88) and comparable to WTs ( Figure 5B bottom right red). Again, cMD did not impact the magnitude of the evoked responses at any stimulus spatial frequency (repeated measures ANOVA, F = 0.33, p = 0.58, n = 5 subjects; Figure 5B bottom right orange).
Despite the significant decrease in amplitude of visually-evoked responses at all SFs in WTs following cMD, SF tuning was unchanged, with~40% of neurons in both NR and cMD conditions preferring 0.05 cpd visual stimuli ( Figure 5C). A similar preference for low spatial frequency visual stimuli was observed in MMP9 −/− mice, which was unchanged by cMD ( Figure 5C). Accordingly, the mean peak neuronal response binned by SF did not differ across the genotypes or visual history (mean ± SEM: 0.17 ± 0.01 cpd, 0.16 ± 0.02 cpd, 0.16 ± 0.04 cpd, 0.11 ± 0.003 cpd for NR WT, cMD WT, NR MMP9 −/− , cMD MMP9 −/− , respectively, one-way ANOVA, F = 0.89, p = 0.47, n = 5 subjects; Figure 5D). Thus, cMD impacts the strength, but not the selectivity, for visual stimulus spatial frequency and contrast in WT, but not MMP9 −/− mice. impact the magnitude of the evoked responses at any stimulus spatial frequency (repeated measures ANOVA, F = 0.33, p = 0.58, n = 5 subjects; Figure 5B, bottom right orange). Despite the significant decrease in amplitude of visually-evoked responses at all SFs in WTs following cMD, SF tuning was unchanged, with ~40% of neurons in both NR and cMD conditions preferring 0.05 cpd visual stimuli ( Figure 5C). A similar preference for low spatial frequency visual stimuli was observed in MMP9 −/− mice, which was unchanged by cMD ( Figure 5C). Accordingly, the mean peak neuronal response binned by SF did not differ across the genotypes or visual history (mean ± SEM: 0.17 ± 0.01 cpd, 0.16 ± 0.02 cpd, 0.16 ± 0.04 cpd, 0.11 ± 0.003 cpd for NR WT, cMD WT, NR MMP9 −/− , cMD MMP9 −/− , respectively, one-way ANOVA, F = 0.89, p = 0.47, n = 5 subjects; Figure 5D). Thus, cMD impacts the strength, but not the selectivity, for visual stimulus spatial frequency and contrast in WT, but not MMP9 −/− mice.

cMD Regulates Visual Response Strength and Selectivity in WT but not MMP9 −/− Mice
We demonstrate that manipulations of postnatal visual experience disassociate MMP9-dependent and -independent forms of plasticity in the murine visual system. In WT mice, chronic monocular deprivation from eye opening to adulthood reduces the

cMD Regulates Visual Response Strength and Selectivity in WT but Not MMP9 −/− Mice
We demonstrate that manipulations of postnatal visual experience disassociate MMP9dependent and -independent forms of plasticity in the murine visual system. In WT mice, chronic monocular deprivation from eye opening to adulthood reduces the strength of deprived eye visually-evoked responses to all stimulus spatial frequencies and contrasts. Furthermore, cMD impacts the neuronal selectivity for orientation and direction, but not spatial frequency. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, including ocular preference and tuning for spatial frequency, orientation, and direction. However, cMD did not impact the strength or the tuning of the neuronal responses evoked by deprived eye stimulation. Nonetheless, other forms of activitydependent plasticity persist in MMP9 −/− mice, including eye specific segregation of RGC axons in the dLGN, the expression and maintenance of contralateral bias of visually-evoked responses, and robust stimulus selective response potentiation induced by repetitive visual stimulation. Thus, MMP9 is obligatory for the plasticity engaged by cMD at eye-opening, but is dispensable for many other forms of plasticity that refine and maintain the mouse visual system.

cMD and MD during the Critical Period Recruit Distinct Forms of Plasticity
Changes in visual response strength induced by cMD were limited to deprived eye responses, and therefore differed from the response to MD at the peak of the critical period, in which deprived eye depression is followed by an increase in the strength and selectivity of non-deprived eye responses [2,35,50,51]. The decrease in the strength of visual responses following cMD was observed across a range of spatial frequencies and contrasts. The absence of a change in SF tuning selectivity following cMD was unexpected, given the significant reduction in deprived eye spatial acuity demonstrated by performance in a visual discrimination task [52]. A disassociation between spatial frequency tuning of individual V1 neurons and spatial acuity has also been described in strabismic macaques [53][54][55].
Tuning for SF is broader than tuning for orientation and direction [56,57]. Although the tuning for different aspects of the visual stimulus can be independently regulated [58], tuning to high spatial frequencies is associated with contralateral eye dominance [37]. cMD decreased orientation and direction selectivity in neurons tuned to high spatial frequencies, indicating co-regulation of eye preference, orientation and direction selectivity by cMD. In contrast, the increase in direction selectivity in neurons tuned to low spatial frequencies observed following cMD is predicted by the increase in binocularity [59].

Assessment of Binocularity in Mouse V1b
The characterization of neuronal responses in mouse V1b as binocular has recently come under scrutiny [37,[59][60][61][62]. Central to this debate is the role of inclusion criteria in the definition of visually-evoked GCaMP activity, as the adaptation of high-threshold inclusion criteria excludes contributions from the non-dominant eye [51]. However, in our hands, increasing the threshold for the inclusion of visually-evoked activity from the non-dominant eye did not reduce the number of neurons categorized as binocular in any experimental populations of adult mice. Our classification of the majority of visually evoked responses in V1b as binocular is consistent with a large body of work utilizing single unit recordings to measure spiking output in response to contra and ipsilateral eye stimulation [63][64][65][66][67][68]. The expression of binocularity in mouse V1b may also be impacted by the strategy for the expression of GCaMP.

Structural and Functional Plasticity Induced by MMP9 Activity
Although MMP9 activity is confined to excitatory synapses, it is differentially expressed across synapse classes in adult mouse V1b. The baseline MMP9 activity is high at cortico-cortical relative to thalamo-cortical synapses, which may contribute to differences in the rates of dendritic spine turnover [18,69]. Importantly, light reintroduction after dark exposure increases the activity of MMP9 preferentially at thalamocortical synapses in adult V1b, and the threshold for the induction of MMP9 activity at these synapses is regulated by the visual experience [30].
The targets of MMP9 that mediate the response to manipulations of visual experience have yet to be defined, but include a number of perisynaptic extracellular molecules that regulate synaptic plasticity [25,70,71]. Potential targets include the dendritic cell adhesion molecule telencephalin [72], which negatively regulates spine maturation [73]. MMP9 deletion prevents the translocation of telencephalin from spine heads to shafts over development [74]. Telencephalin cleavage following tetanic simulation or NMDAR activation, and the spine enlargement that is characteristic of long-term potentiation, are blocked by MMP inhibition [71,75]. However, the long, thin spines characteristic of mouse model of fragile-X-mental-retardation (FMR1 −/− mice) are normalized by MMP9 deletion [76,77]. In MMP9 −/− , dendritic spine density is reduced and extant spines have a normal morphology [18,47], suggesting that a subset of spiny synapses may be subject to regulation by MMP9. Interestingly, cMD in WTs reduced the spine density to the level observed in MMP9 −/− mice, suggesting that visual experience may promote MMP9-sensitive synaptogenesis. Furthermore, in adult mouse V1b, levels of the MMP9 substrate aggrecan are decreased by dark exposure and subsequent light reintroduction, and blocked by the pharmacological inhibition of MMP9 [18,78]. Similarly, the intensity of stained perineuronal nets (PNNs), specialization of the ECM that enshrouds PV+ interneurons, is decreased by brief MD (2 days) during the peak of critical period in WT, but not MMP9 −/− mice [47]. PNN density, in turn, regulates the excitatory drive onto PV INs, suggesting that MMP9 may play a role in the regulation of disinhibition in V1b [1].

Developmental Expression of MMP9
MMP9 expression peaks at P0 [79,80], nonetheless our findings suggest that MMP9 is dispensable for several forms of activity-dependent synaptic plasticity expressed in perinatal development. Eye-specific segregation of RGC axons, which is driven by spontaneous retinal activity prior to eye opening, was normal in MMP9 −/− mice [81,82]. Stimulus selectivity, which is present at eye opening, was grossly normal in adult MMP9 −/− mice. Furthermore, cMD induced a significant decrease in the number of thalamocortical afferents on layer 4 pyramidal neuron cell bodies in WTs but not MMP9 −/− mice.
The presence of normal contralateral bias of neurons in V1b of MMP9 −/− mice demonstrates that the underlying experience-dependent strengthening of contralateral-eye responses is independent of MMP9 activity [2,61], consistent with distinct mechanisms for the development and plasticity of ocular preference [37,62,[83][84][85]. Similarly, robust stimulusselective response potentiation, known to engage mechanisms, distinct from those engaged by MD, also persists in MMP9 −/− mice [2,49,86]. The insensitivity of MMP9 −/− mice to chronic MD initiated at eye opening suggests that the visual system cannot compensate for the absence of MMP9, and is consistent with the observation that MMP2 activity is not elevated in MMP9 −/− mice [18,87].
The contribution of MMP9 to specific forms of synaptic plasticity is predicted to emerge with age, in parallel with the maturation of critical MMP9 substrates. Interestingly, deprived-eye depression in response to MD during the CP persisted in the presence of an MMP9 inhibitor, but the subsequent non-deprived eye strengthening was absent [50]. Non-deprived eye strengthening was also inhibited by manipulations that may reduce MMP9 expression, including deletion of tumor necrosis factor α [88,89]. MMP9 inhibition blocks the enhancement of spatial acuity and contrast sensitivity induced by repetitive engagement of the optomotor reflex in adults [90], although stimulus selective response potentiation is intact in MMP9 −/− mice. Together, this suggests that MMP9 is obligatory for the plasticity engaged by cMD from eye opening, but it is dispensable for other forms of plasticity that refine and maintain the mouse visual system.

Subjects
C57BL/6J and MMP9 −/− (007084, B6 background) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Equal numbers of adult (>P90, up to P170) males and females were used. The animals were raised in a 12 h light/dark cycle. All procedures conformed to the guidelines of the University of Maryland Institutional Animal Care and Use Committee. Experiments were performed (or subjects were sacrificed) 6 h into the light phase of a 12:12 h light/dark cycle.

Chronic Monocular Deprivation
Chronic monocular deprivation was performed at eye opening (P14). Subjects were anesthetized with 2.5% isoflurane in 100% O 2 . The margins of the upper and lower lids of one eye were trimmed and sutured together using a 5-0 suture kit with polyglycolic acid (CP Medical, Norcross, GA, USA). Subjects were returned to their home cage after recovery at 37 • C for 1-2 h. Subjects were disqualified in the event of suture opening.

Immunohistochemistry
Subjects were anesthetized with 4% isoflurane in O 2 and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brain was postfixed in 4% PFA for 24 h, followed by 30% sucrose for 24

Golgi staining and Dendritic Spine Density Analysis
Golgi staining was performed with FD Rapid GolgiStain Kit (FD Neuro Technologies, Columbia, MD, USA), as per the manufacturer's instructions. The brains were immersed in solution A + B for 7 days at room temperature, and then transferred to solution C for 3 days at 4 • C. Coronal sections (100 µm) were made with a Leica VT100S vibrating microtome and mounted on gelatin-coated slides (FD Neuro Technologies). Neurolucida (MBF Bioscience, St. Albans, VT) with an Olympus BX61 light microscope was used to trace the morphologies of the dendrites and spines. Using a 40× lens (Olympus, Center Valley, PA, USA, Plan N, NA = 0.65), the arbors of basolateral dendrites of layer 4 neurons were traced, followed by sholl analysis in a Neuroexplorer (MBF Bioscience, Willston, VT, USA) to identify the region 75 to 100 µm from the soma, which were then traced using a 100× lens (Olympus Plan N Oil, NA = 1.25). Dendritic protrusions with diameters ≥ 50% than the dendritic shaft diameter were classified as spines, and counted in Neuroexplorer.

Intraocular Injections of Anterograde Tracer
Subjects were anesthetized with 2.5% isoflurane in 100% O 2 . Fluorescently-labeled cholera toxin subunit B solutions (CTB-Alexa 488, or CTB-Alexa 555, Invitrogen, 1 mg/mL dissolved in distilled water, 4 µL) were injected into the temporal region of each eye with a Hamilton syringe [91]. Subjects were perfused 48 h after the injection with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brains were post-fixed with 4% PFA for 24 h followed by 30% sucrose for 24  Confocal images were acquired on a Zeiss LSM 710 confocal microscope with a 10× lens (Zeiss Plan-neofluar 10×/0.30, NA = 0.30). Maximal intensity projections (MIP) of z-stack images (three images with 10 µm interval, at a resolution of 512 × 512 pixel representing 772.9 µm × 772.9 µm) were used for the analysis. Three successive sections through the middle of dLGN were used to quantify the areas of contra-and ipsi-lateral projection defined by fluorescence thresholding (autothreshold + 20) in Fiji. dLGN occupancy was calculated as the ratio of each projection to the total dLGN area.
A cranial window consisting of three pieces of coverslips (two 3 mm diameter coverslips glued with optical adhesive (Norland71, Edmund Optics, Barrington, NJ, USA) to a 5 mm diameter coverslip) was implanted following a method described by Goldey et al. [92]. The gap between the skull and glass was sealed with a silicone elastomer (Kwik-Sil). Instant adhesive Loctite 454 (Henkel, Richmond, VA, USA) was used to adhere an aluminum head post to the skull, and to cover the exposed skull, and then black dental cement (iron oxide powder, AlphaChemical, Stoughton, MA, USA, mixed with white powder, Dentsply) was used to coat the surface to minimize light reflections. Subjects were imaged after more than at least 3 weeks of recovery.

Two-Photon Imaging
Awake subjects were placed in a holding tube and immobilized by a head post clamp. Prior to the first imaging session, the subjects were placed in the holding tube at least twice for >30 min for habituation. A shield was placed around the gap between the cranial window and the objective lens to block light from the visual stimuli during imaging. A two-photon microscope (ThorLabs, Sterling, VA, USA) controlled by ThorImageLS software with a 16× NA 0.8 water immersion objective lens (Nikon, Melville, NY, USA) was used to acquire the time lapse fluorescence images. A Chameleon Vision Ti: Sapphire laser (Coherent, Santa Clara, CA, USA) was tuned to 940 nm for excitation, and the emitted photons were directed through a 525/50 nm bandpass filter onto a GaAsP photomultiplier tube. The field of view was 370 × 370 µm (512 × 512 pixels) at 150 to 250 µm from the brain surface. The images were acquired at 30 Hz by bidirectional scanning. Before measuring responses to the visual stimuli, the pattern of the GCaMP signal was evaluated to confirm the absence of toxic expression effects [36]. The neurons expressing GCaMP showed a high cytosolic signal with nucleus exclusion ( Figure 3A). Time after viral injection or the age of subject did not impact ∆F/F, CBI or % of responsive neurons ( Figure 3B, see below for method). If the mean ∆F value during visual stimulation at any direction exceeded 3 × STD of baseline (F 0 ) for 40% or more of trials during either contra or ipsi eye stimulation, the neuron was defined as visually responsive. If this criterion was met in response to stimulation of both eyes, the neuron was designated binocular. Using this criterion, the majority of visually-responsive neurons were binocular in both genotypes. Raising the threshold for the definition of visually responsive did not change the % of neurons defined as binocular ( Figures 1D and 2D).
For ocular dominance measurements, subjects received monocular visual stimuli controlled by the PsychToolBox plugin in MATLAB, (random order of five repeats of 12 orientations of square grating drifting at 1 Hz) at 0.05 cycle/degree (cpd), 100% contrast (28 cd/m 2 ) for 2.5 s interleaved with 2.5 s intensity-matched grey scale) delivered by a 23" display (Acer LCD Monitor, San Jose, CA, USA) 28 cm in front of the eyes. To measure the strength of the visual responses across contrasts and spatial frequencies, visual stimuli were delivered at a range of contrasts (20, 40, 60, 80, and 100%) at 0.05 cycle/degree or a range of spatial frequencies (0.05, 0.10, 0.20, 0.30 and 0.40 cpd) at 100% contrast to the eye contralateral to the cranial imaging window. Movement artifacts were corrected using the Suite2P package [93], using the average intensity of the full image stack as a template. Ring-like regions of interest (ROIs) in the soma area were selected using the averaged image after motion correction, and neuropil subtraction (correction factor, 0.8) was performed to calculate the response amplitude, ∆F/F [31]. To calculate ∆F/F = (F − F 0 )/F 0 , where F corresponds to the fluorescence intensity at a given time point and F 0 corresponds to mean fluorescent intensity during 1 s before the visual stimulus onset.

Chronic In Vivo Recordings for Stimulus-Selective Response Potentiation (SRP)
Adult mice were anesthetized with 2.5% isoflurane in 100% O 2 and a handmade 1.2 mm 16-channel shank electrode was implanted in V1b (stereotaxic coordinates from Bregma: anterior/posterior, 2.8 mm; medial/lateral, 3.0 mm; dorsal/ventral, 1.2 mm). After recovery of the righting reflex, animals were administered buprenorphine (0.1 mg/kg, i.p.) for post-surgical analgesia and returned to their home cage. Awake, head fixed recordings began more than 7 days after implantation. Activity was evoked through passive viewing of 200 × 1 s trials of square-wave gratings (0.05 cpd, 100% contrast, reversing at 1 Hz), via MATLAB with Psychtoolbox extensions [95,96], and a grey screen of equal luminance (26 cd/m 2 ) for spontaneous responses. The initial visual stimulation was presented for 200 s at a single orientation, then 24 h later, the familiar and novel stimuli were presented for 200 s, respectively [32].

Statistical Analyses
An unpaired two-tailed Student's t-test and Mann−Whitney test were used to determine the significance between two independent experimental groups, and a paired Student's t-test was used for two measurements within the same subjects. A one-way ANOVA was used to determine significance between three or more independent experimental groups, as well as repeated measures ANOVA for more than two measures within the same subjects, followed by a Tukey−Kramer post hoc when ANOVA was p < 0.05 (JASP). A Kolmogorov−Smirnov (KS) test was used to determine the significance between the distribution of two independent data sets.

Conclusions
The aim of this study was to identify the contributions of the matrix metalloproteinase MMP9 to postnatal experience-dependent plasticity in the murine visual system. Chronic monocular deprivation initiated at eye opening significantly decreased the strength of the deprived-eye visual responses to all stimulus contrasts and spatial frequencies. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, includ-ing ocular preference, as well as tuning for spatial frequency, orientation, and direction. MMP9 −/− mice were also completely insensitive to plasticity engaged by cMD. Other forms of experience-dependent plasticity, including stimulus selective response potentiation, were normal in MMP9 −/− mice. Thus, the MMP9 activity was dispensable for many aspects of maturation of the visual system, but was obligatory for the plasticity engaged by cMD.