In mammals the development of the visual system may be altered during a sensitive period by modifying the visual input to one or both eyes. These plastic processes are reduced after the end of the sensitive period. It has been proposed that reduced levels of plasticity are at the basis of the lack of recovery from early visual deprivation observed in adult animals. A developmental downregulation of experience-dependent regulation of histone acetylation has recently been found to be involved in closing the sensitive period. Therefore, we tested whether pharmacological epigenetic treatments increasing histone acetylation could be used to reverse visual acuity deficits induced by long-term monocular deprivation initiated during the sensitive period. We found that chronic intraperitoneal administration of valproic acid or sodium butyrate (two different histone deacetylases inhibitors) to long-term monocularly deprived adult rats coupled with reverse lid-suturing caused a complete recovery of visual acuity, tested electrophysiologically and behaviorally. Thus, manipulations of the epigenetic machinery can be used to promote functional recovery from early alterations of sensory input in the adult cortex.
The molecular mechanisms underlying the effects of MD are only partially known. Several factors acting extracellularly and intracellularly at different stages of the plasticity process have been proposed (Medini & Pizzorusso, 2008; Tropea et al., 2009). Large-scale analyses of gene expression in the visual cortex of visually deprived mice, either dark-reared or monocularly deprived, have shown modifications of the expression levels of many genes (Prasad et al., 2002; Lachance & Chaudhuri, 2004; Ossipow et al., 2004; Majdan & Shatz, 2006; Tropea et al., 2006), suggesting that at least part of the consequences of visual deprivations on cortical circuits could involve modifications of mechanisms controlling experience-regulated gene expression. Epigenetic mechanisms regulate gene expression without altering the genetic code itself, and include covalent modifications on histone proteins. It is increasingly clear that epigenetic modifications are very important for neural function. Indeed, alterations of epigenetic mechanisms have been observed in several cognitive disorders (Graff & Mansuy, 2009), and treatments with drugs targeting epigenetic mechanisms showed beneficial effects in animal models of several neural diseases (Tsankova et al., 2007). Among the various histone modifications involved in epigenetic control of gene transcription, histone acetylation has been involved in activation of gene expression in response to drugs of abuse and environmental stimulation in neural cells. Furthermore, experience-dependent histone acetylation has been implicated in synaptic plasticity and multiple aspects of learning and memory (Borrelli et al., 2008; Fagiolini et al., 2009; Graff & Mansuy, 2009; Sweatt, 2009). Experience-dependent histone phosphorylation and acetylation has also been involved in visual cortical plasticity (Putignano et al., 2007). Visually induced histone phosphoacetylation was found to be developmentally downregulated in correlation with the downregulation of plasticity occurring after the SP. Pharmacological increase in histone acetylation was able to enhance the effects of MD in adult mice. This observation prompted us to hypothesize that the increased plasticity obtained with drugs inducing histone acetylation could promote recovery of visual acuity in adult amblyopic animals. To test this possibility we investigated whether treatments with pharmacological agents known to increase histone acetylation coupled with RS could be used to recover the visual acuity deficit induced by long-term MD. We found that adult rats subjected to MD during the SP treated with two different broadly specific inhibitors (valproic acid and sodium butyrate) of histone deacetylases (HDACs) could completely recover the loss of visual acuity assessed electrophysiologically using visual evoked potentials (VEPs). Using a protocol of longitudinal assessment of visual acuity, we found that the deprived eye of adult long-term MD rats treated with valproic acid recovered normal levels of behavioral visual acuity.
Materials and methods
Animals were used in accordance with protocols approved by the Italian Minister for Scientific Research. All experimental procedures conformed to the European Communities Council Directive number 86/609/EEC. Forty-one Long–Evans black hooded rats (Charles River, Italy) were used for the behavioral, electrophysiological and biochemical experiments. The animals were housed in groups of two or three in a room with a temperature of 21°C and a 12-h light–dark cycle, and food and water available ad libitum.
Visual deprivation and animal treatment
Rats were anesthetized with avertin (1 ml/hg) and MD was performed through eyelid suturing at postnatal day (P)21 (Pizzorusso et al., 2006). Lid margins were trimmed and sutured with 6-0 silk. Animals were allowed to recover from anesthesia and were returned to their cages. Eyelid closure was inspected daily until complete cicatrisation. Rats showing occasional lid reopening (observed with a surgical microscope) were not included in the experiments. Adult rats (P120-130) were then subjected to RS, under anesthesia. The long-term deprived eye was reopened using thin scissors, while the other eye was sutured shut. Great care was taken to reopen the eye and to prevent opacities of the reopened eye by topical application (twice daily) of Tobradex cream (tobramycin and dexamethason; Alcon, Italy) onto the cornea during the first 3 days of RS. Again, subjects showing spontaneous lid reopening or eye anomalies were excluded. After 5 days of recovery from RS surgery, rats treated with daily intraperitoneal cronic administration (for an average of 25 days) of valproic acid (300 mg/kg in 0.9% saline at a concentration of 50 mg/mL) or sodium butyrate (1.2 g/kg in 0.9% saline at a concentration of 240 mg/mL) or vehicle (0.9% saline). Behavioral sessions began 2 h after the injection.
After decapitation, brains were removed rapidly and frozen on dry ice. A cortical area corresponding to visual cortex was then homogenized in a hypotonic lysis buffer containing (in mm) Tris (pH 7.5), 10; EDTA, 1; sodium pyrophosphate, 2.5; b-glycerophosphate, 1; sodium orthovanadate, 1; and phenylmethylsulfonylfluoride, 1; with aprotinin, 10 mg/mL; leupeptin (Sigma, Italy), 10 mg/mL; and igepal CA-630, (Sigma Aldrich, Italy) 1%. Histones were extracted from the nuclear fraction by the addition of five volumes of 0.2 m HCl and 10% glycerol, and the insoluble fraction was pelleted by centrifugation (18 000 g; 30 min; 4°C). Histones in the acid supernatant were precipitated with ten volumes of ice-cold acetone followed by centrifugation (18 000 g; 30 min; 4°C). The histone pellet was then resuspended in 9 m urea. Protein concentration was determined by Biorad assay (Biorad, Italy). Each sample was boiled, and 10 mg/lane was loaded into 12% acrylamide gels using the Precast Gel System (Biorad, Italy). Samples were blotted onto nitrocellulose membrane (Amersham, Bucks, UK), blocked in 4% nonfat dry milk in Tris-buffered saline for 1 hr, and then probed with AcH3 and H3 antibodies (Upstate, NY, USA). All antibodies were diluted in Tween Tris Buffered Saline (TTBS) and 2% milk or 2% bovine serum albumin (BSA) and incubated overnight at 4°C. Blots were then rinsed for 20 min in TTBS, incubated in horseradish peroxidase-conjugated antimouse or antirabbit (1 : 3000; Biorad, Italy, in 2% milk or 2% BSA and TTBS), rinsed, incubated in enhanced chemiluminescent substrate (Biorad), and exposed to film (Hyperfilm; Amersham Biosciences, Europe). Films were scanned, and densitometry was analyzed through ImageJ free software (http://rsb.info.nih.gov/ij/).
To minimize variability, each sample was loaded in parallel in two lanes and two gels were run simultaneously on the same apparatus. For each gel, the corresponding filters obtained after blotting were cut in two in order to obtain in each filter a complete series of samples. One of the two filters was reacted with an antibody for the modified protein (AcH3) and the other with an antibody insensitive to the target protein modifications (H3). The densitometric quantification of the band corresponding to AcH3 was then normalized to the value obtained for the total amount of H3 from the same gel (Putignano et al., 2007).
Animals treated with valproic acid or control were anesthetized with urethane (0.6 ml/hg; 20% solution in saline; Sigma) by i.p. injection and placed in a stereotaxic frame allowing full viewing of the visual stimulus. Additional doses of urethane were used, if necessary, to keep the anesthesia level stable throughout the experiment. Body temperature was monitored with a rectal probe and maintained at 37.0°C with a heating pad. Immediately before the recording session the lids were cut, and the eye washed with saline and carefully inspected to verify that the surgical procedure had not caused any damage Both eyes were fixed and kept open by means of adjustable metal rings surrounding the external portion of the eye bulb. A hole was drilled bilaterally in the skull, overlying the binocular portion of the primary visual cortex (binocular area Oc1B) After exposure of the brain surface, the dura was removed. A glass micropipette (4 μm tip, 3 m NaCl filling) was inserted perpendicularly to the stereotaxis plane into the cortex controlateral to the measured eye. Microelectrodes were inserted 4.8–5.1 mm lateral to the intersection between sagittal and lambdoid sutures and advanced 100 μm within the cortex. Electrical signals were amplified (5000- to 20 000-fold), bandpass-filtered (0.3–100 Hz), and averaged (at least 50 blocks of two events each) in synchrony with the stimulus contrast reversal. Transient VEPs in response to abrupt contrast reversal (0.7 Hz) were evaluated in the time domain by measuring the peak-to-baseline amplitude and peak latency of the major component. VEPs in response to a blank stimulus were also frequently recorded to give an estimate of the noise. Visual stimuli were horizontal sinusoidal gratings of different spatial frequency and contrast generated by a VSG2/2 card (Cambridge Research System, Cheshire, UK) and presented on a computer display (mean luminance, 25 cd/m2) placed 20 cm in front of the animal. Visual acuity of each eye was measured in the contralateral cortex. VEP amplitude decreases with increasing stimulus spatial frequency; visual acuity was obtained by extrapolation to zero amplitude of the linear regression through the last four to five data points in a curve where VEP amplitude is plotted against log spatial frequency (Pizzorusso et al., 2006).
Behavioral assessment of visual acuity
Visual acuity was determined with visual water task by following the method of Prusky et al. (2000). The apparatus consisted of a Plexiglas box filled with water, partially divided at one end into two arms by a divider. Visual stimuli, which were generated on computer monitors, were at the end of each arm and consisted of sine-wave vertical gratings of various spatial frequencies or gray fields. Rats are instinctive swimmers and the visual water task capitalizes on their natural inclination to escape from water to a solid substrate, the location of which is directly paired with a visual stimulus. Animals first had to be pretrained to distinguish a low spatial frequency grating (0.117 cycles/deg) from homogeneous gray with high reliability before the limit of this ability could be assessed at higher spatial frequencies. Preventing spatial biases in responses, grating and gray-field positions were alternated by following a pseudorandom sequence. The task rewards animals that take a direct swim path to the monitor displaying the grating, and negatively reinforces animals for choosing the gray stimulus by prolonging the trial. A method-of-limits procedure was used to test the threshold to distinguish the grating from gray, in which incremental changes in the spatial frequency of the grating were made until the ability of animals to distinguish the stimuli fell to chance. An animal was placed in the release chute and allowed to find the platform under the grating; this was a trial of response, and every day rats were subjected to three sessions of 20 trials. If the animal made a correct choice, the spatial frequency of the stimulus was increased by adding one cycle on the screen, and another trial was performed. After reaching approximately half of the animal’s projected threshold, the minimum number of trials to increase stimulus spatial frequency was set to three consecutive correct choices. Once an error was made, additional trials were run until four correct responses were made in sequence, or seven correct choices were made in a block of ten trials. If animal performance did not meet these criteria the spatial frequency of the stimulus was reduced. A preliminary threshold was attained for rats when they failed to achieve 70% accuracy at a spatial frequency. In order to ensure the accuracy of this estimate, spatial frequencies around the threshold were retested until a clear pattern of performance was generated. The highest spatial frequency achieved consistently was recorded as the acuity threshold. Sessions in which the animal was obviously not performing the task were excluded from acuity threshold assessment. Behavioral testing was performed during the light phase of the cycle.
Statistical analysis was performed using Sigma Stat 3.1 (Systat Software, Chicago, IL, USA). Multiple groups were compared by anova followed by post hoc comparisons applying Bonferroni’s correction or the Holm–Sidak test. When two groups were compared a t-test was applied. Normality and omoschedasticity of the data were checked. Data not normally distributed were compared using the nonparametric Kruskal–Wallis anova or rank-sum test. Significance level was equal to 0.05.
To assess whether adult long-term MD rats can recover normal visual acuity with treatments with HDAC inhibitors, we analyzed rats monocularly deprived from P21 until P120-130. These ages are temporally located in correspondence with the beginning of rat SP for MD and well after its closure, respectively (Fagiolini et al., 1994; Guire et al., 1999). This MD protocol is known to cause a strong and spontaneously irreversible amblyopia in rats (Pizzorusso et al., 2006).
Histone deacetylase inhibitors promoted recovery of VEP visual acuity
Long-term MD rats were subjected to RS and, after 5 days, they were treated for 25 days with daily intraperitoneal administration of valproic acid (300 mg/kg; n = 8), sodium butyrate (1.2 g/kg; n = 6) or vehicle (0.9% saline; n = 4) as a control. Finally, visual acuity of the deprived and the nondeprived eye was assessed by means of VEP recordings from the primary visual cortex contralateral to the stimulated eye. Fig. 1 shows the average VEP curve obtained in the three experimental groups. In control rats treated with saline we found a significantly lower VEP acuity for the long-term deprived eye than for the fellow eye (paired t-test, t3 = 4.002, P = 0.028), indicating that the deprived eye remained amblyopic after RS and control treatment. By contrast, both in the group treated with valproic acid and in the group treated with sodium butyrate, VEP acuity of the two eyes did not differ (paired t-test: t7 = −0.739, P = 0.48 for valproic acid; t5 = 1.123, P = 0.31 for sodium butyrate). The recovery of visual acuity induced by HDAC inhibitors was also evident comparing VEP acuity of the deprived eye between the different experimental groups (Fig. 1D). Indeed, VEP acuity of the deprived eye of rats treated with valproic acid or sodium butyrate was significantly higher than VEP acuity of the deprived eye of the control rats (one-way anova: F2,14 = 4.400, P = 0.033; post hoc t-test with Bonferroni correction, valproic acid vs. control, t9 = 2.852, P = 0.019; sodium butyrate vs. control, t8 = 2.946, P = 0.019). These data indicate that two different drugs sharing an inhibitory activity on HDACs promote VEP acuity recovery. Thus, increasing histone acetylation promoted functional recovery in adult long-term MD rats.
Valproic acid promoted recovery of behavioral visual acuity
To investigate whether the recovery of visual acuity assessed electrophysiologically in long-term MD rats treated with HDAC inhibitors was relevant for rat behavior we devised a longitudinal behavioral assessment of the effect of the treatment on visual acuity (Fig. 2A). We chose to asses the effects of valproic acid because it is an FDA-approved molecule well tolerated by animals even for chronic treatments. In addition, valproic acid is soluble in aqueous buffers and easily crosses the blood–brain barrier. Behavioral visual acuity of the nondeprived eye in long-term MD rats was assessed using the Prusky visual water task before RS. After RS at P120, visual acuity of the deprived eye was measured to obtain the pretreatment visual acuity value of the amblyopic eye. This procedure lasted ∼10 days. Subsequently, rats were randomly assigned to the groups of treatment with valproic acid or control saline. Daily treatment was performed for 15 days. Then, visual acuity of the long-term deprived eye was reassessed in the same animals. The treatment was continued during the behavioral experiments, resulting on average in a total treatment duration of 25 days.
Examples of the results obtained in a saline-treated and in a valproic acid-treated rat are shown in Fig. 2B-D, respectively. Fig. 3 reports the average visual acuity of the two groups (valproic acid, n = 4; saline, n = 3). Before the treatment the deprived eye of both groups was clearly amblyopic; indeed, its visual acuity was lower than that of the fellow eye (two-way anova, effect of factor ‘MD’, F1,10 = 59.389, P < 0.001; effect of factor ‘group of treatment’, F1,10 = 1.085 P = 0.322; interaction, F1,10 = 2.861 P = 0.122). After the treatment, the amblyopic eye acuity was significantly improved in the group receiving valproic acid, while it remained unchanged in the group receiving saline: two-way anova for the factors ‘type of treatment’ and ‘before or after treatment’ showed an interaction of the factors (F1,5 = 8.323, P = 0.03); post hoc Holm–Sidak indicated that saline and valproic treated groups did not differ before the treatment (t5 = 0.326, P = 0.75) but they significantly differed after the treatment (t5 = 3.6, P = 0.006). Within treatments, acuity of valproic acid-treated rats was significantly different before and after the treatment (t5 = 3.951, P = 0.011) whereas acuity of saline treated rats was not (t5 = 0.394, P = 0.71). These data show that prolonged treatment with valproic acid coupled with RS in adult rats leads to a functional recovery of behavioral visual acuity of the amblyopic eye after long-term MD. Such recovery appears to be complete, as the acuity of the deprived eyes following treatment is indistinguishable from that typical of a normal eye.
Valproic acid induced histone acetylation in the visual cortex
Finally, we investigated whether the treatment with valproic acid was able to increase histone acetylation in the visual cortex by Western blot using antibodies for histone H3 and its Lys 9 acetylated form. Fig. 4 shows that robust acetylation could be observed in tissue samples of the visual cortex 2 h after an i.p. injection of valproic acid, either in naïve rats or at the end of the protocol of VPA treatment lasting 25 days used for the behavioral experiments (Kruskal–Wallis one-way anova, H2 = 10.677, P = 0.005; post hoc Dunn’s test, chronic valproic versus vehicle, P < 0.05; acute valproic versus vehicle, P < 0.05. Vehicle, n = 6 samples; acute valproic acid, n = 4 samples; chronic valproic acid, n = 6 samples). These data indicate that the amount of histone acetylation induced in the visual cortex by a VPA i.p. injection remained constant for the whole duration of the treatment.
The main finding of this study is that visual acuity of the amblyopic eye recovered to normal values in rats treated with HDAC inhibitors. This effect could be observed both with electrophysiological and behavioral techniques. In saline-treated rats, no spontaneous recovery of visual acuity was present, in agreement with previous studies showing little or no increase in visual acuity after reopening the deprived eye in adult rats (Prusky et al., 2000; Iny et al., 2006; Pizzorusso et al., 2006; He et al., 2007; Sale et al., 2007; Maya Vetencourt et al., 2008; Morishita & Hensch, 2008). Studies performed in kittens have shown that the recovery of deprived eye acuity achieved with RS during the SP can occur in concomitance with an impairment of visual acuity of the previously nondeprived eye (Kind et al., 2002). Intriguingly, our VEP acuity data indicated that visual acuity of the nondeprived eye was not affected by visual deprivation induced by the RS procedure in HDAC inhibitor-treated animals. Although it is not known whether RS during the SP causes an impairment of visual acuity of the previously nondeprived eye also in rats, it could be possible that the increased plasticity induced by HDAC inhibitors do not entirely reinstate the plasticity present during the SP.
What mediates valproic acid and sodium butyrate action on visual acuity?
To inhibit HDACs we used valproic acid, a drug that has different targets in neuronal cells other than HDACs. In particular, valproic acid is a clinically used anticonvulsant and mood stabilizer in bipolar disorder and is known to elevate levels of the inhibitory neurotransmitter GABA by direct inhibition of GABA transaminase and succinic semialdehyde dehydrogenase, which are enzymes responsible for GABA breakdown. In addition, valproic acid can interact with ionic channel function and it has been found to enhance BDNF expression and activation of ERK signaling (Rosenberg, 2007). All these valproic acid effects could exert positive or negative roles on visual cortical plasticity. For instance, recent data indicate that inhibition levels in the adult visual cortex might regulate adult ocular dominance plasticity (Harauzov et al., 2010; Southwell et al., 2010). However, the data also showing a recovery of VEP visual acuity with sodium butyrate, which shares with valproic acid the HDAC inhibitory activity (Tsankova et al., 2007) but has different pharmacological actions, suggest that increased histone acetylation could be the common mechanism mediating the visual acuity recovery induced by valproic acid and sodium butyrate treatments. In keeping with this interpretation, we found a strong increase in histone acetylation in the visual cortex of the valproic acid-treated rats. A key role for histone acetylation in visual acuity recovery is also in line with a previous study showing that administration of trichostatin, another HDAC inhibitor, in adult mice promoted visual cortical plasticity, reactivating a sensitivity to MD similar to that of juvenile mice (Putignano et al., 2007). Importantly, the results in this manuscript indicate that histone acetylation could also be a crucial step in the mechanisms underlying experience-dependent recovery from amblyopia.
Molecular mediators of the action of histone acetylation on visual cortical plasticity
Histone acetylation exerts its effect on transcription either by physical remodeling of chromatin structure or by further recruitment of signaling complexes that drive or repress transcription (Peterson & Laniel, 2004). Histone acetylation is achieved by a histone acetyl transferase adding an acetyl group to a lysine residue. Conversely, HDACs remove these acetyl groups and are generally associated with chromatin inactivation. Therefore, HDAC inhibitors induce histone acetylation and promote gene transcription (Li et al., 2007; Graff & Mansuy, 2009). Increasing evidence, obtained by use of DNA microarrays to profile changes in gene expression of cell lines treated with HDAC inhibitors, demonstrate that the effect of HDAC activity on gene expression is not global because only 1–7% of genes show altered expression (Marks et al., 2000; Glaser et al., 2003), and similar results have also been reported in in vivo studies (Fass et al., 2003; Weaver et al., 2006; Vecsey et al., 2007; Shafaati et al., 2009). In particular, histone acetylation seems to be important for the activation of CREB-regulated genes; indeed CREB activation of gene transcription involves CREB-binding protein, a histone acetyltansferase important for activity-regulated gene expression and synaptic plasticity (Mayr & Montminy, 2001; Vo & Goodman, 2001; Alarcon et al., 2004; Korzus et al., 2004). CREB-mediated gene expression is strongly regulated by visual experience during the SP (Pham et al., 1999; Cancedda et al., 2003; Putignano et al., 2007); however, in adult animals experience-dependent regulation of CREB-mediated gene transcription is strongly reduced (Pham et al., 1999; Putignano et al., 2007). Therefore, HDAC inhibitors might reactivate the expression of plasticity-related genes in the adult cortex by enhancing CREB-mediated gene transcription. This possibility is also supported by the observation that MD in adult mice triggers a labile form of plasticity that can be rendered persistent by the expression of a constitutively active CREB mutant (Pham et al., 2004). Which genes are crucially involved in mediating the action of HDAC inhibitors on visual cortical plasticity, or in other models of brain plasticity, is still poorly known. Further analyses are required to unravel the final effectors of the epigenetic treatment on visual cortical plasticity (Borrelli et al., 2008; Fagiolini et al., 2009).
A comparison between HDAC inhibition and other manipulations enhancing adult visual cortical plasticity
In addition to the manipulation of epigenetic mechanisms, other factors are able to promote a recovery from amblyopia in adult rodents. Environmental enrichment (Sale et al., 2007) and dark rearing (He et al., 2007), coupled with RS or binocular vision, allow the recovery of a long-term deprived eye to normal levels of acuity and ocular dominance. Both protocols lead to a reduction in GABAergic inhibition and probably result in a return to an SP-like balance between excitation and inhibition (Spolidoro et al., 2009). Influencing specific molecular and cellular components was also found to promote recovery from amblyopia in adulthood. Visual cortical plasticity is inhibited by the aggregation of extracellular matrix molecules such as chondroitin sulphate proteoglycans (CSPGs). Enzymatic digestion of CSPGs in combination with RS has been shown to restore visual cortical plasticity in adult rats (Pizzorusso et al., 2002) and to reverse the effects of long-term MD on visual acuity and ocular dominance (Pizzorusso et al., 2006). Also, chronic administration of fluoxetine (which increases extracellular serotonin levels), coupled with RS, allows visual acuity and ocular dominance recovery from long-term MD (Maya Vetencourt et al., 2008); again, a possible mediator of the effect is the lowering of the inhibitory tone (Spolidoro et al., 2009). Intriguingly, environmental enrichment induces histone acetylation, and fluoxetine causes alterations in gene expression overlapping with those induced by HDAC inhibitor treatment (Fischer et al., 2007; Covington et al., 2009); it is therefore possible that epigenetic mechanisms could represent a common endpoint of other treatments enhancing plasticity in the adult visual cortex (Pizzorusso et al., 2007).
In summary, our study demonstrates that targeting HDACs can be an effective pharmacological strategy to promote experience-dependent plasticity in the adult visual cortex and recovery from amblyopia. Although most HDAC inhibitors still show little specificity between different HDACs, resulting in changes occuring not only on histone proteins but also on cytoplasmic proteins with the risk of unwanted side-effects, recent efforts have been made to develop HDAC-type or subclass-specific HDAC inhibitors (Haggarty et al., 2003). When the role of specific HDACs in visual cortical plasticity is clarified, these drugs could be useful, together with behavioral therapy (Levi & Li, 2009), in promoting recovery from amblyopia.
This work was supported by MIUR-PRIN grant, by the EUROV1SION and PLASTICISE FP7 European Union projects and by the EXTRAPLAST IIT project.