Requirement of ERK Activation for Visual Cortical Plasticity Graziella Di Cristo, Nicoletta Berardi, Laura Cancedda, Tommaso Pizzorusso, Elena Putignano, Gian Michele Ratto, and Lamberto Maffei

In vitro recordings. After decapitation the brain was rapidly removed and immersed in ice-cold cutting solution containing: 130 mM NaCl, 3.1 mM KCl, 1.0 mM K₂HPO₄, 4.0 mM NaHCO₃, 5.0 mM dextrose, 2.0 mM MgCl₂, 1.0 mM CaCl₂, 10 mM HEPES, 1.0 mM ascorbic acid, 0.5 mM myo-Inositol, 2 mM pyruvic acid, and 1 mM kynurenate, pH 7.3. A block of visual cortex was sectioned in the coronal plane into 0.4-mm-thick slices using a vibratome (Leica). The slices were transferred to a storage chamber containing cutting solution and maintained at room temperature for at least an hour before recording. The recording solution was composed as follows: 130 mM NaCl, 3.1 mM KCl, 1.0 mM K₂HPO₄, 4.0 mM NaHCO₃, 5.0 mM dextrose, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 10 mM HEPES, 1.0 mM ascorbic acid, 0.5 mM myo-Inositol, 2 mM pyruvic acid, and 0.01 mM glycine, pH 7.3. Chemicals were purchased from Sigma unless otherwise indicated. The experiments were performed on submerged slices continuously perfused at a rate of 2 ml/min with 35°C oxygenated recording solution. Electrical stimulation (20 to 2000 A, 100 s duration) was delivered with a bipolar concentric stimulating electrode (FHC, Bowdoinham, USA) placed either at the border of the white matter and layer VI, or to a site in the middle of the cortical thickness, corresponding to layer IV and upper layer V. Field potentials (FPs) in layer III were recorded by mean of glass micropipettes (1 to 3 megohms) filled with NaCl (3 M). Baseline responses were obtained every 30 s with a stimulation intensity that yielded a half-maximal response. The amplitude of the maximum negative FP in layer III was used as a measure of the evoked population excitatory synaptic current. Changes in the amplitude of the maximum negative FP correlate with changes in the initial slope of EPSPs recorded intracellularly in layer III neurones (1). At least 15 min of stable baseline recordings were made before drugs were applied. U0126 (Promega, Madison, WI or Tocris, Bristol, UK) and PD58059 (Tocris) solutions were freshly prepared for each slice from single-use aliquots of a 1000x stock solution in DMSO.

PhosphoERK immunostaining in electrophysiology slices. Thick cortical slices were prepared and recorded as described above. After achievement of a stable baseline, either in normal recording media or in presence of test drugs (the NMDA-antagonist CPP or U0126, 20 M), the slices received TBS stimulation. Control slices were perfused in normal saline and received only test stimulation. After 5 min the slices were fixed in 6% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Afterwards the tissue was cryoprotected in 30% sucrose overnight. Slices were included face-on in TissueTek and cut at 40-m thickness on a cryostat. PhosphoERK (p-ERK) immunohistochemistry proceeded on the free floating slices with a 1-hour block (PBS containing 10% normal horse serum, 0.3% Triton X-100), incubation with p-ERK (1:400, New England Biolabs) and Neu-N antibody (1:1000, Chemicon), and reaction with biotinylated -horse IgG (1:200, Vector Laboratories, Burlingame, CA) followed by Extravidin-Cy3 (1:1000, Sigma). Slides were coded and confocal images (Olympus FV-300) were acquired blind.

In vivo treatments and recordings. A total of 36 Long-Evans hooded rats were used in this study, which was approved by the Italian Ministry of Public Health. Six normal rats were recorded at postnatal day 28 (P28). Four animals were monocularly deprived for 1 week by eyelid suture under avertin anesthesia at P21. Nineteen rats were implanted with osmotic minipump (model 1007D; Alzet, Palo Alto, CA) and monocularly deprived at P21. Animals were recorded at P28. Minipumps were connected via PE tubing to a stainless steel cannula (30 gauge) implanted in the visual cortex contralateral to the deprived eye. Osmotic minipumps (pumping rate, 0.5 l/hr) were filled with: U0126 250 M (n = 7), PD98059 250 M (n = 4), SB203580 250 M (Tocris; n = 5), DMSO 1% (n = 3). Drugs were solved at the final concentrations from 100x stock solutions in DMSO. Four rats were left undeprived and implanted with osmotic minipumps filled with U0126 250 M and recorded during treatment (2 or 6 days after minipump implantation). Electrophysiological procedures were performed under urethane anaesthesia (0.7 ml/hg i.p., 20% solution in saline; Sigma). For each animal, eight to ten cells were recorded in each of at least three tracks spaced evenly (>200 m) across the binocular primary visual cortex (Oc1B), to avoid sampling bias. None of the treatments apparently altered the number of visually responsive cells in that visually driven cells were recorded at an interdistance of 70 to 100 m from each other in normal animals as well as in all treatment groups. The position of receptive fields of single units were mapped using a hand-held stimulator. Only cells with receptive fields within 20° of the vertical meridian were included in our sample. Spontaneous activity, responsiveness and receptive field (RF) size were determined from peristimulus time histograms recorded in response to computer-generated bars, averaged over at least 20 stimulus presentations (2). Ocular dominance was evaluated according to the classification of Hubel and Wiesel.

Lack of effect of MEK inhibition on TBS response and NMDA mediated synaptic transmission.
One possible cause of the failed induction of LTP during ERK blockage could be a reduced responsiveness to the TBS in presence of MEK inhibitors. We verified this possibility by analyzing the amplitude of the response to the individual stimuli delivered during the TBS. Web fig. 1, A and B, show the normalized amplitude of the response [indicated by the arrowhead in (A)] to the first stimulus of each 100 Hz burst of the two trains. Clearly there is no failure of response in presence of U0126.

The field response mediated by the NMDA receptor was isolated by means of the protocol of Philpot et al. (3). In brief layer IV was stimulated while recording from layer II-III in normal saline solution. After at least 10 min of stable baseline the perfusion was switched to a solution containing nominal [Mg]_° to reduce the voltage-dependent of the receptor, NBQX and bicucullin to block the AMPA and ionotropic GABAergic components of the field response [3 mM CaCl₂, 0.1 mM MgCl₂, 20 µM NBQX (Tocris), 1 µM glycine, and 0.5 µM bicuculline methiodide]. As described in Philpot et al. (3), to avoid the use-dependent depression of the NMDA-mediated response, we tested the slice by means of 4 stimuli delivered every 10 min before and after U0126 perfusion. The NMDA-mediated response, indicated by the arrowhead in fig. 1, C and D, exhibited a long latency, due to the slow kinetic of the NMDA current. After 50 min the MEK inhibitor U0126 (20 M) was added to the perfusion medium, while measuring the slice responsiveness as before. Web fig 1C shows a typical NMDA mediated response recorded after 20 min from the first solution change (Web fig. 1C) and during MEK inhibition (Web fig. 1D). 50 min of incubation in U0126 did not alter the amplitude of the NMDA response (Web fig. 1E, one way ANOVA, P = 0.79).

Up-regulation of pERK staining in the visual cortex of dark reared rats exposed to light for 15 minutes.
We investigated whether visual experience regulates ERK activation in neurons of the visual cortex. A litter of rats was dark reared from birth, then, at P34, some animals were exposed to light for 15 min before sacrifice while the remaining rats were kept in darkness and acted as controls. After immunostaining, fields from the visual cortex of each animal were acquired at the confocal microscope by blind operators. The visual cortex of animals exposed to light showed robust ERK phosphorylation as compared to control, as shown in Web fig. 2A. The confocal images were used to measure the fluorescence intensity and the number of positive cells in the two experimental groups.

Experimental procedure. Animals were dark reared in a climatized environment following a protocol approved by the Italian Ministry of Public Health. All manipulations of the animals during the dark rearing were performed in complete darkness using infrared viewers. At the time of the experiment, the animals were divided in two groups: three animals have been sacrificed and perfused while still in complete darkness, while four animals were exposed to ambient light for 15 min before the onset of perfusion. Transcardial perfusion was executed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were quickly removed and cryoprotected in 30% sucrose overnight, then 25 m coronal sections were cut on a freezing microtome, and processed for immunohistochemistry as described above. Slides were coded and confocal images (Olympus FV-300 or Leica TCS/NT) were acquired blind. From each section a representative field (700 m by 700 m) of the primary cortex was selected and it was reconstructed by imaging eight optical sections covering the entire thickness of the slice. All sections were acquired in random order in a single session to minimise fluctuation in laser output and degradation of fluorescence. The maximum projection was analyzed through a custom-made software to count positive neurons and to measure their fluorescence intensity. The code was broken only at the end of the analysis process.

The ERK pathway is still inhibited 6 days after the beginning of the treatment with U0126.
We investigated whether 6 days after the cortical implant of a cannula connected to an osmotic minipump containing U0126 the ERK pathway could be activated or was still inhibited. To induce phosphorylation of ERK we increased electrical activity by means of a local application of picrotoxin, a GABA_A receptor antagonist. Web fig. 3 shows that picrotoxin is much less effective in activating ERK in the cortex treated for 1 week with U0126 as compared to the contralateral, vehicle treated cortex.

Experimental procedure. To verify whether electrical activity was able to induce ERK activation, picrotoxin 1mM was applied locally on the left and saline on the right visual cortex of three animals. After twenty minutes animals were transcardially perfused as described above. To assess the efficacy of U0126 in reducing ERK activation in vivo, three animals were double implanted with an osmotic minipump filled with U0126 250 M in the left visual cortex and a osmotic minipump filled with DMSO 1% in the right visual cortex. Six days after minipump implantation, picrotoxin 1mM was applied locally directly on both visual cortices; animals were perfused 20 min later and the brain removed and cryoprotected. Immunohistochemistry for pERK was performed as described above. Cortical sections were reconstructed starting from the midline and extending laterally for 5 mm, by composing together several fields acquired at the confocal microscope (Leica, TCS/NT). All sections obtained from the same animal were acquired in a single session to minimize fluctuation in laser output and degradation of fluorescence. Images were processed with a custom-made software to measure the fluorescence of all visible cells in the supra-granular layers. The ratio of average fluorescence between neurones from the control and treated cortices was evaluated by computing the mid-point of the cumulative distribution of fluorescence. Then the intensity of the background fluorescence was subtracted and the ratio between treated side versus control was computed.

MEK inhibitor U0126 does not alter visual acuity.
Visual acuity determined with Visual Evoked Potentials (VEPs) is a sensitive measure for the state of visual cortical development and predicts visual behaviour in humans (4), monkeys (5), cats (4), rats (6, 7) and mice (8-10). We have therefore used visual acuity assessed with VEPs to probe the state of development of the visual cortex after block of ERK activation in animals left undeprived. VEPs were recorded by means of a micropipette (2 to 2.5 megohm impedance) inserted into the binocular portion of the primary visual cortex (Oc1B). Only penetrations where single cell receptive fields were within 20 degrees from the vertical meridian were used to assess VEP acuity. To record VEPs the electrode was positioned at a depth of 450 to 500 m; at this depth, VEPs had their maximal amplitude. Signals were band-pass filtered (0.1 to 100 Hz), amplified and fed to a computer for analysis as previously described (9). Briefly, at least 128 events were averaged in synchrony with the stimulus contrast reversal. Transient VEPs in response to abrupt contrast reversal (0.5 Hz) were evaluated in the time domain by measuring the peak-to-trough amplitude and peak latency of the major negative component. Visual stimuli were horizontal sinusoidal gratings of different spatial frequency and contrast, generated by a VSG2/2 card (Cambridge Research System, Cheshire, UK) running a custom software (kindly provided by C. Orsini) and presented on the face of a Daewoo monitor (20 cm by 22 cm; luminance 15 cd/m²) positioned 20 or 30 cm from the rat eyes and centered on the previously determined receptive fields. Visual acuity was measured as the highest spatial frequency that still evoked a response above noise level at maximum contrast. This frequency coincides with the extrapolation to noise level of the linear regression through the last four to five data points in a curve where VEP amplitude [normalized to the value recorded for the lowest spatial frequency employed, 0.2 cycles per degree (c/deg)] is plotted against log spatial frequency.

Supplemental Figure 1. (A) Sample response during the delivery of the first 100-Hz burst of the first train. The arrowhead indicates the response to the first stimulus of the train. (B) Response amplitude to each stimulus was normalized to the response amplitude of the first stimulus and plotted against time. Error bars, SEM. Data averaged on 9 (control) and 6 (U0126) slices for layer IV stimulation and 10 (control) and 9 (U0126) slices for white matter stimulation. (C and D) Sample wave form showing the NMDA mediated response before and after MEK inhibition. The arrowheads indicate the postsynaptic component of the response. Traces are the average of four sweeps each. (E) Time course of the normalized amplitude of the NMDA component. Data averaged on four different slices from three animals ranging in age from P20 to P26.

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Supplemental Figure 2. (A) pERK immunostaining in the visual cortex of a dark reared rat, and of a rat that was returned to light for 15 min. Calibration bar is 50 m. (B) Exposure to light causes both an increase in fluorescence intensity (left) and an increase in the number of positive cells (right). The two experimental groups were composed by three animals (1133 cells, dark reared controls) and four animals (7053 cells, 15 min exposition to light).

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Supplemental Figure 3. Activation state of ERK after treatments increasing visual cortical cell electrical activity. Electrical activity was increased with local application of picrotoxin; at the dose used (1mM) spontaneous activity increases by a factor of at least three to four. (A) Immunostaining for phosphorylated ERK in the visual cortex after local application of saline (left) or picrotoxin (right), as schematized in the drawing. Twenty minutes after picrotoxin application the level of ERK activation is higher in the picrotoxin treated cortex as compared to the contralateral, vehicle treated cortex. (B) Immunostaining for phospho-ERK after simultaneous local application of picrotoxin on both visual cortices, one infused with U0126 and one with vehicle (left and right respectively) six days after minipump implant (see drawing). ERK activation is unaffected by treatment with vehicle but is clearly reduced by U0126 throughout the entire span of the primary visual cortex (images acquired 5 mm from the medial line). (C) Cumulative distributions of pERK fluorescence intensity after picrotoxin treatment in U0126 (filled symbols, 4321 cells) or vehicle (empty symbols, 5466 cells) infused cortices. The frequency values represent the fraction of cells with fluorescence less than or equal to a given fluorescence value. The cumulative distribution for U0126 is significantly shifted to the left with respect to that for vehicle infused cortex (K-S test). Data from three animals. (D) Mean ± SEM and single results for each of the three animals. For each animal, the fluorescence of neurones in each cortex treated with U0126 is normalised with respect to the contralateral control cortex.

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Supplemental Figure 4. (A) VEP amplitude curve for a normal animal (green dots) and an animal of the same age (P28) after 6 days of implantation with a minipump filled with U0126 (red triangles). Lines through data are a double linear fit. The highest spatial frequency yielding a signal above noise level (visual acuity; data points on the abscissa) was 0.9 c/deg for both animals, and this value coincided with the extrapolation of the linear fit through the last four data points (arrow). (B) Average acuity values found in Normal P28 animals (N = 4) and in P28 animals after 2 or 6 days of U0126 treatment (N = 4, data for 2 and 6 days of treatment pooled together).

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