R E S EA R C H A R T I C L E

Postnatal Accumulation of Intermediate Filaments in the Cat and Human Primary Visual Cortex Seoho Song, Donald E. Mitchell, Nathan A. Crowder, and Kevin R. Duffy* Department of Psychology and Neuroscience, Dalhousie University, Life Sciences Centre, Halifax, Nova Scotia, Canada, B3H 4R2

ABSTRACT A principal characteristic of the mammalian visual system is its high capacity for plasticity in early postnatal development during a time commonly referred to as the critical period. The progressive diminution of plasticity with age is linked to the emergence of a collection of molecules called molecular brakes that reduce plasticity and stabilize neural circuits modified by earlier visual experiences. Manipulation of braking molecules either pharmacologically or though experiential alteration enhances plasticity and promotes recovery from visual impairment. The stability of neural circuitry is increased by intermediate filamentous proteins of the cytoskeleton such as neurofilaments and a-internexin. We examined levels of these intermediate filaments within cat and human primary visual cortex (V1) across development to determine whether they accumulate following a time course consistent with a molecular brake. In both species, levels of intermediate filaments increased con-

siderably throughout early postnatal life beginning shortly after the peak of the critical period, with the highest levels measured in adults. Neurofilament phosphorylation was also observed to increase throughout development, raising the possibility that posttranslational modification by phosphorylation reduces plasticity due to increased protein stability. Finally, an approach to scale developmental time points between species is presented that compares the developmental profiles of intermediate filaments between cats and humans. Although causality between intermediate filaments and plasticity was not directly tested in this study, their accumulation relative to the critical period indicates that they may contribute to the decline in plasticity with age, and may also constrain the success of treatments for visual disorders applied in adulthood. J. Comp. Neurol. 523:2111–2126, 2015. C 2015 Wiley Periodicals, Inc. V

INDEXING TERMS: molecular brake; neurofilament; a-internexin; phosphorylation; plasticity; RRID:AB_94285; RRID:AB_306083; RRID:AB_509998; RRID:AB_305807

Three classes of interconnected filamentous proteins (microfilaments, microtubules, and intermediate filaments) comprise the principal intracellular constituents of the neuronal cytoskeleton. Intermediate filaments contribute to cellular function and maintenance of cell shape, and provide mechanical integrity to neurons (for review, see Goldman et al., 2012). The predominant intermediate filament synthesized by neurons varies according to developmental stage (Lee and Cleveland, 1996). Early in prenatal development the intermediate filaments nestin and vimentin are associated with neural cells (Tapscott et al., 1981; Lendahl et al., 1990); however, the production of these proteins is eventually silenced and replaced by synthesis of a-internexin and the neurofilament triplet proteins: neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) (Kaplan et al., 1990; Fliegner et al., 1994). C 2015 Wiley Periodicals, Inc. V

These later blooming intermediate filaments are among the most plentiful proteins in neurons. Analysis of cytoskeletal polymers in squid giant axon has revealed that almost all (>95%) axonal neurofilaments contribute to a stable cytoskeleton network, and the major protein constituent (>50%) of this network is neurofilament (Morris and Lasek, 1982). The comparatively greater overall stability contributed by

Grant sponsor: the Canadian Institutes of Health Research; Grant number: 102653 (to K.R.D., N.A.C., and D.E.M.); Grant sponsor: Discovery grants from the Natural Sciences and Engineering Research Council; Grant numbers: 298167 (to K.R.D.), 355847 (to N.A.C.), and 7660 (to D.E.M.). *CORRESPONDENCE TO: Kevin Duffy, Department of Psychology and Neuroscience, Dalhousie University, Life Sciences Centre, Halifax, NS, Canada, B3H 4R2. E-mail: [email protected] Received September 19, 2014; Revised March 16, 2015; Accepted March 17, 2015. DOI 10.1002/cne.23781 Published online May 12, 2015 in Wiley Online (wileyonlinelibrary.com)

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neurofilaments and a-internexin has led to the suggestion that these intermediate filaments may not only stabilize the structure of neurons but may also constrain, through their stabilizing properties, the capacity for structural plasticity (Morris and Lasek, 1982; Yuan et al., 2006). It has furthermore been postulated that an accumulation of neurofilaments throughout early postnatal development may contribute to the natural and progressive reduction of neuronal plasticity that occurs with postnatal age (Smith, 1973). Research on the cat and monkey visual cortex has been unambiguous in showing that monocular deprivation implemented in late juvenile animals or adults is less effective at promoting the structural and functional perturbations produced by the same deprivation imposed at younger ages (Wiesel and Hubel, 1963a,b; Hubel and Wiesel, 1970; Giffin and Michell, 1978; LeVay et al., 1980; Olson and Freeman, 1980; Jones et al., 1984; Daw et al., 1992). Likewise, the extent of recovery from the effects of monocular deprivation is much greater when treatment occurs early in postnatal development than when it is initiated in adolescence or in adulthood (Blakemore and Van Sluyters, 1974; Movshon, 1976; Blakemore et al., 1981). Little is currently known about the factors that contribute to this age-related decline in neuronal flexibility, but recent research in rodents on so-called molecular brakes has provided insight into the neural events that diminish plasticity with age (Hensch, 2005; Morishita and Hensch, 2008; Bavelier et al., 2010). According to this account, molecules that act directly to constrain plasticity should be found at comparatively lower amounts early in postnatal life when plasticity is high, and manipulation of their levels should predictably adjust plasticity to the extent of their contribution as a brake on plasticity. Molecules that have been proposed to limit plasticity with age each exhibit a similar developmental profile in which levels are observed to be comparatively low within the critical period, and then accumulate as the capacity for plasticity wanes. Such a profile has been reported for a number of putative braking molecules including a nicotinic acetylcholine receptor binding protein (Morishita et al., 2010), chondroitin sulfate proteoglycans such as neurocan and aggrecan (Pizzorusso et al., 2002; Kind et al., 2013), and myelin and myelin-related proteins (Haug et al., 1976; McGee et al., 2005). Reduction or removal of several of these braking proteins in the adult visual cortex has been shown to restore a juvenile level of plasticity in rodents (Pizzorusso et al., 2002; Morishita et al., 2010; Akbik et al., 2013). In monocularly deprived cats and humans, reduction of neurofilament protein within deprived neurons of the

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lateral geniculate nucleus and within deprived ocular dominance columns of the primary visual cortex (V1) occurs alongside deprivation-induced structural modification, suggesting that removal of neurofilament is permissive for structural plasticity (Kutcher and Duffy, 2007; Duffy et al., 2007; Duffy and Slusar, 2009). In agreement with this notion, extension of the highly plastic critical period through immersion in complete darkness (Cynader and Mitchell, 1980) also causes a lower than normal level of neurofilament in the dorsal lateral geniculate nucleus (dLGN) and V1 (O’Leary et al., 2012; Duffy and Mitchell, 2013). The known stabilizing properties of intermediate filaments and their association with structural plasticity raise the possibility that their postnatal accumulation contributes to the progressive reduction of plasticity with age. In the current study we measured levels of the three neurofilament subunits and the closely associated ainternexin protein (Yuan et al., 2006) within cat and human V1 to evaluate whether each protein accumulates postnatally in a manner congruent with a molecular brake. In comparing developmental profiles between cat and human V1, we also propose an approach to scale postnatal ages between species that could facilitate translation of age-specific treatments to humans with perceptual disorders such as amblyopia.

MATERIALS AND METHODS Animals and tissue The level of four neuronal intermediate filaments was examined from the primary visual cortex of 12 kittens of different postnatal ages and 5 adult cats (>1 year old) that were obtained from a closed breeding colony at Dalhousie University (Halifax, NS, Canada). Measurements were obtained from the left and right V1 of kittens at the following postnatal ages: birth (n 5 2), 7 days (n 5 1), 9 days (n 5 1), 14 days (n 5 1), 25 days (n 5 1), 36 days (n 5 1), 40 days (n 5 1), 47 days (n 5 1) 90 days (n 5 1), 114 days (n 5 1), 150 days (n 5 1). The methods reported here conform to guidelines established by the Canadian Council on Animal Care, and the experiments were conducted according to a protocol approved by the University Committee on Laboratory Animals at Dalhousie University. Parallel sets of measurements were made from samples of human V1 from the left hemisphere of 15 humans at different ages from near birth to adulthood: 2 days, 6 months, 1.7 years, 2 years, 2.8 years, 5.9 years, 6.8 years, 8 years, 8.8 years, 11.5 years, 12.8 years, 15 years, 25.5 years, 35 years, and 62 years (n 5 1 for each age examined). Human samples were obtained from the U.S. National Institute of Child Health and Human

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TABLE 1. Primary Antibodies Used Antigen

Immunogen

Source

Dilution

Neurofilament H

Homogenized rat hypothalamus

a2Internexin

Recombinant full-length protein (rat)

Millipore (Billerica, MA), mouse monoclonal, clone DA2, no. MAB1615. AB_94285 Abcam (Cambridge, MA), mouse monoclonal, clone NF-09, no. ab7794. AB_306083 Covance (Princeton, NJ), mouse monoclonal, clone SMI-32, no. SMI-32R. AB_509998 Abcam (Cambridge, MA), rabbit polyclonal, no. ab7259. AB_305807

1:1,000

Neurofilament M

Enzymatically dephosphorylated pig neurofilaments Pellet of pig brain cold stable proteins

Neurofilament L

Development (NICHD) Brain and Tissue Bank located at the University of Maryland School of Medicine (Baltimore, MD). Research on human brain tissue was approved by the Health Sciences Research Ethics Board at Dalhousie University, which is in accordance with the policy statement on Ethical Conduct for Research Involving Humans.

Histology Tissue preparation Cats were injected with a lethal dose of sodium pentobarbital (Euthanyl, Bimeda-MTC, Cambridge, ON, Canada), after which brain tissue was exsanguinated by transcardial perfusion with 150 ml of phosphate-buffered saline (PBS) followed by 150 ml of formalin made from paraformaldehyde dissolved in PBS. After perfusion, the fixed brain was immediately extracted from the skull and, in preparation for cutting, the left and right primary visual cortex was resected and placed in a PBS solution containing 30% sucrose for 48 hours. Coronal sections of V1 were cut on a freezing microtome at a thickness of 50 mm, and all sections were kept floating in PBS with 0.5% sodium azide until processing.

Antibody characterization Table 1 provides a summary of the primary antibodies used in this study, each of which is documented within the Resource Identification Initiative. Labeling for NF-L was achieved with a mouse monoclonal antibody (MAB1615; Millipore, Billerica, MA), and its specificity was tested in an immunoblot using homogenate from normal adult cat V1. Labeling with this antibody produced a single distinct band at 70 kDa, which is the expected molecular mass for this protein. NF-M protein was examined by using a mouse monoclonal antibody (ab7794; Abcam, Cambridge, MA). The specificity of this antibody was tested with an immunoblot of normal adult cat V1, and labeling revealed a single reacted band at 155 kDa, the expected molecular mass of NF-M. A mouse monoclonal antibody was used to detect NF-H (SMI-32; Covance, Princeton, NJ). The SMI-32 antibody is targeted against nonphosphorylated NF-H.

1:1,000 1:1,000 1:1,000

The specificity of this antibody was assessed with an immunoblot of homogenized normal adult cat visual cortex that was exposed to an alkaline phosphatase solution (P6774; Sigma-Aldrich, Oakville, ON, Canada) before immunolabeling so as to reveal the more plentiful phosphorylated isoforms. Labeling blots for NF-H produced two bands, a distinct band at 180 kDa and a weaker band at 200 kDa. Both bands were consistent with the expected mass of the heavy neurofilament subunit isoforms (Goldstein et al., 1987). Labeling of a-internexin was achieved with a rabbit polyclonal antibody (ab7259; Abcam) whose specificity was tested with an immunoblot of normal adult cat V1 homogenate. Labeling with this antibody revealed a single band at 65 kDa, the appropriate molecular mass for a-internexin.

Tissue processing Sections of cat V1 were first placed in a PBS blocking solution (0.1 M, pH 7.4) that contained normal goat serum (1:250) and were left for 1 hour. Sections were then transferred to a solution containing primary antibody (1:1,000 concentration) and after 12 hours were washed in PBS and placed for 1 hour in PBS with biotinylated goat secondary antibody targeted against either the mouse primary antibody (115-065-003; Jackson ImmunoResearch, West Grove, PA) or rabbit primary antibody (111-065-003; Jackson ImmunoResearch). Immunolabeling was revealed with an avidin and peroxidaseconjugated biotin kit (PK6100; Vector, Burlingame, CA) that was used to make protein labeling visible though turnover of the chromogen substrate, 3,30 -diaminobenzidine tetrahydrochloride. Reacted sections were mounted onto glass slides, allowed to dry overnight, dehydrated in ethanol, and cleared in Histoclear (National Diagnostics, Atlanta, GA); they were then coverslipped using Permount medium (Fisher Scientific, Pittsburgh, PA).

Immunoblotting Cats examined for immnoblotting were injected with a lethal dose of Euthanyl, and the portion of V1 along the medial bank of the postlateral gyrus containing the

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area centralis representation (Tusa et al., 1978) was resected and homogenized at 4 C in a buffer (pH 7.6) containing 20 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 10 mg/ml 4-benzenesulfonyl fluoride hydrochloride (AEBSF), and 5 mg/ml leupeptin. Supernatant from each hemisphere of each animal was extracted by centrifugation of homogenized samples, and a Bradford assay (Bradford, 1976) was then used to equalize protein concentration between samples in preparation for polyacrylamide gel electrophoresis. Samples of solubilized brain protein from left and right V1 from each animal were resolved through a 10% polyacrylamide gel, and each gel lane was loaded with 15 mg of total protein at a concentration of 1 mg/mL. After electrophoresis, separated protein was transferred to nitrocellulose by using a Turbo-Blot transfer system (Bio-Rad, Hercules, CA). The developmental profiles of all four intermediate filaments (NF-L, NF-M, NF-H, and a-internexin) were determined by immunolabeling separate blots with the primary antibodies described above. Before immunolabeling, nitrocellulose blots were immersed in SyproRuby total protein stain (Bio-Rad) for 10 minutes, washed several times in distilled water, and imaged by illumination with 400-nm light using a ChemiDoc imaging system (Bio-Rad). Quantification of total protein staining (Aldridge et al., 2008; Duffy and Mitchell, 2013) was used as a loading control across samples, to overcome the difficulty of finding a single control protein present at the same concentration across the lifespan. After imaging for total protein, blots were transferred into a PBS solution containing 5% skim milk and normal goat serum (1:500) and left for 1 hour. Blots were then placed for 2 hours in PBS with one of the four primary antibodies used in this study, and were then washed with PBS and placed in a PBS solution containing Alexa 647-conjugated secondary antibody (1:1,000; Jackson ImmunoResearch). Blots for each intermediate filament were imaged separately, and these images, as well as the respective total protein images, were used to quantify the amount of each intermediate filament. Tissues from human V1 samples were extracted from the calcarine sulcus and homogenized following the procedures described above. The protocols used for human immunoblotting and blot imaging were the same as those described above for cats.

Quantification Histology The density of neurons labeled for each intermediate filament was measured by using a computerized stereology system (newCAST; VisioPharm, Hørsholm, Denmark). Within the region of cat V1 positioned along the medial

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bank of the postlateral gyrus, random samples were selected, and the optical dissector stereology probe was used to count the number of neurons reactive for each intermediate filament. From the right and left hemisphere, four to five sections of V1 that spanned Horsley–Clarke levels P9.0–P1.8 were selected for sampling. Neurons were counted only if they contained strong cytoplasmic labeling but weak nuclear labeling, which together ensured that only neurons cut through the somal midline were counted, and also ensured that caps of labeled neurons were excluded from our sample. Counts were made at 1,0003 magnification by using a compound microscope (BX51; Olympus, Markham, Canada) fitted with a high-resolution digital camera (Infinity31M; Lumenera, Ottawa, ON, Canada). A guard depth of 5 lm was used for counts, and a total of 50% of each masked region was sampled for counting. The density of labeled neurons was calculated for each section by dividing the total number of counted cells by the size of the region sampled. The density of labeled neurons from left and right V1 of each animal was determined by calculating the average density from the sections sampled from each hemisphere.

Phosphorylation Quantification of the level of phosphorylation across development was assessed for NF-H by calculation of the difference in labeling between tissue sections reacted specifically for nonphosphorylated NF-H and sections reacted for NF-H independent of phosphorylation state by exposure to alkaline phosphatase. Therefore, the same antibody targeted against nonphosphorylated NF-H was used to extrapolate the overall level of NF-H phosphorylation by dephosphorylating tissue sections with alkaline phosphatase and thereby revealing for immunolabeling the extent of phosphorylated NF-H. We reasoned that the difference between labeling conditions (D phosphorylation) would provide a means of measuring the level of phosphorylation for each postnatal age examined. The effect of alkaline phosphatase exposure was quantified by calculating the change in root-meansquared (RMS) contrast of densely labeled layer 5 pyramidal neurons and the surrounding neuropil. Bilateral layer 5 regions of interest were divided into subsamples of approximately equal size, and a phosphorylation metric was calculated for each animal, where D Phosphorylation 5 (Ccon 2 CAP)/(Ccon 1 CAP) and Ccon and CAP are the mean RMS contrast of control and alkaline phosphatase-treated samples, respectively. RMS contrast data were also analyzed with a balanced two-way analysis of variance (ANOVA) with alkaline phosphatase treatment and age as factors. A major advantage of measurement of phosphorylation in fixed tissue sections

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is that, unlike homogenate, fixed tissue does not require that active kinases and phosphatases be inhibited throughout the extraction and homogenization process.

Immunoblotting Quantification of the levels of intermediate filaments throughout postnatal development in cats and humans was achieved by measurement of the volume of fluorescently labeled protein bands using the software Image Lab4 (Bio-Rad) to analyze the digital blot images collected after immunolabeling. From the same lanes in which intermediate filament bands were measured, the volume of bands produced by SyproRuby total protein fluorescent stain was measured as loading controls, and then each intermediate filament volume was normalized to total protein volume calculated from the same lane. This process was repeated for each hemisphere and from each animal, as has been described previously (Duffy and Mitchell, 2013). Finally, the proportion of each intermediate filament to total protein was normalized relative to the results obtained from the same hemisphere of the adult average.

Curve fitting Histological and western blot measures of the accumulation of intermediate filaments with age were fit to Naka–Rushton curves (Naka and Rushton, 1966): Iðt Þ5

Imax 3t n 1 B t n 1t50 n

where I(t) is the measure of intermediate filament accumulation at time t, B is the baseline of the curve, n is the exponent that determines the steepness of the curve, Imax is the maximum elevation above the baseline, and t50 is the age at which intermediate filament accumulation has reached half of Imax. The value of t50 also indicates the age at which filament accumulation rate is maximal because the derivative of a sigmoid is a Gaussian-shaped function that peaks at t50.

RESULTS Postnatal development of NF-L Levels of the four intermediate filaments within cat V1 were measured throughout postnatal development by using immunoblot and immunohistochemical methods that were applied to separate groups of animals. Results from immunoblots of V1 homogenate showed that the level of NF-L (Fig. 1A; magenta fluorescence) at birth was extremely low, and remained that way until about postnatal day (P)50, at which time levels started to increase. The amount of NF-L continued to rise through to P90, and then increased even further into

adulthood when levels were observed to be at their highest. Staining for total protein from the same blot shown in Figure 1A demonstrated about equal protein loading across lanes, indicating that the low level of NF-L observed in samples from early postnatal life were not the consequence of unequal total protein loading (Fig. 1B, green fluorescence). Immunolabeling of tissue sections from V1 at birth revealed low NF-L labeling intensity and few immunopositive cell bodies within the developing cortical plate, although some labeled pyramidal shaped perikarya were observed in putative infragranular layers (Fig. 1C). Within the next few weeks, NF-L labeling intensity increased, and was accompanied by the emergence of heavily labeled pyramidal neurons located mostly within the infragranular and supragranular layers 2 and 5 weeks after birth (Fig. 1D,E). Noticeably absent in this period were reacted cells within layer 4, which appeared as a light band that separated supragranular and infragranular layers. Labeling in tissue sections from adult V1 was noticeably more intense than at earlier ages, particularly within the neuropil (Fig. 1F). As a consequence of the increase in neuropil labeling intensity in adults, perikaryal reactivity appeared less distinct from the dark background compared with earlier ages. The high level of extraperikaryal reactivity in the adult appeared to originate predominantly from an increase in labeled processes. Quantification of NF-L across the lifespan from immunoblots (Fig. 1G) and from tissue sections (Fig. 1H) revealed similar sigmoidal accumulations during postnatal V1 development. Measurements from immunoblots and from tissue sections demonstrated that NF-L levels were low at the time of birth, and then increased to reach a peak accumulation rate (50% of adult levels) at P80 from immunoblots, and slightly earlier at P50 when measured from tissue sections. Adult levels of NF-L were reached at approximately 4 months of age and were maintained thereafter.

Postnatal development of NF-M The level of NF-M examined from immunoblots was very low at the time of birth, but showed a noticeable increase at P47 and P90, and even higher levels were observed in adulthood (Fig. 2A; loading controls shown in Fig. 2B). Tissue sections from V1 revealed weak NF-M immunoreactivity at the time of birth, with a small population of labeled pyramidal shaped cells within the developing infragranular layers (Fig. 2C), as was observed with NF-L labeling. The intensity of labeling for NF-M increased primarily within large pyramidal cells in infragranular layers during the first few weeks after birth, and by 6 weeks after birth there were many more immunoreactive neurons, most obviously within the infragranular layers but also at this time there was an emergence

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Figure 1. Development of NF-L in cat V1. A: Multiplexed immunoblot of homogenate from cat V1 showed postnatal accumulation of NF-L (magenta fluorescent bands) from birth to adulthood. These fluorescent bands were observed at 70 kDa. B: The same immunoblot lanes shown in A stained for total protein (green fluorescence) reveal near equal protein loading across lanes. C–F: Sections of V1 reacted for NF-L demonstrate a large postnatal increase in labeled neurons from birth (P0) to adulthood. G: Quantification of band volume from immunoblots of NF-L show a progressive increase in protein across postnatal development that is well characterized by a sigmoid function. H: Measurement of the density of V1 neurons reactive for NF-L shows a sigmoidal increase in immunopositive density with age. In G and H, all measurements are normalized to the highest adult value for each hemisphere separately; squares and circles represent measurements from the left and right hemisphere, respectively; the solid triangle in each graph represents the age at which levels reached 50% of adult values. Scale bar 5 100 mm in F (applies to C–F).

of reactive cells within supragranular layers (Fig. 2D,E). Sections of V1 from adults showed a marked increase in labeling compared with what was observed earlier, with a large number of reactive processes that produced dense labeling product throughout V1 layers (Fig. F), as was also observed with NF-L. The dense NF-M labeling in adults made it more difficult to resolve embedded perikarya compared with NF-M labeling from younger animals. Quantification of the level of NF-M from immunoblots of V1 (Fig. 2G) and the density of immunopositive neurons in sections of V1 (Fig. 2H) across the lifespan both showed low levels at birth that remained low for the following several weeks, after which levels

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began to rise. Quantification of NF-M from both methods showed a sigmoidal increase that had similar peak accumulation rates (P51 from immunoblots) and (P31 from tissue sections). Adult levels of NF-M were reached at approximately P100 and were maintained.

Postnatal development of NF-H Immunoblots reacted for NF-H revealed very low levels at the time of birth that remained low though to P47 (Fig. 3A; loading controls in Fig. 3B). At P90, NF-H levels were considerably higher compared with earlier ages; levels continued to rise and were highest in adults.

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Figure 2. Development of NF-M in cat V1. A: Homogenate from cat V1 examined for NF-M with an immunoblot (magenta fluorescence) revealed a progressive increase from low levels at birth that increased to reach much higher levels in adulthood. These fluorescent bands were observed at 155 kDa. B: Total protein staining of the same blot shown in A confirmed a similar quantity of protein was loaded across lanes. C–F: Labeling for NF-M in sections of V1 was weak at birth but increased across development and was strong in adults. G: Measurement of NF-M from immunoblots showed a sigmoidal rise from low levels early in postnatal life to much higher and stable levels in adulthood. H: The density of NF-M positive neurons measured from sections of V1 was low early in life, and then increased beyond the critical period into adulthood when density stabilized. In G and H, all measurements are normalized to the highest adult value for each hemisphere separately; squares and circles represent measurements from the left and right hemisphere, respectively; the solid triangle represents the points at which levels reached 50% of adult values. Scale bar 5 100 mm in F (applies to C–F).

Tissue sections of V1 labeled for NF-H showed weak labeling at the time of birth, with few labeled perikarya even within the lower layers of the developing cortex (Fig. 3C). At P14 and P36, there was an increase in immunolabeling in the infragranular and supragranular layers due to the emergence of darkly labeled pyramidal neurons that were particularly distinct at P36 (Fig. 3D,E). Sections from adult V1 had many darkly labeled neurons that were evident throughout the supragranular and infragranular layers but not within layer 4, where few labeled cells were observed (Fig. 3F). Quantification of NF-H with immunoblots showed a sigmoi-

dal accumulation that was delayed relative to levels measured for NF-L and NF-M. Levels of NF-H were low at birth but began to rise quickly at about P60, reaching about 50% of adult levels at P101, and stabilizing at adult levels by about P150 (Fig. 3G). Quantification of immunopositive NF-H neuron density (Fig. 3H) showed a similar sigmoidal increase to that observed for NF-L and NF-M but was slightly left-shifted relative to the NF-H immunoblot data. The density of NF-H– reacted neurons was low at the time of birth, increased to reach 50% of adult levels at P42, and reached and sustained adult values by about P100.

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Figure 3. Development of NF-H in cat V1. A: Immunoblot of homogenized cat V1 labeled for NF-H (magenta fluorescence) revealed low levels at birth that increased considerably into adulthood. These fluorescent bands were observed at 180 kDa. B: Total protein staining (green fluorescence) provided a loading control for NF-H bands in A, which revealed a similar quantity of protein was loaded across lanes. C–F: Sections labeled for NF-H examined from cats that ranged from birth to adulthood showed weak reactivity at the time of birth, which increased considerably with age and was very strong into adulthood. G: Quantification of NF-H levels in V1 from immunoblots showed a sigmoidal age-dependent increase from low levels at birth to high and stable levels in adulthood. H: Sections of V1 assessed for density of neurons reactive for NF-H revealed low density early in postnatal life that increased with age according to a sigmoidal function, and reached a much high density in adulthood that remained stable. In G and H, all measurements are normalized to the highest adult value for each hemisphere separately; squares and circles represent measurements from the left and right hemisphere, respectively; the solid triangle represents the age at which levels reached 50% of adult values. Scale bar 5 100 mm in F (applies to C–F).

Postnatal development of NF-H phosphorylation Although all neurofilaments exhibit a susceptibility for posttranslational modification by phosphorylation, the NF-H subunit contains many more repeats of the motif lys-ser-pro (KSP) that offer potential phosphorylation sites (Myers et al., 1987) and provide NF-H with a much greater capacity for phosphorylation than the other neurofilament subunits (Julien and Mushynski, 1982). The phosphorylation of neurofilament is believed to confer an enhanced degree of protein stability by

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decreasing the rate of protease-mediated turnover (Pant, 1988; Gong et al., 2003). In the current study, the appearance of labeling for NF-L, NF-M, and NF-H was similar in sections of V1 during early postnatal development, but sections from adult animals labeled for NF-L and NF-M were much more strongly reacted than adult sections labeled for NF-H (compare C–F in Figs. 1–3). Whereas for NF-L and NF-M there was a pronounced increase in extraperikaryal labeling in V1 from older animals, a similar increase was not observed in sections labeled for NF-H. The antibody that was

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used to identify NF-H (SMI-32) is specific to the nonphosphorylated form of the protein, whereas the antibodies used to label NF-L and NF-M targeted both phosphorylated and nonphosphorylated states. That the labeling intensity and characteristics between neurofilament subunits were comparable at early ages but not

at older ages suggested that in addition to a developmentally regulated accumulation of neurofilament there may also be an age-dependent increase in posttranslational phosphorylation of neurofilament. In other words, we believed that the considerable increase observed with NF-L and NF-M labeling intensity observed in adults may have been the consequence of a developmentally regulated increase in neurofilament phosphorylation. We therefore examined the extent of neurofilament phosphorylation across development by dephosphorylating NF-H with an alkaline phosphatase solution that was applied directly to tissue sections of V1. We reasoned that for each postnatal age the difference in labeling intensity produced by exposure to alkaline phosphatase would represent the extent of NF-H phosphorylation. Early in postnatal development, we observed little difference in the pattern and intensity of NF-H labeling before (Fig. 4A) and after dephosphorylation (Fig. 4B), indicating that phosphorylated neurofilament contributes little to the labeling signal early in development. In contrast, sections of V1 from older animals that were exposed to alkaline phosphatase were substantially more reactive compared with sections from the same animals that were not dephosphorylated, indicating that in older animals a much greater proportion of NF-H is phosphorylated. The extent of NF-H phosphorylation across development was quantified by measurement of contrast in sections labeled for nonphosphorylated NF-H relative to sections exposed to alkaline phosphatase that labeled nonphosphorylated and phosphorylated NF-H (also see Materials and Methods). Early in postnatal development, phosphorylated NF-H accounted for a small amount of the labeling signal in V1; however, in older animals, a much larger portion of the labeling signal was driven by phosphorylated NF-H (Fig. 4C). RMS contrast data for phosphorylated and dephosphorylated sections (D phosphorylation) was

Figure 4. Phosphorylation of NF-H in cat V1 across postnatal development. A: Labeling of nonphosphorylated NF-H was localized to neuron perikarya and dendrites, and the number of reactive neurons increased with postnatal age. B: The extent of NF-H phosphorylation in separate sections from the animals shown in A was revealed by exposure of sections to alkaline phosphatase before NF-H immunohistochemistry. Note that in comparison with sections in A, sections from younger animals (P14 and P36) in B look similar; however, sections from older animals in B look considerably darker relative to those in A due to increased phosphorylation. C: Measurement of change in phosphorylation of NF-H across postnatal development indicated that a low proportion of NF-H was phosphorylated early in development, but increased substantially beyond the critical period and was highest in adulthood. Scale bar 5 100 mm in B (applies to A,B).

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Figure 5. Development of a-internexin in cat V1. A: Examination of a-internexin (magenta fluorescence) with an immunoblot of homogenized V1 showed low levels at the time of birth that increased with postnatal age to reach high and stable levels in adulthood. These fluorescent bands were observed at 65 kDa. B: Total protein staining (green fluorescence) was used as a loading control and revealed that a similar quantity of protein was loaded across the immunoblot lanes shown in A. C–F: Sections of V1 labeled for a-internexin showed weak reactivity at birth, but there was a progressive increase throughout postnatal development and the strongest labeling was observed in adulthood. G: Measurement of the level of a-internexin from immunoblots revealed low levels early in postnatal life that increased sigmoidally with age to ultimately reach high levels in adulthood. H: The density of a-internexin–positive neurons in V1 was also low early in life but increased following a sigmoid function to reach high densities in adulthood. In G and H, all measurements are normalized to the highest adult value for each hemisphere separately; squares and circles represent measurements from the left and right hemisphere, respectively; the solid triangle represents the age at which levels reached 50% of adult values. Scale bar 5 100 mm in F (applies to C–F).

analyzed with a balanced two-way ANOVA with alkaline phosphatase treatment and age as factors. Both main effects were significant (both P < 0.0001), and a significant interaction between age and alkaline phosphatase treatment indicated that difference in phosphorylation state significantly increased with age (P < 0.0003).

Postnatal development of a-internexin The level of a-internexin revealed by immunoblots was low in V1 at the time of birth and for about the

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first 50 postnatal days, and at P90 it was considerably higher than at earlier ages but still less than in adults (Fig. 5A; loading controls in Fig. 5B). Sections of V1 labeled for a-internexin showed a similar age-related accumulation, with a small number of immunopositive neurons observed at birth, which were located within the deep layers of the developing cortex (Fig. 5C). An increase in the number of immunopositive neurons emerged within the supragranular and infragranular layers between 2 and 5 weeks after birth (Fig. 5D and E). Sections of V1 from adults showed a high level of

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a-internexin labeling (Fig. 5F), within perikarya but also within processes that gave sections a much darker appearance overall compared with early ages. Quantification of a-internexin levels from immunoblots (Fig. 5G) or from tissue sections (Fig. 5H) showed low levels at birth and for the first few weeks thereafter; as with the neurofilament subunits, levels rose in a sigmoid fashion to reach about 50% of adult levels by between P80 (measured from immunoblots) and P41 (measured from tissue sections). Sustained adult levels of a-internexin measured from immunoblots or tissue sections were reached at about P100.

Postnatal development of intermediate filaments in human V1 To determine whether the neurofilament subunits and a-internexin in human V1 are postnatally regulated as they are in cat V1, we used the immunoblot method on homogenized samples of human V1 to quantify separately the levels of intermediate filaments thoughout development. Assessment of tissue quality across human samples was made with a total protein stain (green fluorescence, Fig. 6A–D) that demonstrated samples contained a similar level of total protein. Both the immunoblot images (Fig. 6A–D, magenta fluorescence; adjacent green fluorescence represents respective loading control for each filament) and the volume analysis (Fig. 6E–H) show an increase in protein level similar to that observed in cats. Near the time of birth, levels of each intermediate filament were barely detected, but each rose progressively over the next 7–10 years, at which time levels of each neurofilament reached about half of what was observed in adults. The accumulation of a-internexin was slightly delayed relative to neurofilament subunits, and reached 50% of adult levels about 1 year later (Fig. 6D,H). By about 15 years of age, intermediate filament levels reached adult values.

DISCUSSION Results from this study show that NF-L, NF-M, NF-H, and a-internexin, the predominant intermediate filaments found in postnatal neurons, are present in the cat primary visual cortex at very low levels at the time of birth and for the first postnatal month. Levels of all four filaments increased rapidly after the peak of the critical period for ocular dominance plasticity at about 4–5 weeks of age (Olson and Freeman, 1980), and then high and stable adult levels of each were reached between 3 and 4 postnatal months, when plasticity in V1 is known to be low (Olson and Freeman, 1980; Jones et al., 1984). For each intermediate filament we found a similar sigmoidal postnatal accumulation profile

in human V1, but the increase followed a much slower time course compared with what was measured from cats. We also observed that phosphorylation of NF-H increased with age, suggesting that this posttranslational modification, which enhances neurofilament stability, may represent a posttranslational influence on the capacity for structural plasticity beyond early postnatal life. Taken together, these results offer insight into the postnatal maturation of neurons in the visual system, and they are consistent with the view that accumulation of intermediate filaments contributes to the age-related decline of plasticity in cat and human V1. Knowledge of the myriad cellular events that contribute to the decline of plasticity with maturation and the ability to manipulate them may improve responsiveness to treatments aimed at promoting neural recovery in adults. The natural and progressive reduction of plasticity observed in the visual system during postnatal development presumably acts to stabilize and secure experience-driven modifications to neuron structure and function. Among the collection of critical periods that underlie development of the cat primary visual cortex, the most widely studied is the susceptibility of ocular dominance to a period of monocular deprivation. The physiological shift in cortical ocular dominance caused by a period of monocular deprivation (Wiesel and Hubel, 1963b) reaches peak sensitivity in kittens at about 4–5 weeks of age, after which susceptibility to deprivation quickly declines so that little alteration occurs if started beyond 6–8 months of age (Olson and Freeman, 1980; Jones et al., 1984; Daw et al., 1992). The developmental profiles of all three neurofilament subunits and ainternexin in cat V1 were consistent with the idea that these proteins could act to attenuate plasticity, and that their accumulation contributes to the natural agerelated decline in the capacity for neural plasticity. For each intermediate filament we found low levels during the peak of the critical period (P30) when plasticity is high, but shortly thereafter levels of each filament increased rapidly. All four filaments reached stable adult levels at 3–4 months of age when the capacity for plasticity is low. We found a similar relationship in our examination of human V1 that extended to humans the possibility of intermediate filaments acting as brakes on neural plasticity. The critical period of susceptibility to unilateral congenital or traumatic cataract in humans (a medial opacity that resembles experimentally induced monocular deprivation in kittens) extends from 6–8 weeks after birth to about 10 years of age (Vaegan and Taylor, 1979; Birch et al., 1998; Daw, 2013). At 6–8 weeks after birth, we found barely detectable levels of intermediate filaments in human

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Figure 6. Postnatal accumulation of intermediate filaments in human V1. A–D: Respectively, immunoblots of NF-L, NF-M, NF-H, and ainternexin (magenta fluorescence) in human V1 across the lifespan all demonstrate a substantial increase with age. Total protein staining (green fluorescence), used as the loading control for labeling of each intermediate filament, revealed that a similar quantity of protein was loaded across immunoblot lanes. E–H: Respectively, quantification of the levels of NF-L, NF-M, NF-H, and a-internexin from immunoblots reveal a sigmoidal rise in the level of each intermediate filament from low levels shortly after birth, to high and stable levels beyond about 10–15 years. Levels of each intermediate filament were normalized to the highest adult value; the filled triangle in each graph represents the point at which the level of protein reached 50% of adult values.

V1, whereas peak accumulation rates were observed at about 8 years, with adult values reached at about 13– 15 years of age. The stability that intermediate filaments confers to the structural integrity of neurons derives from characteristics such as their long half-life, which is about 3 weeks (Nixon and Logvinenko, 1986; Yuan et al., 2009), their polymerization into stable stationary intracellular networks (Yuan et al., 2009), and their resistance to proteolysis through susceptibility to posttranslational phosphorylation (Pant, 1988; Gong

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et al., 2003). These protein characteristics, in conjunction with the complementary relationship that we report between the critical period profile and the accumulation of intermediate filaments in cat and human V1, provide support for the suggestion that these proteins contribute to the progressive attenuation of plasticity within the developing mammalian visual system (Smith, 1973). Our examination of the level of neurofilament phosphorylation within developing cat V1 revealed a considerable increase with age, even when total neurofilament

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levels at each age were taken into account. Beginning after the peak of the critical period, there was a much larger proportion of NF-H that was phosphorylated compared with measurements at earlier ages. The increase in phosphorylation across postnatal development measured in this study is in agreement with a qualitative assessment of axons in cat V1 labeled specifically for phosphorylated NF-H that showed weak labeling at birth and during the critical period, and then a progressive increase thereafter with the strongest labeling observed in adulthood (Liu et al., 1994). The enhanced stabilization of neurofilament that derives from phosphorylation (Pant, 1988; Gong et al., 2003) raises the intriguing possibility that this modification represents a posttranslational means to regulate plasticity with age. In addition, neurofilament accumulation and phosphorylation may facilitate the progressive postnatal increase in signal conduction velocity in the visual system (Tsumoto and Suda, 1982), an axonal characteristic that is influenced by factors that promote axonal growth such as myelination and the presence of neurofilaments (Yamasaki et al., 1992; Ohara et al., 1993; Zhu et al., 1997). Notably, the spacing between neurofilaments is larger within regions of axons where neurofilaments are phosphorylated, and at these sites axon diameter is larger, implicating neurofilament phosphorylation as a mechanism involved in axonal growth (Hsieh et al., 1994). Taken together, the increase in phosphorylation with age may further the braking ability of neurofilament through enhanced stabilization, and may additionally contribute to the increase in signal conduction velocity that occurs with maturation. A relationship between intermediate filaments and structural plasticity has been established in the visual system of cats (Bickford et al., 1998; Duffy and Sluar, 2009), monkeys (Duffy and Livingstone, 2005), and humans (Duffy et al., 2007), whereby structural modification has been shown to be coincident with a reduction in neurofilament protein. Adult cats and monkeys subjected to monocular eyelid closure do not show the substantial structural changes observed when closure happens during the critical period (Wiesel and Hubel, 1963a; LeVay et al., 1980), and in neither species is there a reduction in neurofilament with adult deprivation (Duffy and Livingstone, 2005; Duffy and Slusar, 2009). Furthermore, the enhanced neural plasticity that occurs with dark rearing (Cynader and Mitchell, 1980; He et al., 2007; Duffy and Michell, 2013) is accompanied by a reduction in neurofilament within the lateral geniculate nucleus (O’Leary et al., 2012) and visual cortex (Duffy and Mitchell, 2013). These results suggest that the loss of neurofilament is permissive for the production of neural plasticity. Reduction of neurofilaments and a-internexin can occur via pro-

teolysis by a calcium-dependent cysteine protease called calpain (Schlaepfer and Zimmerman, 1985; Yang et al., 2013), which has recently been postulated as a molecular brake that regulates neural plasticity in the hippocampus of mice (Wang et al., 2014). It will be interesting to determine whether alteration of neurofilament levels in the context of visual deprivation is catalyzed by calpain activity. Although a causal relationship between neurofilament and plasticity was not evaluated in this study, the results provide additional supportive evidence that intermediate filaments observed in mature neurons act to constrain the capacity for neural plasticity in the visual system. However, it remains possible that the postnatal accumulation of intermediate filaments simply marks the developmental state of neurons and V1, and is not directly related to critical period plasticity. A direct manipulation of intermediate filament levels through genetic techniques or more locally with injection of suitable proteases may provide a useful path for revealing a causal relationship with plasticity; however, the existence of myriad other braking proteins could limit the ability to enhance plasticity by their removal alone. The enhanced plasticity and reduction of neurofilament that occurs with monocular deprivation or dark rearing likely derives from the alteration of a constellation of molecules that synergistically act to promote the observed increase in neural plasticity. The age-related accumulation of intracellular and extracellular molecules has been suggested to contribute to the limited success of treatment for amblyopia in adulthood (Sugiyama et al., 2008; Bavelier et al., 2010; Morishita et al., 2010; Maurer and Hensch, 2012). Within the visual system, the progressive stabilization of cell structure by intracellular and extracellular factors (Pizzorusso et al., 2002; Sugiyama et al., 2008; Duffy and Mitchell, 2013), as well as by adjustments in receptor signaling during postnatal development (Morishita et al., 2010), provides examples of events that occur presumably pari passu with the decreased capacity for plasticity beyond the critical period. Manipulation of these braking molecules has revealed an exciting opportunity to enhance recovery from amblyopia by increasing the capacity for plasticity (Pizzorusso et al., 2006; Morishita et al., 2010; Beurdeley et al., 2012; Duffy and Mitchell, 2013). Investigation of developmentally regulated factors in the cat and human visual systems, such as those detailed in the current study, provides a means of extrapolating the optimal timing of treatments from animal models to humans. A large body of research has investigated the translation of developmental time points between model species and humans (Robinson and Dreher, 1990; Clancy et al., 2001; Workman et al., 2013). Of particular interest is

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cats and 9 years for humans. Within the linear part of the sigmoidal curve shown in Figure 7, we calculated the following conversion equation to extrapolate age between species: ½cat age in months2:3  ½human age in years

Figure 7. Graphical representation depicting an approach to scaling ages between cats and humans that is based on the developmental profile of the accumulation of intermediate filaments measured from cat and human V1. The t50 and n parameters extracted from sigmoidal fits to the western blot data were averaged across intermediate filaments for both cats and humans to generate a summary timeline of intermediate filament accumulation across species. The vertical dashed line indicates for both species the age at which intermediate filament levels reach 50% of adult values.

the “neuroinformatics” approach to equating brain development across species (Berardi et al., 2000; Clancy et al., 2001, 2007; Workman et al., 2013), which allows extrapolation of age based on multiple shared events between species. Despite the many strengths and advantages of this approach to scaling, the limited available data regarding postnatal development of the human visual system have presented an obstacle to translation of postnatal ages from model species with respect to V1 development. With acknowledgement that the current data set is limited to the postnatal measurement of intermediate filaments, we present a possible approach to scaling postnatal ages between cats and humans through comparison of the developmental profiles for each species. This approach to linking developmental time points across species is similar to one that makes use of observable anchoring events such as eye opening that are shared across species but occur at different times (Robinson and Dreher, 1990). In our approach to scaling, the developmental profiles of intermediate filaments from cat and human V1 can be used to predict postnatal ages between the two species (Fig. 7). For instance, the age at which 50% of adult levels was reached was about 3 months for

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The age at which cats are most susceptible to the effects of monocular deprivation by measurement of the magnitude of an ocular dominance shift is about 1.25 postnatal months (Olson and Freeman, 1980), which translates to about 1.7 human years when converted using the above formula. The peak susceptibility of the human visual system to monocular deprivation by unilateral cataract is estimated to be between 1 and 4 postnatal years (extracted from Vaegan and Taylor, 1979), indicating that on at least this measure our age translation is reasonably accurate. The current study, along with others that examine additional factors related to the maturation of the human visual system, such as those that have plotted the development of plasticity mechanisms in human visual cortex (Murphy et al., 2005; Pinto et al., 2010), will collectively provide a more accurate means of scaling ages between species, and will provide a better template to evaluate how closely animal models represent plasticity mechanisms in humans. Ultimately, these comparisons could provide critical data to appropriately scale developmental events relevant to the translation of age-sensitive therapies such as the possible use of darkness in treating amblyopia (Duffy and Mitchell, 2013).

ACKNOWLEDGMENTS We are grateful to the NICHD Brain and Tissue Bank for providing all human brain samples.

CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest.

ROLE OF AUTHORS All authors had full access to all the data in the study and take full responsibility for the integrity of the data and the accuracy of the data analysis. The study was conceived and designed by all authors. Histology and biochemistry was performed by KRD and SS. Data collection was completed by KRD and SS. Data analysis and figure composition was completed by KRD and NAC. All authors contributed to the writing and editing.

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