Altered glial gene expression, density, and architecture in the visual cortex upon retinal degeneration

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Research Report

Altered glial gene expression, density, and architecture in the visual cortex upon retinal degeneration Ashley Cornett a , Joseph F. Sucic a , Dylan Hillsburg b , Lindsay Cyr b , Catherine Johnson b , Anthony Polanco b , Joe Figuereo b , Kenneth Cabine a , Nickole Russo a , Ann Sturtevant a, 1 , Michael K. Jarvinen b,⁎ a

Biology Department, University of Michigan-Flint, Flint, MI 48502 USA Psychology Department, Emmanuel College, Boston, MA 02115 USA

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Genes encoding the proteins of cytoskeletal intermediate filaments (IF) are tightly

Accepted 7 September 2011

regulated, and they are important for establishing neural connections. However, it

Available online 14 September 2011

remains uncertain to what extent neurological disease alters IF gene expression or impacts cells that express IFs. In this study, we determined the onset of visual deficits in

Keywords:

a mouse model of progressive retinal degeneration (Pde6b− mice; Pde6b+ mice have normal

GFAP

vision) by observing murine responses to a visual task throughout development, from

Vimentin

postnatal day (PND) 21 to adult (N = 174 reliable observations). Using Q-PCR, we evaluated

Astrocyte

whether expression of the genes encoding two Type III IF proteins, glial fibrillary acidic pro-

Intermediate filament

tein (GFAP) and vimentin was altered in the visual cortex before, during, and after the onset

Retinal implant

of visual deficits. Using immunohistochemical techniques, we investigated the impact of

S100

vision loss on the density and morphology of astrocytes that expressed GFAP and vimentin in the visual cortex. We found that Pde6b− mice displayed 1) evidence of blindness at PND 49, with visual deficits detected at PND 35, 2) reduced GFAP mRNA expression in the visual cortex between PND 28 and PND 49, and 3) an increased ratio of vimentin:GFAP-labeled astrocytes at PND 49 with reduced GFAP cell body area. Together, these findings demonstrate that retinal degeneration modifies cellular and molecular indices of glial plasticity in a visual system with drastically reduced visual input. The functional consequences of these structural changes remain uncertain. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

The mammalian visual system relies on patterns of spontaneous neural activity that are received from the retina to establish correct visual pathways. As development proceeds, a combination of spontaneous and stimulus-evoked activities must occur to shape neural connections in the visual cortex

(c.f., Goodman and Shatz, 1993); most major developmental events related to the establishment of neural circuitry in the mouse visual cortex are completed by postnatal day (PND) 35 (for review, see Hooks and Chen, 2007). Manipulations to the visual pathway during this developmental period in rodents clearly alter the maturation of neuronal physiology, resulting in immature neuronal firing characteristics (c.f., Fagiolini

⁎ Corresponding author at: Psychology Department, Emmanuel College, 400 The Fenway, Boston, MA 02115, USA. Fax: + 1 617 735 9751. E-mail address: [email protected] (M.K. Jarvinen). 1 Current address: Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.09.011

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et al., 1994; Hooks and Chen, 2007). One potentially important cellular player in this process is the astrocyte. Astrocytes comprise approximately 28% of all cells and 60% of all glia in the visual cortex (Gabbott and Stewart, 1987; Stichel et al., 1991). They reach adult-like numbers relatively early in postnatal development (Caviness, 1982; Taft et al., 2005) and have putative roles in synaptogenesis (for review, see Ullian et al., 2004), neural plasticity (Rudge et al., 1992; Muller et al., 1995, for review, see Barres, 2008), and modification of neurotransmitter release (Smith, 1992, c.f., Paixao and Klein, 2010). Subpopulations of astrocytes in the brain, including the visual cortex, can be identified by the presence of two intermediate filament (IF) proteins, vimentin and glial fibrillary acid protein (GFAP) (Argandona et al., 2003; Dahl et al., 1981; Kafitz et al., 1999; Messing and Brenner, 2003; Missler et al., 1994; Muller, 1992; Privat, 2003). IFs are 8–11 nm in size (Fuchs and Weber, 1994; Herrmann and Aebi, 2004; Parry and Steinert, 1992) and are comprised of IF proteins assembled on microtubule tracks in a kinesin-dependent manner in mammals (Goldman et al., 1996). Five classifications of IF genes exist, based upon similarities in coding sequences (Fuchs and Weber, 1994: Herrmann and Aebi, 2004). Type III IF proteins include vimentin, GFAP, desmin, and peripherin, with only vimentin and GFAP expressed in the CNS (for review, see Sihag et al., 2007). The expression of genes coding for IFs is tightly regulated and likely plays a role in the establishment of neural connections. A number of strategies, including mouse gene knockouts for GFAP and/or vimentin (Colucci-Guyon et al., 1994; Pekny et al., 1995, 1999), have provided evidence that GFAP and vimentin may serve different functions in the brain. In particular, the presence of vimentin IFs, but not GFAP, may signal the existence of an optimal environment for neurite extension, in the absence of injury (Wilhelmsson et al., 2004; for review, see Pekny, 2001). S100β, a calcium-binding protein that modulates GFAP polymerization, may be a mediator of IF function (Bianchi et al., 1993; Ziegler et al., 1998). Given the potential impact of IFs on neural outgrowth, a progressively compromised visual system provides an excellent opportunity to evaluate the expression profiles of IF genes and the influence of these profiles on the cells in which they are expressed. In this study, we used a mouse model of retinal degeneration caused by a naturally-occurring mutation in the phosphodiesterase 6 beta subunit (Pde6b) gene (Pittler and Baehr, 1991; for review, see Chang et al., 2002) expressed in rod photoreceptors (Bowes, et al., 1990). This mutation is lethal to photoreceptors and causes significant remodeling in the retina, such that 90% of rods are lost by PND 21 and 50% of cones by PND 56 (Carter-Dawson et al., 1978; for review, see Jones and Marc, 2005). While significant loss of photoreceptors has been demonstrated for these mice early in development, it is unknown whether there are corresponding neurological changes in the visual cortex throughout postnatal development. This question is particularly relevant and timely for the promising work of therapeutic interventions and treatment of vision loss. We hypothesized that progressive retinal degeneration would result in profiles of elevated vimentin and reduced GFAP gene expression and, thereby, result in similarly altered

47

astrocyte architecture. To this end, we first measured Pde6b− mouse responses to a visual task to identify whether visual deficiencies could be detected throughout postnatal development. We then used Q-PCR to evaluate whether the expression of GFAP or vimentin was altered in the visual cortex in response to retinal degeneration. Lastly, immunohistochemistry was used to explore changes in the density and morphology of GFAP- and vimentin-expressing subpopulations of astrocytes. Our data demonstrate that Pde6b− mice failed the visual task by PND 49; additionally, GFAP gene expression, along with astrocyte density and morphology, was significantly modified at PND 49. These findings are discussed in terms of the putative roles for Type III IFs in neural plasticity, and implications for optimizing retinal transplants.

2.

Results

2.1.

Time-course of vision impairment

We investigated mouse responses to a visual task to determine the onset of vision deficits. Chi-square tests of independence (using 174 valid cases; 75 Pde6b+ observations and 99 Pde6b− observations) revealed no significant association between landing platform recognition and genotype at ages PND 21 and 28 (p > 0.05). Significant associations were observed (Fig. 1B) between landing platform recognition and genotype at PND 35 (χ2(1) = 9.02, p = .003), PND 42 (χ2(1) = 17.036, p < 0.0001), and PND 49 (χ2(1) = 31.395, p < 0.0001). At these later ages, Pde6b− mice were significantly less likely to elevate their hind legs prior to contact with the landing platform. Animals that failed to detect the platform also had a strong tendency to draw their limbs into their body upon descent.

2.2.

Changes in gene expression resulting from vision loss

We evaluated whether gene expression in the visual cortex was altered in mice with progressive retinal degeneration. QPCR was conducted on samples of visual cortex from PND 21 to adult. Sequences, optimal annealing temperatures and primer concentrations are reported in Table 1. We ran samples in triplicate, and calculated the mean CT values of the two closest samples as a representation of the true data point for that animal. The two closest samples were always significantly positively correlated (data not shown). We calcu+ − ΔCTPde6b−) and then compared lated the ΔΔCT value (ΔCTPde6b the means for each age group. There was a significant effect of age on expression levels of GFAP (F(5, 19) = 5.035, p = 0.008), S100β (F(5, 19) = 3.297, p = 0.036), and vimentin (F(5, 19) = 3.144, p = 0.041) in Pde6b− mice relative to Pde6b+ mice. GFAP mRNA expression was reduced in Pde6b− mice relative to Pde6b+ mice from PND 28 to PND 49 (p < 0.05) and later increased (p < 0.05) from PND 49 to adult age (Fig. 2). Statistically significant post-hoc differences were not found for S100β and vimentin. This is likely the result of a combination of several factors including 1) our modest sample size, 2) an unbalanced design, 3) large numbers of comparison groups, and 4) interanimal differences in the progression of retinal degeneration. This last factor is also likely the cause of elevated variability in a portion of the Q-PCR data set, particularly at PND 35.

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A

B

Fig. 1 – Visual test. A: The set-up, in which the experimenter lowered each mouse down from an elevated platform (dotted rectangle) to a landing platform (square grid). Two digital camcorders (rectangles) captured the descent of each mouse from both the side and belly perspectives and drop duration (stop watches; filled black rectangles). B: Line graph showing percentage of Pde6b− and Pde6b+ mice that elevated their hind limbs prior to contact with the landing platform different developmental ages. Number of independent mouse observations (Pde6b−:Pde6b+) for each developmental age: PND 21 (24:15), PND 28 (13:15), PND 35 (16:14), PND 42 (23:15), and PND 49 (23:16). **p<0.05.

2.3. Changes in astrocyte density and morphology resulting from vision loss

Fig. 2 – Changes in gene expression in Pde6b − mice (relative to Pde6b +mice) across ages for GFAP, S100β, and vimentin (**p < 0.05). Expression levels are plotted as ΔΔCT (see Experimental procedures). GFAP panel: Pde6b − mice showed significant reductions from PND 28 to PND 49 (**), followed by a significant increase to adult age (**). Note that a ΔΔCT of one is equivalent to a 100% increase in mRNA level. Error bars represent SEM in all figures.

Trained researchers used a representative sampling strategy to quantify cellular density and morphology from labeled cells in sections of the visual cortex (Figs. 3, 4A, and 5A). A Pearson correlation coefficient was calculated to evaluate the inter-rater

reliability, with highly significant positive correlations found (data not shown). There were significant effects of age on the density of vimentin-labeled (F(1, 11) = 8.183, p = 0.024) and

Table 1 – Real-time PCR primer sequences, product sizes, optimal concentrations, and annealing temperatures. Gene GFAP S100β Vimentin GAPDH

Sequence

Product Size

[Primer]

Anneal (°C)

Up-5′-TTGCAGACCTCACAGACGCTGCGT-3′ Down-5′-GCATGGCGCTCTTCCTGTT-3′ Up-5′-TAAGAATCAAGGCAGACTACCAA-3′ Down-5′-GTCTGTCTACTTTCTGGAGCAT-3′ Up-5′-GCCAAATCCCCTATGCCCAAATCA-3′ Down-5′-CCTTCTTTTTATCTGCAACATCTT-3′ Up-5′-GGCAAGGTCATCCCAGAGC-3′ Down-5′-CCTTCAGTGGGCCCTCAGATGC-3′

172 bp

250 nM 500 nM 500 nM 1000 nM 500 nM 1000 nM 1000 nM 1000 nM

53.5

173 bp 193 bp 163 bp

58.0 53.5 56.5

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A

49

B 1

3

2

4

C

Fig. 3 – Sampling strategy. A: Schematic diagram showing sampling area of the primary visual cortex; commissure of fornix is also shown for reference. B: Low magnification photomicrograph (200×; scale bar=100 μm) of GFAP-labeled cells showing the four sampling grids used to obtain cell density counts. Note that each sampling grid overlapped with other grids to provide anatomical landmarks to minimize doublecounting cells. Pia mater (angled arrows) and commissure of fornix (straight arrows) are indicated. C: An example of sample grid #3 at higher magnification (400×; scale bar=50 μm). Commissure of fornix is indicated by the arrow.

GFAP-labeled (F(1, 11) = 11.914, p = 0.011) cells in the visual cortex. Significant interactions between age and genotype were observed for vimentin (F(1, 11) = 8.973, p = 0.02) and GFAP (F(1, 11) = 6.367, p = 0.04). Pde6b− mice showed increased density compared to Pde6b+ mice (p < 0.05) in vimentin- and GFAP-labeled cells in the visual cortex at PND 49 compared to PND 28 (see Table 2). GFAP results were validated by S100β cell counts (data not shown). Data were transformed into vimentin:GFAP ratios to highlight the strength of the effect of retinal degeneration on astrocytes that express these IFs (see Fig. 4B). There were significant effects of age on GFAP-labeled cell body areas in the visual cortex (F(1, 11) = 11.764, p = 0.009). A significant interaction between age and genotype were also observed for GFAP (F(1,11) = 5.979, p = 0.04). Pde6b− mice showed decreased cell body area compared to Pde6b+ mice (p < 0.05) at PND 49 in GFAP-labeled cells in the visual cortex (see Fig. 5B). This effect was also observed in S100β-labeled cells (data not shown).

3.

Discussion

The goal of this study was to determine whether visual impairment caused by a neurological disease altered the expression of astrocyte-specific intermediate filament (IF) genes, and whether

Fig. 4 – Cell density. A: Low power photomicrograph showing vimentin-labeled cells in the visual cortex of both Pde6b+ and Pde6b− mice at PND 28 and PND 49. Pia mater (angled arrows) and commissure of fornix (straight arrows) are indicated. Scale bar = 100 μm. B: Astrocyte cell number ratio, Vimentin:GFAP.

this led to a change in the density or structure of specific subpopulations of astrocytes in the visual cortex. First, we investigated the progression of visual impairment in Pde6b− mice by administering a visual task and found that they consistently failed the task by PND 49. Second, we evaluated the effect of retinal degeneration on Type III IF gene expression (GFAP and vimentin) in the visual cortex by using Q-PCR. Our data showed transient changes in GFAP expression between PND 28 and PND 49 in Pde6b− mice. Third, we investigated the relationship between IF gene expression and astrocyte density/structure. Cell counts of vimentinlabeled astrocytes were elevated more strongly compared to GFAP-labeled astrocytes at PND 49, and cell body size (an indirect measure of astrocyte processes) for GFAP-labeled astrocytes was significantly decreased, suggesting the possibility that astrocytic processes had retracted. Together, these findings support the conclusion that loss of sensory inputs to the visual cortex transiently altered astrocyte-specific IF gene expression which reduced the sphere of influence of each astrocyte.

3.1.

Time-course and relevance of vision loss in Pde6b − mice

Our data indicated that adolescent Pde6b− mice performed normally to stimuli (visual platforms) that required a photopic visual system, but their performance declined progressively

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Fig. 5 – Cell area size. A: Photomicrograph showing GFAP-labeled cells in the visual cortex. Numbers identify cells that met the criteria for cell counts. Arrows identify cells that met the criteria for quantification of cell body area. Scale bar = 15 μm. B: Quantified cell area for vimentin and GFAP. **p < 0.05.

until there was no measureable response to these stimuli at PND 49. This deficit was maintained into adulthood (data not shown) and therefore permanent. We believe the 2-week period of time, from PND 35 (early symptom onset, only some Pde6b− mice affected) to PND 49 (complete symptom onset; all Pde6b− mice affected), reflects the progressive nature of this neurological disease in Pde6b− mice. Visual deficits have been reported before in adult FVB/NJ (Pde6b−) mice; they perform no better than chance on a variety of visual acuity tests (c.f., Wong and Brown, 2006). However, to our knowledge, PND 35 represents the earliest reported deficiency in performance on a visual task in this strain of mice. This presented to us a very interesting opportunity to investigate how the visual cortex responded to retinal cell death in a

Table 2 – Number of Type III IF cells/mm2 ± SD in the visual cortex of Pde6b+ and Pde6b− mice at different developmental ages. **p < 0.05.

PND 28 Pde6b+ Pde6b− PND 49 Pde6b+ Pde6b−

GFAP

Vimentin

567 ± 180 473 ± 77

785 ± 177 553 ± 108

634 ± 42 906 ± 99**

761 ± 59 1,593 ± 500**

clinically relevant and reliable model of vision loss caused by a naturally-occurring mutation.

3.2. Retinal degeneration alters Type III IF gene expression: functional implications We found no large differences in Type III IF gene expression at PND 21 to suggest that rod degeneration caused changes in the visual cortex. However, GFAP mRNA expression increased approximately 500% in Pde6b− mice from PND 21 to PND 28 (ΔΔCT = 2.3). This may be instructive, given that nearly all rod photoreceptors have degenerated during the first postnatal month of life (Carter-Dawson et al., 1978) causing dramatic retinal reorganization in Pde6b− mice (for review, see Jones and Marc, 2005). Future studies that use scotopic vision tests may yield important progressive deficits related to rod function that may correspond with gene transcription studies in the visual cortex. Despite the fact that cone photoreceptors comprise only approximately 3% of the photoreceptors in the mouse retina (Jeon et al., 1998), we found that progressive loss of photopic visual inputs after PND 21 lead to a multiphasic response in Type III IF gene expression in the visual cortex. In Phase 1 (“Generalized IF reduction”), from PND 28 to PND 35, GFAP and vimentin mRNA expression in Pde6b− mice were each reduced 36% and 27%, respectively. Genes encoding GFAP and vimentin have similar promoter sequences and coordinated regulation of these genes could decrease the availability of

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both IF proteins. This could have functional implications. Complete elimination of Type III IFs has definitive outcomes in GFAP−/−Vim−/− knockout mice, resulting in increased neuron survival and dendritic branching following transplantation of neural precursor cells into the hippocampus (Widestrand et al., 2007), increased synaptic complexes following a cortical lesion (Wilhelmsson et al., 2004), and increased axonal sprouting and functional recovery following injury to the spinal cord (Menet et al., 2003). In Phase 2 (“Vimentin compensation”), from PND 35 to PND 42, vimentin expression was elevated approximately 800% (although not statistically significant) at a time when visual deficits are evident in mice with progressive retinal degeneration. This very large, albeit transient, increase in vimentin IF mRNA levels may have compensatory biological relevance, since neurons cocultured in the absence of GFAP (and presence of vimentin) IFs showed enhanced survival and neurite outgrowth (Menet et al., 2001). The presence of vimentin, lack of GFAP, or the modulation of factors that influence both gene products, may be important for creating conditions that support brain plasticity. Given the progressive loss of visual inputs to the visual cortex in this model of retinal degeneration, and the existence of cross-modal plasticity reported in other models of sensory loss (c.f., Van Brussel et al., 2011; Newton and Sur, 2005), it is tempting to speculate on the different roles Type III IFs may play in the establishment of such cross-modal plasticity. This is important to note because in Phase 3 (“GFAP reduction”), at PND 49, GFAP expression was further significantly reduced to 4% of PND 28 expression levels. We note that S100β, a mediator of IF protein assembly, showed decreased expression at PND 49 to 20% of PND 42 expression levels, perhaps to compensate for reduced GFAP. Typically, S100β, upon calcium activation, prevents the assembly of IFs by sequestering individual subunits and/or inhibiting their phosphorylation (Donato, 2003; Frizzo et al., 2004; Rothermundt et al., 2003; Ziegler et al., 1998). Phase 4 (“Generalized IF restoration”, seen in adult mice), was characterized by IF (and S100β) expression levels returning to or exceeding Pde6b+ mice (wildtype) levels. This observation suggests that any changes to the neural milieu in the visual cortex have stabilized by adult age. We believed that significant reduction in Type III IF transcription levels might be reflected in astrocyte cell death, since they are expressed preferentially in these cells. After vision impairments became obvious, IF-labeled astrocytes were more numerous, with a much higher ratio of vimentin:GFAP astrocytes. This ratio has been described before in a model of avian seasonal song behavior, noting that a higher ratio of vimentin:GFAP astrocytes was exhibited upon loss of song behavior as compared to gain of song behavior (Kafitz et al., 1999). These authors suggested that increased numbers of vimentin-containing astrocytes may promote plasticity in song regions of the bird brain whereas increased numbers of GFAP-containing astrocytes may inhibit plasticity in order to maintain existing neurological connections. In mammals, several lines of evidence suggest that the density of specific subclasses of astrocytes is increased in the visual cortex by factors known to induce plasticity, such as environmental enrichment (increased numbers of GFAP-labeled cells; c.f., Jones and Greenough, 1996) and physical exercise (increased S100-

51

labeled cells; Argandona et al., 2009), most notably in conjunction with a number of indices of neuronal plasticity (for review, see Nithianantharajah and Hannan, 2006). However, it is unclear whether the vimentin:GFAP ratio is higher in these models because the density of vimentin-labeled cells was not sampled. The cause of increased vimentin:GFAP ratio in our study, after visual impairment, is uncertain. Vimentin expression is often associated with functionally immature astrocytes since vimentin-labeled cells are observed early in postnatal development before brain maturation has taken place (Dahl, 1981; Dahl et al., 1981; Stichel et al., 1991). It is unlikely that mature astrocytes reverted to an immature state, since there was good agreement in the number of GFAP- and S100β-labeled cells (each conventionally used as a marker for mature astrocytes) at PND 28 and at PND 49. It is unknown whether retinal degeneration influences astrogenesis; however, there are examples where damage to the visual cortex results in astrogenesis. Neural stem/progenitor cells rapidly proliferate in vivo after laser-induced lesions in adult mouse visual cortex. When cells from the lesion area were placed into culture, they were able to differentiate into astrocytes ex vivo (Sirco et al., 2009). One possible mechanism for astrogenesis is the absence of CD81, an integrin-associated tetraspan protein, which strongly increases astrocyte proliferation during development (Geisert et al., 1996, 2002). Our data also indicated the possibility of diminished astrocyte differentiation. GFAP-labeled cells in Pde6b− mice were 86% of the size of control mice, but only after obvious visual impairment was evident. We used cell body size as an indirect measure of astrocytic processes because it is difficult, if not impossible, to take measurements of and make reasonable conclusions regarding process length in situ. There are several reasons for this, including 1) the variability of IF antibodies in labeling astrocytic components, 2) the lack of IF proteins in distal astrocytic processes (Bushong et al., 2002), and 3) the challenges inherent in using thin brain sections (process truncation). In vitro, others have demonstrated that the diameter of GFAP-positive cell bodies is positively correlated with both number and length of cell processes; this cellular architecture can be modulated significantly and quickly, within a 30-min period of time (Muranyi et al., 2006). IF transcription is required for these changes in process length as shown by GFAP antisense inhibition in vitro (Chen and Liem, 1994; Weinstein, et al., 1991). At the surface, animal models of visual deprivation that utilize dark-rearing to induce visual deprivation would appear to be good candidates to approximate findings from our model of retinal degeneration. In these paradigms, visual deprivation often precedes eye opening and is maintained for weeks or months. Changes to the visual cortex are then measured. Data from these models show, among other things, modified astrocyte density (Argandona et al., 2003; Corvetti et al., 2006; Gabbott et al., 1986; Muller, 1990; Stewart et al., 1986). With regard to astrocytes, the length and timing of dark-rearing appear to be critical determinants of GFAP expression in the visual cortex. Animals dark-reared, prior to eye-opening until PND 24, showed no differences in GFAP protein level or cell counts (Corvetti et al., 2003), whereas animals dark-reared until PND 40 showed reduced number of GFAP-

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labeled cells (80%) and protein level (50%); the latter also exhibited altered cell shape and process length. S100β was reduced to a similar extent. Notably, changes in GFAP protein expression and cell structure were reported in the absence of differences in GFAP mRNA expression (Corvetti et al., 2006). Together, data from these studies suggest, like ours, that GFAP mRNA expression levels may not accurately predict translational outcomes or features related to cell number or architecture. The Pde6b− mice used in our study differ from animals that are dark-reared in that they displayed no evidence of altered photopic vision until PND 35, approximately 3 weeks after eye opening. In short, Pde6b− mice had visual experiences for a period of time that overlapped with known critical periods in the visual cortex. Our data underscore the importance of visual experience in an organism that is becoming blind, in that the ratio of vimentin:GFAP astrocytes increased at postnatal ages that corresponded to vision loss, and these cells likely had diminished astrocytic processes. Anatomically, this could create a more compact sphere of influence, as defined by the shape and extent of astrocytic processes. A recent review underscored the relationship between astrocytic polyhedral shape, contact spacing, and relative nonoverlap with adjacent astrocytes (Nedergaard et al., 2003). The outcome suggested by our data would be a smaller functional unit, the implications of which could be substantial; the stellation of astrocytic processes is known to influence extracellular ion homeostasis, neurotransmission, and likely brain function (reviewed in Theodosis et al., 2008).

3.3.

Considerations for transplant strategies

Retinal transplantation has the capability to improve the lives of millions of people worldwide. MacLaren et al. (2006) showed promising evidence that retinal transplants can result in some restoration of pupillary dilation and light sensitivity in adult mice with retinal degeneration. These observations suggest that photoreceptor cells have become incorporated functionally at the level of the retina, a critical first step in the process of sight restoration. Additional factors will likely increase the success rate of transplants (c.f., West et al., 2008). Still, very little is known about how changes in the visual cortex, resulting from retinal degeneration, affect the encoding of incoming visual signals. We suggest that the timing of the transplant might be optimal at the age when vision loss is first experienced; in our model of retinal degeneration, this would be between PND 35 and PND 49, when vimentin and GFAP expression were substantially modified in the visual cortex. Interestingly, mice deficient in GFAP and vimentin have been used as hosts for retinal transplantations. The transplanted cells were able to integrate into the host retina, maintain neuronal identity, and also project to the appropriate neural area. No functional validation of these projections was reported (Kinouchi et al., 2003).

3.4.

Conclusion

In conclusion, this study used a visual task to identify postnatal time points when photopic vision became impaired in a mouse model of retinal degeneration. This visual impairment

occurred concomitantly with a multiphasic pattern of altered expression of GFAP and vimentin IF genes in the visual cortex, an increased ratio of vimentin:GFAP astrocytes, as well as possibly decreased astrocyte process differentiation. In other animal models, data patterns similar to the ones we describe suggest the possibility for the formation of a more permissive environment for neural connectivity. Our data indicate that this effect, however, is likely transient. Future studies will be needed to determine the functional implications of these findings and to test whether the changes in these IF genes believed to affect brain permissivity present an opportunity to optimize therapeutic retinal implant interventions.

4.

Experimental procedures

4.1.

Animals

All mice were housed individually in standard cages in colony rooms with a 12:12 (L:D) cycle, with food and water provided ad libitum. Animals were bred for the purpose of this study, and all protocols were approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan and Emmanuel College. Separate groups of mice were used for each phase of the experiment. This study utilized a commercially available (Jackson Laboratories; Stock #003257) transgenic mouse strain, FVB/N-Tg (GFAP-GFP)14Mes/J (Zhuo et al., 1997), hereafter called Pde6b− mice. The Pde6b−mouse has a naturally-occurring nonsense mutation in exon 7 of the phosphodiesterase 6 beta subunit (Pde6b) gene (Pittler and Baehr, 1991) expressed in rod photoreceptors (Bowes, et al., 1990). This causes cGMP levels to accumulate (Farber and Lolley, 1974) and leads to a moderate rate of retinal degeneration that occurs in several welldefined stages (Marc et al., 2003). An appropriate behavioral control for the Pde6b− mice is the congenic mouse strain, FVB.129P2-Pde6b + Tyrc-ch/AntJ, hereafter called Pde6b+ mice (Jackson Laboratories; Stock #004828). This strain originated on a FVB;129P2 background and is homozygous for the 129P2/OlaHsd wildtype Pde6b allele. Backcrossed for at least 11 generations on the FVB background, Pde6b+ mice do not show any evidence of retinal degeneration. 4.2.

Procedure

4.2.1. Vision loss in Pde6b− mice during development The visual task used in this study was a variation on the Irwin (1968) protocol, used originally to test the effects of pharmacological agents on behavior. In our task, we archived mouse behavior with digital camcorders equipped with a slow playback feature thereby allowing later unambiguous quantification of murine limb movements. Ten litters of mice (five litters of Pde6b+ mice and five litters of Pde6b− mice) were tested weekly from postnatal day (PND) 21 to 49, and then again as adults (PND 140). Litters were culled to 10 pups at birth to control for developmental effects. All trials were conducted between 1100 and 1400 h; these behavioral tests only occurred in a lit room, necessary to evaluate the physiological relevance of remaining cones. Individual

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litters of mice were brought to the test room 1 h prior to testing to minimize anxiety associated with a novel environment. The experimenter lowered each mouse from a starting platform located 20 cm above a 400 cm2 landing platform (see Fig. 1A for schematic of set-up). We used heavy grit sandpaper to create a three-dimensional contrast. We quantified whether a mouse splayed its front limbs or elevated its hind limbs to better position its body for contact. A mouse that is blind has no limb response. Two digital camcorders captured the descent of each mouse from both the side and belly perspective to provide multiple views to unequivocally measure limb behavior. Stop watches, accurate to the 100th of a second, were also positioned in the field of view to validate drop time (each drop took approximately 0.5 s). Only three trials were made per mouse to control for fatigue and learning. Sheets of sandpaper were changed for each litter to minimize the effect of novel scents on motivation. Only mice that maintained a steady body orientation and visualization of all limbs were included in subsequent analyses. 4.2.2. Type III IF mRNA expression in the visual cortex during development Forty male Pde6b− (N = 20) and Pde6b+ (N = 20) mice were euthanized weekly between PND 21 and PND 49, and also at PND 140 (representative adult age). Brain tissue from the left visual cortex was quickly placed into ice-cold phosphate buffered saline (PBS; pH 7.4) and homogenized. RNA was extracted using the PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen; Carlsbad, CA). Contaminating genomic DNA was removed by treating RNA with DNase I (Invitrogen). The DNased RNA samples were stored at −70 °C. cDNA synthesis (SuperScript™ III First-Strand Synthesis System for RT-PCR; Invitrogen) was carried out for every DNase treated RNA sample. Primer sets were designed using sequences of mouse genes obtained from GenBank (Accession numbers NM_010277.2 [GFAP], NT_039510.2 [S100β], NM_008691.2 [vimentin], and XR_031086.1 [GAPDH]). Each primer set resulted in a single product with no primer-dimer formation; amplification of the correct product was verified independently by the DNA Sequencing Core at the University of Michigan (Ann Arbor, MI). Standard and efficiency curves were plotted for both the Pde6b− and Pde6b+ strains of mice and evaluated using well-established criteria (Yuan et al., 2006). Since the slope of each line for each gene was not significantly different from −1, and the slope of Pde6b− and Pde6b+ lines for the same gene was not significantly different from each other, the efficiency values for each gene were accepted as optimal. Reactions were done in triplicate for each animal. Each reaction for Q-PCR contained 12.5 μL of SYBR Green master mix (QIAGEN), 1 μL upstream primer, 1 μL of downstream primer, 9.5 μL of RNase-free water, and 1 μL of appropriate cDNA. Each sample was run in triplicate along with no-template controls. Cycling parameters (Mastercycler ep Realplex4; Eppendorf; Westbury, NY) were: 1 cycle at 95 °C for 5 min, 40 cycles at 95 °C for 15 s, annealing temperature for 15 s, 72 °C for 20 s, and 1 cycle of 95 °C for 15 s. Q-PCR fluorescence measurements were taken after every cycle, and the cycle threshold (CT; designated as fluorescence intensity 10 standard deviations above the noise of the

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baseline) was recorded for each reaction. We analyzed the closest two of the triplet values, as long as they were within one amplification cycle. If the data did not meet this criterion, the reaction was repeated in triplicate. 4.2.3. Astrocyte immunohistochemistry The immunohistochemistry procedure was used to investigate whether changes in the abundance of intermediate filament mRNA corresponded to altered astrocyte number or structure. Mice (N = 12) were sacrificed with CO2 and then perfused transcardially with 25 ml of 0.1 M phosphate-buffered saline (PBS, pH 7.4) with heparin followed by 50 ml of 4% paraformaldehyde in 0.1 M PBS. Brains were extracted, post-fixed overnight at 4 °C, and then cryoprotected for 24 h in 30% sucrose/PBS. A freezing slide microtome was used to obtain 30 μm thick coronal sections from the primary visual cortex. Serial sections were cut in series of three and placed in wells containing PBS, three wells per animal (i.e., Well #1 received sections 1, 4, 7, etc.; Well #2 received sections 2, 5, 8, etc.; Well #3 received sections 3, 6, 9, etc.). The immunohistochemistry protocol was begun immediately thereafter, and all steps were conducted on an orbital shaker table. Sections in each well were incubated in 0.3% hydrogen peroxide in potassium-buffered saline (KPBS) to quench endogenous peroxidase activity. All sections were blocked in goat serum overnight at 4 °C and incubated the next day for 24 h in antibodies specific for either GFAP or vimentin (Thermo Fisher Scientific; Pittsburgh, PA). Additional sections were incubated in antibodies specific for S100β (Abcam; Cambridge, MA). The next day, sections in each well were incubated in goat anti-rabbit biotinylated secondary antibody for 2 h at room temperature followed by incubation in horseradish-peroxidaseconjugated avidin–biotin complex (Elite Standard kit, Vector Laboratories, Inc.; Burlingame, CA) for 1.5 h. The peroxidase complex was visualized using 3,3-diaminobenzidine tetrachloride (DAB kit, Vector). Sections were dehydrated in graded ethanol rinses, cleared in xylene, mounted on glass slides, and coverslipped using Permount (Fisher Scientific). Following immunohistochemistry, sections that were stained consistently, with an absence of tears or folds, were chosen randomly to provide representative estimates of GFAP, vimentin, and S100β cell counts. All sections in our sample were matched to corresponding regions of the primary visual cortex (approximate to − 0.40 to 0 mm Interaural and −4.20 to −3.80 mm from Bregma) (Fig. 3A). Each section was evaluated using light microscopy, and four images of the primary visual cortex from viable sections were captured at 200× magnification (QImaging camera, Ludl Electronic Products, Ltd.; Hawthorne, NY). These overlapping images, each approximately 400 μm × 300 μm, were used to delineate the sampling area, and represented the entire lamination of the visual cortical region, from pia mater to white matter (Fig. 3, panels B and C). For each digital image, Image J software was used to identify and quantify cells that expressed GFAP, S100β, and vimentin. To identify labeled cells, each digital image was inverted and a background pixel value was calculated by taking the average of three representative background points within the tissue section that were not labeled cells. An “identification” threshold value was then calculated that was 10%

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above this mean background pixel value. All pixel values lower than the identification threshold were eliminated and operationally defined as “noise”. Trained researchers, blind to the goals of the study, marked all unambiguous cell bodies that remained in the image. This image was then compared to the original digital image. Only cells that were more than 50% labeled were counted. Our use of the identification threshold was designed to eliminate false positives (cells with a label intensity no greater than background) and “partial” cells (truncated from the sectioning procedure) from our sample (Fig. 3C). Landmarks within each overlapping image ensured that no cell was missed in the counting procedure (Fig. 4A). In a subset of quantified cells, Image J was used to measure cell body size (an operational estimate of process complexity). These measurements only occurred in cells with clear cell body profiles (Fig. 5A). 4.3.

Statistical analyses

All behavioral and gene expression data were analyzed with SPSS for Windows (Version 18) statistical software program. To evaluate behavioral data from the vision task, Chi-square tests of independence were used to test for the statistical significance of the association between landing platform recognition and genotype. To evaluate differences in Q-PCR gene expression, we calculated the ΔCT value by subtracting GAPDH from each target gene for each animal at each age (i.e., GFAPPND 28 − GAPDHPND 28). In order to calculate the ΔΔCT value for each gene at each time point, ΔCT values for Pde6b− were ordered and then subtracted from ordered ΔCT values for Pde6b+ (i.e., highest ΔCT Pde6b+PND28 − highest ΔCT Pde6b−PND28; lowest ΔCT Pde6b+PND28 − lowest ΔCT Pde6b−PND28). Means were evaluated by one-way ANOVAs for each gene with Age as the between-subject variable. Differences in astrocyte cell counts and cell body area were evaluated by twoway ANOVAs with Genotype and Age as between-subject variables. For all statistical tests, p < 0.05 was considered statistically significant. Post-hoc pairwise comparisons using a conservative Bonferroni adjustment were conducted to determine differences between means, and to assist with interpreting any significant interactions.

Acknowledgments We thank Dr. Todd Williams for a critical evaluation of this manuscript. This work was supported by funding from the Office of the Vice President of Academic Affairs at Emmanuel College, and the Office of Research at the University of MichiganFlint.

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