Dendritic spine abnormalities in the occipital cortex of C57BL6 Fmr1 knockout mice.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 136B:98 –102 (2005) Dendritic Spine Abnormalities in the Occipital Cortex of C57BL/6 Fmr1 Knockout Mice Brandon C. McKinney,1,4 Aaron W. Grossman,2,3,4 Nicholas M. Elisseou,4 and William T. Greenough2,4,5* 1 Department of Biology, University of Illinois, Urbana, Illinois Neuroscience Program, University of Illinois, Urbana, Illinois 3 Medical Scholars Program, University of Illinois, Urbana, Illinois 4 Beckman Institute, University of Illinois, Urbana, Illinois 5 Departments of Psychology, Psychiatry and Cell and Structural Biology, University of Illinois, Urbana, Illinois 2 Fragile X syndrome (FXS) is the most common form of inherited mental retardation. Observed neuropathologies associated with FXS include abnormal length, morphology, and density of dendritic spines, reported in individuals with FXS and in Fmr1 knockout (KO) mice, an animal model of FXS. To date, however, these neuropathologies have been studied in Fmr1 KO mice bred in a FVB background (a strain with genetic mutations that complicate interpretation of results) and findings have been inconsistent. Here, Golgi–Cox impregnation was used to investigate length, morphology, and density of dendritic spines on layer V pyramidal neurons in visual cortices of Fmr1 KO and wildtype (WT) mice bred in a C57BL/6 background. We report that spine abnormalities in these animals parallel abnormalities reported in humans with FXS, perhaps to a greater degree than KO mice bred in an FVB background. Specifically, Fmr1 KO mice bred in a C57BL/6 background exhibited significantly more longer dendritic spines and fewer shorter spines, as well as more spines with immature-appearing morphology and fewer with mature-appearing morphology than WT littermates. Spine length abnormalities were demonstrated to be largely independent of spine morphology abnormalities, as the length phenotype was observed in KOs even within a morphological category. Fmr1 KO mice also had a greater overall spine density than WTs. These findings provide powerful support for the essence of the dendritic spine abnormalities in the absence of FMRP, now found to be largely consistent with human data across two mouse backgrounds. ß 2005 Wiley-Liss, Inc. Grant sponsor: NIH; Grant numbers: MH35321, HD37175; Grant sponsor: FRAXA Foundation. Brandon C. McKinney’s present address is University of Michigan, Ann Arbor, Michigan. Nicholas M. Elisseou’s present address is University of Chicago, Chicago, Illinois. *Correspondence to: William T. Greenough, University of Illinois at Urbana-Champaign, Beckman Institute, 405 N. Mathews Avenue, Urbana, IL 61801. E-mail: email@example.com Received 5 October 2004; Accepted 3 February 2005 DOI 10.1002/ajmg.b.30183 ß 2005 Wiley-Liss, Inc. KEY WORDS: mental retardation; knockout mice; background strain differences; Golgi BODY OF TEXT Fragile X Syndrome (FXS) is the most common form of inherited mental retardation affecting 1 in 4,000–6,000 individuals [de Vries et al., 1997]. Behavioral features of FXS include moderate to severe mental retardation, as well as autistic-like and hyperactive behaviors. Common physical features include large and prominent ears, a long, narrow face, and macroorchidism [Hagerman and Hagerman, 2002]. FXS is an X linked disorder caused by an expanded CGG repeat in the 50 -untranslated region of the FMR1 gene, resulting in transcriptional silencing and absence of fragile X mental retardation protein (FMRP) [Verkerk et al., 1991]. The neuropathological feature most consistently reported in individuals with FXS is abnormal dendritic spines. Qualitative observations of cortical neurons in post-mortem tissue from individuals with FXS have described longer, thinner dendritic spines with a smaller synaptic contact area than in control tissue [e.g., Hinton et al., 1991]. A quantitative study of dendritic spines on Golgi-stained layer V pyramidal neurons compared cortical tissue from individuals with FXS with tissue from control subjects [Irwin et al., 2001]. These authors reported (1) significantly more dendritic spines with immature-appearing morphology (thin spines) and fewer spines with mature-appearing morphology (mushroom or stubby-shaped; see Fig. 1A); (2) more longer and fewer shorter dendritic spines; and (3) a higher density of spines (number per unit dendrite length) in tissue from individuals with FXS. These findings suggest a requirement for FMRP in normal dendritic spine development and/or maturation. Knockout (KO) mouse models of FXS have also been examined for similar dendritic spine abnormalities. Like humans with FXS, KO mice in which the FMR1 gene has been disrupted produce no functional FMRP, making them valuable models for studying the pathobiology of FXS [Bakker et al., 1994]. Initially, Comery et al.  observed elevated dendritic spine density and increased spine length on apical shafts of Golgi-stained layer V pyramidal neurons of adult Fmr1 KO mouse visual cortex compared to wildtype (WT) mice, paralleling findings in individuals with FXS. Following this early report, other laboratories began to study dendrites and dendritic spines in Fmr1 KO mice using a variety of techniques. Braun and Segal , for example, reported that dendrites on cultured hippocampal neurons from Fmr1 KO mice were shorter and had fewer dendritic spines. Fluorescence imaging of GFP-infected neurons in slices and in culture revealed that dendritic spines were longer and more dense early in Fmr1 KO mouse development, but that these differences had virtually disappeared at 4 weeks of age [Nimchinsky et al., 2001]. Galvez Dendritic Spines in C57BL/6 Fmr1 KO Mice 99 Fig. 1. Dendritic spine morphology in Fmr1 KO mice bred in a C57BL/6 background. A: Spine morphology categories. Each dendritic spine was assigned the letter corresponding to the shape it most closely resembled. Spine categories A–E were considered immature-appearing, and categories F–H were considered mature-appearing. Adapted from Irwin et al. [2001, 2002]. B: Representative images of Golgi-stained apical shaft dendrites from WT and Fmr1 KO mice. Exemplar spines of morphologies A/B, C/D, E, and F/ G are indicated. Scalebar ¼ 5 mm. Fmr1 KO mice exhibited significantly more immature-appearing dendritic spines and significantly fewer matureappearing dendritic spines along apical shaft (C), apical oblique (D), and basilar dendrites (E) than WT mice. Error bars represent SEM *, P < 0.05. and Greenough (in press) found similarly that the spine phenotype disappeared at this age and then re-emerged as mice matured to adulthood. Collectively, these studies provide further support for involvement of FMRP in normal dendritic spine development and/or maturation. Mice used by Comery et al.  were bred in an FVB background and may have exhibited a recessive form of retinal degeneration [Taketo et al., 1991]. Irwin et al.  therefore repeated the study by Comery et al., using Fmr1 KO and WT mice bred in an FVB background but unaffected by retinal degeneration. In these animals, the normal alleles from the 129 strain that provided embryonic stem cells for the KO procedure had been retained in favor of the Pde6b or rd mutation alleles found in WT FVB mice [Gimenez and Montoliu, 2001]. Using these sighted animals, Irwin et al.  found that adult Fmr1 KO mice still had more longer and fewer shorter dendritic spines on apical shaft, apical oblique, and basilar dendrites of layer V pyramidal cells in visual cortex compared to littermate controls, although the effect appeared to be less pronounced than what had been reported in humans. Whereas Fmr1 KO mice studied by Irwin et al.  also displayed more dendritic spines with immature-appearing morphologies than littermate controls, they did not exhibit the statistically significant overall increase in spine density that had been observed by Comery et al.  and in FXS patients [Irwin et al., 2001]. Although they were sighted, the Fmr1 KO mice bred in an FVB background that were used by Irwin et al. , were albino (homozygous for the tyrc allele), and therefore may have exhibited a number of visual system deficits associated with albinism [Jeffery, 1997]. Given the extensive literature on effects of monocular deprivation and dark rearing on structure and function of the visual cortex [reviewed by Greenough and Chang, 1988], results of previous analyses of dendritic spines in Fmr1 KO mice bred in an FVB background may in part reflect the interaction between FMRP’s absence and intrinsic visual deficits of FVB mice. Mouse background has been demonstrated previously to affect other aspects of Fmr1 KO phenotype [e.g., Paradee et al., 1999; Dobkin et al., 2000]. The dependence of some aspects of Fmr1 KO phenotypes on mouse background may model the contribution of genetic background to the variability in expression of symptoms among individuals 100 McKinney et al. with FXS [Hagerman and Hagerman, 2002]. In the present study, we therefore examine dendritic spine length, morphology, and density in Fmr1 KO mice bred in a C57BL/6 background to more clearly interpret the effect of FMRP on dendritic spine morphology. Seven adult (60–90 days) male Fmr1 KO (B6.129P2Fmr1tm1Cgr; backcrossed six times) and seven WT (WT with respect to the Fmr1 locus) littermate control mice bred in a C57BL/6 background were used. Fmr1 genotypes were determined by PCR analysis of DNA extracted from tail samples [WT PCR protocols described by Bakker et al., 1994; KO PCR protocol–NEOTD protocol # 701 found at www.jax.org]. Mice were anesthetized with sodium pentobarbital (85 mg/kg) and transcardially perfused with 100 ml PBS (pH 7.4). Brains were removed and placed in 25 ml of Golgi–Cox solution (1% potassium dichromate, 1% mercuric chloride, 0.8% potassium chromate in distilled water) for 20–30 days [Glaser and Van der Loos, 1981]. Following staining, brains were embedded in Parlodion (Mallinckrodt; Phillipsburg, NJ) and sectioned at 120 mm. Sections were dehydrated, developed, fixed, mounted on slides, and coverslipped [Irwin et al., 2001]. Slides were assigned codes so that raters were blind to group identity. Apical shaft, apical oblique, and basilar dendrites on layer V pyramidal neurons from primary visual cortex were evaluated (see Fig. 1B). For each branch type, one randomly chosen branch on each of ten randomly chosen, fully impregnated neurons per animal was analyzed for dendritic spine length, morphological categorization, and density at an optical magnification of 1,200. To be analyzed, apical shafts dendrites had to be 175 mm long, apical oblique dendrites 87.5 mm long, and basilar dendrites 62.5 mm long. For each apical shaft, lengths and morphological categories of the first 10 dendritic spines in each successive 25 mm segment were recorded, starting 50 mm from the soma and continuing to the end of the apical shaft. For each apical oblique and basilar dendrite, lengths and morphologies of up to 10 dendritic spines in each successive 12.5 mm segment were recorded, starting 25 mm from apical shaft or soma, respectively, and continuing to the end of the dendrite. Morphological categories were assigned to dendritic spines based on the morphology that they most resembled (see Fig. 1A); categories were the same as those used by Irwin et al. [2001, 2002] and by Galvez and Greenough (in press). Morphological category A was pooled with category B (A/B), C with D (C/D), and F with G (F/G) for analysis. Dendritic spine length was defined as the length from the distal surface of the spine head to the dendrite; lengths were measured with an ocular reticule aligned parallel to the dendritic spine, and rounded up to the nearest 0.5 mm. For each branch type, the number of spines of each length and morphological category was calculated across all neurons and animals, and across all distances from the soma, and Chi-Square analysis was used to compare the distribution of spines in these categories between genotypes. Then the average proportion of spines in each length and morphological category was calculated for each animal (n ¼ 7 mice per group), and the difference between genotypes was analyzed by Student’s t-test. To measure spine density on apical shaft dendrites, the number of spines on each successive 25 mm segment was counted starting at the soma and continuing to the end of the dendrite. Dendritic diameter was measured every 50 mm by positioning the reticule perpendicular to the dendrite shaft to ascertain whether differential diameter could be affecting spine number counts. For apical oblique and basilar dendrites, the number of spines on each successive 12.5 mm segment was counted starting 25 mm from the apical shaft or soma, respectively, and continuing to the end of the dendrite. Dendritic diameter was measured every 25 mm. Densities for each segment and for each neuron were pooled to get an average spine density per animal for each branch type; the difference between genotypes was analyzed by Student’s t-test. Fmr1 KO mice exhibited more dendritic spines with immature-appearing morphology and fewer dendritic spines with mature-appearing morphology than littermate controls, as shown in Figure 1. This was demonstrated by Chi-Square analysis of spines along apical shafts (w2 ¼ 172.2, 4 df, P < 0.01; 9,542 spines); along apical oblique dendrites (w2 ¼ 118.5, 4 df, P < 0.01; 6,369 spines); and along basilar dendrites (w2 ¼ 115.1, 4 df, P < 0.01; 4,692 spines). Students t-test demonstrated that a significantly larger proportion of dendritic spines in Fmr1 KO mice were of A/B morphology along basilar dendrites (t(1,12) ¼ 5.42, P < 0.05); a significantly larger proportion of dendritic spines in Fmr1 KO mice were of C/D morphology along apical shaft (t(1,12) ¼ 25.02, P < 0.01), apical oblique (t(1,12) ¼ 12.71, P < 0.01), and basilar (t(1,12) ¼ 15.57, P < 0.01) dendrites; and a significantly smaller proportion of dendritic spines in KO mice were of F/G morphology along apical shaft (t(1,12) ¼ 88.78, P < 0.01), apical oblique (t(1,12) ¼ 23.67, P < 0.01), and basilar (t(1,12) ¼ 34.32, P < 0.01) dendrites. As Figure 2 illustrates, Fmr1 KO mice exhibited more longer and fewer shorter dendritic spines than littermate controls along apical shaft (w2 ¼ 99.1, 4 df, P < 0.01; 9,542 spines), apical oblique (w2 ¼ 36.1, 4 df, P < 0.01; 6,361 spines), and basilar dendrites (w2 ¼ 36.1, 4 df, P < 0.01; 4,691 spines). A significantly smaller proportion of dendritic spines in Fmr1 KO mice were of length 0.5 mm along apical shaft (t(1,12) ¼ 12.61, P < 0.01), apical oblique (t(1,12) ¼ 7.05, P < 0.05), and basilar (t(1,12) ¼ 9.77, P < 0.01) dendrites and a significantly larger proportion of dendritic spines in Fmr1 KO mice were of length 1.5 mm along basilar dendrites (t(1,12) ¼ 4.93, P < 0.05). As seen in Figure 3, Fmr1 KO mice exhibited greater dendritic spine density than littermate controls along apical Fig. 2. Fmr1 KO mice bred in a C57BL/6 background exhibited significantly more longer and significantly fewer shorter spines than WT controls along apical shaft (A), apical oblique (B), and basilar dendrites (C). Error bars represent SEM *, P < 0.05. Dendritic Spines in C57BL/6 Fmr1 KO Mice 101 Fig. 3. Fmr1 KO mice bred in a C57BL/6 background exhibited greater overall dendritic spine density than WT mice, along apical shafts and apical oblique dendrites (A). Fmr1 KOs exhibited greater dendritic spine density than WT mice at many distances from the soma along apical shaft dendrites (B). Fmr1 KOs exhibited greater dendritic spine density than WT mice along apical oblique dendrites more proximal to the apical shaft (C). Spine density in KO mice was not abnormal along basilar dendrites (D). Error bars represent SEM *, P < 0.05. shaft dendrites (t(1,12) ¼ 28.78, P < 0.01) and apical oblique dendrites (t(1,12) ¼ 6.60, P < 0.05). Dendritic spine densities were not significantly different along basilar dendrites (t(1,12) ¼ 3.03, P ¼ 0.11). Beginning from the cell body (for apical and basilar dendrites) or from the apical shaft (for apical oblique dendrites), we also compared the spine density between KO and WT mice every 25 or every 12.5 microns along the dendrite. In Figure 3B–D, distances from the soma or apical shaft at which spine density was elevated in KO mice are indicated (*, P < 0.05). Dendrite diameter did not differ between genotypes, indicating that this parameter did not affect spine counts. It is possible that the increased number of long dendritic spines and increased spines with immature morphology observed in Fmr1 KO mice represent the same phenomenon. That is, dendritic spines of the immature-appearing morphology C/D tend to be longer than spines of mature-appearing morphology F/G and any spine length difference between genotypes may actually represent a shift in morphology. Therefore, we tested whether the frequency of long and short spines within category C/D differed between Fmr1 KO and WT mice. As Figure 4 illustrates, Fmr1 KO mice had more longer C/ D spines and fewer shorter C/D spines on apical shaft dendrites (w2 ¼ 20.8, 4 df, P < 0.01; 5,915 spines) and apical oblique dendrites (w2 ¼ 10.7, 4 df, P < 0.05; 3,815 spines). On basilar dendrites, the effect was in the same direction, but fell short of statistical significance (w2 ¼ 9.3, 4 df, P ¼ 0.06; 2,709 spines). This finding indicates that the overall tendency for KO mice to have greater spine length does not merely reflect a shift in relative frequency toward a particular (often longer) dendritic Fig. 4. On apical shaft and apical oblique dendrites, Fmr1 KO mice had more longer C/D spines and fewer shorter C/D spines than WT mice. Error bars represent SEM. 102 McKinney et al. spine morphology. Instead, elevated dendritic spine length in KOs appears to reflect a true genotype difference in spine length within the morphological categories used here. In the present study, we describe systematic replication of previously reported dendritic spine length and morphology abnormalities in Fmr1 KO mice bred in a different background from that used in prior work. We further demonstrate that the length and morphology phenotypes observed in Fmr1 KO mice are largely independent and that the length phenotype is not simply a product of there being more spines in morphological categories in which spines tend to be longer. These findings suggest that the dendritic spine length and morphology abnormalities observed here in Fmr1 KO mice (1) parallel findings from individuals with FXS, (2) correlate with the absence of FMRP and (3) are not exclusively a direct or interactive effect of background phenotype. It is of note that, whereas spine morphological abnormalities were observed along basilar dendrites in the present study as well as in individuals with FXS [Irwin et al., 2001], this difference was not observed in Fmr1 KO mice bred in an FVB background [Irwin et al., 2002]. Perhaps more importantly, the significantly increased dendritic spine density observed in individuals with FXS and here in adult Fmr1 KO mice bred in a C57BL/6 background also did not reach statistical significance in Fmr1 KO mice bred in an FVB background [Irwin et al., 2002]. In the occipital cortex, therefore, at least in terms of spine morphology along basilar dendrites and in terms of spine density, Fmr1 KO mice bred in a C57BL/6 background may more closely model the human FXS condition than those bred in an FVB background. These findings provide powerful support for the essence of the dendritic spine abnormalities observed in the absence of FMRP, now found to be largely consistent across two mouse backgrounds. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Ben Oostra, Erasmus University, Rotterdam for contributing the C57BL/6 Fmr1 KO mice, and Andrea Beckel-Mitchener, Lisa Foster, Dack Shearer and Holly Fairfield for their assistance in maintaining the colony. We also thank Roberto Galvez and Julie Markham for technical assistance and for valuable comments on this article. REFERENCES Bakker CE, Verheij C, Willemsen R, van der Helm R, Oerlemans F, Vermey M, Bygrave A, Hoogeveen AT, Oostra BA, Reyniers E, et al. 1994. Fmr1 knockout mice: A model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium. Cell 78(1):23–33. Braun K, Segal M. 2000. FMRP involvement in formation of synapses among cultured hippocampal neurons. Cereb Cortex 10(10):1045–1052. Comery TA, Harris JB, Willems PJ, Oostra BA, Irwin SA, Weiler IJ, Greenough WT. 1997. Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits. Proc Natl Acad Sci USA 94(10): 5401–5404. de Vries BB, van den Ouweland AM, Mohkamsing S, Duivenvoorden HJ, Mol E, Gelsema K, van Rijn M, Halley DJ, Sandkuijl LA, Oostra BA, et al. 1997. Screening and diagnosis for the fragile X syndrome among the mentally retarded: An epidemiological and psychological survey. Collaborative Fragile X Study Group. Am J Hum Genet 61(3):660–667. Dobkin C, Rabe A, Dumas R, El Idrissi A, Haubenstock H, Brown WT. 2000. Fmr1 knockout mouse has a distinctive strain-specific learning impairment. Neuroscience 100(2):423–429. Galvez R, Greenough WT. Sequence of abnormal dendritic spine development in primary somatosensory cortex of a mouse model of the fragile X mental retardation syndrome. Am J Med Genet A. (In press). Gimenez E, Montoliu L. 2001. A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdeb(rd1)) in FVB/Nderived transgenic mice. Lab Anim 35(2):153–156. Glaser EM, Van der Loos H. 1981. Analysis of thick brain sections by obverse-reverse computer microscopy: Application of a new, high clarity Golgi-Nissl stain. J Neurosci Methods 4(2):117–125. Greenough WT, Chang FL. 1988. Plasticity of synapse structure and pattern in the cerebral cortex. In: Peters A, Jones EG, editors. Cerebral cortex: Development and maturation of cerebral cortex. New York: Plenum Pub. Corp. Hagerman RJ, Hagerman PJ. 2002. Physical and behavioral phenotype. In: Hagerman RJ, Cronister A, editors. Fragile X syndrome: Diagnosis, treatment, and research. Third edition. Baltimore: Johns Hopkins University Press. pp 3–109. Hinton VJ, Brown WT, Wisniewski K, Rudelli RD. 1991. Analysis of neocortex in three males with the fragile X syndrome. Am J Med Genet 41(3):289–294. Irwin SA, Patel B, Idupulapati M, Harris JB, Crisostomo RA, Larsen BP, Kooy F, Willems PJ, Cras P, Kozlowski PB, et al. 2001. Abnormal dendritic spine characteristics in the temporal and visual cortices of patients with fragile-X syndrome: A quantitative examination. Am J Med Genet 98(2):161–167. Irwin SA, Idupulapati M, Gilbert ME, Harris JB, Chakravarti AB, Rogers EJ, Crisostomo RA, Larsen BP, Mehta A, Alcantara CJ, et al. 2002. Dendritic spine and dendritic field characteristics of layer V pyramidal neurons in the visual cortex of fragile-X knockout mice. Am J Med Genet 111(2):140–146. Jeffery G. 1997. The albino retina: An abnormality that provides insight into normal retinal development. Trends Neurosci 20(4):165–169. Nimchinsky EA, Oberlander AM, Svoboda K. 2001. Abnormal development of dendritic spines in FMR1 knock-out mice. J Neurosci 21(14):5139– 5146. Paradee W, Melikian HE, Rasmussen DL, Kenneson A, Conn PJ, Warren ST. 1999. Fragile X mouse: Strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience 94(1): 185–192. Taketo M, Schroeder AC, Mobraaten LE, Gunning KB, Hanten G, Fox RR, Roderick TH, Stewart CL, Lilly F, Hansen CT, et al. 1991. FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci USA 88(6):2065–2069. Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, et al. 1991. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65(5):905–914.