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Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines
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Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines Aaron W. Grossman a,b,c , Nicholas M. Elisseou a , Brandon C. McKinney a , William T. Greenough a,b,d,⁎ a
Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Neuroscience Graduate Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA c Medical Scholars Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Departments of Psychology, Psychiatry and Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of
Accepted 7 February 2006
functional fragile X mental retardation protein (FMRP). FXS is associated with elevated
Available online 30 March 2006
density and length of dendritic spines, as well as an immature-appearing distribution profile
Keywords:
similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine
Plasticity
maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal
of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a
Hippocampus
subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a
Spine shape
morphology profile that, while essentially opposite of that described in the Fmr1 knockout
Activity dependent
neocortex, appears similarly immature. This finding strongly suggests that FMRP is required
Development
for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.
Abbreviations:
© 2006 Elsevier B.V. All rights reserved.
FMRP, fragile X mental retardation protein LTP, long-term potentiation LTD, long-term depression FXS, Fragile X syndrome
1.
Introduction
A common inherited form of mental retardation, Fragile X syndrome (FXS) is characterized by the absence of functional FMRP (Hagerman and Hagerman, 2002). Postmortem tissue from the neocortex of FXS patients reveals pathology of dendritic spines (Hinton et al., 1991). Specifically, spines are longer and more dense along dendrites of cortical neurons in FXS patients than controls (Irwin et al., 2001). These neurons from FXS patients
also exhibit a higher proportion of thin spines and a lower proportion of stubby or mushroom-shaped spines than controls. Similar cortical neuropathology has been observed in a murine model for FXS that also lacks FMRP, the Fmr1 knockout mouse (Bakker et al., 1994; Irwin et al., 2002; McKinney et al., 2005). Across several species, maturation of the neocortex has been associated with dendritic spine overproduction followed by pruning of a subset of these spines and with a shift in distribution from a greater proportion of longer thinner spines
⁎ Corresponding author. Beckman Institute, University of Illinois, 405 N. Mathews, Urbana, IL 61801, USA. Fax: +1 217 244 5180. E-mail address: [email protected] (W.T. Greenough). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.044
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to more shorter stubbier ones (Galofre and Ferrer, 1987; Horner, 1993; Murphy and Magness, 1984; Schuz, 1986). Recent findings indicate that, whereas dendritic spines in the somatosensory cortex of wildtype mice appear to follow this normal developmental course, adult Fmr1 knockout mice exhibit elevated spine density and a profile of spine lengths and morphologies similar to younger subjects (Galvez and Greenough, 2005). In light of these data and others, we have hypothesized that FMRP is required for the activity-dependent processes of spine shape maturation and pruning and that without FMRP dendritic spines that would normally either mature or be pruned tend to persist into adulthood (Bagni and Greenough, 2005; Churchill et al., 2002). FMRP is highly expressed in the hippocampus, a brain region important for learning and memory (Eichenbaum and Cohen, 2001; Hinds et al., 1993). Patients with FXS exhibit bilaterally enlarged hippocampal volumes, and physiological abnormalities have been reported in hippocampal subfield CA1 of Fmr1 knockout mice, suggesting that the absence of FMRP may lead to structural and functional deficits in the hippocampus (Huber et al., 2002; Reiss et al., 1994). Several groups have examined hippocampal connectivity in Fmr1 knockout mice or have studied cultured hippocampal neurons
159
from these animals (Braun and Segal, 2000; Ivanco and Greenough, 2002; Mineur et al., 2002), but there have been no reports of dendritic spine morphology in the Fmr1 knockout mouse hippocampus in vivo. During normal development, dendritic spines on hippocampal pyramidal cells appear to follow a progression distinct from that observed in the neocortex. Harris et al. (1992), for example, have found by examining three-dimensional reconstructions of serial electron micrographs that the distribution profile of spines along apical shafts of pyramidal cells in hippocampal subfield CA1 shifts from an abundance of stubby or mushroom-shaped spines in young animals to predominantly thin spines by adulthood. If FMRP is involved in spine maturation, rather than associated with the development of a particular spine shape per se, one would expect to see more stubby or mushroom-shaped spines along these hippocampal neurons in adult Fmr1 knockout than control mice—essentially the opposite of the morphology profile in the neocortex of these animals and yet similarly suggestive of an immature synaptic state in the hippocampus. To test this hypothesis, Golgi-impregnated dendritic spines were examined along apical shafts of hippocampal area CA1 pyramidal cells of adult Fmr1 knockout and control mice
Fig. 1 – Dendritic spine morphology is altered in hippocampal area CA1 of Fmr1 knockout mice. (A) Morphology categories to which spines were assigned. (B) Representative dendritic segments from wildtype and Fmr1 knockout (KO) mice with exemplar spines identified. Scale bar represents 3 μm. (C) In area CA1, Fmr1 knockout mice exhibit more stubby, mushroom-shaped spines and fewer thin spines than wildtype controls. (D) In the visual cortex of these same animals, by contrast, Fmr1 knockout mice exhibit more thin spines and fewer stubby, mushroom-shaped spines than controls. (Part D of this figure is adapted from McKinney et al., 2005.)
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text-dependent deficit in the brain-region-specific maturation of dendritic spines in Fmr1 knockout mice.
2.
Fig. 2 – Dendritic spine length is elevated in hippocampal area CA1 of Fmr1 knockout (KO) mice, as has been observed in the neocortex. (A) In CA1, knockout mice exhibit more longer spines and fewer shorter spines than wildtype controls. (B) In the visual cortex of these same animals, a similar trend is observed. (Part B of this figure adapted from McKinney et al., 2005.)
(wildtype with respect to the Fmr1 locus). Although there was no difference in the density of spines between the genotypes, there were more stubby, mushroom-shaped spines and fewer thin spines in knockout mice than in controls. Furthermore, Fmr1 knockout mice had more longer and fewer shorter dendritic spines than controls overall and for each spine morphology type analyzed. This phenotype suggests a con-
Results
Assigning each dendritic spine to a morphological category (used previously by our laboratory; Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005; see Fig. 1A) revealed that the profile of spine morphologies along CA1 pyramidal cell apical shafts in Fmr1 knockout mice was significantly different overall from that of controls (χ2 = 22.5, 4 df, P < 0.01; >7100 spines; see Fig. 1C). There were more stubby or mushroom-shaped spines (morphological category F/G) and fewer thin spines (category A/B) in knockout mice than in controls. The difference in spine morphology profiles was most pronounced 50–100 and 150–200 μm from the soma (see Supplementary data). When compared with the spine morphology profile that has been described in the visual cortex of these same animals (Fig. 1D, adapted from McKinney et al., 2005), it is clear that these profiles are brain-region-specific and that genotype differences are context-dependent. The morphology profiles in adult Fmr1 knockout CA1 and neocortex are both suggestive of profiles observed in younger wildtype animals (Galvez and Greenough, 2005; Harris et al., 1992). As shown in Fig. 2A, there were more longer and fewer shorter spines along CA1 apical shafts in knockout mice than in controls (χ2 = 17.04, 4 df, P < 0.01, >7100 spines). The genotype difference in spine length was most pronounced 100–150 μm from the soma (see Supplementary data). A similarly elevated dendritic spine length has been observed in the visual cortex of these same animals (Fig. 2B, adapted from McKinney et al., 2005). This finding of elevated spine length in area CA1 of Fmr1 knockout mice may seem inconsistent with knockout mice having fewer thin spines (which tend to be long) and more stubby or mushroomshaped spines (which tend to be short) in this brain region. In fact, when spine length is analyzed within each major spine morphology type, Fmr1 knockout mice have longer A/B spines, longer C/D spines and longer F/G spines than controls (see Fig. 3).
Fig. 3 – For each spine type analyzed (A/B, C/D and F/G spines), Fmr1 knockout mice have more longer and fewer shorter spines in area CA1 than wildtype controls. A/B spines: (χ2 = 39.58, 4 df, P < 0.0001, 1296 spines); C/D spines (χ2 = 14.76, 4 df, P < 0.01, 2291 spines); F/G spines (χ2 = 11.41, 4 df, P < 0.01, 3452 spines).
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in the neocortex. For example, one study of dendritic spine density along hippocampal dendrites across development and into adulthood observed spine overproduction followed by a pruning effect of approximately 10–15% (Meyer and FerresTorres, 1978), compared with a ∼20–30% reduction in the neocortex (Galofre and Ferrer, 1987; Murphy and Magness, 1984). Another more recent study of CA1 dendrites did not observe a substantial pruning effect (Norrholm and Ouimet, 2000). While it is possible that the age range of adult animals used in the present study was too broad to suggest any deficit in spine pruning in Fmr1 knockout mice, these observations are compatible with our previous work indicating that the spine density phenotype is more subtle and more variable in Fig. 4 – The density of spines along CA1 pyramidal cell apical shafts of Fmr1 knockout (KO) mice did not differ from wildtypes. This was true overall and along the apical shaft at various distances from the soma. Error bars represent SEM.
No difference was detected in the overall density of dendritic spines along CA1 apical shafts of adult Fmr1 knockout mice nor was a spine density difference observed at any distance from the soma (see Fig. 4).
3.
Discussion
The dendritic spine phenotype associated with Fragile X syndrome has been well characterized in the neocortex, and we have hypothesized that the elevated density, the longer spines and the abnormal morphology profile observed in cerebral cortical tissue from FXS patients and Fmr1 knockout mice indicate a role for FMRP in spine pruning and maturation. Here, in a separate brain region (hippocampal subfield CA1), we describe a distinct dendritic spine phenotype of Fmr1 knockout mice and we suggest that, because spine maturation follows a course specific to each brain region, the absence of FMRP will have neuroanatomical consequences that are also specific to each brain region. In hippocampal subfield CA1, the relative profile of spine morphologies differs from that observed in the neocortex. The profile of spines along CA1 apical shafts of adult Fmr1 knockout mice resembles the morphology profile in immature rodents that has been described by Harris et al. (1992), with more stubby and mushroom-shaped spines (morphological category F/G) and fewer thin spines (category A/B) than control mice. Fmr1 knockout mice also have more longer and fewer shorter spines of morphology categories A/B, C/D and F/G along these dendrites than control mice, similar to the spine length phenotype in FXS and in Fmr1 knockout neocortex. Together, these findings strongly support the hypothesis that, in the absence of FMRP, dendritic spines fail to mature properly (Weiler and Greenough, 1999) and that the specific pathology observed will depend on the brain region and its normal developmental trajectory (see Fig. 5). The absence of a difference in hippocampal dendritic spine density between wildtype and Fmr1 knockout mice may reflect the fact that the process of overproduction followed by pruning appears less pronounced in the hippocampus than
Fig. 5 – Schematic of dendritic spine development in neocortex and hippocampus of wildtype and Fmr1 knockout mice. Top left: During maturation of the neocortex, the initial overproduction of dendritic spines is followed by pruning of a subset of these spines (Galofre and Ferrer, 1987; Galvez and Greenough, 2005). The initial profile of many long, thin spines also exhibits a shift toward a profile that includes many short, stubby and mushroom-shaped spines (Galofre et al., 1987; Galvez and Greenough, 2005). Bottom left: In mice that lack FMRP (Fmr1 knockout mice), these developmental processes do not occur normally, and adults exhibit spine density, spine length and a spine morphology profile that resembles immature animals (Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005). Top right: Studies in rats suggest that the initial morphology profile in hippocampal subfield CA1 reveals an abundance of stubby, mushroom-shaped spines, which shifts toward more thin spines in adult animals (Harris et al., 1992, see also Tarelo-Acuna et al., 2000). Bottom right: In adult Fmr1 knockout mice, we have observed more long spines and a greater proportion of stubby and mushroom-shaped spines than in wildtype mice, suggesting that FMRP is required generally for the maturation of spines and not for development of a particular spine shape per se. As these hypotheses have not been tested directly in wildtype and Fmr1 knockout mice at both age groups, question marks have been added to the right two panels. Furthermore, no inferences can be made at this time about dendritic spine characteristics in area CA1 of young Fmr1 knockout mice.
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knockout mice than in human patients (see McKinney et al., 2005). Our findings also vary from the report of reduced spine density on cultured knockout hippocampal neurons (Braun and Segal, 2000), though it is difficult to draw meaningful conclusions from comparisons between Golgi staining of neurons in situ and fluorescence imaging of cultured neurons. As sites of biochemical compartmentalization and sources of electrical resistance, the shape of dendritic spines appears to be integrally tied to their function (Sorra and Harris, 2000). The immature-appearing spine morphology and length are likely, therefore, to generate a physiological pattern that is different in the knockout hippocampus from the wildtype hippocampus and probably from the knockout and wildtype neocortex. It is also possible that the abnormal morphology of hippocampal dendritic spines reported here in Fmr1 knockout mice is the consequence of prolonged exposure to synaptic depression as enhanced long-term depression (LTD) has been reported in area CA1 of these animals (Huber et al., 2002). LTD (albeit a different variety) has been associated with spine shrinkage (Zhou et al., 2004), and it is reasonable to predict that prolonged exposure of Fmr1 knockout mice to synaptic depression in vivo might result in smaller spines, perhaps evidenced by an increase in the proportion of stubby, mushroom-shaped spines. The present findings, however, indicate that spines in Fmr1 knockout mouse CA1 are, in fact, longer than in wildtype mice, suggesting that other mechanisms may be involved. The functional consequences of the dendritic spine phenotypes in Fmr1 knockout mice may be related to the physiological differences reported between the cortex and hippocampus of these animals; in the hippocampus, for example, enhanced LTD has been reported but no differences in long-term potentiation (LTP) have been observed, whereas reduced LTP has been reported in the neocortex (Godfraind et al., 1996; Huber et al., 2002; Li et al., 2002). One recent paper has suggested that these physiological differences are age-dependent (Larson et al., 2005). As with regional differences in physiological plasticity, we see here that the specific pathology of synaptic morphology that arises from the functional loss of FMRP depends upon the cellular, and presumably the molecular, context. As an mRNA binding protein, FMRP appears to be involved in the transport and translation of a subset of mRNAs near synapses (Ling et al., 2004; Weiler et al., 2004). Normal regulation of this subset of messages may be critical for normal synaptic function and, as the findings suggest here, for normal activity-dependent maturation of synapses (Grossman et al., in preparation). Further characterization of the role of these mRNAs in the development of spine morphology and in adult synaptic plasticity in Fragile X syndrome will be important in understanding the pathobiology of this disease.
control mice (also backcrossed 6 times, and wildtype with respect to the FMR1 locus) bred in a C57BL/6 background were used. At postnatal days 60–90, mice were deeply anesthetized and transcardially perfused with 100 ml PBS (pH 7.4). Brains were submerged in 25 ml of Golgi–Cox solution for 20–30 days (Glaser and Van der Loos, 1981), embedded and sectioned at 120 μm. After processing and dehydration, sections were mounted on slides which were coded so that raters were blind to group identity. In area CA1 of the dorsal hippocampus of each animal, apical shafts of ten randomly selected pyramidal neurons were examined. To be analyzed, apical shafts of fully impregnated neurons had to be ≥175 μm long. All analysis was done by a single rater on a Zeiss Standard Universal at an optical magnification of 1200×. Dendrites in Fig. 1B are printed at a magnification of ∼1280×.
4.2.
Dendritic spine analysis
For each neuron, the morphology and length were determined of the first 10 spines in every 25 μm bin along the apical shaft. For spine morphology, spines were assigned the morphological category (used previously by our laboratory; Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005; see Fig. 1A) that most resembled the shape of that spine. Morphological category A was pooled with category B (A/B), C with D (C/D) and F with G (F/G) for analysis. The length of each dendritic spine evaluated for morphology was also measured. Length was defined as the distance from the distal surface of the spine head to the dendrite, rounded up to the nearest 0.5 μm, so that spines fell into one of five length categories. Finally, the density of spines (number per length of dendrite) in each 25 μm bin was measured.
4.3.
Statistical analysis
The number of spines of each morphology and length category was summed across all neurons and all animals, and Chisquare analysis was used to compare the profile (i.e. frequency distribution) of spines in these categories between genotypes. Chi-square analysis was subsequently performed on spine profiles along the apical dendrite at various distances from the soma (see Supplementary data). Chi-square analysis of the profile of dendritic spine lengths (overall and for each morphology type) was performed in the same way. We also tested for group differences in spine density overall along the apical shaft and at 50-μm-long distances from the soma (this was computed as the average of the two 25-μm-long bins). For these comparisons, Student's t tests were performed overall and on each 50-μm-long distance using the mean spine density per animal, averaged across all neurons.
4.
Experimental procedures
Acknowledgments
4.1.
Subjects and tissue processing
The authors would like to thank Roberto Galvez for his technical assistance as well as Julie Markham and Georgina Aldridge for their thoughtful comments on this manuscript. We also thank Kathy Bates, Lisa Foster and Dack Shearer for their valuable contributions to this research. N.M. Elisseou is currently enrolled at the Pritzker School of Medicine at the
The tissue examined for this study is the same as was used by McKinney et al. (2005), where a more thorough description of methods can be found. Briefly, seven adult Fmr1 knockout (B6.129P2-Fmr1tm1Cgr; backcrossed 6 times) and four adult
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University of Chicago, and B.C. McKinney is enrolled in the University of Michigan's Medical Scientist Training Program. This work was supported by FRAXA, NIH Grants MH35321, HD07333, and the Spastic Paralysis and Allied Diseases of the Central Nervous System Research Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.brainres.2006.02.044.
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Research Report
Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines Aaron W. Grossman a,b,c , Nicholas M. Elisseou a , Brandon C. McKinney a , William T. Greenough a,b,d,⁎ a
Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Neuroscience Graduate Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA c Medical Scholars Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Departments of Psychology, Psychiatry and Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of
Accepted 7 February 2006
functional fragile X mental retardation protein (FMRP). FXS is associated with elevated
Available online 30 March 2006
density and length of dendritic spines, as well as an immature-appearing distribution profile
Keywords:
similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine
Plasticity
maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal
of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a
Hippocampus
subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a
Spine shape
morphology profile that, while essentially opposite of that described in the Fmr1 knockout
Activity dependent
neocortex, appears similarly immature. This finding strongly suggests that FMRP is required
Development
for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.
Abbreviations:
© 2006 Elsevier B.V. All rights reserved.
FMRP, fragile X mental retardation protein LTP, long-term potentiation LTD, long-term depression FXS, Fragile X syndrome
1.
Introduction
A common inherited form of mental retardation, Fragile X syndrome (FXS) is characterized by the absence of functional FMRP (Hagerman and Hagerman, 2002). Postmortem tissue from the neocortex of FXS patients reveals pathology of dendritic spines (Hinton et al., 1991). Specifically, spines are longer and more dense along dendrites of cortical neurons in FXS patients than controls (Irwin et al., 2001). These neurons from FXS patients
also exhibit a higher proportion of thin spines and a lower proportion of stubby or mushroom-shaped spines than controls. Similar cortical neuropathology has been observed in a murine model for FXS that also lacks FMRP, the Fmr1 knockout mouse (Bakker et al., 1994; Irwin et al., 2002; McKinney et al., 2005). Across several species, maturation of the neocortex has been associated with dendritic spine overproduction followed by pruning of a subset of these spines and with a shift in distribution from a greater proportion of longer thinner spines
⁎ Corresponding author. Beckman Institute, University of Illinois, 405 N. Mathews, Urbana, IL 61801, USA. Fax: +1 217 244 5180. E-mail address: [email protected] (W.T. Greenough). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.044
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to more shorter stubbier ones (Galofre and Ferrer, 1987; Horner, 1993; Murphy and Magness, 1984; Schuz, 1986). Recent findings indicate that, whereas dendritic spines in the somatosensory cortex of wildtype mice appear to follow this normal developmental course, adult Fmr1 knockout mice exhibit elevated spine density and a profile of spine lengths and morphologies similar to younger subjects (Galvez and Greenough, 2005). In light of these data and others, we have hypothesized that FMRP is required for the activity-dependent processes of spine shape maturation and pruning and that without FMRP dendritic spines that would normally either mature or be pruned tend to persist into adulthood (Bagni and Greenough, 2005; Churchill et al., 2002). FMRP is highly expressed in the hippocampus, a brain region important for learning and memory (Eichenbaum and Cohen, 2001; Hinds et al., 1993). Patients with FXS exhibit bilaterally enlarged hippocampal volumes, and physiological abnormalities have been reported in hippocampal subfield CA1 of Fmr1 knockout mice, suggesting that the absence of FMRP may lead to structural and functional deficits in the hippocampus (Huber et al., 2002; Reiss et al., 1994). Several groups have examined hippocampal connectivity in Fmr1 knockout mice or have studied cultured hippocampal neurons
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from these animals (Braun and Segal, 2000; Ivanco and Greenough, 2002; Mineur et al., 2002), but there have been no reports of dendritic spine morphology in the Fmr1 knockout mouse hippocampus in vivo. During normal development, dendritic spines on hippocampal pyramidal cells appear to follow a progression distinct from that observed in the neocortex. Harris et al. (1992), for example, have found by examining three-dimensional reconstructions of serial electron micrographs that the distribution profile of spines along apical shafts of pyramidal cells in hippocampal subfield CA1 shifts from an abundance of stubby or mushroom-shaped spines in young animals to predominantly thin spines by adulthood. If FMRP is involved in spine maturation, rather than associated with the development of a particular spine shape per se, one would expect to see more stubby or mushroom-shaped spines along these hippocampal neurons in adult Fmr1 knockout than control mice—essentially the opposite of the morphology profile in the neocortex of these animals and yet similarly suggestive of an immature synaptic state in the hippocampus. To test this hypothesis, Golgi-impregnated dendritic spines were examined along apical shafts of hippocampal area CA1 pyramidal cells of adult Fmr1 knockout and control mice
Fig. 1 – Dendritic spine morphology is altered in hippocampal area CA1 of Fmr1 knockout mice. (A) Morphology categories to which spines were assigned. (B) Representative dendritic segments from wildtype and Fmr1 knockout (KO) mice with exemplar spines identified. Scale bar represents 3 μm. (C) In area CA1, Fmr1 knockout mice exhibit more stubby, mushroom-shaped spines and fewer thin spines than wildtype controls. (D) In the visual cortex of these same animals, by contrast, Fmr1 knockout mice exhibit more thin spines and fewer stubby, mushroom-shaped spines than controls. (Part D of this figure is adapted from McKinney et al., 2005.)
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text-dependent deficit in the brain-region-specific maturation of dendritic spines in Fmr1 knockout mice.
2.
Fig. 2 – Dendritic spine length is elevated in hippocampal area CA1 of Fmr1 knockout (KO) mice, as has been observed in the neocortex. (A) In CA1, knockout mice exhibit more longer spines and fewer shorter spines than wildtype controls. (B) In the visual cortex of these same animals, a similar trend is observed. (Part B of this figure adapted from McKinney et al., 2005.)
(wildtype with respect to the Fmr1 locus). Although there was no difference in the density of spines between the genotypes, there were more stubby, mushroom-shaped spines and fewer thin spines in knockout mice than in controls. Furthermore, Fmr1 knockout mice had more longer and fewer shorter dendritic spines than controls overall and for each spine morphology type analyzed. This phenotype suggests a con-
Results
Assigning each dendritic spine to a morphological category (used previously by our laboratory; Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005; see Fig. 1A) revealed that the profile of spine morphologies along CA1 pyramidal cell apical shafts in Fmr1 knockout mice was significantly different overall from that of controls (χ2 = 22.5, 4 df, P < 0.01; >7100 spines; see Fig. 1C). There were more stubby or mushroom-shaped spines (morphological category F/G) and fewer thin spines (category A/B) in knockout mice than in controls. The difference in spine morphology profiles was most pronounced 50–100 and 150–200 μm from the soma (see Supplementary data). When compared with the spine morphology profile that has been described in the visual cortex of these same animals (Fig. 1D, adapted from McKinney et al., 2005), it is clear that these profiles are brain-region-specific and that genotype differences are context-dependent. The morphology profiles in adult Fmr1 knockout CA1 and neocortex are both suggestive of profiles observed in younger wildtype animals (Galvez and Greenough, 2005; Harris et al., 1992). As shown in Fig. 2A, there were more longer and fewer shorter spines along CA1 apical shafts in knockout mice than in controls (χ2 = 17.04, 4 df, P < 0.01, >7100 spines). The genotype difference in spine length was most pronounced 100–150 μm from the soma (see Supplementary data). A similarly elevated dendritic spine length has been observed in the visual cortex of these same animals (Fig. 2B, adapted from McKinney et al., 2005). This finding of elevated spine length in area CA1 of Fmr1 knockout mice may seem inconsistent with knockout mice having fewer thin spines (which tend to be long) and more stubby or mushroomshaped spines (which tend to be short) in this brain region. In fact, when spine length is analyzed within each major spine morphology type, Fmr1 knockout mice have longer A/B spines, longer C/D spines and longer F/G spines than controls (see Fig. 3).
Fig. 3 – For each spine type analyzed (A/B, C/D and F/G spines), Fmr1 knockout mice have more longer and fewer shorter spines in area CA1 than wildtype controls. A/B spines: (χ2 = 39.58, 4 df, P < 0.0001, 1296 spines); C/D spines (χ2 = 14.76, 4 df, P < 0.01, 2291 spines); F/G spines (χ2 = 11.41, 4 df, P < 0.01, 3452 spines).
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in the neocortex. For example, one study of dendritic spine density along hippocampal dendrites across development and into adulthood observed spine overproduction followed by a pruning effect of approximately 10–15% (Meyer and FerresTorres, 1978), compared with a ∼20–30% reduction in the neocortex (Galofre and Ferrer, 1987; Murphy and Magness, 1984). Another more recent study of CA1 dendrites did not observe a substantial pruning effect (Norrholm and Ouimet, 2000). While it is possible that the age range of adult animals used in the present study was too broad to suggest any deficit in spine pruning in Fmr1 knockout mice, these observations are compatible with our previous work indicating that the spine density phenotype is more subtle and more variable in Fig. 4 – The density of spines along CA1 pyramidal cell apical shafts of Fmr1 knockout (KO) mice did not differ from wildtypes. This was true overall and along the apical shaft at various distances from the soma. Error bars represent SEM.
No difference was detected in the overall density of dendritic spines along CA1 apical shafts of adult Fmr1 knockout mice nor was a spine density difference observed at any distance from the soma (see Fig. 4).
3.
Discussion
The dendritic spine phenotype associated with Fragile X syndrome has been well characterized in the neocortex, and we have hypothesized that the elevated density, the longer spines and the abnormal morphology profile observed in cerebral cortical tissue from FXS patients and Fmr1 knockout mice indicate a role for FMRP in spine pruning and maturation. Here, in a separate brain region (hippocampal subfield CA1), we describe a distinct dendritic spine phenotype of Fmr1 knockout mice and we suggest that, because spine maturation follows a course specific to each brain region, the absence of FMRP will have neuroanatomical consequences that are also specific to each brain region. In hippocampal subfield CA1, the relative profile of spine morphologies differs from that observed in the neocortex. The profile of spines along CA1 apical shafts of adult Fmr1 knockout mice resembles the morphology profile in immature rodents that has been described by Harris et al. (1992), with more stubby and mushroom-shaped spines (morphological category F/G) and fewer thin spines (category A/B) than control mice. Fmr1 knockout mice also have more longer and fewer shorter spines of morphology categories A/B, C/D and F/G along these dendrites than control mice, similar to the spine length phenotype in FXS and in Fmr1 knockout neocortex. Together, these findings strongly support the hypothesis that, in the absence of FMRP, dendritic spines fail to mature properly (Weiler and Greenough, 1999) and that the specific pathology observed will depend on the brain region and its normal developmental trajectory (see Fig. 5). The absence of a difference in hippocampal dendritic spine density between wildtype and Fmr1 knockout mice may reflect the fact that the process of overproduction followed by pruning appears less pronounced in the hippocampus than
Fig. 5 – Schematic of dendritic spine development in neocortex and hippocampus of wildtype and Fmr1 knockout mice. Top left: During maturation of the neocortex, the initial overproduction of dendritic spines is followed by pruning of a subset of these spines (Galofre and Ferrer, 1987; Galvez and Greenough, 2005). The initial profile of many long, thin spines also exhibits a shift toward a profile that includes many short, stubby and mushroom-shaped spines (Galofre et al., 1987; Galvez and Greenough, 2005). Bottom left: In mice that lack FMRP (Fmr1 knockout mice), these developmental processes do not occur normally, and adults exhibit spine density, spine length and a spine morphology profile that resembles immature animals (Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005). Top right: Studies in rats suggest that the initial morphology profile in hippocampal subfield CA1 reveals an abundance of stubby, mushroom-shaped spines, which shifts toward more thin spines in adult animals (Harris et al., 1992, see also Tarelo-Acuna et al., 2000). Bottom right: In adult Fmr1 knockout mice, we have observed more long spines and a greater proportion of stubby and mushroom-shaped spines than in wildtype mice, suggesting that FMRP is required generally for the maturation of spines and not for development of a particular spine shape per se. As these hypotheses have not been tested directly in wildtype and Fmr1 knockout mice at both age groups, question marks have been added to the right two panels. Furthermore, no inferences can be made at this time about dendritic spine characteristics in area CA1 of young Fmr1 knockout mice.
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knockout mice than in human patients (see McKinney et al., 2005). Our findings also vary from the report of reduced spine density on cultured knockout hippocampal neurons (Braun and Segal, 2000), though it is difficult to draw meaningful conclusions from comparisons between Golgi staining of neurons in situ and fluorescence imaging of cultured neurons. As sites of biochemical compartmentalization and sources of electrical resistance, the shape of dendritic spines appears to be integrally tied to their function (Sorra and Harris, 2000). The immature-appearing spine morphology and length are likely, therefore, to generate a physiological pattern that is different in the knockout hippocampus from the wildtype hippocampus and probably from the knockout and wildtype neocortex. It is also possible that the abnormal morphology of hippocampal dendritic spines reported here in Fmr1 knockout mice is the consequence of prolonged exposure to synaptic depression as enhanced long-term depression (LTD) has been reported in area CA1 of these animals (Huber et al., 2002). LTD (albeit a different variety) has been associated with spine shrinkage (Zhou et al., 2004), and it is reasonable to predict that prolonged exposure of Fmr1 knockout mice to synaptic depression in vivo might result in smaller spines, perhaps evidenced by an increase in the proportion of stubby, mushroom-shaped spines. The present findings, however, indicate that spines in Fmr1 knockout mouse CA1 are, in fact, longer than in wildtype mice, suggesting that other mechanisms may be involved. The functional consequences of the dendritic spine phenotypes in Fmr1 knockout mice may be related to the physiological differences reported between the cortex and hippocampus of these animals; in the hippocampus, for example, enhanced LTD has been reported but no differences in long-term potentiation (LTP) have been observed, whereas reduced LTP has been reported in the neocortex (Godfraind et al., 1996; Huber et al., 2002; Li et al., 2002). One recent paper has suggested that these physiological differences are age-dependent (Larson et al., 2005). As with regional differences in physiological plasticity, we see here that the specific pathology of synaptic morphology that arises from the functional loss of FMRP depends upon the cellular, and presumably the molecular, context. As an mRNA binding protein, FMRP appears to be involved in the transport and translation of a subset of mRNAs near synapses (Ling et al., 2004; Weiler et al., 2004). Normal regulation of this subset of messages may be critical for normal synaptic function and, as the findings suggest here, for normal activity-dependent maturation of synapses (Grossman et al., in preparation). Further characterization of the role of these mRNAs in the development of spine morphology and in adult synaptic plasticity in Fragile X syndrome will be important in understanding the pathobiology of this disease.
control mice (also backcrossed 6 times, and wildtype with respect to the FMR1 locus) bred in a C57BL/6 background were used. At postnatal days 60–90, mice were deeply anesthetized and transcardially perfused with 100 ml PBS (pH 7.4). Brains were submerged in 25 ml of Golgi–Cox solution for 20–30 days (Glaser and Van der Loos, 1981), embedded and sectioned at 120 μm. After processing and dehydration, sections were mounted on slides which were coded so that raters were blind to group identity. In area CA1 of the dorsal hippocampus of each animal, apical shafts of ten randomly selected pyramidal neurons were examined. To be analyzed, apical shafts of fully impregnated neurons had to be ≥175 μm long. All analysis was done by a single rater on a Zeiss Standard Universal at an optical magnification of 1200×. Dendrites in Fig. 1B are printed at a magnification of ∼1280×.
4.2.
Dendritic spine analysis
For each neuron, the morphology and length were determined of the first 10 spines in every 25 μm bin along the apical shaft. For spine morphology, spines were assigned the morphological category (used previously by our laboratory; Galvez and Greenough, 2005; Irwin et al., 2002; McKinney et al., 2005; see Fig. 1A) that most resembled the shape of that spine. Morphological category A was pooled with category B (A/B), C with D (C/D) and F with G (F/G) for analysis. The length of each dendritic spine evaluated for morphology was also measured. Length was defined as the distance from the distal surface of the spine head to the dendrite, rounded up to the nearest 0.5 μm, so that spines fell into one of five length categories. Finally, the density of spines (number per length of dendrite) in each 25 μm bin was measured.
4.3.
Statistical analysis
The number of spines of each morphology and length category was summed across all neurons and all animals, and Chisquare analysis was used to compare the profile (i.e. frequency distribution) of spines in these categories between genotypes. Chi-square analysis was subsequently performed on spine profiles along the apical dendrite at various distances from the soma (see Supplementary data). Chi-square analysis of the profile of dendritic spine lengths (overall and for each morphology type) was performed in the same way. We also tested for group differences in spine density overall along the apical shaft and at 50-μm-long distances from the soma (this was computed as the average of the two 25-μm-long bins). For these comparisons, Student's t tests were performed overall and on each 50-μm-long distance using the mean spine density per animal, averaged across all neurons.
4.
Experimental procedures
Acknowledgments
4.1.
Subjects and tissue processing
The authors would like to thank Roberto Galvez for his technical assistance as well as Julie Markham and Georgina Aldridge for their thoughtful comments on this manuscript. We also thank Kathy Bates, Lisa Foster and Dack Shearer for their valuable contributions to this research. N.M. Elisseou is currently enrolled at the Pritzker School of Medicine at the
The tissue examined for this study is the same as was used by McKinney et al. (2005), where a more thorough description of methods can be found. Briefly, seven adult Fmr1 knockout (B6.129P2-Fmr1tm1Cgr; backcrossed 6 times) and four adult
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University of Chicago, and B.C. McKinney is enrolled in the University of Michigan's Medical Scientist Training Program. This work was supported by FRAXA, NIH Grants MH35321, HD07333, and the Spastic Paralysis and Allied Diseases of the Central Nervous System Research Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.brainres.2006.02.044.
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