American Journal of Medical Genetics 135A:155 –160 (2005)
Sequence of Abnormal Dendritic Spine Development
in Primary Somatosensory Cortex of a Mouse Model
of the Fragile X Mental Retardation Syndrome
Roberto Galvez1,5* and William T. Greenough1,2,3,4,5
1
Neuroscience Program, University of Illinois, Urbana, Illinois
Department of Psychology, University of Illinois, Urbana, Illinois
3
Department of Psychiatry, University of Illinois, Urbana, Illinois
4
Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois
5
Beckman Institute, University of Illinois, Urbana, Illinois
2
Anatomical analyses of occipital and temporal
cortex of patients with fragile X mental retardation syndrome (FXS) and in a mouse model of the
syndrome (FraX mice) compared to controls have
suggested that the fragile X mental retardation
protein (FMRP) is important for normal spine
structural maturation and pruning. However, a
recent analysis of spine properties in somatosensory cortex of young FraX mice has suggested that
this region may not exhibit spine abnormalities.
While spine abnormalities were present 1 week
after birth in somatosensory cortex, by 4 weeks
almost all spine abnormalities had disappeared,
suggesting that adult spine abnormalities observed in other cortical regions may not persist
post-developmentally in somatosensory cortex.
To resolve this discrepancy we examined spine
properties in somatosensory cortex of young (day
25) and adult (day 73–76) FraX compared to wildtype (WT) mice. Spine properties in young FraX
and WT mice did not consistently differ from each
other, consistent with the recent analysis of
developing somatosensory cortex. However, adult
FraX mice exhibited increased spine density,
longer spines, more spines with an immatureappearing structure, fewer shorter spines, and
fewer spines with a mature structure, a pattern
consistent with prior analyses from other adult
cortical brain regions in humans and mice. These
findings (1) support the previous report of the
absence of major spine abnormalities in the fourth
postnatal week, (2) demonstrate normal spine
development in WT mice, (3) demonstrate abnormal spine development after the fourth postnatal
week in FraX mice, and (4) demonstrate spine
Grant sponsor: FRAXA Research Foundation; Grant sponsor:
NIH/NICHD; Grant number: HD37175; Grant sponsor: NIH/
NIMH; Grant number: MH35321; Grant sponsor: Society for
Neuroscience Minority Neuroscience Fellowship Program—NIH/
NIMH; Grant number: 1T32MH20069; Grant sponsor: Illinois—
Eastern Iowa District of Kiwanis International Spastic Paralysis
and Allied Diseases of the Nervous System Research Foundation.
*Correspondence to: Roberto Galvez, Department of Physiology, Northwestern University Feinberg School of Medicine, 303
East Chicago Avenue, Chicago, IL 60611.
E-mail: [email protected]
Received 10 August 2004; Accepted 10 February 2005
DOI 10.1002/ajmg.a.30709
ß 2005 Wiley-Liss, Inc.
abnormalities in somatosensory cortex of adult
FraX compared to adult WT mice. In doing so,
these findings resolve a potential conflict in the
literature and more thoroughly describe the role
of FMRP in spine development.
ß 2005 Wiley-Liss, Inc.
KEY WORDS: development; fmr1; FraX; FMRP;
somatosensory cortex
INTRODUCTION
The fragile X mental retardation syndrome (FXS) is the most
common form of inherited mental retardation with an
incidence of 1:4,000 in males and 1:8,000 in females [Crawford
et al., 2001]. Although some exceptions exist [Siomi et al.,
1994], the syndrome has been shown to result most commonly
from transcriptional silencing of Fmr1, the gene that codes for
the fragile X mental retardation protein (FMRP) [Oberle et al.,
1991; Pieretti et al., 1991; Gedeon et al., 1992; Sutcliffe et al.,
1992; Devys et al., 1993]. Histological analyses of neuronal
properties in both FXS patients and fmr1 knockout (FraX) mice
[Dutch–Belgian Fragile X Consortium] have strongly suggested a possible role for FMRP in neuronal development.
Anatomical analyses of dendritic spine properties in adult FXS
patients and FraX mice in occipital and temporal cortex have
consistently demonstrated various abnormalities in the length
and shape of dendritic spines. Analysis of spine properties in
these cortical regions has shown the absence of FMRP to be
associated with a higher abundance of long, thin spines, fewer
short, more robust appearing spines, and a greater density
of spines overall [Rudelli et al., 1985; Hinton et al., 1991;
Wisniewski et al., 1991; Comery et al., 1997; Irwin et al., 2000,
2001, 2002; McKinney et al., 2002]. This pattern of differences
has been proposed to arise as a result of a failure in normal
developmentally regulated pruning and/or maturation of
cortical dendritic spines in FXS patients and FraX mice
compared to controls [Rudelli et al., 1985; Hinton et al., 1991;
Wisniewski et al., 1991; Comery et al., 1997; Irwin et al., 2000,
2001, 2002; McKinney et al., 2002]; thus, suggesting a role for
FMRP in cortical spine development and pruning.
However, one report found that these spine abnormalities
largely disappeared in the somatosensory cortex during
development in FraX mice [Nimchinsky et al., 2001]. Analyses
of dendritic spine properties on layer V pyramidal neurons in
somatosensory cortex indicated that, while spine length and
density were significantly greater 1 week after birth in FraX
mice compared to controls, by age 4 weeks (p27), the oldest time
point that could be examined due to potential technical
problems with the intracellular fluorescent label enhanced
green fluorescent protein (EGFP), there was no significant
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Galvez and Greenough
difference in spine density and only a slight difference in thirdorder basilar dendritic spine length between FraX and wildtype (WT) mice [Nimchinsky et al., 2001]. To account for the
discrepancy between their findings and prior spine analyses,
the authors offered four possible explanations: (1) Since prior
in vivo spine analyses in FraX mice were conducted in occipital
and not somatosensory cortex [Comery et al., 1997; Irwin et al.,
2002; McKinney et al., 2002], the authors suggested that
dendritic maturation may differ in these two cortical areas.
However Nimchinsky et al. [2001] correctly pointed out that
prior analyses of spine development in these regions have
demonstrated comparable time courses [Wise et al., 1979;
Miller, 1981], making this possibility unlikely. (2) Since
transfection efficiency with EGFP labeling decreases with
age, they suggested that labeling of ‘‘neurons with abnormal
spines’’ may selectively decrease more rapidly with age, thus
making it appear as if the spine abnormalities disappear with
age. (3) Since different visualization procedures were used
(Golgi vs. EGFP), they suggested that the observed discrepancy may reflect differences between these methods. (4) Since
normal spine density decreases after 1 month of age in layer V
pyramidal neurons in somatosensory cortex [Wise et al., 1979;
Miller, 1981; Galofre and Ferrer, 1987], they suggested that
the abnormalities observed in other cortical regions may reflect
a selective abnormality in this later pruning process and thus
would not have been observed even at the last time point
[Nimchinsky et al., 2001] examined (p27). Nimchinsky et al.
[2001] noted that the limitations of EGFP staining in mature
tissue precluded adequately evaluating these alternatives—
thus, they were unable to determine a specific reason for the
discrepancy between their results and all prior and subsequently reported results for effects of the absence of FMRP on
neocortical spines in both mice and humans. In an attempt to
resolve this apparent discrepancy, utilizing Golgi–Cox
impregnation so that both young and adult time points could
be visualized, the present study examined spines of layer V
pyramidal neurons in primary somatosensory cortex of young
and adult FraX and control mice.
Fig. 1. Spine structure and morphological classification. Photomicrographs of spines on apical dendrites of (A) WT young, (B) FraX young, (C)
WT adult, (D) FraX adult mice in primary somatosensory cortex. E: Spine
classification scheme, adopted from Irwin et al. [2002], used for characterizing spine morphologies.
MATERIALS AND METHODS
To generate the mice used in the present experiment, fmr1
knockout mice, originally obtained from the Dutch-Belgian
Fragile X Consortium [1994] were backcrossed six times with
inbred C57BL/6J mice, selecting for the knockout. Eight of
these young (postnatal day 25; 4 WT and 4 FraX) and eight
adult (postnatal day 73–76; 4 WT and 4 FraX) C57BL/6J
(129P2-fmr1tm1Cgr) mice were transcardially perfused with
0.9% saline, pH 7.4. The brains were immediately processed for
Golgi–Cox analysis using a standard protocol [Glaser and Van
Der Loos, 1981], embedded in celloidin [Irwin et al., 2002] and
sectioned in a coronal plane at 175 mm. Seven to eight
completely impregnated layer V pyramidal neurons in primary
somatosensory cortex with apical dendrites greater than
250 mm in length, apical oblique dendrites greater than
75 mm, and basilar dendrites greater than 50 mm were analyzed
for each animal. Spine analysis was conducted as outlined by
Irwin et al. [2002]. Briefly, the number, length, and categorical
morphology (Fig. 1) of spines were determined in four 50 mm
dendritic segments between 50 and 250 mm above the soma
along the apical dendrite, and in two 25 mm dendritic segments
along the first 25–75 mm of apical oblique dendrites and the
first 25–50 mm of basilar dendrites.
Prior to conducting statistical analysis, mean values for each
animal were analyzed to remove neurons exhibiting characteristics two standard deviations from the individual animal
means. A maximum of only one neuron was removed per animal. Statistical analysis was then conducted using a general
linear model ANOVA in SAS on an IBM compatible computer
(SPSS Science, Chicago, IL). Specifically genotype and age
were treated as between subject independent variables with
the two ages analyzed separately and then combined in a
factorial design for evaluation of age effects. The number of
spines, proportion of spines with specific lengths, or proportion
of spines exhibiting specific morphologies for each 50 mm
(apical) or 25 mm (oblique and basilar) bin were then treated as
within subject repeating dependent variables.
RESULTS
Analysis of spine density in young mice (p25) largely showed
no significant differences between FraX and WT mice (Fig. 2,
young), consistent with findings of Nimchinsky et al. [2001].
However, analysis of spine density in adult mice (p73–76)
demonstrated a consistent pattern of significant group effects
(F(1,6) ¼ 84.50, P < 0.05 apical; F(1,6) ¼ 15.36, P < 0.05 apical
oblique; F(1,6) ¼ 35.81, P < 0.05 basilar), with FraX mice having
significantly more spines than WT mice (Fig. 2, adult). This
adult result is consistent with prior findings from adult
occipital and temporal cortex of FXS patients and FraX mice
[Rudelli et al., 1985; Hinton et al., 1991; Wisniewski et al.,
1991; Comery et al., 1997; Irwin et al., 2000, 2001, 2002;
McKinney et al., 2002]. Furthermore, this difference between
adult FraX and WT mice appears to be predominantly due to a
decrease in spine density from young to adult in WT mice
(young vs. adult: F(1,6) ¼ 7.70, P < 0.05 apical; F(1,6) ¼ 7.55,
P < 0.05 apical oblique; F(1,6) ¼ 6.49, P < 0.05 basilar) paired
Developmental Spine Abnormalities in S1 of Frax Mice
157
Fig. 2. Spine density for FraX and WT young and adult mice in
somatosensory cortex. The number of spines per unit dendritic length in
FraX and WT young and adult mice on (A) apical, (B) apical oblique, and
(C) basilar dendrites at fixed intervals from the soma (*P < 0.05).
with the lack of a similar developmental decline in FraX mice
(Fig. 2), consistent with impaired pruning.
Analysis of spine length similarly showed predominantly an
absence of significant differences between young FraX and WT
mice (Figs. 3 and 4, young), again consistent with the findings
of Nimchinsky et al. [2001]. In contrast, analysis of spine
length in adult mice uncovered a consistent pattern of significant effects of genotype on spine length (F(19,114) ¼ 10.81,
P < 0.05 apical; F(9,54) ¼ 16.31, P < 0.05 apical oblique;
F(4,24) ¼ 7.15, P < 0.05 basilar; Fig. 3), with post-hoc analyses
demonstrating that FraX mice had significantly more longer
and fewer shorter spines than WT mice (Figs. 3 and 4, adult).
This observation is consistent with prior findings from adult
occipital and temporal cortex of FXS patients and FraX mice
[Rudelli et al., 1985; Hinton et al., 1991; Wisniewski et al.,
1991; Comery et al., 1997; Irwin et al., 2000, 2001, 2002;
McKinney et al., 2002]. This difference between adult FraX
and WT mice, as in the spine density analyses, appears to be
primarily due to normal developmental shortening of spines in
WT mice (young vs. adult: F(19,114) ¼ 9.32, P < 0.05 apical;
F(9,54) ¼ 7.65, P < 0.05 apical oblique; F(4,24) ¼ 5.79, P < 0.05
basilar), and a lack of such development in FraX mice (Figs. 3
and 4).
Analysis of spine morphology categories in young mice again
showed few significant differences between FraX and WT mice
(Figs. 5 and 6, young). However, these spine morphology
categories in adult mice showed a significant effect of genotype
Fig. 3. Spine length on the apical shaft for FraX and WT young and adult
mice in somatosensory cortex. The proportion of spines with various spine
lengths in FraX and WT young and adult mice (A) 50–100 mm from the soma,
(B) 100–150mm from the soma, (C) 150–200 mm from the soma, and (D) 200–
250 mm from the soma (*P < 0.05).
(F(23,138) ¼ 11.24, P < 0.05 apical; F(11,66) ¼ 14.35, P < 0.05
apical oblique; F(5,30) ¼ 3.29, P < 0.05 basilar) with post-hoc
analyses demonstrating, despite some differences among
branch types, that FraX mice contain significantly more spines
with an immature structure and fewer spines with a mature
structure (Fig. 1e; Figs. 5 and 6 adult). These adult spine
abnormalities are again consistent with observations of
spine properties from occipital and temporal cortex of adult
FXS patients and FraX mice [Rudelli et al., 1985; Hinton et al.,
1991; Wisniewski et al., 1991; Comery et al., 1997; Irwin et al.,
2000, 2001, 2002; McKinney et al., 2002]. This difference
between adult FraX and WT mice, as in the spine length and
density analyses, appears to be predominantly due to a
developmental shift in WT mice from an immature to a more
mature structure (Fig. 1e; young vs. adult: F(23,138) ¼ 13.71,
P < 0.05 apical; F(11,66) ¼ 6.14, P < 0.05 apical oblique; F(5,30) ¼
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Galvez and Greenough
Fig. 4. The proportion of spines with various spine lengths on apical
oblique and basilar dendrites for FraX and WT young and adult mice in
somatosensory cortex. Values are presented for FraX and WT young and
adult mice on (A) apical oblique dendrites 25–50 mm from the apical shaft,
(B) apical oblique dendrites 50–75 mm from the apical shaft, and (C) basilar
dendrites 25–50 mm from the soma (*P < 0.05).
3.54, P < 0.05 basilar), and a lack of such development in FraX
mice (Figs. 5 and 6).
Fig. 5. Spine morphology categories on apical shafts for FraX and WT
young and adult mice in somatosensory cortex. The proportion of spines
exhibiting various structures in FraX and WT young and adult mice (A) 50–
100 mm from the soma, (B) 100–150 mm from the soma, (C) 150–200 mm from
the soma, and (D) 200–250 mm from the soma (*P < 0.05).
DISCUSSION
Analyses of dendritic spines in FXS patients and FraX mice
have suggested that FMRP is essential for normal development of spine density, length, and structure in the occipital and
temporal cortex [Rudelli et al., 1985; Hinton et al., 1991;
Wisniewski et al., 1991; Comery et al., 1997; Irwin et al., 2000,
2001, 2002; McKinney et al., 2002]. These analyses suggest
that such spine abnormalities resulting from an absence of
FMRP are most probably a general abnormality found
throughout the neocortex. However, analysis of spine properties in intracellular EGFP-filled somatosensory cortical neurons demonstrated that spine abnormalities largely disappear
on layer V pyramidal neurons by age 27 days [Nimchinsky
et al., 2001]. Although the authors offered several possible
explanations for the discrepancy between their findings in
young animals and other results in adults, limitations of their
methodology (intracellular EGFP) did not allow them to
determine the cause of this discrepancy. The present study
examined spine properties on layer V pyramidal neurons in
somatosensory cortex, using Golgi–Cox staining so that young
and adult time points could be compared.
The first proposed explanation for this discrepancy was the
possibility that dendritic maturation may differ between
previously examined neocortical regions and somatosensory
cortex. As Nimchinsky et al. [2001] pointed out, prior analyses
of spine development in various cortical regions have demonstrated comparable time courses [Wise et al., 1979; Miller,
1981], rendering different paths of maturation unlikely. Hence
this hypothesis was not tested here.
The second proposed explanation for this discrepancy was
the possibility that as the subjects’ age increased, EGFP
labeling might selectively label ‘‘normal’’ neurons. This would
create the appearance that the spine abnormalities diminish
by p27, the oldest age examined by Nimchinsky et al. [2001].
However, we also found minimal spine abnormality at p25
using Golgi staining, a finding consistent with Nimchinsky
et al. [2001]. Thus, possible selective labeling of ‘‘normal’’
Developmental Spine Abnormalities in S1 of Frax Mice
159
chinsky et al. [2001] results and all reported spine analyses of
adult FraX mice.
In addition to resolving a potential conflict in the literature,
the current study also clearly demonstrates a lack of spine
maturation and pruning following the fourth week of life in
FraX mice. Prior analyses of adult spine properties in FXS
patients and FraX mice had largely assumed that development
was abnormal based on the adult morphology, but offered no
insight as to when these abnormalities occurred. Our results,
for the first time, link these previously characterized spine
abnormalities to abnormal spine development occurring after
the first month of life. In contrast to WT mice, FraX mice
exhibit no evident spine pruning (as indicated by the lack of
reduction in spine number) and reduced morphological
modification (shortening and head-widening). WT mice show
a reduction in overall spine number combined with apparent
spine shape frequency changes (more shorter spines with
larger heads) that may reflect both pruning and shape
transformation. These data clearly demonstrate that FMRP
plays a pivotal role in this post 1-month phase of spine pruning
and maturation. Furthermore, unlike early spine differences
that largely disappear by age 25/27 days [Nimchinsky et al.,
2001], spine abnormalities that arise after 1 month of age
persist into adulthood, paralleling the anatomical [Irwin et al.,
2000, 2001] and functional [Hagerman and Hagerman, 2003]
deficits observed in adult FXS patients. The fact that adult
synaptic abnormalities develop between age 4 weeks and
adulthood in the mouse suggests that, if the human developmental process involves late pruning comparable to that of the
mouse (see, e.g., Huttenlocher and Dabholkar, 1997), there
may be a long period during which therapeutic intervention
might be effective, when such interventions become available.
ACKNOWLEDGMENTS
Fig. 6. Spine morphology categories on apical oblique and basilar
dendrites for FraX and WT young and adult mice in somatosensory cortex.
The proportion of spines exhibiting various structures in FraX and WT
young and adult mice on (A) apical oblique 25–50 mm from the apical shaft,
(B) apical oblique 50–75 mm from the apical shaft, and (C) basilar 25–50 mm
from the soma (*P < 0.05).
The authors thank Aaron W. Grossman and Teresita Briones
for technical assistance.
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