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Dendritic spine abnormalities in the occipital cortex of C57BL6 Fmr1 knockout mice.

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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: wgreenou@s.psych.uiuc.edu
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. [1997] 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 [2000], 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. [1997] were bred in an FVB
background and may have exhibited a recessive form of retinal
degeneration [Taketo et al., 1991]. Irwin et al. [2002] 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. [2002] 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. [2002] 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. [1997] 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. [2002], 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.
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