Journal of Neuroscience Research 73:19 (2003)

Insulin-Like Growth Factor 1 Is Essential for

Normal Dendritic Growth
Clara M. Cheng,1 Ronald F. Mervis,2 Shui-Lin Niu,3 Norman Salem Jr.,3
Lee A. Witters,4 Victor Tseng,1 Rickey Reinhardt,1 and Carolyn A. Bondy1*
1
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland
2
NeuroStructure Research Laboratories, Columbus, Ohio
3
Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, Bethesda, Maryland
4
Department of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire

This study evaluated somatic and dendritic growth of

neurons in the frontoparietal cortex of Igf1/ brains.
Pyramidal neuron density was increased by 25% (P
.005) and soma size reduced by 10% (P .001). Golgi
staining revealed that cortical layer IIIII neurons exhibited a signicant reduction in dendritic length and complexity in Igf1 null mice. Dendritic spine density and
presumably synaptic contacts were reduced by 16%
(P .002). Similar ndings were obtained for cortical
layer V and piriform cortex pyramids. Supporting a reduction in synapses, synaptotagmin levels were reduced
by 30% (P .02) in the Igf1 null brain. Investigation of
factors critically involved in dendritic growth and synaptogenesis showed an 50% reduction in cortical CDC42
protein expression (P .001) and an 10% reduction in
brain cholesterol levels (P .01) in Igf1 null mice. Evidence is presented that Igf1 deletion causes disruptions
in lipid and microtubule metabolism, leading to impaired
neuronal somatic and dendritic growth.
Published 2003 Wiley-Liss, Inc.

Key words: cholesterol; synaptogenesis; phospholipid;

cdc42; synaptotagmin; cholesterol; mental retardation

Insulin-like growth factor 1 (IGF1) has pleiotropic

effects on neural tissue in vitro that have been reviewed
elsewhere (Feldman et al., 1997). Studies of the murine
Igf1-targeted-deletion brain have elucidated IGF1s neurotrophic effects in vivo. We have demonstrated that
glucose utilization is signicantly reduced in the Igf1/
brain during early postnatal development, particularly in
those structures where IGF1 is normally highly expressed
(Cheng et al., 2000). The reduction in glucose use is
associated with reduced phosphorylation of Akt and
GSK3, decreased GLUT4 expression and hexokinase
activity, and reduced glycogen accumulation in growing
neurons that normally express IGF1 (Cheng et al., 2000).
These ndings suggest that IGF1 serves an anabolic,
insulin-like role promoting neuronal glucose utilization
and growth during brain development (Cheng et al.,
2000). IGF1 expression is most abundant in growing proPublished 2003 Wiley-Liss, Inc. This article is a US Government work
and, as such, is in the public domain in the United States of America.

jection neurons, which become the largest neurons

equipped with the most extensive dendritic trees in the
brain (Bondy, 1991). Also, IGF1 is highly expressed during the time in early postnatal development when dentritic
process growth is most exuberant (Bartlett et al., 1991;
Bondy, 1991). Because neuronal processes or neuropil
occupy a large percentage of brain volume, it seemed
possible that impaired dendritic development in the absence of IGF1 may contribute to the reduced brain size
and cognitive impairment noted in the human with Igf1
deletion (Woods et al., 1996, 1997). To evaluate this
hypothesis, we have used Golgi histological staining and
biochemical analyses to evaluate dendritic growth and
complexity in the Igf1/ brain and wild-type (WT) littermates.
MATERIALS AND METHODS
Mice
The Igf1/ mouse line used in this study, which was
approved by the NICHD Animal Use and Care Committee,
was generated at Genentech, Inc. (San Francisco, CA; PowellBraxton et al., 1993). This mutation has been bred into a CD1
outbred line for more than 40 generations. Igf1/ and WT
littermate mice were genotyped as described previously (PowellBraxton et al., 1993). For biochemical studies, mice were sacriced by decapitation after CO2 anesthesia; then, brains were
dissected, snap frozen immediately, and stored at 70C. For
morphological study, mice were anesthetized with sodium pentobarbital and perfused through the ascending aorta with 4%
paraformaldehyde in phosphate-buffered saline (PBS) buffer (pH
7.4) for 45 min. These brains were then postxed in 10%
formalin in PBS buffer overnight. Brains were bisected sagitally,
and a half brain from each animal was used for morphological
*Correspondence to: Carolyn A. Bondy, MD, Bldg. 10/10N262, 10 Center
Dr., NIH, Bethesda, MD 20892. E-mail: bondyc@exchange.nih.gov
Received 13 February 2003; Revised 27 February 2003; Accepted 7 March
2003

Cheng et al.

observation and cell density measurement, with the other half

used for Golgi staining.
Golgi Analysis
Blocks of sagitally cut, 10% formalin-xed mouse brain
encompassing the prefrontal and piriform cortices were stained
with the rapid Golgi method as detailed elsewhere (Valverde,
1976). Briey, the blocks were impregnated using osmium
tetroxide plus potassium dichromate, followed by immersion in
silver nitrate. After dehydration, the blocks were embedded in
nitrocellulose, and coronal sections were cut at 120 m. Camera
lucida drawings of the basilar tree were made using a 40
long-working-distance oil-immersion lens, a 1.6 Zeiss optivar
intermediate lens setting, and a 10 ocular. For dendritic analyses, ve pyramidal neurons in each of cortical layers IIIII and
V and the pyramidal layer of the piriform cortex from each
animal were randomly selected for assessment (N 7 for WT
controls and N 6 for Igf1/ mice). A prole for representative neurons in each region of individual animal was generated, with data averaged for each animal. The randomly selected
neurons were located in a cortical region starting medially from
the midsagittal ssure (but not including the cingulate cortex)
and extending laterally to the rhinal ssure. Also, the selected
neurons had to meet the following criteria: a soma located in the
middle one-third of the thickness of the slide; fully impregnated
basilar dendritic tree and spines as ascertained by microscopic
observation under high magnication; and no obscuring of
branches in the neuropil by glia, blood vessels, or precipitate.
The extent and distribution of dendritic branching of these
neurons were evaluated by Sholl analysis [method of concentric
circles (Sholl, 1953)]. Briey, a transparent overlay of increasingly larger concentric circles at 10-m intervals was superimposed on the camera lucida drawing of each basilar tree and the
number of dendritic branch intersections (e.g., hits) with each
progressively larger circle counted to a distance of 200 m from
the soma of each neuron. The total dendritic length for the
basilar tree of each neuron was calculated by tracing camera
lucida drawings with a planimeter. Radial tip analysis measured
the straight distance between the tips of branches from the
center of the cell body. The soma size was measured from the
area of a neuron cell body obtained by camera lucida drawings.
For spine analysis, only visible anking dendritic spines were
counted along 30-m terminal-tip segments of four total segments for each of ve randomly selected neurons per animal
under a magnication of 1,200.
Cell Density Measurement
Formalin-xed brains were embedded in parafn and cut
into 10-m sagittal sections. Anatomically matched sections
1 mm lateral to the midline were stained with toluidine blue
for morphological analysis. Neurons were identied as palely
stained, ovoid cells with a diameter at least 15 m and more
darkly staining nucleus fully within the plane of section (see Fig.
1). Neuron cell numbers in medial parietal association cortex
(from 1.25 to 2.5 Bregma) were counted by a blinded observer at 400 in a eld containing the full thickness of the
cortex. Two sections were scored for each mouse, and data were
meaned prior to pooling to obtain group means. Data are
expressed as cell number per 5 103 m2 and represent the
mean SEM for nine mice per group.

Immunoblotting
Immunoblotting was performed according to Cheng et al.
(1998), except for sample preparation. To prepare total-brain
homogenate, the mouse brains (approximately 0.4 g) were homogenized in a boiling solution containing 10 mM Tris (pH
7.4), 1 mM sodium orthovanadate, and 1% sodium dodecyl
sulfate (SDS) at a ratio of 1 g tissue to 17.5 ml solution. An equal
volume of total-brain homogenates from each sample (10 l)
was loaded and resolved on NuPAGE 10% Bis-Tris Gels (Invitrogen Life Technologies, Carlsbad, CA ) and transferred to
nitrocellulose membranes using electrophoretic transfer cell
(Bio-Rad, Hercules, CA). The primary antibodies were purchased from BD Transduction Laboratories (San Diego, CA):
anti-CDC42 (dilution factor 1:125), anti-Rac1 (1:1,000), antiRho (1:250), and antisynaptotagmin (1:500). After incubation
with horseradish peroxidase-linked secondary antibodies, protein bands were visualized by SuperSignal West Pico detection
reagents (Pierce, Rockford, IL) on a Kodak 1D Image machine
(Kodak, Rochester, NY). A digital image of the immunoblot
was made, and the relative amounts of proteins in Igf1/ (N
4 6) and WT (N 4 6) brains were compared using Kodak
1D image-analysis software.
Cholesterol and Phospholipid Analysis
Brains were rst homogenized in a boiling buffer (1 g/
17.5 ml) containing 10 mM Tris (pH 7.4), 1 mM sodium
orthovanadate, and 1% SDS. The brain homogenate (100 l)
was diluted vefold in Tris-buffered saline (TBS) buffer and
then assayed for cholesterol, total phospholipids, and total proteins (N 6 for each group).
Cholesterol assay. Cholesterol was determined by an
enzymatic colorimetric method (Allain et al., 1974) using the
Cholesterol CII kit from Wako (Richmond, VA). The assay was
performed in a 96-well plate in triplicate. A standard curve was
generated using a buffer blank and cholesterol standards covering
the concentration range of 0 0.2 mg/ml. Briey, 10 l of each
standard or sample was mixed with 200 l of color reagent in a
well and incubated at 37C for 5 min for color development.
Cholesterol concentration was determined by measuring the
absorbance at 505 nm in a plate reader. Data were expressed as
milligram cholesterol per gram brain protein. Total-brain protein was assayed using the BCA protein assay reagent kit from
Pierce Chemical Co. (Rockford, IL). Statistical signicance was
evaluated using ANOVA, followed by a Fishers PLSD test.
Phospholipid assay. Total phospholipids were determined by the method of Bartlett (1959), with slight modications. Briey, 200 l of each standard (KH2PO4, 0 1 mM) or
100 l of each sample diluted with 100 l of water was mixed
with 200 l of concentrated H2SO4 and heated at 300C for
35 min to convert phospholipids into inorganic phosphate. The
mixture was then heated at 300C for an additional 30 min.
After brief cooling, the reaction mixture was added to 5 ml
water, 200 l 5% (w/v) ammonium molybdate, and 200 l
Fiske Subbarow Reagent (Sigma, St. Louis, MO) and heated at
100C for 10 min. The phosphate concentration was determined by measuring the absorbance at 660 nm.
Enzyme Activity Assay
After decapitation, brains were rapidly dissected and immediately homogenized (Teon/glass) in ice-cold 0.32 M su-

IGF1 and Dendritic Growth

Fig. 1. Neuron cell density, size, and dendritic proles of cortical pyramids from layer IIIII of WT
(A,C) and Igf1/ (B,D) mice. A and B are representative micrographs taken from anatomically
matched sagittal sections of adult P50 mice. The neuron cell density is increased in Igf1/ brain, and
the soma is signicantly smaller. C and D show representative camera lucida drawings of the basilar
tree of Golgi-stained pyramidal neurons from WT and Igf1/ brains.

crose (25 mg tissue wet wt/ml). All sucrose solutions contained

1 TE buffer and a protease inhibitor solution (10 g/ml
pepstatin A, 10 g/ml leupeptin,10 g/ml aprotinin). A crude
mitochondria fraction was prepared according to the method of
Gray and Whittaker (1962) by an initial centrifugation at 1,000g
for 10 min at 4C. The resultant supernatant was centrifuged at
12,000g for 20 min at 4C to provide the mitochondria pellet.
The pellet was resuspended in the 0.32 M sucrose solution, and
small aliquots of the mitochondria suspension were subjected to
enzyme activity assays. Hexokinase, phosphofructokinase, pyruvate kinase, malate dehydrogenase, D-3-hydroxybutyrate dehydrogenase, and lactate dehydrogenase activity assays were performed according to Clark and Lai (1989), with N 4 for each
group. Data were presented as nanomoles of product formed per
minute per milligram protein added.
Statistical Analysis
For all biochemical analyses in this study, statistical significance was evaluated using ANOVA, followed by Fishers PLSD
test. For dendritic analysis by Golgi stain, statistical signicance
was examined by ANONA, followed by Mann-Whitney test,
unless otherwise specied.

RESULTS
Morphometry
We compared neuronal cell density and soma size in
the neocortex of postnatal day 50 (P50) Igf1/and WT
mice. At this young adult stage, dendritic development
was largely mature. Cell density was increased and soma
size decreased in Igf1/ compared with WT mice (Fig.
1A,B). Mean cortical cell density was increased by approximately 23% in Igf1/ brain compared with WT (2.11
0.06 vs. 2.58 0.14 cells per 5 1,000 m2 for WT vs.
Igf1/, P .005), whereas soma size was decreased by
about 10% (111.2 16.9 m2 vs. 98.7 22.0 m2 for
WT vs. Igf1/, P .001). The decreased space between
nerve cell bodies in Igf1/ brains represents decreased
neuropil, which consists mainly of neuronal and glial cell
processes, so we turned our attention to an investigation of
neuronal process development in the Igf1/ and WT
mice.
To investigate the effect of Igf1 deletion on dendritic
structure, Golgi staining was used to visualize and measure
these neuronal processes. Pyramidal neurons were exam-

Cheng et al.

ined in cortical layers IIIII and V of the prefrontal cortex

and in the piriform cortex. The results of dendritic analyses
by Golgi staining were similar for all three regions but
were most profound for cortical layer IIIII pyramids.
Igf1/ cortical pyramidal dendrites were shorter and less
complex than those of WT (Fig. 1C,D). The amount of
dendritic material per neuron was measured by Sholl analysis, using a series of concentric circles of increasing diameter superimposed on camera lucida drawings of each
neuron. This analysis showed that Igf1/ neurons had
signicantly (P .0002) fewer dendritic intersections
with the concentric circles, particularly at increasing distance from the soma (Fig. 2A), indicative of signicantly
less dendritic material in Igf1/ brains. Branch-point

analysis measured the number of branch points at each

branch order on the camera lucida drawings for Igf1/
and WT neurons. This analysis showed that Igf1/ neurons had fewer branch points at each branch order, with
statistically signicant reductions in the rst and fourth
branch orders (Fig. 2B). Measurement of dendritic terminal tips also showed signicant reduction in Igf1/ neurons (P .005; Fig. 2C).
To address whether the extension of dendrites was
also affected by Igf1 deletion, dendritic length was compared between Igf1/ and WT brains (Fig. 3A). Igf1/
dendrites were approximately 25% shorter than WT dendrites (P .001). This result was supported by radial tip
analysis, which measured the straight distance of branch
tips from the center of the cell body (Fig. 3B). All these
results showed that Igf1/ neurons had signicant less
dendritic material with less complexity compared with
WT. The number of dendritic spines was also signicantly
reduced in the Igf1/ brain (16% in cortical layer IIIII,
P .002; 11% in both cortical layer V and piriform
cortex, P .03; Fig. 3C). Figure 4 shows representative
Golgi-stained pyramidal neurons and basilar dendrites of
Igf1/ and WT littermates at a high magnication, allowing visualization of dendritic spines.
Expression of Proteins Involved in Neurite
Outgrowth and Synapse Formation
There was also a decrease in the level of a synapsespecic protein, synaptotagmin, which was reduced by
approximately 30 40% (Fig. 5). We also evaluated expression of Rho GTPases thought to be involved in dendritic extension (Dickson, 2001; Redmond and Ghosh,
2001). RhoA and Rac1 were equally concentrated in
Igf1/ and WT brains, but CDC42 was reduced by
40 50%.
Biochemical Analyses
Cholesterol is a critical factor in stimulation of neurite growth and synaptogenesis (Mauch et al., 2001; Fan et
al., 2002). Brain cholesterol and phospholipid concentra

Fig. 2. Dendritic branching and complexity in WT and Igf1/ pyramidal neurons of cortical layers IIIII. A: Cumulative sholl analysis
shows that Igf1/ pyramidal neurons have signicantly less dendritic
material than WT, and the decit is more profound as the measurement
moves away from the soma (P .0002). The number of dendritic
intersections was obtained by superimposing a series of concentric
circles (shells) with increasing diameters on the camera lucida drawing
of the basilar dendritic tree. B: Branch-point analysis revealed that there
are fewer branch points at each branch order measured in the Igf1/
neurons compared with WT. Statistically signicantly fewer branch
points were seen in the rst (P .014 by Mann Whitney test and P
.012 by unpaired t-test) and fourth (P .025 by unpaired t-test and not
signicant by Mann-Whitney test) branch orders. This indicates that
the Igf1/ brains have less complex dendritic arbors. C: Terminal-tip
measurement shows that there are signicant fewer tip segments in the
neurons of the Igf1/ brains (P .005 by Mann-Whitney test). Five
cortical pyramidal neurons were analyzed for each animal. N 7 for
Igf1/ mice and N 6 for WT controls; Data are presented as
mean SE for each group.

IGF1 and Dendritic Growth

Fig. 3. Dendritic length and spine of WT and Igf1/ pyramidal

neurons of cortical layers IIIII. A: Estimated dendritic length of
Igf1/ neurons is approximately 25% shorter than that of WT neurons
(P .0022 by Mann-Whitney test and P .001 by unpaired t-test).
B: Radial-tip analysis, which measures the straight distance from the
dendritic branch tips to the center of the cell body, conrms that the
Igf1/ neurons have shorter dendrites (P .0448 by Mann-Whitney
test and P .0415 by unpaired t-test). C: Spines from ve terminal-tip
segments per neuron were counted and estimated for the average spine
density. Results show that there were signicantly fewer dendritic
spines in the Igf1/ neurons of all three brain regions examined. Data
are shown as percentage of WT control. N 7 for Igf1/ mice and
N 6 for WT controls.

tions were each reduced by approximately 10% in the

IGF1 null brain (cholesterol: 160 2.5 mg/g protein for
WT vs. 145 3.2 mg/g for Igf1/, P .01; phospholipid: 6.5 0.15 mM for WT vs. 5.9 0.2 mM for
Igf1/, P .05). Because of this reduction, we investigated aspects of lipid metabolism in the Igf1/ brain.

Analysis of the fatty-acid prole revealed a small but

signicant reduction in saturated and monounsaturated,
longer chain (18C) fatty acids (data not shown). Interestingly, there was a small but signicant increase (8%, P
.04) in docosahexaenoic acid (DHA, 22:6n3) in the
Igf1/ brain. The rate-limiting enzyme involved in cholesterol synthesis is HMG-CoA reductase, and this enzyme, at least at the protein level, was slightly though not
statistically signicantly increased in the Igf1/ brain (data
not shown). The polypeptide concentrations of other enzymes involved in lipogenesis, including fatty acid synthase, ATP-citrate lyase, and acetyl CoA carboxylase were
also within normal limits in the Igf1/ brain (data not
shown). Activities of critical lipogenic enzymes, pyruvate
kinase and malate dehydrogenase, were signicantly reduced in Igf1/ brains, whereas the activities for enzymes
not involved in lipogenesis, phosphofructokinase (Table I)
and D-3-hydroxybutyrate dehydrogenase, were equal in
Igf1/ and WT brains. These abnormalities in enzyme
activity were clearly evident at postnatal day 10, a time of
peak IGF1 expression in the normal mouse, but were no
longer evident by P40, when IGF1 expression was greatly
reduced (data not shown). This was similar to the observations of impaired glucose utilization in the Igf1/ brain,
which was most profound at P10 but barely evident by
P40 (Cheng et al., 2000).
DISCUSSION
This study has shown that dendritogenesis is significantly impaired in the Igf1 null brain. Dendrite extension/
length is reduced by approximately 25%, and branching is
reduced by 10 20%, with a similar decrease in the density
of dendritic spines. We have not shown directly, but infer
from the reduction in spine numbers and synaptotagmin
levels in the Igf1 null brain, that synapses are also signicantly reduced. The present study also shows that pyramidal neuron soma size is signicantly reduced, and it has
previously been shown that Purkinje cells are signicantly
smaller in Igf1 null brain (Cheng et al., 1998). Taken
together, these observations indicate that IGF1 serves as an
anabolic factor for neuronal somatic and dendritic growth
during brain development. In support of the ndings of
the present in vivo study, IGF1 stimulates neurite outgrowth in vitro in a number of different model systems
(Kim et al., 1997; Torres-Aleman, 1999; Kimpinski and
Mearow, 2001; Rind and von Bartheld, 2002). For example, a recent study using IGF1 treatment of cortical
slices showed signicantly increased dendritic growth
(Niblock et al., 2000). Moreover, in transgenic mice overexpressing IGF1 in brain, neuropil volume (Carson et al.,
1993) and synaptogenesis are increased (OKusky et al.,
2000).
The fact that IGF1 appears to regulate cell size within
the brain is consistent with this growth factors role in
other tissues and indeed an evolutionarily conserved role
in other organisms. The role of the insulin/IGF signaling
pathway in cell size regulation has been clearly established
in Drosophila melanogaster (Birnbaum, 2002), and we have
shown that short stature in Igf1-decienct mice is due
mainly to reduced somatic size of growth plate chondro-

Cheng et al.

Fig. 4. Photomicrographs of Golgi-stained neurons in layers IIIII of the parietal cortex show a
reduction in basilar dendritic spines in the Igf1/ mice (B,D,F) compared with WT littermates
(A,C,E) at postnatal day 50. E and F show the dendrite spines at higher magnication, clearly
demonstrating lower spine density in the Igf1/ sample (F). The apparent difference in dendrite
diameter seen in E and F is because WT dendrite is more distal than the Igf1/ sample. Scale bar
20 m for AD; 5 m for E,F.

cytes (Wang et al., 1999). To elucidate the mechanism(s)

whereby IGF1 contributes to neuronal/dendritic growth,
we investigated elements of lipid and microtubule pathways that are important for neurite growth and synapse
formation. An important nding in this context is the
reduction in cholesterol production in the Igf1 null brain.
Cholesterol is an essential building block for neuronal
membranes, which undergo extraordinary elaboration
during dendritic growth. The ability of neurons to elaborate dendrites and form synapses is limited by the availability of cholesterol (Mauch et al., 2001; Fan et al., 2002),
so reduced cholesterol production in the Igf1 null brain

may contribute to the observed reduction in dendritic

growth.
The cause of reduced cholesterol production in the
Igf1 null brain is not clear from the present study. Most of
the enzymes immediately involved in lipogenesis appeared
to be present in normal concentration, e.g., fatty acid
synthase, ATP-citrate lyase, and acetyl CoA carboxylase.
The activity of the latter enzyme is the rate limiter in
cholesterol biosynthesis, and its activity, judging by phosphorylation status, was not appreciably altered in knockout compared with WT brains (our unpublished data).
Glucose serves as the major carbon source for brain lipo-

IGF1 and Dendritic Growth

genesis by glycolytic transformation to actetyl-CoA, and

its oxidation provides essential reducing power for lipid
biosynthesis. Thus, decreased glycolytic ux, leading to
decreased TCA cycle generation of mitochondrial citrate,
and decreased NADPH generation at malic enzyme could
certainly explain decreased lipogenesis. In support of this
possibility, there were signicantly reduced activities of
malate dehydrogenase and pyruvate kinase, which are
critically involved in early, glucose-dependent steps in
lipogenesis. Insofar as we have previously shown major
reductions in glucose utilization by the developing Igf1
null brain (Cheng et al., 2000), it is possible that glucoprivation leads to reduced cholesterol production. Alternatively, IGF1s effect on dendrite growth could be more
direct, and the observed changes in lipids could be secondary to decreased dendritic material.
An additional system is implicated in disrupted microtubule metabolism in the Igf1 null brain. Rho GTPase
signal transduction pathways are involved in dendritic
morphogenesis (Luo, 2000). Rho A inhibits dendritic
growth, whereas Rac and cdc42 promote dendritogenesis
(Threadgill et al., 1997), with cdc42 controlling growth
and branching and Rac controlling stability and spine
formation (Luo, 2000; Scott and Luo, 2001). We have
demonstrated that, in Igf1/ brains, although the protein
levels of RhoA and Rac are not altered, the expression of
cdc42 is reduced by 40 50% compared with control littermates. To our best knowledge, this is the rst evidence
linking IGF1s action in dendritic development to a Rho
GTPase pathway. Interestingly, a recent study showed that
disruption of Rho GTPase signaling resulted in reduced
cell, organ, and animal size (Sordella et al., 2002), similar
to the Igf1 null phenotype, implicating Rho GTPase pathways in mediating IGF1s growth-promoting actions.
The dendritic growth decit in the Igf1 null brain is
expected to result in impaired cognitive function. Limited
behavioral testing of Igf1/ mice has not shown major
neurological decits (Cheng et al., 1998), but testing in
this murine model is limited by the fact that the knockout
mice are only about half the size of WT, making the use
of standard maze- and platform-based tests of higher level
functions invalid. Only a single individual has been described with homozygous Igf1 deletions, and he demonstrates a global reduction in brain size and mental retardation (Woods et al., 1996). Additional indirect evidence
suggests that brain IGF1 promotes process growth and
synapse formation during brain development, thus enhancing cognitive potential. For example, environmental
and/or nutritional exposures that reduce IGF1 expression,
e.g., ethanol exposure, are associated with reduced dendritic development and synapse formation and decreased

Fig. 5. Expression of dendritic proteins during early postnatal development in Igf1/ and WT brains. A and B show immunoblot analyses
of Rho-family proteins cdc42, Rac1, and Rho and the synaptic protein
synaptotagmin (Syt) from P10 (A) and P40 (B) total-brain homogenates. cdc42 And Syt are signicantly reduced at both stages, whereas
Rac1 and Rho are equal in Igf1/ and WT mice.

Cheng et al.

TABLE I. Comparison of Some Metabolic Enzyme Activities in Igf1/ and Wild-Type Brains*

Igf1/
WT
Changea
Statistical signicance

PFK

PK

LDH

MDH

96.7 4.7
106.7 3.3

P .13

446 11
553 15
19% Decrease
P .001

482 8
539 14
11% Decrease
P .01

1,516 31
1,830 30
17% Decrease
P .0004

*Brain enzyme activities were assayed as described in Materials and Methods. Values are means SEM (nmole/mg/min) of N 4 for each group.
Statistical differences between two groups were determined by ANOVA, followed by Fishers least signicant different test.
PFK, phosphofructokinase; PK, pyruvate kinase; MDH, malate dehydrogenase; LDH, lactate dehydrogenase.
a
Decrease refers to a decrease in Igf/ brains vs. wild type.

cognitive function (Cui et al., 1997; Luo and Miller, 1998;

Lynch et al., 2001).
In summary, this work shows that neuronal somatic
and dendritic growth is impaired in the Igf1 null mouse
brain, resulting in hypoplastic processes that are shorter
and less branched, with fewer spines per segment. The
anatomic defect is associated with selective reductions in
brain lipids and reductions in the activities of enzymes
involved in converting glucose into lipid, suggesting that
glucoprivation may contribute to the impaired growth.
Igf1 deletion is also associated with decreased CDC42
expression, which may also impair neurite growth. Whatever the mechanism(s) whereby IGF1 normally stimulates
neuronal and dendritic growth, these clearly are important
effects with signicantly functional implications. Further
research is needed to elucidate mechanisms regulating
IGF1 expression and activity during brain development.
ACKNOWLEDGMENT
We are grateful to Gene C. Ness for helpful discussion and providing an anti-HMG CoA antibody.
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