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Cheng, Mervis, Bondy (2003) PDF IGF1 Dendritic Growth
Cheng, Mervis, Bondy (2003) PDF IGF1 Dendritic Growth
Cheng et al.
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-
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.
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.
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.
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.
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.