Download Quantitative Non-radioactive In Situ Hybridization Study of GAP

Quantitative Non-radioactive In Situ
Hybridization Study of GAP-43 and SCG10
mRNAs in the Cerebral Cortex of Adult and
Infant Macaque Monkeys
We performed non-radioactive in situ hybridization histochemistry in
several areas that include both the association areas and the lower
sensory areas of monkey cerebral cortex, and investigated the
localization of neurons expressing two growth-associated proteins:
GAP-43 and SCG10. Both GAP-43 and SCG10 mRNAs were observed
in both pyramidal and non-pyramidal neurons. Prominent hybridization signals for GAP-43 mRNA were observed in layers II–VI of the
adult association areas: the prefrontal areas (FD), the temporal (TE)
and the parietal (PG) association areas. The signals for GAP-43
mRNA were weak in layers I–III of the adult primary somatosensory
area (PB) and primary (OC) and secondary (OB) visual areas, and
cells with prominent signals were observed in layers IV–VI. The
prominent signals for SCG10 mRNA were observed in layers IV–VI
of all areas examined. These results suggest that the expression
pattern of GAP-43 mRNA, but not that of SCG10 mRNA, may be
related to the functional difference between association and lower
sensory areas of adult cortex. In the infant cortex (postnatal days 2,
8 and 31), the signals for both mRNAs were intense in layers II–VI
of all areas. Therefore, layer-specific expressions of GAP-43 and
SCG10 mRNAs are established after the first postnatal month.
Differentiation between association areas and primary areas is a
prominent feature of the primate cerebral cortex. The growthassociated protein GAP-43 is a protein that characterizes the
association areas. It participates in signal transduction during
axonal elongation (Akers and Routtenberg, 1985; Alexander et
al., 1987; Strittmatter et al., 1990; for review see Benowitz and
Routtenberg, 1997). GAP-43 and its mRNA are abundant in the
association areas of the mature cerebral cortex, whereas they
exist at only low levels in the primary areas (Neve et al., 1987,
1988; Benowitz et al., 1989; Oishi et al., 1998). Phosphorylation
of GAP-43 occurs more frequently in the higher order visual
cortices (Nelson et al., 1987), and the levels of GAP-43 are
selectively increased in the association cortices in schizophrenic
patients (Perrone-Bizzozero et al., 1996). These results, however,
came from studies using Northern blot analysis (Neve et al.,
1987, 1988; Oishi et al., 1998), Western blot analysis (PerroneBizzozero et al., 1996), two-dimensional gel electrophoresis
(Nelson et al., 1987) and immunohistochemistry (Benowitz et
al., 1989). The exact localization of the cell bodies synthesizing
GA P-43 cannot be revealed with these non-histological techniques or by immunohistochemical methods because GAP-43
is localized in nerve terminals of the neuropil, but not in the
cell bodies (Nelson and Routtenberg, 1985; Meiri et al., 1986,
1988; Skene et al., 1986). Therefore, we performed an in situ
hybridization study in various areas of the monkey cerebral
cortex, including both association and lower sensory areas,
to determine the distribution and the types of cells expressing
GA P-43 mRNA, and we investigated the differences between
GAP-43 mRNA-containing structures in the association and
lower sensory areas. Because GAP-43 serves as an intrinsic deter-
© Oxford University Press 1999
Noriyuki Higo1,2, Takao Oishi1, Akiko Yamashita3,
Keiji Matsuda1 and Motoharu Hayashi4
1
Neuroscience Section, Information Science Division,
Electrotechnical Laboratory, Umezono, Tsukuba, Ibaraki
305-8568, 2Department of Physiology, University of Tsukuba
School of Medicine, Tennodai, Tsukuba, Ibaraki 305-0006,
3
Department of Anatomy, Nihon University School of
Medicine, Oyaguchi-Kamimachi, Itabashi, Tokyo 173-8610 and
4
Department of Cellular and Molecular Biology, Primate
Research Institute, Kyoto University, Kanrin, Inuyama, Aichi
484-0081, Japan
minant of synaptic growth and plasticity even in the adult brain
(Aigner et al., 1995; Holtmaat et al., 1995; Kapf hammer, 1997;
for review see Benowitz and Routtenberg, 1997), the present
results provide insight into the neuronal components that have
high levels of plasticity in the association areas.
We also performed an in situ hybridization study of another
growth-associated protein, SCG10. Although SCG10 (Stein et
al., 1988a) and GAP-43 (Basi et al., 1987; Karns et al., 1987;
Rosenthal et al., 1987) have dissimilar amino acid sequences,
the subcellular localization of SCG10 is quite similar to that of
GAP-43 (Stein et al., 1988a). SCG10 is thought to participate
in axonal elongation (Stein et al., 1988b; McNeill et al., 1992;
Hannan et al., 1996) by regulating the instability of microtubules
(Riederer et al., 1997). Therefore, we compared the expression
patterns of SCG10 with those of GAP-43.
Furthermore, we investigated the expression of these growthassociated proteins in the cerebral cortex of infant monkeys.
Both GAP-43 mRNA (Neve and Bear, 1989; Mower and Rosen,
1993; Sugiura and Mori, 1995; Kanazir et al., 1996; Oishi et al.,
1998) and SCG10 mRNA (Anderson and Axel, 1985; Stein et
al., 1988a; Sugiura and Mori, 1995; Higo et al., 1996) are highly
expressed in the infant nervous system. There are no data available, however, on the localization of GAP-43 and SCG10 mRNAs
in the cerebral cortex of infant primate. Therefore, we compared
the localization of GAP-43 and SCG10 mRNAs between mature
and infant monkey cerebral cortices to determine whether the
region-specific expression of these growth-associated proteins is
established postnatally. Preliminary results have been reported
elsewhere (Higo et al., 1998b,c).
Materials and Methods
Animals and Tissue Preparation
Brain tissue was obtained from seven Japanese monkeys (Macaca
fuscata): four adults and three infants (postnatal days 2, 8 and 31). Adult
monkeys were purchased from a local provider. Infant monkeys were
bred as follows: 2-day-olds in the Tokyo Metropolitan Institute for
Neuroscience, 8-day-olds in Juntendo University School of Medicine
and 31-day-olds in the Primate Research Institute of Kyoto University. All
animal experiments were carried out in accordance with the Guide for
the Care and Use of Laboratory Animals established by the Institute
of Laboratory Animal Resources and the Guide for the Care and Use of
Laboratory Primates established by the Primate Research Institute, Kyoto
University.
The animals were pretreated with an i.m. injection of ketamine
hydrochloride (10 mg/kg) and deeply anesthetized by i.v. administration
of pentobarbital sodium (Nembutal; 30 mg/kg). They were then perfused
through the ascending aorta with 0.5 l of ice-cold saline containing 2 ml
(2000 units) of heparin sodium, followed by 1–2 l of ice-cold fixative [2%
paraformaldehyde (PFA) and 0.5% glutaraldehyde in 0.15 M phosphate
buffer (pH 7.4) or 2% PFA, 0.5% glutaraldehyde and 0.2% picric acid in
0.15 M phosphate buffer]. During perfusion, the heads were chilled with
Cerebral Cortex Jun 1999;9:317–331; 1047–3211/99/$4.00
crushed ice. After perfusion, the brains were immediately removed and
blocked in the coronal plane (5 mm thick). Then they were immersed
in a post-fixative solution containing 4% PFA and 5% sucrose in 0.15 M
phosphate buffer for several hours, followed by successive immersions in
10, 20 and 30% sucrose in 0.15 M phosphate buffer. The brain blocks
were mounted in O.C.T. compound (Miles Inc., Elkhart, IN) and frozen
rapidly in a dry ice/acetone bath, then stored at –80°C until dissection.
Synthesis of Probes
For the detection of GAP-43 and SCG10 mRNAs, digoxigenin-labeled DNA
probes were used. Human cDNA clones for GAP-43 (pG#20, 500 bp;
a gift from Dr R.L. Neve of Har vard Medical School, Boston) and
SCG10 (pSG601, 550 bp; a gift from Dr N. Mori of National Institute for
Longevity Sciences, Japan) were labeled with digoxigenin using a random
priming method according to the manufacturer’s instructions (DIG-DNA
Labeling Kit, Boehringer Mannheim Biochemica, Germany). Before use,
the labeled probes were precipitated with ethanol, then washed to
remove unincorporated digoxigenin-labeled nucleotides.
Pretreatment of Sections
The blocks including cortical areas prefrontal area (FD), the temporal
association area (TE), the parietal association area (PG), the primary
somatosensory area (PB) and primar y (OC) and secondary (OB) visual
areas (Fig. 1) of von Bonin and Bailey (1947) were sectioned at a 10 µm
thickness on a cr yostat (CRYOCUT 3000, Leica, Nussloch, Germany).
Because of the condition of the preservation and the need to distribute
the preparations among other researchers, we could not investigate all
cortical areas of every animal. Three adult and two infant (postnatal days
2 and 8) cortices were used for FD, PG and PB; three adult and three
infant (postnatal days 2, 8 and 30) cortices were used for TE; and four
adult and three infant (postnatal days 2, 8 and 30) cortices were used for
OB and OC.
The sections were mounted on slides coated with Vectabond
Reagent (VECTOR Laboratories, Burlingame, CA), dried and pretreated
for in situ hybridization by successive incubations in: 4% PFA in 0.15 M
phos-phate buffer for 15 min at room temperature; 30 µg/ml Proteinase K
(Boehringer Mannheim Biochemica; pH 8.0) for 30 min at 37°C; and 4%
PFA in 0.15 M phosphate buffer for 10 min at room temperature. After a
wash of 0.15 M phosphate buffer, the sections were dehydrated through
70, 80, 90 and 100% ethanol series (1 min at each concentration), then
dried.
Hybridization and Detection
Sections were prehybridized in 45% formamide, 4× SSC (standard saline
citrate), 1× Denhardt’s solution, 0.25% SDS, 10 mM Tris–HCl (pH 7.6) and
500 µg/ml denatured salmon sperm DNA for 3 h at 45°C. Following
prehybridization, sections were transferred to fresh hybridization buffer
containing an additional 10% dextran sulfate and 200 ng/ml digoxigeninlabeled DNA probe, then covered with a cover glass, heated on a dry
block at 95°C for 10 min and chilled on ice for 10 min. Hybridization was
performed for at least 16 h at 45°C.
The hybridized sections were incubated in 2× SSC, 0.2% SDS to
remove the cover glasses, rinsed four times in 2× SSC, 0.2% SDS to remove
the hybridization mixture (room temperature), then washed four times in
pre-heated 0.5× SSC, 0.2% SDS at 55°C, each time for 15 min.
The buffer was changed to 0.1 M maleic acid, 0.15 M NaCl and 0.2%
Tween 20 (pH 7.5) for 10 min at room temperature. Sections were then
incubated in 2% blocking reagent (DIG Nucleic Acid Detection Kit,
Boehringer Mannheim Biochemica), 0.1 M maleic acid, 0.15 M NaCl
(pH 7.5) for 1 h at room temperature, then incubated in diluted (1:500)
anti-digoxigenin Fab-fragments conjugated with alkaline phosphatase
(DIG Nucleic Acid Detection Kit, Boehringer Mannheim Biochemica),
2% blocking reagent, 0.1 M maleic acid and 0.15 M NaCl (pH 7.5) for 3 h
at room temperature. Unbound antibody conjugate was removed by
washing three times for 10 min each with 0.1 M maleic acid, 0.15 M NaCl
and 0.2% Tween 20 (pH 7.5).
Finally, the sections were preincubated in 0.1 M Tris–HCl buffer (pH
9.5) containing 0.1 M NaCl and 0.05 M MgCl2 for 5 min, then incubated in
the same buffer containing the substrates nitroblue tetrazolium (NBT,
340 µg/ml; Boehringer Mannheim Biochemica) and 5-bromo-4-chloro-3indolyl phosphate (BCIP, 170 µg/ml; Boehringer Mannheim Biochemica)
318 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
Figure 1. Sampled areas are shown on the left hemisphere of the cerebral cortex.
Areas are FD (the prefrontal area), TE (the temporal association area), PG (the parietal
association area), PB (the primary sensory area), OB (the secondary visual area) and OC
(the primary visual area), named after von Bonin and Bailey (1947).
for 20 h in the dark. Color development was stopped by incubation in
0.1 M Tris–HCl buffer (pH 7.5) containing 0.01 M EDTA for 10 min. The
sections were incubated in 4% PFA in 0.15 M phosphate buffer for 10 min
at room temperature to prevent fading. Following this, sections were
dehydrated through 70, 80, 90 and 100% ethanol series (1 min at each
concentration), transferred to xylene for three washes of 5 min each,
then covered with a coverslip and Permount histological mounting
medium (Fisher Scientific, Fair Lawn, NJ).
Quantification
The relative amount of mRNA in each cortical layer was evaluated by
measuring optical density rather than by cell counting because the
intensity of the hybridization signals for both GAP-43 mRNA and SCG10
mRNA were highly variable among the neurons.
Images were captured with a microscope (BX60; Olympus, Tokyo,
Japan) using a 3CCD color video camera (DXC-950; Sony, Tokyo, Japan)
and digitized by an image analysis system (MCID; Imaging Research Inc.,
St. Catharines, Canada). Using this image analysis system, we overlaid in
situ hybridized sections onto the adjacent Nissl (cresyl violet)-stained
sections, and identified each cortical layer. Optical density was measured
in each layer of a 300-µm-wide column that sampled all layers of cortex
(Fig. 3), and optical density of the background staining was measured
in the subjacent white matter. As presented by Meberg and Routtenberg
(1991) in their study of the rat hippocampus, the optical density of a
given cortical layer, expressed as a percentage of the optical density of
the background staining, was used to define the staining intensity (SI).
Twelve columns from each cortical area (four columns from each of three
sections or six columns from each of two sections) were measured for
each monkey. SIs from in situ hybridized sections of GAP-43 mRNA and
SCG10 mRNA (in situ-SI) were compared with SIs from Nissl-stained
sections (Nissl-SI), which represent the cell density in each layer of each
cortical area (Tables 1 and 2).
To clarify the difference in the expression patterns of each cortical
area, we compared the SI of the outer pyramidal layer (layer III) and the
inner pyramidal layer (layer V) of each cortical area. We first normalized
each in situ-SI with reference to its cellular density (Nissl-SI), then
determined the ratio:
IIIOD/VOD = (in situ-SI in layer III/Nissl-SI in layer III)/(in situ-SI in layer
V/Nissl-SI in layer V)
When the expression of GAP-43 mRNA or SCG10 mRNA is higher in layer
V than in layer III, this ratio is <1. Because we could not fix the boundary
between layers II and III of area OC, we used the SI from layers II–III
instead of the SI from layer III in the study of area OC. This ratio was
useful for confirming any difference in expression patterns for GAP-43
mRNA or SCG10 mRNA among cortical areas and between adult and
infant cortices (Figs 7 and 10).
Results
Control Experiments
The specificity of the probes was confirmed by Northern blot
Figure 2. Control experiments to confirm the specificity of the signals from GAP-43 (A,C,E) and SCG10 (B,D,F) mRNAs. Six adjacent coronal sections of cortical area TE were studied.
Normal reactions for GAP-43 mRNA (A) and SCG10 mRNA (B) mRNAs produced positive hybridization signals. Only background levels of signals were observed in the sections
pretreated with ribonuclease A (C,D), and signals were dramatically reduced in the sections of the competition control (E,F). Scale bar = 500 µm.
analysis, in which specific bands for both GAP-43 and SCG10
mRNAs were observed (Higo et al., 1998a). In addition, two
kinds of control experiments were carried out in the same way
as the previous in situ hybridization study in the monkey
hippocampus (Higo et al., 1998a). A series of adjacent sections
that included area TE were treated with ribonuclease A before in
situ hybridization (Fig. 2C,D). The sections were incubated with
ribonuclease A (20 µg/ml) for 30 min at 37°C, then processed
through the hybridization as described above. Another series
of adjacent sections were used as a competition control (Fig.
2E,F), in which >800-fold unlabeled probe was added to the
hybridization buffer along with the digoxigenin-labeled probe
(200 ng/ml). The sections that were treated normally with the
digoxigenin-labeled probe (Fig. 2A,B) had positive hybridization
signals in the cytoplasm and the proximal dendrites of the
neuron. Only background levels of signals were observed in the
sections pretreated with ribonuclease A (Fig. 2C,D), and signals
were dramatically reduced in the sections of the competition
control (Fig. 2E,F). These results confirmed that the signals
observed in the normal reaction were specific to each mRNA.
Expression Patterns of GAP-43 mRNA in the Adult
Cerebral Cortex
In areas FD, TE and PG, many GAP-43 mRNA-positive cells with
moderate to intense hybridization signals were observed in
layers II–VI (Figs 3B,E,H and 4B). Intense hybridization signals
were frequently observed in the larger pyramidal cells in
layers III, V and VI (Fig. 5A, 15–30 µm in diameter), but we also
observed intense hybridization signals in the smaller pyramidal
cells (Fig. 5C, 5–15 µm in diameter) and the non-pyramidal cells
in these layers. Some of them resembled the bipolar cells that
had vertically oriented dendrites (Fig. 5E; 15–30 µm in length;
Jones, 1975; Fairén et al., 1984; Peters, 1984; Lund and Lewis,
1993; Gabbott and Bacon, 1996; Peters and Sethares, 1997). The
intensities of the hybridization signal were almost the same in
outer pyramidal layer (layer III) and inner pyramidal layer (layer
V), and the IIIOD/VOD ratios were close to 1 (1.03 ± 0.02 in area
FD, 0.99 ± 0.04 in area TE and 1.06 ± 0.11 in area PG; Fig. 7A).
In areas PB, OB and OC, the hybridization signals for GAP-43
mRNA were weak in layer II (Fig. 6B,E,H). Though signals were
generally weak in layer III, gradients of signal intensity were
Cerebral Cortex Jun 1999, V 9 N 4 319
Figure 3. The areas FD (A–C), TE (D–F) and PG (G–I) of the adult cortex. (A, D, G) Nissl-stained sections. (B,E,H) Localization of GAP-43 mRNA. (C,F,I) Localization of SCG10 mRNA.
To quantify the hybridization signal, the optical density was measured in a 300-µm-wide column sampling all layers of the cortex, and the optical density of the background staining
was measured in the subjacent white matter (A–C; see Materials and Methods for detail). Scale bar = 200 µm.
320 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
Figure 4. Three adjacent sections of the area TE of the adult cortex. (A) Nissl-stained sections. (B) Localization of GAP-43 mRNA. (C) Localization of SCG10 mRNA. The same blood
vessels are indicated by arrowheads. Scale bar = 100 µm.
obser ved (Fig. 6B,E,H): the lower part of layer III contained
more intense hybridization signals than the upper part of layer
III. The larger pyramidal cells at the lowermost part of layer III
often had more intense signals than any cells in layer IV. In layers
V and VI, we observed a large number of cells with moderateto-intense hybridization signals. Intense signals were frequently
observed in the larger pyramidal cells. As in the association
areas, intense hybridization signals were also observed in the
smaller pyramidal cells and the non-pyramidal cells in layers V
and VI. The hybridization signals were weaker in layer III than in
layer V (Fig. 6B,E,H). In areas PB and OB, there were only slight
differences between the in situ-SI of layer III and the in situ-SI of
layer V (Table 1). Because the neuronal densities were higher in
layer III than in layer V (see Nissl-SI in Table 1), the IIIOD/VOD
ratios were <1 (0.92 ± 0.06 in area PB and 0.87 ± 0.06 in area OB;
Fig. 7A). The IIIOD/VOD ratio for area OC (0.77 ± 0.07) may have
been underestimated because the neurons in layer II tended to
contain less intense signals than the neurons in layer III, and we
used the SI from layers II–III instead of the SI from layer III in the
study of area OC (see Materials and Methods). The IIIOD/VOD
ratios were significantly lower in the lower sensory areas (areas
PB and OB; we excluded area OC because the IIIOD/VOD ratio for
Cerebral Cortex Jun 1999, V 9 N 4 321
Figure 5. High-magnification photomicrographs of cortical neurons that have hybridization signals for GAP-43 mRNA (A,C,E,G,I,K) and SCG10 mRNA (B,D,F,H,J,L). (A,B) The large
pyramidal cells in layer V of area TE. The arrows show the apical dendrites of the pyramidal cells. (C,D) The small pyramidal cells in layer V of area TE. The arrows show the apical
dendrites of the pyramidal cells. (E,F) The bipolar cells in layer V of area TE. The arrows show the vertically oriented dendrites of the bipolar cells. (G,H) The outer Meynert cells in
layer IVB of area OC. (I,J) The inner Meynert cells in layer VI of area OC. (K,L) The small neurons that had weak to moderate signals for GAP-43 mRNA (the arrows in K) and weak
signals for SCG10 mRNA (the arrows in L) in layer I of area TE. Scale bar = 20 µm.
area OC may have been underestimated) than in the association
areas (areas FD, TE and PG: P < 0.01, Mann–Whitney U-test;
Fig. 7A). In area OC, intense hybridization signals were often
observed in the large cells in layer IVB (Fig. 5G, outer Meynert
cell; Lund, 1973; Valverde, 1985; Peters and Sethares, 1991;
Peters, 1994) and layers V and VI (Fig. 5I, inner Meynert cell;
Lund, 1973; Chan-Palay et al., 1974; Winfield et al., 1981;
Valverde, 1985; Payne and Peters, 1989; Peters and Sethares,
1991; Peters, 1994).
In layer I of all areas examined, weak-to-moderate GAP-43
mRNA-positive signals were obser ved in the small (5–10 µm
in diameter), round neurons (Fig. 5K), but we could not detect
GAP-43 mRNA-positive Cajal–Retzius neurons in this layer
(Marin-Padilla, 1984). We also observed GAP-43 mRNA-positive
neurons with weak hybridization signals in the white matter.
322 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
Expression Patterns of SCG10 mRNA in the Adult
Cerebral Cortex
Intense hybridization signals for SCG10 mRNA were preferentially observed in layers V and VI of all areas examined (Figs
3C,F,I and 6C,F,I). As shown in Figure 4, SCG10 mRNA as well as
GAP-43 mRNA was localized in most neurons in layers V and VI,
suggesting that a number of neurons in these layers contain both
GAP-43 and SCG10 mRNAs. In layer II, the signals for SCG10
mRNA were weak. In layer III, the signals were generally weak
and gradients of signal intensity were observed: the lower part of
layer III contained more intense hybridization signals than the
upper part of layer III. The larger pyramidal cells at the
lowermost part of layer III often had more intense signals than
any cells in layer IV. Intense signals tended to be localized in
larger pyramidal cells in layers V and VI (Fig. 5B), but intense
Figure 6. The areas PB (A–C), OB (D–F) and OC (G–I) of the adult cortex. (A,D,G) Nissl-stained sections. (B,E,H) Localization of GAP-43 mRNA. (C,F,I) Localization of SCG10 mRNA.
Scale bar = 200 µm.
Cerebral Cortex Jun 1999, V 9 N 4 323
Table 1
The results of staining intensity ± SD in each area of the adult cortex
Nissl
GAP-43
SCG10
FD
II
III
IV
V
VI
123.6 ± 3.8
128.9 ± 7.7
135.9 ± 8.1
132.9 ± 6.4
125.3 ± 6.9
133.7 ± 10.0
143.2 ± 20.8
149.7 ± 23.8
144.6 ± 22.5
139.8 ± 20.0
116.1 ± 5.12
126.9 ± 13.8
136.3 ± 19.3
134.1 ± 17.3
131.2 ± 20.1
TE
II
III
IV
V
VI
121.5 ± 6.8
122.5 ± 7.5
132.8 ± 16.3
126.5 ± 8.3
124.9 ± 7.7
125.4 ± 6.8
132.4 ± 11.5
140.8 ± 13.5
137.6 ± 13.2
138.5 ± 10.8
112.0 ± 4.1
123.8 ± 8.0
134.9 ± 12.0
137.5 ± 15.5
135.6 ± 12.6
PG
II
III
IV
V
VI
129.7 ± 11.1
135.0 ± 8.8
146.0 ± 17.9
136.7 ± 11.5
136.1 ± 15.8
132.2 ± 2.1
145.6 ± 6.9
152.1 ± 7.9
140.1 ± 1.5
135.7 ± 7.1
111.9 ± 5.3
129.1 ± 4.4
144.1 ± 8.8
139.6 ± 6.4
135.2 ± 6.6
PB
II
III
IV
V
VI
134.3 ± 14.4
153.1 ± 13.4
155.2 ± 10.3
141.3 ± 8.4
131.6 ± 5.7
114.0 ± 6.5
133.9 ± 9.4
139.0 ± 13.6
136.8 ± 12.7
131.9 ± 11.3
113.0 ± 11.8
128.8 ± 7.5
133.0 ± 5.4
132.7 ± 5.3
128.8 ± 5.6
OB
II
III
IV
V
VI
146.2 ± 7.8
145.8 ± 7.0
152.5 ± 8.8
131.3 ± 3.8
125.6 ± 6.3
122.7 ± 10.2
134.3 ± 7.7
145.8 ± 7.2
139.1 ± 2.8
138.2 ± 7.0
123.3 ± 4.1
135.7 ± 5.5
143.3 ± 10.3
136.4 ± 6.1
134.7 ± 5.6
OC
II–III
IVA
IVB
IVCα
IVCβ
V
VI
138.4 ± 12.1
149.4 ± 15.9
143.8 ± 16.1
153.3 ± 20.1
162.3 ± 21.1
139.1 ± 18.1
143.7 ± 14.3
111.1 ± 11.3
134.3 ± 22.1
141.9 ± 26.6
149.6 ± 30.5
153.2 ± 31.4
143.5 ± 24.3
145.7 ± 17.1
114.7 ± 6.7
136.9 ± 12.9
141.5 ± 16.5
147.1 ± 17.6
152.1 ± 17.9
144.8 ± 13.9
145.8 ± 8.7
Three cortices were used for FD, PG, TE and PB. Four cortices were used for OB and OC.
Figure 7. The IIIOD/VOD ratio was calculated to clarify the differences in the expression
patterns between the outer pyramidal layer (layer III) and the inner pyramidal layer
(layer V) of each cortical area (see Materials and Methods for detail). (A) The IIIOD/VOD
ratio (± SD) of GAP-43 mRNA in the adult cortices. (B) The IIIOD/VOD ratio of SCG10
mRNA in the adult cortices. #The IIIOD/VOD ratio for area OC may have been underestimated because the neurons in layer II tended to contain less intense signals than
the neurons in layer III, and we used the SI from layera II–III instead of the SI from layer
III in the study of area OC. *A Mann–Whitney U-test revealed a significant difference (P
< 0.01) between the IIIOD/VOD ratio in the association areas (areas FD, TE and PG) and
that in the lower sensory areas (areas PB and OB; we excluded area OC because the
IIIOD/VOD ratio for area OC may have been underestimated).
324 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
hybridization signals were also observed in the smaller pyramidal cells (Fig. 5D) and the non-pyramidal cells in layers V and
VI (Fig. 5F). In all areas examined, the hybridization signals were
weaker in layer III than in layer V (the IIIOD/VOD ratios were: 0.97
± 0.05 in area FD, 0.93 ± 0.04 in area TE, 0.94 ± 0.02 in area PG,
0.90 ± 0.09 in area PB, 0.91 ± 0.05 in area OB and 0.79 ± 0.03 in
area OC; Fig. 7B). In areas PB and OC, as we found for GAP-43
mRNA, there were only slight differences between the in situ-SI
for SCG10 mRNA of layer III and the in situ-SI of layer V (Table 1).
As with GAP-43 mRNA, because of the differences in neuronal
densities between layer III and layer V, the IIIOD/VOD ratios were
<1 also in these areas. The IIIOD/VOD ratio for area OC may have
been underestimated because the neurons in layer II tended to
contain less intense signals than the neurons in layer III. The
difference between the association and lower sensory areas was
not significant (P > 0.05, Mann–Whitney U-test). In area OC, the
outer Meynert cells (Fig. 5H) and inner Meynert cells (Fig. 5J)
contained intense signals. The hybridization signals for SCG10
mRNA in these Meynert cells were more intense than those in
neighboring cells (Fig. 5H, J).
Weak SCG10 mRNA-positive signals were sometimes observed
in the small neurons in layer I (Fig. 5L) and in the white matter of
all areas examined.
Expression Patterns of GAP-43 mRNA in the Infant
Cerebral Cortex
Intense hybridization signals for GAP-43 mRNA were observed
in layers II–VI of all areas of the infant cortex examined (Figs
8B,E,H and 9B,E,H). Intense signals for GAP-43 mRNA tended to
be observed in the pyramidal cells, but were sometimes observed in non-pyramidal cells. The expression pattern was identical
among the infant cortices of three different ages (postnatal days
2, 8 and 31), so we combined quantifying data from these three
monkeys (Table 2, Fig. 10A). The hybridization signals in layer
III were higher than those in layer V in most cases (Figs
8B,E,H, 9B,E,H and 10A, Table 2). The IIIOD/VOD ratio for
area OC (0.95 ± 0.05; Fig. 10A) may have been underestimated
because the neurons in layer II tended to contain less intense
signals than the neurons in layer III. In layer I and the white
matter of all areas examined, moderate signals for GAP-43 mRNA
were observed.
In areas FD, TE and PG, in situ-SIs for GAP-43 mRNA in all
layers of the infant cortex were higher than those of the adult
cortex (Tables 1 and 2). In layers II–IV of areas PB and OB, and
layers II–IVA of area OC, in situ-SIs for GAP-43 mRNA were
higher in the infant cortex than in the adult cortex (Tables 1 and
2), whereas in layers V and VI of areas PB and OB, and layers IVB
to VI of area OC, in situ-SIs for GAP-43 mRNA were almost the
same between infant and adult cortices (Tables 1 and 2).
Expression Patterns of SCG10 mRNA in the Infant
Cerebral Cortex
Intense hybridization signals for SCG10 mRNA were observed
from layers II–VI of all areas of the infant cortex examined (Figs
8C,F,I and 9C,F,I). As we saw with GAP-43 mRNA, intense
signals for SCG10 mRNA were frequently observed in the
pyramidal cells. In the study of SCG10 mRNA, the pyramidal
cells tended to be more densely stained than those in the study of
GAP-43 mRNA (Figs 8C,F,I and 9C,F,I). The expression pattern
was identical among the infant cortices of three different ages
(postnatal days 2, 8 and 31), so we combined quantifying data
from these three monkeys (Table 2, Fig. 10B). The hybridization
signals in layer III were higher than those in layer V in most
Figure 8. The areas FD (A–C), TE (D–F) and PG (G–I) of the infant cortex (postnatal day 8). (A,D,G) Nissl-stained sections. (B,E,H) Localization of GAP-43 mRNA. (C,F,I) Localization
of SCG10 mRNA. Scale bar = 200 µm.
Cerebral Cortex Jun 1999, V 9 N 4 325
Figure 9. The areas PB (A–C), OB (D–F) and OC (G–I) of the infant cortex (postnatal days 8). (A,D,G) Nissl-stained sections. (B,E,H) Localization of GAP-43 mRNA. (C,F,I) Localization
of SCG10 mRNA. Scale bar = 200 µm.
326 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
Table 2
The results of staining intensity ± SD in each area of the infant cortices
+
Nissl
GAP-43
SCG10
FD
II
III
IV
V
VI
136.7 ± 13.9
140.7 ± 12.8
152.2 ± 19.3
144.3 ± 16.8
139.9 ± 14.3
166.1 ± 8.5
169.2 ± 9.2
174.9 ± 0.4
163.7 ± 0.5
152.1 ± 1.5
158.7 ± 6.3
166.9 ± 21.0
170.0 ± 11.4
166.8 ± 17.8
155.7 ± 23.7
TE
II
III
IV
V
VI
138.1 ± 15.4
138.1 ± 13.0
153.6 ± 14.1
137.1 ± 10.8
133.6 ± 9.1
155.4 ± 1.8
158.1 ± 9.4
167.0 ± 14.1
148.0 ± 8.3
143.7 ± 10.5
154.4 ± 11.1
171.4 ± 6.4
170.3 ± 3.8
169.3 ± 11.8
164.6 ± 16.6
PG
II
III
IV
V
VI
139.8 ± 13.9
140.4 ± 7.0
161.5 ± 4.3
141.7 ± 2.2
138.9 ± 2.2
157.1 ± 8.4
161.5 ± 15.2
178.4 ± 17.4
161.0 ± 11.6
155.9 ± 7.2
166.6 ± 20.1
189.8 ± 26.5
194.9 ± 16.1
181.2 ± 20.2
172.8 ± 14.3
PB
II
III
IV
V
VI
129.7 ± 8.8
128.3 ± 6.4
135.8 ± 4.8
126.1 ± 3.0
127.7 ± 1.3
145.2 ± 3.2
152.4 ± 4.4
155.6 ± 7.6
142.7 ± 6.2
140.1 ± 13.4
143.4 ± 2.5
161.2 ± 2.8
163.9 ± 8.2
152.6 ± 8.2
149.1 ± 8.3
OB
II
III
IV
V
VI
136.2 ± 9.3
126.7 ± 7.4
152.5 ± 8.8
123.5 ± 7.2
124.1 ± 5.6
150.5 ± 12.6
153.3 ± 9.9
145.8 ± 7.2
145.1 ± 8.6
151.8 ± 12.9
169.4 ± 16.4
180.5 ± 12.0
143.3 ± 10.3
166.7 ± 9.8
171.1 ± 16.1
OC
II–III
IVA
IVB
IVCα
IVCβ
V
VI
138.5 ± 6.2
134.7 ± 5.2
128.0 ± 1.3
135.6 ± 0.6
146.2 ± 6.8
122.2 ± 1.8
131.7 ± 1.6
147.7 ± 6.7
152.2 ± 5.5
149.2 ± 8.7
153.6 ± 8.8
161.9 ± 9.4
137.9 ± 8.1
153.5 ± 11.3
181.0 ± 9.5
185.1 ± 12.7
178.1 ± 14.4
192.4 ± 16.5
193.9 ± 15.9
162.3 ± 22.6
183.9 ± 17.2
Two cortices (postnatal days 2 and 8) were used for FD, PG and PB. Three cortices (postnatal days
2, 8 and 30) were used for TE, OB and OC.
cases (Figs 8C,F,I, 9C,F,I and 10B, Table 2). As we found with
GAP-43 mRNA, the IIIOD/VOD ratio for area OC (1.00 ± 0.13; Fig.
10B) may have been underestimated because the neurons in
layer II tended to contain less intense signals than the neurons
in layer III. Nevertheless, the ratio for area OC was close to 1
because of the intense signals in layer III. In layer I and the white
matter of all areas examined, moderate signals for SCG10 mRNA
were observed.
In all areas examined, in situ-SIs for SCG10 mRNA in all layers
of infant cortex were higher than those of adult cortex (Tables 1
and 2).
Discussion
This report describes the exact location and types neurons
labeled positively for GAP-43 mRNA and SCG10 mRNA in the
monkey cerebral cortex. Our results revealed that the distribution patterns of both GAP-43 and SCG10 mRNAs were different
between infant and adult cortices. We also showed that the
distribution patterns of GAP-43 mRNA were different between
the association areas and the lower sensory areas of the adult
cortex.
Comparison with Northern Blot Study in Monkeys
Previously, we had investigated the developmental changes of
Figure 10. (A) The IIIOD/VOD ratio of GAP-43 mRNA in the infant cortices. (B) The
IIIOD/VOD ratio of SCG10 mRNA in the infant cortices. #The IIIOD/VOD ratio for area OC
may have been underestimated because the neurons in layer II tended to contain less
intense signals than the neurons in layer III, and we used the SI from layer II–III instead
of the SI from layer III in the study of area OC.
GAP-43 mRNA (Oishi et al., 1998) and SCG10 mRNA (Higo et al.,
1996) by Northern blot analyses. We have shown that both
GAP-43 and SCG10 mRNAs are more abundant in all examined
areas of infant monkeys (postnatal days 1, 8 and 30) than in those
of the adult monkey (Higo et al., 1996; Oishi et al., 1998). These
results are consistent with the present results: more intense
hybridization signals were observed in all cortical areas of all
three infant monkeys than in adult cortical areas. We have also
shown in Northern blot analyses that the amount of SCG10
mRNA decreases more steeply from the infant cortex to the adult
cortex than does the amount of GAP-43 mRNA (N. Higo et al.,
unpublished observation). In the present results, the differences
in signal intensity between infant and adult cortices were
remarkable, especially for SCG10 mRNA (Tables 1 and 2). Therefore, the results from the previous Northern blot analyses and
the results from the present in situ hybridization studies are also
consistent on this point.
In the present study, we showed that the amount of GAP-43
mRNA decreased in every layer of areas FD, TE and PG during
postnatal development (Figs 3B,E,H and 8B,E,H, Tables 1 and 2).
In contrast, the amount of GAP-43 mRNA decreased selectively
in layers II–IV of areas PB, OB and OC (Figs 6B,E,H and 9B,E,H).
The amount of GAP-43 mRNA did not decrease much in layers V
and VI of areas PB, OB and OC, and the SIs in these layers of
the adult cortex were almost the same as those in the same layers
of the infant cortex (Tables 1 and 2). The amount of SCG10
mRNA decreased in every layer of all areas examined during
postnatal development (Figs 3C,F,I, 6C,F,I, 8C,F,I and 9C,F,I).
The amount of SCG10 mRNA decreased more steeply in layers
II–IV than in layers V and VI (Tables 1 and 2).
In a Northern blot analysis of the adult cortex, we have shown
that the amount of GAP-43 mRNA is higher in the association
areas (areas FD, TE and PG) than in the lower sensory areas
(areas PC, OB and OC; Oishi et al., 1998). The present results
showed that the distribution patterns of GAP-43 mRNA were
also different between the association and lower sensory areas:
prominent hybridization signals for GAP-43 mRNA were restricted to layers IV–VI of the lower sensory areas (Fig. 6B,E,H),
but were observed from layers II–VI of the association areas (Fig.
3B,E,H) of the adult cortex. Therefore, the difference in the
amount of GAP-43 mRNA between the association areas and the
lower sensory areas may be due to the differential distribution
patterns of GAP-43 mRNA between them.
There was no difference in the amount of SCG10 mRNA
Cerebral Cortex Jun 1999, V 9 N 4 327
among adult cortical areas in the Northern blot analysis (Higo
et al., 1996). The present results showed no difference in the
laminar distribution of SCG10 mRNA among cortical areas (Figs
3C,F,I and 6C,F,I). Thus, results from these two studies are
consistent.
From Northern blot analyses and in situ hybridization studies,
we confirmed that both the quantity and the distribution pattern
of GAP-43 mRNA, but not those of SCG10 mRNA, are different
between the association and lower sensory areas of the adult
cortex. Nelson et al. (1987) reported that the phosphorylation
of GAP-43 is higher in the visual association area (TE) than in
the primary (OC) and secondary (OB) visual areas. The present
result suggests that this gradient of the phosphorylation is regulated, at least in part, by the level of mRNA.
Comparison with Studies of Other Animals
Several in situ hybridization studies of the rat cerebral cortex
have examined GAP-43 mRNA (Kruger et al., 1993; Yao et al.,
1993; Sugiura and Mori, 1995) and SCG10 mRNA (Himi et al.,
1994b; Sugiura and Mori, 1995). Both GAP-43 mRNA (Kruger et
al., 1993; Yao et al., 1993) and SCG10 mRNA (Himi et al., 1994b)
are preferentially expressed in layer IV, V or VI of most of
the mature rat cerebral cortex. The observation that GAP-43
mRNA is expressed in layer II as well as layer VI of the anterior
limbic (cingulate) field of the rat cortex (Kruger et al., 1993)
corresponds with our findings in monkeys that GAP-43 mRNA is
expressed in the supragranular layers as well as the infragranular
layers only in the associative regions (Fig. 7A). In the postnatal
development of the rat cerebral cortex, the hybridization signals
for both GAP-43 and SCG10 mRNAs are more intense in the
supragranular layers than the infragranular layers (Sugiura and
Mori, 1995), which are similar to the present findings in the
monkey cerebral cortex (Fig. 10A,B). Previous studies revealed
that GAP-43 mRNA exists in the hippocampal dentate granule
cells of the monkey (Higo et al., 1998a), but not in that of the rat
(Meberg and Routtenberg, 1991; Jacobs et al., 1993; Kruger et
al., 1993; Yao et al., 1993). Conversely, SCG10 mRNA exists in
the granule cells of the rat (Himi et al., 1994a), but not in that of
the monkey (Higo et al., 1998a). We did not detect these kinds of
striking differences between the monkey and the rat cerebral
cortex.
In humans, the distribution of neurons expressing GAP-43
mRNA has been studied in only a few cortical areas (Neve et
al., 1988; Perrone-Bizzozero et al., 1996). In one human study,
Neve et al. (1988) showed that the most intense hybridization
signals were concentrated in layer II of both the association area
(Brodmann’s area 20, corresponding to area TE) and the primary
area (area 17, corresponding to area OC); this is not consistent
with our results in monkeys. Perrone-Bizzozero et al. (1996)
showed, however, that the expression of GAP-43 mRNA was
robust in layers II–VI of the association area (area 10), while a
few GAP-43 mRNA-positive cells were also detected in layer I
of the area. These results are consistent with our results in the
association areas of monkeys (Fig. 3B,E,H). The laminar distribution patterns of GAP-43 mRNA in some areas might be
different between monkeys and humans. Because human data
are currently scarce and only qualitative, further experiments in
the various areas of human cerebral cortex are necessary to
confirm the laminar distribution of GAP-43 mRNA.
Benowitz et al. (1989) performed an immunohistochemical
study of GAP-43 protein in several areas that included both the
association areas and the primary sensory areas of the adult
human cerebral cortex. Because GAP-43 immunoreactivity is
328 GAP-43 and SCG10 mRNAs in the Monkey Cerebral Cortex • Higo et al.
localized primarily in the nerve terminals of the neuropil, the
result tells nothing about the density and the distribution of cell
bodies expressing GAP-43. However, GAP-43 immunoreactivity
also showed specific laminar distribution and more localization
in restricted layers of the lower sensory areas than in the
association areas (Benowitz et al., 1989).
Comparison with Other Molecules Regarding Changes
in Laminar Distribution during Postnatal Development
In this study, we showed the laminar distribution patterns for
both GAP-43 and SCG10 mRNAs in both infant and adult cerebral cortices. There are several kinds of molecules whose laminar
distribution patterns change during development.
At the newborn stage, the somatostatin-immunoreactive cells
are transiently distributed at a high density in the supragranular
layers of many cortical areas, especially in layer II of areas FD
and PE (Yamashita et al., 1989). The expression of somatostatin
and its mRNA decrease during postnatal development (Hayashi
and Oshima, 1986; Yamashita et al., 1989; Hayashi et al., 1990).
These results are similar to our results for GAP-43 and SCG10
mRNAs. Somatostatin has been reported to enhance neurite
outgrowth in cultured cells (Ferriero et al., 1994), molluscan
neurons (Bulloch, 1987; Grimm-Jørgensen, 1987) and cerebellar
granule cells of the rat (Taniwaki and Schwartz, 1995). Thus,
somatostatin, as well as GAP-43 and SCG10, may be involved
in the axonal elongation in the supragranular layers of infant
monkey cortex.
The molecule DA RPP-32, a dopamine- and cA MP-regulated
phosphoprotein, also showed differential expression patterns
between infant and adult cerebral cortices (Berger et al., 1990).
Between postnatal days 3 and 42, pyramidal cells containing
DARPP-32 are distributed in layers II–VI of various cortical areas
of the macaque monkey. The immunoreactivity for DARPP-32
remains prominent in the adult inferior temporal gyrus and the
paralimbic structure in most of the layers. The immunoreactivity
for DARPP-32 is faint in most of the other areas, and localized in
more restricted layers of these areas. These results are consistent
with our results of GAP-43 mRNA: highly plastic cortical areas
keep an extensive laminar distribution, a characteristic of infant
cerebral cortex, even in the adult cortex. Further research into
the layer-specific development of various molecules will increase
our understanding of the maturation of neuronal circuits in the
cerebral cortex of primates.
Intensity of mRNA Signals among Cell Types
For both GA P-43 and SCG10 mRNAs, intense hybridization
signals were frequently observed in the larger pyramidal cells
in layers III, V and VI (Fig. 5A,B). The results suggest that these
molecules are highly expressed in the long projection neurons.
We also observed intense signals for both GAP-43 and SCG10
mRNAs in the non-pyramidal cells in layers III, V and VI. Although it was difficult to identify the type of non-pyramidal cells
because both GAP-43 and SCG10 mRNA were localized in the
cytoplasm and the proximal dendrites of neurons, we observed
GAP-43 or SCG10 mRNA-positive cells which have the shape of
a bipolar cell (Fig. 5E,F; Jones, 1975; Fairén et al., 1984; Peters,
1984; Lund and Lewis, 1993; Gabbott and Bacon, 1996; Peters
and Sethares, 1997). These cells were found especially in layers
III, V and VI. These bipolar cells have intrinsic axon terminals
within the cortical area. It is likely that the hybridization signals
for both mRNAs in these non-pyramidal neurons indicate the
plastic nature of their axon terminals.
In area OC, both mRNAs were highly expressed in the outer
Meynert cells in layer IVB (Fig. 5G,H) and the inner Meynert cells
in layer V or VI (Fig. 5I, J). Various studies previously showed
that some of these Meynert cells project to area MT of the cortex
or the superior colliculus (Lund and Boothe, 1975; Tigges et al.,
1981; Fries and Distel, 1983; Sipp and Zeki, 1989; Peters, 1994).
The results might ref lect functional specialization regarding the
plasticity of these projections.
Intensity of mRNA Signals among Anatomical Circuits in
the Adult Cortex
Intense hybridization signals for both GAP-43 and SCG10 mRNAs
were frequently observed in the pyramidal neurons in layers V
and VI of all areas of adult cortex examined. The neurons in
layers V and VI extend to various subcortical projections that
include corticothalamic or corticostriatal projections (Jones
and Wise, 1977; Jones et al., 1977; Catsman and Kuypers, 1978;
Arikuni et al., 1983; Arikuni and Kubota, 1986; Goldman-Rakic
and Selemon, 1986; Giguere and Goldman-Rakic, 1988; Lund,
1988). Northern blot analysis revealed that the thalamus of the
macaque monkey contained a higher level of GAP-43 mRNA than
the cerebral cortex and the same level of SCG10 mRNA as the
cerebral cortex (T. Oishi et al., unpublished observation). The
caudate nucleus and the putamen also contained the same levels
of GAP-43 and SCG10 mRNAs as the cerebral cortex. These
results indicate the plastic nature of the projections among the
cerebral cortex and these subcortical structures.
We detected high levels of GAP-43 mRNA in the supragranular
layers (layers II and III) as well as in the infragranular layers of
the association areas: areas FD, TE and PG (Figs 3B,E,H and 7A).
The neurons in the supragranular layers of those cortical areas
supply both cortico-cortical connections (Barbas and Mesulam,
1981; Schwartz and Goldman, 1984; Friedman et al., 1986;
Shiwa, 1987; Vogt and Pandya, 1987; Barbas, 1988; Johnson et
al., 1989; Seltzer and Pandya, 1989; Andersen et al., 1990) and
intrinsic connections (A mir et al., 1993; Levitt et al., 1993;
Kritzer and Goldman-Rakic, 1995; Fujita and Fujita, 1996).
Intralaminar local connections in the supragranular layers of the
association areas are more laterally widespread than in the
primary and secondary visual areas (Kisvarday et al., 1989; Amir
et al., 1993; Levitt et al., 1993; Kritzer and Goldman-Rakic,
1995). The abundance of GAP-43 mRNA in the supragranular
layers of the association areas may suggest that these intrinsic
connections spread laterally within the association areas are
functionally specialized regarding plasticity.
Notes
This work was supported by grants from Agency of Industrial Science and
Technology, Ministry of International Trade and Industry, Japan and the
Cooperation Research Program of the Primate Research Institute, Kyoto
University. We are grateful to Drs T. Arikuni and K. Kawano for their
valuable discussions and continuous encouragement during this study.
We thank Dr R.L. Neve for providing the cDNA clone for GAP-43 and Dr
N. Mori for providing the cDNA clone for SCG10. We also thank Ms M.
Okui for help with tissue preparation.
Address correspondence to T. Oishi, Neuroscience Section, Information Science Division, Electrotechnical Laboratory, Umezono, Tsukuba,
Ibaraki 305-8568, Japan. Email: [email protected].
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