Download Development of GAP-43 mRNA in the macaque cerebral cortex

Developmental Brain Research 109 Ž1998. 87–97
Research report
Development of GAP-43 mRNA in the macaque cerebral cortex
Takao Oishi
a
b
a,)
, Noriyuki Higo a , Yumiko Umino
a,1
, Keiji Matsuda a , Motoharu Hayashi
b
Neuroscience Section, DiÕision of Information Science, Electrotechnical Laboratory, 1-1-4 Umezono, Tsukuba, Ibaraki 3058568, Japan
Department of Molecular and Cellular Biology, Primate Research Institute, Kyoto UniÕersity, Kanrin, Inuyama, Aichi 4840081, Japan
Accepted 31 March 1998
Abstract
To estimate the extent of axonal growth in various areas of the cerebral cortex, we measured the amount of GAP-43 mRNA in the
cerebral cortex of developing macaque monkeys. In four areas, i.e., the prefrontal area ŽFDD ., the temporal association area ŽTE., the
primary somatosensory area ŽPC., and the primary visual area ŽOC., the amount of GAP-43 mRNA was measured from the intermediate
fetal period wembryonic day 120 ŽE120.x to the adult stage. In two other areas, i.e., the parietal association area ŽPG. and the secondary
visual area ŽOB., the amount of GAP-43 mRNA was measured during the postnatal period. The amount of GAP-43 mRNA was highest at
E120, decreased roughly exponentially, and approached the asymptote by postnatal day 70 ŽP70.. The amount of GAP-43 mRNA was
higher in the association areas ŽFDD, TE, and PG. than in the primary sensory areas ŽPC and OC. during development and at the adult
stage. These findings suggest that axonal growth in the cerebral cortex is most exuberant before or during the intermediate fetal period
and approximately ends by P70. Furthermore, axonal growth is evidently more intensive in the association areas than in the primary
sensory areas during the stage following the intermediate fetal period. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Growth-associated protein; Association area; Primary sensory area; Monkey; Northern blotting
1. Introduction
The cerebral cortex is composed of several functionally
different areas. Development of the cerebral cortex has
been studied from various aspects, e.g., proliferation and
migration of neuronal cells, arborization of dendrites, axonal elongation, synaptogenesis, and myelination Žfor review, see w20x.. Few studies, however, focus on the differences between the cortical areas. Some aspects of development are concurrent among the cortical areas, while others
are not. In the human, myelination occurs in the primary
areas earlier than in the association areas w17x. It is widely
accepted that the primary sensory and motor areas develop
earlier than the association areas, because development of
behavioral function, e.g., vision, movement, and memory,
generally corresponds to the order of myelination, though
it is risky to directly correlate cortical development and
behavioral development. Rakic et al. w49x, however, have
shown in a series of primate studies that cortical develop)
Corresponding author. Fax: q 81-298-54-5849; E-mail:
[email protected]
1
Present address: Department of Forensic Medicine, National Defense
Medical College, 3-2 Namiki, Tokorozawa, Saitama 3590042, Japan.
0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 0 6 7 - 4
ment does not necessarily follow this pattern: the primary
areas develop earlier than the association areas. There are
two other types of cortical development. One type displays
a similar time course of development among the involved
areas. Developmental changes in the density of synapses in
each area are representative phenomena. Rakic et al., by
counting the synapses on a band of an electromicrograph,
reported that the densities of synapses in the prefrontal
area, the primary motor area, and the primary visual area
reach their peaks concurrently, at 2–4 months after birth,
then decrease gradually w49x. These authors have continued
detailed observation in those areas w11,12,58,59x. The binding of the ligands to receptors of several neurotransmitters
in different areas also reaches a peak concurrently at 2–4
months w32,33x. In the other type of development, the
association areas develop earlier than the primary areas.
Final division of neuronal cells finishes earlier in the
secondary visual area ŽE90. than in the primary visual area
ŽE102. w46,47x.
To understand the maturation of cortical circuits, it is
important to determine when extensive elongation of axons
occurs and whether the degrees of elongation during development differ among areas. Quantitative analyses of axonal development in the central nervous system have been
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
88
performed only in the fiber bundles, e.g., optic nerve w48x,
corpus callosum ŽCC. w29x, and anterior commissure ŽAC.
w29x. With morphological methods, it is difficult to study
axonal development in various brain regions other than
fiber bundles. Thus, we focus on mRNA of GAP-43, a
representative growth-associated protein that increases in
accordance with axonal elongation.
The amounts of mRNA, protein, and phosphorylation of
GAP-43 increase during regeneration after injury and also
during normal development of the central and the peripheral nervous systems Žfor review, see w6,7x.. GAP-43 exists
in the growth cones and axon terminals w36,51x, is phosphorylated with protein kinase C w3x, can bind calmodulin
when GAP-43 is not phosphorylated w4x, and can interact
with a GTP-binding protein, Go, in the growth cone w52x.
These data suggest that GAP-43 is involved in signal
transduction in the growth cones and axon terminals. Furthermore, an overexpression study w2x and a knockout
study w53x indicate that GAP-43 is a key molecule in
pathfinding during axonal elongation. Together, these findings suggest that GAP-43 is instrumental in axonal elongation.
This study is the first detailed demonstration of the
developmental change in the amount of GAP-43 mRNA in
the cerebral cortex of the primate. Addition of these findings to the morphological findings will shed light on
cortical maturation of the primate. Preliminary results have
been reported elsewhere w42–44x.
2. Materials and methods
2.1. Experimental animals and tissue preparation
Macaque monkeys at ages ranging from embryonic day
120 ŽE120. to adult were used ŽTable 1.. As there was no
apparent difference in measurements between E120 to
E125, we combined data from these three monkeys as the
data of E120. All monkeys, except those adults purchased
from a local provider, were bred in the Primate Research
Table 1
Number of subjects for each developmental age and each cortical area
E120 E122 E125 E142 P1 P8 P30 P70 P183 Adult
FDD
TE
PC
OC
PG
OB
1a
1a
1a
1a
a
1
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
2b
1c
2b
2c
2c
2c
2c
2c
2c
2d
2d
2d
2d
2d
2d
2c
2c
2c
2c
2c
2c
2b
2b
2b
2b
2b
2b
3e
2b
2b
3e
2b
2b
E120–E142: embryonic day 120–142, P1–P183: postnatal day 1–183.
a
One Macaca fascicularis.
b
One Macaca fuscata and one Macaca mulatta.
c
One or two Macaca mulatta.
d
Two Macaca fuscata.
e
Two Macaca fuscata and one Macaca mulatta.
Fig. 1. Dissected areas are shown on the left hemisphere of the cerebral
cortex. Areas are FDD Žthe prefrontal area., TE Žthe temporal association
area., PC Žthe primary somatosensory area., OC Žthe primary visual area.,
PG Žthe parietal association area., and OB Žthe secondary visual area.,
named after von Bonin and Bailey w57x.
Institute, Kyoto University. Because of the limited availability, we used three species of macaque monkeys, i.e.,
Macaca fascicularis, M. mulatta, and M. fuscata and the
number of subjects in each stage was small. To determine
the embryonic stage of fetal monkeys, we applied the
timed mating method and measured the length of head axis
and crown-rump length using the ultrasonic transmission
method ŽSonolayergraph, Toshiba, Tokyo, Japan.. Embryonic monkeys were obtained from halothane-anesthetized
pregnant monkeys by Cesarean section. All monkeys, except adults, were deeply anesthetized with an overdose of
sodium pentobarbital Ž35 mgrkg, i.p., Nembutal, Abbot,
North Chicago, IL. and killed by bloodletting from the
carotid artery. Adult monkeys were perfused with ice-cold
saline after an overdose injection of pentobarbital. All
procedures were executed in accordance with The Guide
for the Care and Use of Laboratory Animals established by
NIH Ž1985. and The Guide for the Care and Use of
Laboratory Primates Ž1986. established by the Primate
Research Institute, Kyoto University.
Macaque brains were dissected on crushed ice as quickly
as possible into the cortical areas using the classification
by von Bonin and Bailey w57x determined from sulcal
patterns ŽFig. 1.. Area FDD was taken from the upper and
lower banks of the middle one-third portion of the principal sulcus in postnatal monkeys. As for embryonic monkeys, the dorsolateral portion of the prefrontal area was
used. Area PC was taken from the posterior bank of the
central sulcus. Area PG was taken from the exposed
convexity between the intraparietal sulcus, the superior
temporal sulcus, and the medial end of the Sylvian sulcus.
Area TE was taken from the inferior temporal gyrus
anterior to the posterior middle temporal sulcus, posterior
to the posterior end of the inferior temporal sulcus, and
dorsal to the anterior middle temporal sulcus. Area OB
was taken from the posterior bank of the lunate sulcus.
Area OC was taken from the exposed surface of the
dorsolateral occipital cortex, more than 3 mm posterior to
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
the lunate sulcus and more than 3 mm dorsal to the inferior
occipital sulcus. Dissected tissues were immediately frozen
with dry ice and stored at y808C until use.
2.2. RNA extraction and Northern blotting
Total RNA was prepared by the method of Chomczynski and Sacchi w13x. Determination of amount and confir-
89
mation of purity of the RNA sample was performed with
the spectroscopic measurements at 260, 280, and 230 nm.
Extracted RNA was divided into 0.31–2.5 m g samples and
denatured by incubating at 608C for 15 min with 50%
formamide, 2.2 M formaldehyde, and 0.5 = MOPS buffer
ŽpH 7.0., then stored at y308C until use. The samples
were electrophoresed on a 0.9% agarose gel containing 2.2
M formaldehyde and blotted onto a nylon membrane ŽHy-
Fig. 2. ŽA.: Autoradiograms of GAP-43 mRNA and G3PDH mRNA. Diluted standards and samples were electrophoresed on the same gel. Standards were
serially diluted from 10.0 m g to 0.613 m g. As the amount of GAP-43 mRNA was comparatively large during embryonic days, samples from fetal monkeys
ŽE120–E142. were diluted four times. With this dilution of samples, the signal intensities of both GAP-43 mRNA and G3PDH mRNA were within the
linear region of standard curve. ŽB.: The amount of G3PDH mRNA did not change during development Ž r 2 s 0.017, p ) 0.6.. The amount of G3PDH
mRNA was represented as a multiple of G3PDH mRNA in the same amount of standard total RNA.
90
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
bond-N, Amersham, UK. by capillary blotting with 20 =
SSC ŽStandard Saline Citrate: 3 M sodium chloride and 0.3
M sodium citrate.. After UV-irradiation, membranes were
prehybridized at 428C overnight in 250 m grml sheared
salmon sperm DNA, 50% formamide, 5 = SSC, 50 mM
phosphate buffer ŽpH 6.5., and 1 = Denhardt’s solution
Ž0.02% Ficoll, Type 400, Pharmacia, Piscataway, NJ,
0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin..
Human GAP-43 cDNA wŽpGA3A., Neve et al. w39x, 1.1
kb Ža generous gift from Dr. Neve, Harvard Medical
School.x, and human glyceraldehyde-3X-phosphate dehydrogenase ŽG3PDH. cDNA Ž1.1 kb, Clontech, Palo Alto,
CA. were labeled with 32 P y dCTP using the random
primer method ŽBoehringer Mannheim Biochemica, Germany..
Hybridization was performed in 2.5 ng Ž10 5 cpm.rml
of radiolabeled probe, 250 m grml sheared salmon sperm
DNA, 50% formamide, 5 = SSC, 50 mM phosphate buffer
ŽpH 6.5., and 1 = Denhardt’s solution at 428C for
overnight. Then, membranes were rinsed at room temperature with 2 = SSC and 0.2% SDS several times, rinsed at
558C twice with 0.1 = SSC and 0.2% SDS, sealed in a
plastic bag, and the radioactivity was measured with an
image analyzing system ŽBAS 1500, Fuji Film, Tokyo,
Japan..
To calibrate the amount of applied total RNA, we
employed a two-step standardization method ŽFig. 2A.. In
the first step, to compensate for the differences of signal
intensity caused by different specific activity in each experiment, we electrophoresed serially-diluted standard total
RNA, extracted from a large amount of cerebral cortex,
and experimental samples on the same plate. The amount
of GAP-43 mRNA in each sample was represented as a
multiple of it in the standard total RNA. In the second
step, to compensate for the error in the amount of total
RNA applied, we used G3PDH mRNA as an internal
control, since G3PDH is a housekeeping gene and frequently used as an internal control of GAP-43 mRNA
measurement w37,39,55,56x. As shown in Fig. 2B, the
amount of G3PDH mRNA did not show obvious tendencies with regard to age. In this study, we divided the value
of GAP-43 mRNA by the value of G3PDH mRNA. Thus,
the normalized values of GAP-43 mRNA are indicated as
a multiple of the ratio of GAP-43 mRNA to G3PDH
mRNA in the standard total RNA from brain homogenate.
Measurements were performed at least twice for each
RNA sample.
3. Results
3.1. General
The amount of GAP-43 mRNA was measured between
the intermediate fetal period and the adult stage in FDD,
TE, PC, and OC. In two additional areas, i.e., PG and OB,
GAP-43 mRNA was measured only during the postnatal
period. The amount of GAP-43 mRNA was calibrated with
the amount of G3PDH mRNA.
3.2. FDD
In area FDD, the amount of GAP-43 mRNA was the
highest at E120 Ž15.0 " 0.8., decreased sharply until P1
Ž2.9 " 0.6., decreased moderately until P70 Ž0.8 " 0.0.,
then slightly increased until the adult stage Ž1.6 " 0.4.
ŽFig. 3A..
3.3. TE
In area TE, the amount of GAP-43 mRNA was also the
highest at E120 Ž14.4 " 2.2., decreased sharply until P8
Ž3.3 " 0.4., decreased moderately until P70 Ž1.2 " 0.5.,
then remained at the same level until the adult stage
Ž1.3 " 0.6. ŽFig. 3B..
3.4. PC
In area PC, the amount of GAP-43 mRNA was the
highest at E120 Ž11.8 " 0.2., though slightly lower than in
area FDD or TE. It decreased sharply until P1 Ž1.9., then
gradually decreased until the adult stage Ž1.0 " 0.4. ŽFig.
3C..
3.5. OC
In area OC, the amount of GAP-43 mRNA was also the
highest at E120 Ž8.4 " 1.0., though much lower than in
areas FDD or TE. It decreased sharply until P1 Ž2.2 " 0.1.,
then gradually decreased until the adult stage Ž0.9 " 0.2.
ŽFig. 3D..
3.6. PG
In area PG, the amount of GAP-43 mRNA was highest
at P8 Ž2.7 " 0.1., the earliest measurement. It was as high
as that of FDD or TE. Then, it decreased moderately until
P183 Ž0.9 " 0.0.. There were large individual variations at
the adult stage ŽFig. 3E..
2.3. Statistics
3.7. OB
To test the differences between age or area, a two-way
analysis of variance ŽANOVA. was performed using
StatView 4.5 ŽAbacus Concepts, Berkeley, CA..
In area OB, the amount of GAP-43 mRNA was also the
highest at P8 Ž3.3 " 0.5., the earliest measurement. It was
as high as that of FDD, TE, or PG. Then, it decreased
moderately until P70 Ž0.9 " 0.3. and remained at approxi-
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
91
Fig. 3. Developmental time course of the amount of GAP-43 mRNA in six cortical areas. An open circle indicates the mean value of an individual subject
of repeated measurements. A closed circle indicates the mean value of the subjects of the same age. ŽA.: FDD Žthe prefrontal area.; ŽB.: TE Žthe temporal
association area.; ŽC.: PC Žthe primary somatosensory area.; ŽD.: OC Žthe primary visual area.; ŽE.: PG Žthe parietal association area.; and ŽF.: OB Žthe
secondary visual area..
mately the same level until the adult stage Ž0.8 " 0.2.. It
was as high as that of PC or OC after P70 ŽFig. 3F..
3.8. Summary of all areas
Fig. 4A shows the composite data of the developmental
change of GAP-43 mRNA in four areas starting from the
intermediate fetal stage ŽE120.. The overall time course of
the developmental change in the amount of GAP-43 mRNA
was similar among all areas. The amount of GAP-43
mRNA was higher in the association areas, i.e., FDD and
TE, than in the primary sensory areas, i.e., PC and OC
from the intermediate fetal stage ŽE120. to the adult stage,
except at P70. This tendency was confirmed with two-way
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T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
93
GAP-43 mRNA in six areas during the postnatal period are
shown separately in Fig. 4B. Generally, the change in the
amount of GAP-43 mRNA was quite small after P70.
4. Discussion
4.1. General
Fig. 5. Comparison of the amount of GAP-43 mRNA between the
association areas and the primary sensory areas during the embryonic
period ŽA. and the postnatal period ŽB.. An open and a hatched bar
indicate the mean amount of GAP-43 mRNA in the association areas and
the primary sensory areas, respectively. An error bar indicates the standard deviation. During the embryonic period, FDD and TE were included
in the association areas, whereas during the postnatal period, PG was also
included in the association areas. During all developmental periods, PC
and OC were included in the primary sensory areas. Note that at any
developmental period, the amount of GAP-43 mRNA was higher in the
association areas than in the primary areas.
ANOVA both during the embryonic period ŽFig. 5A. and
during the postnatal period ŽFig. 5B.. During the embryonic period, the amount of GAP-43 mRNA was 14.6 " 2.0
at E120 and 10.6 " 2.9 at E140 in the association areas
including FDD and TE, while it was 10.1 " 2.0 at E120
and 6.0 " 0.9 at E140 in the primary sensory areas including PC and OC. The association areas were significantly
different from the primary sensory areas: n s 1, F s 14.9,
p - 0.01. During the postnatal period, the amount of GAP43 mRNA was 2.9 " 0.9 at P1, 2.9 " 0.4 at P8, 2.3 " 0.4
at P30, 1.1 " 0.4 at P70, 1.1 " 0.2 at P183, and 1.6 " 0.9
at the adult stage in the association areas including FDD,
TE, and PG, while it was 2.1 " 0.2 at P1, 1.7 " 0.3 at P8,
1.6 " 0.3 at P30, 0.9 " 0.3 at P70, 0.8 " 0.2 at P183, and
0.9 " 0.3 at the adult stage in the primary sensory areas
including PC and OC. The association areas were also
significantly different from the primary sensory areas:
n s 1, F s 24.8, p - 0.01. There was also a significant
difference among ages both during the embryonic period
Žbetween E120 and E140: n s 1, F s 12.0, p - 0.01. and
during the postnatal period Žamong 6 ages: n s 5, F s 17.0,
p - 0.01..
As the amount of GAP-43 mRNA was much lower
during the postnatal period than during the embryonic
period, the composite data of the developmental change of
The amount of GAP-43 mRNA was highest at E120 in
the macaque cerebral cortex, then decreased exponentially
to reach the asymptote by P70, at which time it was nearly
equal to that at the adult stage. These patterns were similar
among areas, however, the amount of GAP-43 mRNA was
higher in the association areas than in the primary sensory
areas during development and even after maturation. These
results are discussed in the sections below.
Because of the limited availability of monkeys, we used
three species of macaque monkeys. The three species are
especially close in genus Macaca and body sizes are
similar in early postnatal days. Moreover, there were no
obvious differences in the amount of GAP-43 mRNA
between individuals of two species at the same age. Therefore, development of GAP-43 mRNA may be similar
among them.
4.2. Comparison between the association areas and the
primary sensory areas
A protein phosphorylation study in the occipito-temporal system of the monkey w38x, immunohistochemistry in
the adult human cortex w8x, Western blotting study in both
normal and schizophrenic human cortex w45x and Northern
blotting experiments in the human cortex w40,41x indicate
that GAP-43 and GAP-43 mRNA are more abundant in the
association areas than in the primary sensory areas of the
adult cerebral cortex. Our present results confirm these
observations and extend them to the developmental stage
between the intermediate fetal days and P183. At the adult
stage, there may be higher potential of terminal reorganization in the association areas than in the primary sensory
areas w7,8,40,41x.
There is a possibility that the greater amount of GAP-43
mRNA, compared with G3PDH mRNA, in the association
areas than in the primary sensory areas results from a
larger ratio of neurons to glial cells in the association area.
This, however, is not plausible because the ratio of neurons among total cells, i.e., neurons and glial cells, is quite
similar during development Ž0.93 and 0.89 at P5 in area 3
ŽPC. and area 20 ŽTE., respectively. and at the adult stage
Fig. 4. ŽA.: Composite data of the time course of the mean amount of GAP-43 mRNA in four areas from P120 to the adult stage. Colors of the lines
correspond to that of the areas in the insert. ŽB.: Postnatal development of the mean amount of GAP-43 mRNA in six areas. Colors of the lines correspond
to that of the areas in the insert.
94
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
Ž0.81 and 0.83 at P180 in area PC and area TE, respectively. in mouse Žcalculated from the data of Heumann et
al. w24x, and Leuba et al. w31x..
The amount of GAP-43 mRNA during embryonic period was much larger than that at the adult stage and net
elongation of axons occurs during development, especially
during the embryonic period. Therefore, levels of GAP-43
mRNA may, at least in part, reflect degrees of axonal
growth including both elongation and terminal modification during the embryonic period and early postnatal period Žbefore P70.. If so, we can interpret our results in two
ways. One is that the peaks of axonal elongation in the
primary sensory areas occur earlier than that in the association areas ŽFig. 6A.. The other is that axonal elongation is
consistently more extensive in the association areas than in
the primary sensory areas ŽFig. 6B., leading to much more
exuberant axons or to a higher rate of degeneration of
axons in the association areas in comparison with the
primary sensory areas. Although there are no reports regarding the areal difference of the net length of intrinsic
and efferent axons or degree of degeneration of axons, it is
not likely that the net length of axons is much longer or
that degeneration occurs at a much higher rate in the
association areas. Thus, we believe the first interpretation
is more plausible.
There remains another possibility that axonal elongation, which does not involve GAP-43, is consistently more
extensive in the primary sensory areas during develop-
ment. To test each hypothesis, measurement of GAP-43
mRNA before E120 and measurement of other growth-associated proteins during development remain to be performed in future experiments.
4.3. Comparison with the deÕelopment of commissural
axons
The development of commissural axons was investigated in detail with an electromicroscopic method in CC
w29x and AC w30x. Most neocortical areas, such as FDD,
PC, PG, and OB are interconnected bilaterally with CC
w28x, while area TE is mainly interconnected with AC
w27,60x. The number of axons in CC w29x and AC w30x
increases during the fetal period and reaches the peak at
P0, then decreases during the postnatal development,
though timing and magnitude of axon overproduction and
elimination are different between these two commissural
systems. The ratio of growth cones against axons remains
constant from E65 to E128 in CC w29x and AC w30x,
sharply decreases by E138 in AC and by E144 in CC, then
diminishes by P0 in CC, suggesting that axon addition
occurs in CC only in the fetal period w29x. In the current
results, the amount of GAP-43 mRNA in areas FDD, PC,
and TE was highest at E120 and sharply decreased to P1.
These results are in accordance with the developmental
change of the amount of growth cones in CC and AC,
suggesting that GAP-43 in these areas is involved in the
Fig. 6. Two hypotheses regarding axonal elongation in the development of the association areas and the primary sensory areas. The first hypothesis is that
the peak of the axonal elongation occurs earlier in the primary sensory areas than in the association areas. This hypothesis postulates that the profile of the
peak is similar among areas ŽA.. The second hypothesis is that the peak of the axonal elongation is similar at each developmental age, but that the extent of
the axonal elongation is constantly greater in the association areas than in the primary sensory areas ŽB.. Solid line: axonal elongation estimated with the
amount of GAP-43 mRNA, hatched line: supposed axonal elongation, thick line: axonal elongation in the association areas, thin line: axonal elongation in
the primary sensory areas.
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
development of commissural axons. Though developmental pattern is different between CC and AC w29,30x, we
could not find any obvious differences in the temporal
pattern, or individual differences in the amount of GAP-43
mRNA between area FDD and area TE.
Even in the postnatal development, we believe that
axon addition occurs at a low but significant rate even
after birth because the amount of GAP-43 mRNA was
significantly higher during the earlier period of the postnatal development ŽP1 to P30. than during the later period
ŽP70 to adult.. GAP-43 in the pre- and postnatal development of the neocortex may be involved in the axonal
elongation not only in the commissural systems but also in
other systems, e.g., intracortical local circuit, association
fibers, and descending projections. Determination of the
laminar distribution of the cells expressing GAP-43 mRNA
in the developing neocortex is necessary to address this
issue. We are now in course of in situ hybridization study
in the cerebral cortex.
4.4. Comparison with other mammals in reference to
commissural deÕelopment and synaptogenesis
So far, developmental change of GAP-43 or its mRNA
in the cerebral cortex has been studied in cat, rat, and
human.
In the cat, the amount of GAP-43 w35x and its mRNA
w37x shows an exponential decrease in the primary visual
area during the postnatal period, which is similar to our
results in the monkey. Thus, the peak in the amount of
GAP-43 and its mRNA may occur during the fetal period.
The number of axons in CC reaches the peak at P0 and
decreases exponentially w9x. Therefore, the peak in the
amount of GAP-43 and its mRNA occurs before the
number of the commissural axons reaches the peak, similar
to the monkey.
In the rat, the level of GAP-43 w26x and its mRNA
w5,15,34,50x increases to reach its peak at approximately
P7 and decreases significantly by P14. The number of
axons in CC reaches a plateau at P5 and remains constant
until P60 w18x, and the number of axons in AC reaches the
plateau at P4 and remains constant until P389 w19x. Thus,
the peak in the amount of GAP-43 and its mRNA occurs
after the number of the commissural axons reaches the
plateau, in contrast to the monkey and the cat. Reason of
this discrepancy is unclear.
The peak in the amount of GAP-43 mRNA precedes the
peak density of synapses in the cerebral cortex of all
animals studied. In the monkey, the densities of synapses
reach their peaks at 2-4 months after birth w49x. In the cat,
the density of synapses in the visual cortex reaches a peak
at approximately P40 w14x. In the rat, the density of cortical
synapses reaches a plateau at P26 in the parietal cortex w1x
and at P16 in the visual cortex w10x, at which time the
amount of GAP-43 mRNA is lower than that at P7. As for
95
the human cerebral cortex, developmental study of GAP-43
mRNA was preliminary. The amount of GAP-43 mRNA is
reported to be higher at fetal week 19 or new born stage
than at the adult stage in TE, PC, OB, OC, and some other
areas w39,41x. The density of synapses in the visual cortex
ŽOC. is greatest at 8-12 months postnatally w25x. As the
peak in the amount of GAP-43 mRNA precedes the peak
density of synapses in all animals including primate, the
peak in the amount of GAP-43 mRNA may occur at latest
postnatal 8 months in the human.
4.5. Comparison with other molecules
So far, the amounts of several kinds of neurotransmitters, neuromodulators, receptors, synthesizing enzymes
of neurotransmitters, and other signal transduction
molecules have been shown to be regulated during the
development of the monkey cerebral cortex. Some of these
molecules have been studied with regard to areal differences: neuropeptides wsomatostatin ŽSRIF. and its mRNA,
cholecystokinin ŽCCK., vasoactive intestinal peptide ŽVIP.
and substance P ŽSP.x, the activity of glutamate decarboxylase ŽGAD. w21–23x, and receptors of neurotransmitters
Ždopamine, serotonin, norepinephrine, acetylcholine, and
GABA. w32,33x. There is only a small amount of these
peptides at E120 except SP in OC, and it dramatically
increases around the perinatal period w21x. Therefore, the
development of these peptides is preceded by the development of GAP-43 mRNA. As SRIF has been reported to
enhance neurite outgrowth in vitro using PC12 w16x and
cerebellar granule cell w54x, it is difficult to interpret the
discrepancy that the amount of SRIF mRNA increases
from E120 to E140 w23x and SRIF increases from E120 to
the newborn stage w21x while GAP-43 mRNA decreases
during the same period. CCK-8 is similar to GAP-43 with
respect to abundance in the association areas relative to the
primary sensory areas w22x, but the relationship between
them is not clear. In all areas, GAD activity is very low
Ž9–20% of that of the adult. at E120 and increases gradually to maturation w22x; this is contrary to the development
of GAP-43 mRNA. Quantitative in vitro autoradiography
studies of binding of several ligands reveals that receptors
of neurotransmitters, i.e., dopamine, serotonin, norepinephrine, acetylcholine, and GABA, develop synchronously in
each cortical area, containing FDD, PC, and OC w32,33x.
The peak amount of the binding occurs at 2–4 months
after birth, corresponding with the peak of synaptic density
in cortical areas. Among these receptors, the binding of
D 2-doperminergic receptors, 5-HT2-serotonergic receptors,
a 1-adrenergic receptors, and b-adrenergic receptors is
higher in FDD than in PC and OC during development,
similar to the amount of GAP-43 mRNA. Accumulation of
knowledge about the development of various molecules
will help us to understand the functional maturation of
neuronal circuits in the central nervous system of primate.
96
T. Oishi et al.r DeÕelopmental Brain Research 109 (1998) 87–97
Acknowledgements
This work was supported by the grant of AIST, MITI,
Japan and Cooperation Research Program of Primate Research Institute, Kyoto University. We appreciate Dr. R. L.
Neve for generously providing the cDNA clone of GAP-43.
We thank Dr. K. Kawano and Dr. S. Yamane for valuable
discussions, and Ms. M. Okui for excellent technical assistance.
w18x
w19x
w20x
w21x
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