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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 92 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. 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