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The Ontogeny of Cerebral and Cerebellar Nitric Oxide Synthase in the Guinea Pig and Rat

Abstract

The appearance of nitric oxide synthase (NOS, EC 1.14.13.39) activity in the brain of fetal and neonatal guinea pigs and rats was studied. In the guinea pig, NOS increased from an almost undetectable level at 0.49 of gestation (31 d), reaching adult levels before birth and peaking at 140% of the adult activity (forebrain) or 250% of the adult activity (cerebellum) in the week after birth. The rise in fetal NOS activity followed the reported rise in the estrogen receptor concentration in the brain and could be reduced by treatment of the guinea pig at full term with tamoxifen, implicating estrogens in the expression of fetal NOS activity. In the rat, brain NOS activity did not rise significantly until after birth, reaching adult levels approximately 2 wk after birth, and rising to 150 or 130% of the adult activity in the forebrain and cerebellum, respectively, at 4 wk after birth. The appearance of NOS activity in the rat also followed the reported appearance of estrogen receptors in the brain. In both species the appearance of high NOS activity in the brain immediately precedes the period in which maximal synaptogenesis occurs: immediately before birth in the guinea pig and 2-3 wk after birth in the rat. Thus the appearance of a functional estrogen-estrogen receptor system in the brain may be responsible, at least in part, for the expression of a high activity of NOS, which in turn may play important roles in promoting cerebral blood flow and synaptogenesis in the developing brain.

Main

NO is an important messenger molecule implicated in multiple physiologic and pathophysiologic events in both the peripheral and central nervous system(1, 2). NOS (EC 1.14.13.39) catalyzes the oxidation of L-arginine to NO plus citrulline(3). Although the calcium-dependent NOS which are normally present in the brain and other tissues have been described as constitutive, there is evidence for the regulation of their expression by estrogen(4). Proposed roles for NO within the CNS include synaptic plasticity and memory via long-term potentiation in the hippocampus and depression in the cerebellum(57), control of blood flow by neural activity(8, 9), and the establishment and activity-dependent refinement of axonal projections during the later stages of development(10). Proposed pathophysiologic roles for NO in the CNS include brain dysfunction associated with inflammation(11), the mediation of glutamate toxicity in cerebral ischemia(1, 12, 13), and epilepsy(1417). Each of these roles could be important in the fetus and newborn, but there is no published data on the development of NOS activity in the brain before birth and little on its activity in the neonate. Quantitative data on the appearance of functional NOS may be particularly important in understanding the potential role of NO in brain ischemia. Qualitative immunohistochemical data on the rat brain show that there is a redistribution of NOS protein expression within the brain during the neonatal period, but do not show whether the overall activity changes during this period(18), whereas NOS activity measurements suggest that the development of functional NOS in this species is predominantly postnatal(19).

In the present study we have determined the ontogeny of functional NOS by measuring its activity in fetal and neonatal forebrain and cerebellum from two species with very different developmental profiles: the guinea pig and the rat. We also provide evidence for the involvement of the estrogen-estrogen receptor system in the ontogeny of NOS.

METHODS

Pregnant Duncan Hartley guinea pigs with a known date of conception (term 63 d average) and Wistar rats (term 21 d average) were obtained from a commercial breeder (Charles River). Fetal and neonatal brains were rapidly removed under general anesthesia (pentobarbitone 120 mg/kg intraperitonally for the mother or 30 mg intraperitonally for the neonate), divided into sections of forebrain and cerebellum, freeze-clamped in liquid nitrogen, and stored at -70°C until studied. Whole guinea pig fetal brain was used at 31 d (0.49 gestation) because of the small size.

Estrogen receptor blockade. To test the role of estrogen in changing NOS activity during gestation, guinea pigs at full term (either at 58 or 61 d of gestation) received four doses of the estrogen-receptor partial agonist tamoxifen (250 μg intraperitonally twice a day), and the fetal brains were removed 6 h after the last injection (on d 60). Neonatal brains were removed within 16 h of birth. Animals of the same gestational age were used for controls. Although tamoxifen can have both agonist and antagonist activities in different tissues, it acts predominantly as an antagonist in the brain(20). Tamoxifen treatments longer than 2 d were not attempted because of the risk of premature termination of pregnancy.

NOS activity. The frozen tissue was homogenized (with a Ystral homogenizer) at 0°C in 5 volumes of buffer containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1 mM DL-DTT, 100 μg/mL phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL soybean trypsin inhibitor, and 2 μg/mL aprotinin brought to a pH 7.0 at 20°C with HCl. The crude homogenate was centrifuged at 0°C at 15,000 × g for 20 min, and the pellet was discarded. NOS activity was determined in the postmitochondrial supernatant within 1 h of preparation by measuring in duplicate the conversion of L-[U-14C]arginine to L[U-14C]citrulline as previously described in detail(21). Total activity was determined by calculating the difference between the [14C]citrulline produced from control incubations and the incubations containing both 1 mM EGTA to bind calcium and 1 mM Nω-monomethyl L-arginine to inhibit NO synthase(21). The activity of calcium-independent NO synthase was determined by calculating the difference between the incubations containing 1 mM EGTA alone and the incubations containing both 1 mM EGTA and 1 mM Nω-monomethyl L-arginine. Calcium-dependent activity was calculated by subtracting calcium independent activity from total activity. Measurements are presented as picomoles of citrulline/min/mg of protein. The protein was measured in an aliquot of the crude homogenate using bicinchoninic acid(22). Intra- and interassay variations were each less than 8%.

Chemicals and statistical analyses. L-[U-14C]Arginine was obtained from Amersham Corp.; all other chemicals were from Sigma Chemical Co. The results are presented as the mean ± SEM. The day of copulation was considered to be the first day of pregnancy. Statistical significance was assessed by the t test. A p < 0.05 was considered to indicate either a significant correlation or difference among means.

RESULTS

Guinea pig . Cerebellum. Whole brain NOS activity was at the lower limit of detection at 0.49 gestation (1.9 ± 0.9 pmol/min/mg of protein). Thereafter, the calcium-dependent NOS activity increased rapidly, reaching the activity present in the adult by 0.62 gestation (24 d before delivery). NOS continued to increase until d 6 after birth, reaching 250% of the adult activity, and declining thereafter (Fig. 1).

Figure 1
figure1

NO synthase activity in the forebrain (closed squares) and cerebellum (open circles) of the guinea pig fetus and neonate. NO synthase activity was measured by the conversion of L-arginine to L-citrulline and is shown as mean ± SEM (3-8 animals/group).

Forebrain. The calcium-dependent NOS activity increased rapidly after 0.49 gestation, reaching the activity present in the adult by 0.95 (5 d before delivery). In contrast to the cerebellum, NOS activity increased between 0.95 gestation and 1 d of age. Thereafter, activity plateaued at 140% of the adult level until d 6 after birth. By d 10, NOS activity had declined to the adult level (Fig. 1). Calcium-independent NOS activity was observed in both tissues, but on average it was <5% of the total activity and was unaltered during gestation.

Estrogen receptor blockade. Two days of treatment with tamoxifen in the guinea pig caused significant reductions in forebrain and cerebellum calcium-dependent NOS activities in the fetus and in the neonate (Fig. 2).

Figure 2
figure2

Effect of tamoxifen treatment on NO synthase activity in the forebrain and cerebellum of the guinea pig fetus (60 d) and neonate (1 d). Pregnant animals received four doses of tamoxifen (250 μg intraperitonally). The data are shown as means ± SEM (3-8 animals/group) *p < 0.05 (t test).

Rat. NOS activity was low at 0.8 gestation (forebrain 16± 1.1, cerebellum 9 ± 1.3 pmol/min/mg of protein,Fig. 3). By 12 d (forebrain) or 20-30 d (cerebellum) the activity was similar to that of the adult. Thereafter, NOS increased to 130-150% of the adult level by 30 d of age. Calcium-independent NOS activity was low and was unaltered in both tissues.

Figure 3
figure3

NO synthase activity in the forebrain (closed squares) and cerebellum (open circles) of the rat fetus and neonate. NO synthase activity was measured by the conversion of L-arginine to L-citrulline and are shown as means ± SEM (3-8 animals/group).

DISCUSSION

We have observed a maturational process which begins after 0.49 gestation in the guinea pig but which does not begin until after 0.8 gestation in the rat. Adult NOS activity was reached by 0.62 gestation in the cerebellum and 0.95 in the forebrain in the guinea pig but was not reached until later(approximately 10-30 d after birth) in the rat.

Little is known about the regulation of the expression of the constitutives NOS (neuronal and endothelial)(3). Pregnancy is characterized by a high concentration of circulating estrogen and this steroid is known to increase the transcription of a number of enzymes. We have recently shown that estrogen induces neuronal and endothelial NOS in a range of tissues, including the brain(4). Estradiol crosses both the blood-brain barrier and the placenta, resulting in increased NOS activity in maternal tissues early in pregnancy, reaching a maximum value by 0.3 of gestation(23). Therefore maternal estrogen could alter fetal brain NOS activity if either placental transport of estrogen or the number of fetal estrogen receptors increased with advancing gestation.

The quantity of estrogen receptors within the fetal guinea pig brain increases with advancing gestation and then decreases after birth(24). We plotted the concentration of cytosolic estradiol-specific receptors in the fetal guinea pig whole brain(24) against the NOS activity measured in the current study The two curves are almost superimposable (Fig. 4), so that, as the concentration of available receptor sites increases, so does the NOS activity after a short delay. Thus, it is reasonable to hypothesize that estrogen enhances the synthesis of NOS in the fetal guinea pig brain. A prediction which would follow from this hypothesis is that the treatment of pregnant animals with tamoxifen (which acts in the brain as an estrogen antagonist)(20) at the end of gestation should reduce NOS activity in the fetal brains, and our results show that this does occur. Thus the increase in calcium-dependent NOS activity in the fetal guinea pig brain during pregnancy is, at least in part, mediated by estrogen. These results suggest that, despite the presence of the highly specificα-feto/neonatal estradiol-binding plasma protein and the related fetal/neonatal estradiol-binding protein in brain cytosol(25), sufficient free estradiol must be present in the fetus to exert biologic effects.

Figure 4
figure4

NO synthase activity (closed squares) of the guinea pig fetus and neonate forebrain plotted against the number of estradiol cytosolic receptors (open circles) as reported by Pasqualiniet al.(24).

In the rat brain, the estrogen receptor concentration increases at a later age than in the guinea pig brain. Whereas the most rapid increase in estrogen receptors occurs between 0.5 and 0.8 of gestation in the guinea pig(24), it occurs at around term in the rat(26, 27). Thus in both species the estrogen receptor concentration rises immediately (4-6 d) before the rise in NOS activity in the brain. Furthermore, in early neonatal rat brain, the estrogen receptors are most abundant in the cerebral cortex(26). In the present study, we have found approximately 2-fold more NOS activity in the forebrain than in the cerebellum at this stage (postnatal d 3,Fig. 3). Our data on the ontogeny of NOS activity in the rat forebrain is consistent with that of Matsumoto et al.(19) and with recent reports that the development of NADPH diaphorase-positive neurones (presumed to reflect NOS-containing neurones) and NOS immunostaining occur predominantly during the first 2 wk of postnatal life(18, 28, 29).

The increase in NOS may play several roles in the fetus and neonate. It has been shown that estrogen can be a growth factor to central nervous cells(3033). The increase in NOS activity in rat and guinea pig correlates with the development of the estrogen receptor population, as we have seen above, and precedes the development of synaptic structures in these two species. In the guinea pig, studies using a range of methods show that the most rapid increase in the number of synaptic junctions occurs during the last days of gestation and the 1st wk of life(3436). In contrast to the guinea pig, the number of synapses in various regions of the rat brain does not increase markedly until 2 and 3 wk postnatally(3438). The predominantly prenatal and postnatal synaptogenesis, in the guinea pig and rat, respectively, is reflected in the general neurologic maturity of these two species at birth. The guinea pig is born essentially functional and with its eyes open, whereas the rat is born with limited mobility and with its eyes closed. Our studies, demonstrating that the increase in NOS activity in brain occurs approximately 10 d before the rapid phase of synapse formation in both guinea pigs and rats, support the hypothesis that NO formation may be important in the physiology and development of the synapse(10). Very recent data showing that NOS appears postnatally in the mouse brain and again preceding the rapid phase of synaptogenesis(39) are also consistent with this hypothesis. In the present study we have not demonstrated that the brain NOS activity measured can be attributed to neuronal NOS, although studies of adult brain NOS in several species and of developing brain NOS in the mouse make this likely(39).

NO is involved in the regulation of cerebral blood flow(9, 40), increasing local cerebral blood flow in response to neuronal activation by dilating resistance vessels. Although the normal fetal environment is not characterized by profound hypoxemia, it is one of relative hypoxemia which intensifies as gestational age advances. It is attractive to hypothesize that the elevation of NOS above adult levels may be a mechanism to cope with the demand of brain regions which are metabolically or physiologically highly active, providing a neuroprotective effect by enhancing blood flow. It is known that cerebral vascular resistance decreases and blood flow increases with advancing gestational age. The increased production of NO around blood vessels in the brain could be responsible for the decreased resistance and increased volume of flow.

Another possible role of NO that our findings might impact upon relates to memory. The newborn must form a large number of memories critical for survival. We observed high levels of NOS activity in both the forebrain and cerebellum during the first days of life in the guinea pig and at 3 wk of age in the rat. A growing body of evidence suggests that NO may act as a retrograde messenger for long-term potentiation and thus memory formation(57). The high levels of NOS activity that we have seen may therefore play a role in memory formation. It is not clear how the ontogeny of NOS will relate to the pathophysiologic roles suggested for NO, such as ischemic damage or epilepsy. The data presented will clearly provide the basis of studies to elucidate these roles, for example by examining the susceptibility of animals of different ages to ischemic brain damage.

In conclusion NOS activity increases in both the forebrain and cerebellum predominantly prenatally in the guinea pig and predominantly postnatally in the rat. The sequence observed in both species, of increased estrogen receptors followed by increased NOS activity followed by synaptogenesis, is consistent with the hypothesis that estrogen may play a crucial role in the ontogeny of NOS and suggests that NO formation may play an important role in synaptogenesis. The high levels of NOS activity may also have important functions in promoting cerebral blood flow and memory formation during the periods of most rapid brain development.

Abbreviations

NO:

nitric oxide

NOS:

nitric oxide synthase

References

  1. 1

    Moncada S, Palmer RMJ, Higgs EA 1989 Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem Pharmacol 38: 1709–1715.

    CAS  Article  Google Scholar 

  2. 2

    Moncada S, Palmer RMJ, Higgs EA 1991 Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109–142.

    CAS  PubMed  Google Scholar 

  3. 3

    Knowles RG, Moncada S 1994 Nitric oxide synthases in mammals. Biochem J 298: 249–258.

    CAS  Article  Google Scholar 

  4. 4

    Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S 1994 Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91: 5212–5216.

    CAS  Article  Google Scholar 

  5. 5

    Bohme GA, Bon C, Stutzmann JM, Doble A, Blanchard JC 1991 Possible involvement of nitric oxide in long-term potentiation. Eur J Pharmacol 199: 379–381.

    CAS  Article  Google Scholar 

  6. 6

    Shibuki K, Okada D 1991 Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum. Nature 349: 326–328.

    CAS  Article  Google Scholar 

  7. 7

    Bliss TVP, Collingridge GL 1993 A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361: 31–39.

    CAS  Article  Google Scholar 

  8. 8

    Iadecola C 1993 Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link?. Trends Neurosci 16: 206–214.

    CAS  Article  Google Scholar 

  9. 9

    Iadecola C, Pelligrino DA, Moskowitz MA, Lassen NA 1994 Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab 14: 175–192.

    CAS  Article  Google Scholar 

  10. 10

    Gally JA, Montague PR, Reeke GN, Edelman GM 1990 The NO hypothesis: Posible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system. Proc Natl Acad Sci USA 87: 3547–3551.

    CAS  Article  Google Scholar 

  11. 11

    Koprowski H, Zheng YM, Heber-Katz E, Fraser N, Rorke L, Fang Fu Z, Hanlon C, Dietzschold B 1993 In vivo expression inducible nitric oxide synthase mRNA in experimentally induced neurologic diseases. Proc Natl Acad Sci USA 90: 3024–3027.

    CAS  Article  Google Scholar 

  12. 12

    Bruhwyler J, Chleide E, Liegeois JF, Carreer F 1993 Nitric oxide: a new messenger in the brain. Neurosci Biobehav Rev 17: 373–384.

    CAS  Article  Google Scholar 

  13. 13

    Lafon-Cazal M, Culcasi M, Gaven F, Pietri S, Bockaert J 1993 Nitric oxide, superoxide and peroxynitrite: putative mediators of NMDA-induced cell death in cerebellar granule cells. Neuropharmacology 32: 1259–1266.

    CAS  Article  Google Scholar 

  14. 14

    De Sarro GG, Donato Di Paola E, De Sarro A, Vidal MJ 1991 Role of nitric oxide in the genesis of excitatory amino acid-induced seizures from the deep prepiriform cortex. Fundam Clin Pharmacol 5: 503–511.

    CAS  Article  Google Scholar 

  15. 15

    De Sarro GG, Donato Di Paola E, De Sarro A, Vidal MJ 1993 L-Arginine potentiates excitatory amino acid-induced seizures elicited in the deep prepiriform cortex. Eur J Pharmacol 230: 151–158.

    CAS  Article  Google Scholar 

  16. 16

    Mollace V, Bagetta G, Nistico G 1991 Evidence that L-arginine possesses proconvulsant effects mediated through nitric oxide. Neuroreport 2: 269–272.

    CAS  Article  Google Scholar 

  17. 17

    Bagetta G, Iannone M, Scorsa AM, Nistico G 1992 Tacrine-induced seizures and brain damage in LiCl-treated rats can be prevented by Nω-nitro-L-arginine methyl ester. Eur J Pharmacol 213: 301–304.

    CAS  Article  Google Scholar 

  18. 18

    Bredt DS, Snyder SH 1994 Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium. Neuron 13: 301–313.

    CAS  Article  Google Scholar 

  19. 19

    Matsumoto T, Pollock JS, Nakane M, Forstermann U 1993 Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain. Dev Brain Res 73: 199–203.

    CAS  Article  Google Scholar 

  20. 20

    McKenna SE, Simon NG, Cologer-Clifford A 1992 An assessment of agonist/antagonist effects of tamoxifen in the female mouse brain. Horm Behav 26: 536–544.

    CAS  Article  Google Scholar 

  21. 21

    Salter M, Knowles RG, Moncada S 1991 Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett 291: 145–149.

    CAS  Article  Google Scholar 

  22. 22

    Hill HD, Straka JG 1988 Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents. Anal Biochem 170: 203–208.

    CAS  Article  Google Scholar 

  23. 23

    Weiner CP, Knowles RG, Moncada S 1994 Induction of nitric oxide synthases early in pregnancy. Am J Obstet Gynecol 171: 838–843.

    CAS  Article  Google Scholar 

  24. 24

    Pasqualini JR, Sumida C, Gelly C, Nguyen BL, Tardy J 1978 Specific binding of estrogens in different fetal tissues of guinea pig during fetal development. Cancer Res 38: 4246–4250.

    CAS  PubMed  Google Scholar 

  25. 25

    Plapinger L, McEwen BS 1975 Immunochemical comparison of estradiol-binding molecules in perinatal rat brain cytosol and serum. Steroids 26: 255–265.

    CAS  Article  Google Scholar 

  26. 26

    MacLusky NJ, Chaptal C, McEwen B 1979 The development of estrogen systems in the rat brain and pituitary: postnatal development. Brain Res 178: 143–160.

    CAS  Article  Google Scholar 

  27. 27

    Vito CC, Fox TO 1982 Androgen and estrogen receptors in embryonic and neonatal rat brain. Dev Brain Res 2: 97–110.

    Article  Google Scholar 

  28. 28

    Tomic D, Zobundzija M, Meaugorac M 1994 Postnatal development of nicotinamide adenine dinucleotide phosphate diaphorase(NADPH-d) positive neurons in rat prefrontal cortex. Neurosci Lett 170: 217–220.

    CAS  Article  Google Scholar 

  29. 29

    Yan XX, Garey LJ, Jen LS 1994 Development of NADPH-diaphorase activity in the rat neocortex. Dev Brain Res 79: 29–38.

    CAS  Article  Google Scholar 

  30. 30

    Nishizuka M, Arai Y 1982 Synapse formation in response to estrogen in the medial amygdala developing in the eye. Proc Natl Acad Sci USA 79: 7024–7027.

    CAS  Article  Google Scholar 

  31. 31

    Lustig RH, Sudol M, Pfaff DW, Federoff HJ 1991 Estrogenic regulation and sex dimorphism of growth-associated protein 43 kDa(GAP-43) messenger RNA in the rat. Brain Res Mol Brain Res 11: 125–132.

    CAS  Article  Google Scholar 

  32. 32

    Honjo H, Tamura T, Matsumoto Y, Kawata M, Ogino Y, Tanaka K, Yamamoto T, Ueda S, Okada H 1992 Estrogen as a growth factor to central nervous cells. J Steroid Biochem Mol Biol 41: 633–635.

    CAS  Article  Google Scholar 

  33. 33

    Shughrue PJ, Dorsa DM 1993 Estrogen Modulates the growth-associated protein GAP-43 (neuromodulin) mRNA in the rat preoptic area and basal hypothalamus. Neuroendocrinology 57: 439–447.

    CAS  Article  Google Scholar 

  34. 34

    Jones DG, Dittmer MM, Reading LC 1974 Synaptogenesis in guinea-pig cerebral cortex: a glutaraldehyde-PTA study. Brain Res 70: 245–259.

    CAS  Article  Google Scholar 

  35. 35

    Lohmann SM, Ueda T, Greengard P 1978 Ontogeny of synaptic phosphoproteins in brain. Proc Natl Acad Sci USA 75: 4037–4041.

    CAS  Article  Google Scholar 

  36. 36

    Lennon AM, Francon J, Fellous A, Nunez J 1980 Rat, mouse, and guinea pig brain development and microtubule assembly. J Neurochem 35: 804–813.

    CAS  Article  Google Scholar 

  37. 37

    Crain B, Cotman C, Taylor D, Lynch G 1973 A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat. Brain Res 63: 195–204.

    CAS  Article  Google Scholar 

  38. 38

    Tallant EA, Cheung WY 1983 Calmodulin-dependent protein phosphatase: a developmental study. Biochemistry 22: 3630–3635.

    CAS  Article  Google Scholar 

  39. 39

    Ogilive P, Schilling K, Billingsley ML, Schmidt HHHW 1995 Induction and variants of neuronal nitric oxide synthase type I during synaptogenesis. FASEB J 9: 799–806.

    Article  Google Scholar 

  40. 40

    Beckman JS 1991 The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 15: 53–59.

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank E. A. Higgs for her critical reading of the manuscript.

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Correspondence to Ignacio Lizasoain.

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Supported in part by grants from the Ministerio de Educación y Ciencia, Spain (PF91/05378715), the Commission of the European Communities(Human Capital and Mobility Programme IS/ERB-4001GT-921179), and the United States National Institutes of Health HD24494 (C.P.W.), HL49041 (C.P.W.), and HL51735 (C.P.W.).

1 Visiting Scientist while this work was performed. Permanent address: Departamento de Farmacologia, Facultad de Medicina, Universidad Complutense de Madrid, Spain.

2 Visiting Scientist while this work was performed. Current address: Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Perinatal Research Laboratory, University of Iowa College of Medicine, Iowa City, IA 52242.

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Lizasoain, I., Weiner, C., Knowles, R. et al. The Ontogeny of Cerebral and Cerebellar Nitric Oxide Synthase in the Guinea Pig and Rat. Pediatr Res 39, 779–783 (1996). https://doi.org/10.1203/00006450-199605000-00006

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