Brain Research, 625 (1993) 177-179
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25860
The effect of lactation on nitric oxide synthase gene expression S. Ceccatelli
M. Eriksson b
Departments of a Histology and Neurobiology and b Pharmacology, Karolinska Institute, Box 60400, 10401 Stockholm, Sweden
(Accepted 13 July 1993)
Key words: Nitric oxide; In situ hybridization; Paraventricular nucleus
Nitric oxide-synthesizing(NOS) enzyme has been identified in several neural populations, including the hypothalamic paraventricular nucleus (PVN). The PVN plays a major role in regulating milk ejection. In the present study, using in situ hybridization, the effect of lactation on NOS mRNA in the PVN was investigated. A significant increase was seen in the PVN of lactating rats, indicating a possible involvementof nitric oxide in the hypothalamic-pituitaryregulation of milk ejection.
Nitric oxide (NO), a gas formed from L-arginine by the C a 2 + / c a l m o d u l i n - d e p e n d e n t enzyme nitric oxide synthase (NOS) 4'14, has b e e n proposed as a possible messenger in the CNS and peripheral nervous system. Since the purification of the enzyme 5 and the cloning of the gene from rat brain 2, histochemistry for NOS has been used to study localization of N O synthesis. Moreover, since NOS is identical with neuronal nicotinamide adenine dinucleotide phosphate diaphorase ( N A D P H d ) , it has been possible to localize NOS also with N A D P H d histochemistry 12'21. The physiological role of N O in the nervous system is still unknown. N O is a potent vasodilator and seems to be involved in neurogenic relaxation of cerebral arteries 7'n'13'16'2°. It might also mediate the biological effect of other neurotransmitters through an increase in c G M P l°. Immunohistochemical and in situ hybridization studies have shown that N O S and N A D P H d 1'3'21 are present in cell bodies of the hypothalamic paraventricular nucleus (PVN). Since it is well established that the PVN plays a major role in controlling milk ejection 22, this study was designed to examine the effect of lactation on NOS m R N A levels in the PVN. Nine lactating female (10 days after delivery) and nine non-lactating female albino S p r a g u e - D a w l e y rats (300-350 g; ALAB, Stockholm, Sweden) were used.
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Animals were housed under standard conditions (light on: 06.00-20.00 h) in individual cages. Each litter was limited to 8 - 1 0 pups. After decapitation, the brains were rapidly removed, the hypothalami dissected out and frozen on dry ice. 14-mm-thick sections through the P V N were cut in a cryostat (Microm, Heidelberg, Germany), thaw-mounted onto precleaned microscope glass slides (ProbeOn, Fisher Scientific, Pittsburgh, PA) and hybridized with an ot-[35S]dATP-labeled oligonucleotide probe with sequences complementary to m R N A for rat NOS (aminoacids 151-164) (Scandinavian G e n e Synthesis). After the hybridization procedure, the slides were placed in a cassette, covered with Hyperfilm /3-max X-ray film (Amersham, UK) and exposed at - 2 0 ° C for 15 days. The films were developed using D19 developer (Kodak) and H y p a m fixative (Ilford). Sections were then dipped in NTB2 emulsion (Kodak) diluted 1 : 1 with distilled water, exposed at - 2 0 ° C for 2 weeks, which was an optimal time to show the difference between the two groups, developed with D 19 (Kodak) and fixed with G333 (Agfa Gevaert), coverslipped with g l y c e r o l / P B S (3:1). The mean optical density of autoradiographs was measured on a Macintosh II × (Apple Computer, Cupertino, CA) with Image Software provided by W. Rasband ( N I M H , Washington, DC). The PVN was identified after toluidine-staining of the sections. Using an adjustable window to sample the labeled areas of the nucleus, five rostro-caudal matching sections of the PVN were measured and the m e a n + S.D. was first
Fig. 1. A - D : darkfield photomicrographs of sections showing NOS mRNA-labeling in PVN of control (con) (A,B) and lactating (lact) (C,D) rats. Note upregulation of NOS m R N A in lactating rats (C,D). Arrowheads in D point to strongly labeled parvocellular neurons. Star indicates 3rd ventricle, mp, medial parvocellular, pro, posterior magnocellular. Bar = 50 ram.
calculated per each animal. The Student's t-test was used for the statistical analysis. The distribution of NOS m R N A in the PVN of lactating and non-lactating rats is shown in Fig. 1. The quantitative analysis data are presented in Fig. 2. In control female non-lactating rats, NOS m R N A was present in both the magnocellular and parvocellular component throughout the rostro-caudal extension of the PVN (Fig. 1A). When lactating rats were examined, a significant increase in NOS m R N A was observed (Figs. 1C,2). The increase was seen in all subdivisions of the magnocellular and parvocellular regions. In the magnocellular component, the most dramatic difference was in the posterior part (Fig. 1C) while, in the parvocellular region, the most strongly labeled cells could be seen in the medial part (Fig. 1D). When film autoradiographs were analysed for quantification, a 40% increase was evident (Fig. 2).
The present results describe an increase in NOS m R N A in the magnocellular and parvocellular PVN of lactating rats. The hypothalamic PVN, with both its components, has a crucial role in control of milk ejec2OO P (=
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Fig. 2. Pmalysis o f effect o f lactation on NOS m R N A in P V N o f control (con) and lactating (lact) rats. Data are expressed as percentage o f control. Statistical analysis was carried out with Student's
tion during lactation. The magnocellular part, together with the supraoptic nucleus, is the main site of oxytocin production TM with a major projection to the posterior pituitary. There is also an oxytocinergic autonomic-related projection from the P V N to the medulla oblongata 17 which seems to modulate autonomic functions 8. Both systems are involved in milk ejection 9. The parvocellular P V N is the site of production of several neuropeptides 19 and for some of them, such as TRH, a role in control of PRL secretion has clearly been established 15. The presence of NOS in a nucleus of importance for several neuroendocrine functions suggests that NO might be involved in controlling pituitary hormone secretion. The effect of NO might be related to its well-established vasodilatatory action which could be exerted near the portal blood vessels in the median eminence or in the posterior pituitary. In this way, the amount of hormones secreted by the posterior pituitary or secreted at the median eminence level and then reaching the anterior pituitary could be modulated. NO could also control hormone release from the pituitary gland acting directly as a co-releasing factor or indirectly through an influence on PVN cells in a autocrine/paracrine fashion. Another possibility is that NO may influence local blood circulation in the PVN. The hypothesis of NO regulating hypothalamic-hypophyseal blood flow is supported by the well-known vasodilatory action of N O 16 and by the presence of a dense NOS innervation around blood vessels at the median eminence level 7. The upregulation of NOS gene expression seen during lactation could then be related to the increase in PVN and pituitary hormone secretion. Moreover, lactation is not the only physiological stimulus causing an increase in NOS since it has been shown that also stress upregulates NOS mRNA in the PVN 6. This could indicate that the increase in NOS mRNA is part of a general response to stimuli causing an increase in PVN activity. The present data suggest a possible involvement of NO in controlling milk ejection. Whether the mechanism is directly related to hormone secretion and/or to blood flow regulation remains to be clarified. 1 Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615624. 2 Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed,
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