- Email: [email protected]
Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons
Brain Research 839 Ž1999. 164–172 www.elsevier.comrlocaterbres
Research report
Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons Marco Virgili, Barbara Monti, Anna LoRusso, Monia Bentivogli, Antonio Contestabile
)
Department of Biology, UniÕersity of Bologna, Via Selmi 3, 40126 Bologna, Italy Accepted 15 June 1999
Abstract The diffusible chemical messenger nitric oxide ŽNO. is involved in neuronal plasticity and it is, therefore, supposed to play a role in brain development. A shortage of NO during the critical period of brain maturation may theoretically have long-lasting consequences on the organization of the adult brain. We have performed in neonatal rats a chronic inhibition of the enzyme responsible for NO production, nitric oxide synthase ŽNOS., from postnatal day 3 to postnatal day 23, through administration of the competitive antagonist N-nitro-L-arginine methylester ŽL-NAME.. The calcium-dependent catalytic activity resulted almost completely inhibited throughout the period of treatment and it took more than 4 days after its suspension to get a full recovery. The expression of the neuronal isoform of the enzyme ŽnNOS., revealed by immunoblotting, was unchanged during the treatment and after it. The histochemical reaction for NADPH diaphorase was reduced at the end of the treatment and recovered in concomitance with the recovery of the catalytic NOS activity. No gross structural alterations were detected in brain morphology. The levels of three neurotransmitter-related and one astrocytic marker were unchanged in the cerebellum, hippocampus and cortex of 60-day-old rats which had been neonatally treated. A similar lack of significant effects on neurochemical brain maturation was also noticed in a parallel series of experiments, in which a short pulse of NOS inhibition was performed at a critical prenatal time of brain development, from gestational day 14 to gestational day 19. In vitro, chronic exposure of cerebellar granule cells to L-NAME Ž500 mM. resulted in slight decrease of surviving neurons after 8 days in culture and in better resistance to the challenge of stressful culture conditions. The present results suggest that the basic plan of brain organization can be achieved despite an almost complete NOS inhibition during the maturation period. In vitro, NOS inhibition may bring to more pronounced consequences on neuronal viability and function. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Brain development; Nitric oxide; Nitric oxide synthase inhibitor; Cerebellar granule cell culture; Neurochemistry
1. Introduction The diffusible free radical nitric oxide ŽNO. is a cell-derived chemical messenger in several systems, including the immune, vascular and nervous ones. The role of NO as a novel biological messenger w4,22x and its involvement in brain development and function w13x is, at present, substantiated by a rather extensive evidence. NO-dependent processes in the nervous system include synaptogenesis, modulation of neurotransmitter release, synaptic plasticity and cell death, suggesting that this simple molecule plays important roles in the development, maintenance and functional modifications of brain circuits w7,13–15,33x. Experimental evidence for a role of NO in brain development is based on profiles of NO-related markers during normal ) C orresponding author. [email protected]
Fax:
q 39-51-251208;
E-m ail:
development w4,18,19,24,32,42x as well as in conditions of experimental manipulation of the development w37,38, 41,42x. Surprisingly enough, knockout mice for the neuronal isoform of the synthesizing enzyme, nitric oxide synthase ŽnNOS., do not show any patent alteration in brain structure and function w17x even if they are abnormal in aggressive and sexual behaviour w26x and their neurons are less sensitive to excitotoxic damage w21x. Contrasting results have also arisen from studies based on pharmacological manipulation of NO–NOS system during critical periods of brain development. In both mammals and in the chicken, some aspects of the normal pattern of retinal projections to the brain were altered upon chronic treatment with NOS inhibitors at critical times for synaptic refinement w6,44x. However, a recent study based on pharmacological NOS inhibition associated with knockout technology w9x concluded that NO was not essential for the patterning of either visual or somatosensory thalamocorti-
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 7 5 0 - 3
M. Virgili et al.r Brain Research 839 (1999) 164–172
cal connections. Furthermore, other reports were not able to demonstrate clear effects of chronic NOS blockade during development on projection topography w25x, learning and memory w43x and cell survival w39x. Very recently w30x chronic administration of L-NAME to rat pups from birth to 24 days of age, was reported to have no effects on brain morphology nor on animal behaviour in adults. In most cases, however, the actual extent of the inhibition of the catalytic NOS activity was not measured during the treatment and the recovery of it was not assessed upon suspension of the treatment. In the present study, we have performed a chronic long term inhibition of NOS during the critical period of structural, functional and neurochemical maturation of the brain and we have checked for the effective block of the catalytic activity as well as for the expression of the neuronal NOS isoform during the period of its functional inactivation. Both parameters, as well as NADPH histochemistry, were also determined after suspension of the treatment in order to assess the recovery of the enzyme function. Having established the extent of the inhibition of NOS catalytic activity, we have performed a regional survey of some neurochemical markers whose possible alteration in the adult could have been taken as a long-term effect of the neonatal treatment. The same neurochemical markers were evaluated in young–adult rats subjected to a short pulse of NOS inhibition during a critical period for brain gestational development. In parallel experiments, we have estimated the effect of the chronic exposure to a NOS inhibitor on the survival and differentiation of cerebellar granule cells in primary culture.
2. Materials and methods 2.1. In ÕiÕo experiments Wistar rat pups were treated from postnatal day 3 to postnatal day 23 Žday of birth being counted as postnatal day 1. with a total dosage of 60 mg kgy1 dayy1 of N-nitro-L-arginine methylesther ŽL-NAME., dissolved in saline and delivered subcutaneously divided into two daily administrations at 8 AM and 8 PM. Control pups received an equivalent volume of the saline only. At different intervals from the beginning of the treatment, at the end of it and at different times after the end of it, animals were killed by decapitation, the cerebellum and the whole forebrain were rapidly dissected out and stored in the deep freezer until assayed. During and at the end of the treatment, samples were always collected 10–12 h after the last administration of L-NAME. For the determination of the calcium-dependent NOS activity w3x, the tissue was homogenized in 50 mM Tris– HCl buffer at pH 7.4 containing 2 mM EDTA and centrifuged at 11,000 = g for 15 min. Aliquots of the supernatant, containing 10–20 mg of protein in 25 ml, were
165
added with a solution containing 3 H-arginine Žspecific activity 40 Cirmmol, 19 nM final concentration; NEN Dupont., b-NADPH Ž0.7 mM final concentration. and CaCl 2 Ž0.4 mM final concentration. in a total volume of 135 ml. Incubation was started by addition of CaCl 2 , lasted for 15 min at 258C and was blocked by addition of excess 20 mM HEPES buffer ŽpH 5.5. containing 2 mM EDTA. Blanks were incubated without adding calcium. The product of the enzymatic reaction, 3 H-citrulline was collected in scintillation vials by chromatography on Dowex 50 W = 8 resin Žsodium form. and counted in 5 ml of an appropriate scintillation cocktail. Protein content of the samples was determined colorimetrically w23x. For detection of nNOS protein through immunoblotting, the tissue was homogenized in 50 mM Tris–HCl buffer containing 1 mM EDTA, trypsin inhibitor, aprotinin and leupeptin Ž10 mgrml each. and centrifuged at 30,000 = g for 30 min at 48C. Aliquots of centrifugation supernatant, containing 100 mg of protein, were loaded in each lane of a 10% acrylamide gel in SDS for electrophoretic separation. The samples were then transferred to a Hybond-C-extra nitrocellulose membrane ŽAmersham. and incubated overnight at 48C with a polyclonal antibody raised in rabbits against rat nNOS ŽEuro-Diagnostica, B 220-1. diluted 1:2000. The immunoreaction was visualized by the ABC technique and intensified. The cerebellum, hippocampus and cerebral cortex, freshly dissected from the brain of young adult rats Ž60 days old., belonging to the neonatally L-NAME- or salinetreated groups, were homogenized in 0.32 M sucrose Ž10 strokes at 1000 rpm., in glass tubes with a teflon pestle. Aliquots were used to measure 3 H D-aspartate high affinity uptake w20x. The remaining homogenate was added with Triton X-100 Ž0.5% final concentration. and used to assay choline acetyltransferase ŽChAT. w10x, glutamate decarboxylase ŽGAD. w11x and glutamine synthetase ŽGS. w29x. In a parallel series of experiments, the same neurochemical markers were measured in the cerebellum, hippocampus and cerebral cortex of 40-day-old rat, whose mother had been subjected to NOS inhibition for a short period during gestation. To this aim, pregnant females were injected subcutaneously with 60 mg kgy1 dayy1 L-NAME, divided into two daily administrations, or saline, from gestational day 14 to gestational day 19. The beginning of pregnancy of these rats was ascertained from the presence of sperms in the vaginal smear after mating. For the histochemical localization of NADPH-diaphorase activity, L-NAME- and saline-treated rats at 23, 29 and 60 days of age were anaesthetized with ether and perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. Brains were removed, post-fixed overnight in the same fixative and washed in buffered 15% sucrose until sunk to the bottom. Parasagittal sections of the cerebellum and transverse sections of the forebrain were cut with a freezing microtome at 40-mm thickness. After permeabilization for 5 min with 0.1% Triton X-100
166
M. Virgili et al.r Brain Research 839 (1999) 164–172
in order to prevent proliferation of non-neuronal cells. While doing so, half of the cultures were added with a concentrated solution of L-NAME to bring it to a final concentration of 500 mM and the other cultures were added with the same volume of the solvent. A further addition of L-NAME was made at the end of the fourth day in culture and treated and control cultures were maintained up to the 8th day. At this stage, some of the cultures were shifted for 16 h to a fresh serum-free medium containing 5 mM KCl with L-NAME Žfinal concentration 500 mM. being added to those cultures which had been previously exposed chronically to the drug. At various stages of culture, viable cells were stained with fluorescein diacetate, observed and photographed with a fluorescence microscope. Survival of cells was evaluated by counting stained granule neurons in standard photographic prints.
3. Results 3.1. In ÕiÕo experiments
Fig. 1. Developmental pattern of calcium dependent NOS activity in the cerebellum and the forebrain of control rats and effect of the chronic treatment with L-NAME from postnatal day 3 to postnatal day 23. Each point is the mean of 6–10 experiments"S.E.
The chronic neonatal L-NAME treatment had a modest effect on body weight gain, treated animals showing a 10% decrease in body weight as compared to controls on day 23. This difference was completely recovered in young– adult rats Ž60 days old.. Furthermore, no patent alterations of behaviour or motor activity were noticed in the treated rats. The schedule of L-NAME administration adopted for the present study provided an almost complete inhibition of the calcium-dependent NOS activity in both the cerebellum and the forebrain throughout the entire period of treatment ŽFig. 1.. The inhibitory potency of the treatment was essentially the same, or even higher, as compared to
in PBS, NADPH-diaphorase activity was revealed by incubation for 45 min at 378C in PBS containing 0.1 mgrml nitro blue tetrazolium and 1 mgrml b-NADPH w36x. Some of the sections were Nissl-stained in order to appreciate the general architecture of the various brain regions. Experiments were performed according to the Italian law on the use of experimental animals after having been approved by a local bioethical committee, under the supervision of a veterinary committee to control animal care and comfort. 2.2. In Õitro experiments Cerebella were explanted from 8-day-old rat pups and cultures of dissociated cells were prepared as described w12,31x and plated at a density of 2.4 = 10 6rdish in polyL-lysine-coated 35 mm dish in basal modified Eagle’s medium ŽBME. containing 10% heat inactivated calf serum, 2 mM glutamine, gentamicin Ž100 mgrml. and 25 mM KCl. Cultures were placed in a humidified incubator at 378C under 95% airr5% CO 2 atmosphere. Cytosine arabino-furanoside Ž10 mM. was added 16 h after plating
Fig. 2. Immunoblotting for nNOS extracted from the forebrain of control ŽC. and L-NAME-treated ŽN. rats at postnatal days 23 Ž23d. and 29 Ž29d..
M. Virgili et al.r Brain Research 839 (1999) 164–172
167
Fig. 3. NADPH-diaphorase positive neurons in the striatum and the cortex of control and L-NAME-treated rats at postnatal days 23 and 29. ŽA. Striatum of a control rat at postnatal day 23; ŽB. striatum of a treated rat at postnatal day 23; ŽC. cortex of a control rat at postnatal day 23; ŽD. cortex of a treated rat at postnatal day 23; ŽE. striatum of a control rat at postnatal day 29; ŽF. striatum of a treated rat at postnatal day 29; ŽG. cortex of a control rat at postnatal day 29; ŽH. cortex of a treated rat at postnatal day 29. Calibration bar, 80 mm.
M. Virgili et al.r Brain Research 839 (1999) 164–172
168
Table 1 Neurochemical markers after neonatal NOS inhibition Brain regions
Cortex Hippocampus Cerebellum
ChAT Žmmol Žg prot.y1 hy1 .
GAD Žmmol Žg prot.y1 hy1 .
GLN SYNTH Žmmol Žg prot.y1 hy1 .
D-Asp
uptake Žpmol Žmg prot.y1 Ž3 min.y1 .
Control
NAME
Control
NAME
Control
NAME
Control
NAME
39.9 " 1.0 71.1 " 6.4 4.7 " 0.3
45.6 " 3.7 65.5 " 3.3 4.7 " 0.3
170.0 " 10.1 131.6 " 11.1 106.9 " 4.0
167.4 " 7.5 150.8 " 9.9 111.9 " 6.1
1.84 " 0.05 1.47 " 0.05 1.86 " 0.03
2.05 " 0.14 1.59 " 0.08 1.77 " 0.08
471.5 " 23.6 517.9 " 40.7 62.2 " 6.2
478.5 " 34.0 632.8 " 19.0 56.2 " 3.3
Samples were taken from brains of 60-day-old rats subjected to the neonatal ŽP3–P23. treatment with L-NAME or saline-injected controls. Values are the mean " S.E. of six experiments.
adult rats in which 6 days of administration of the same dosage of L-NAME resulted in comparable inhibition of calcium-dependent NOS activity Žcerebellum: controls, 91.5 " 1.8, treated, 7.2 " 0.5; forebrain: controls 24.5 " 3.4, treated, 1.3 " 0.1 pmol Žmg prot.y1 hy1 ; N s 6.. At the end of the pharmacological treatment, the recovery of the enzymatic activity was relatively slow and it took more than 4 days before that the values of control animals were reached by treated rats ŽFig. 1.. In young–adults, no differences were noticed in the calcium-dependent NOS activity measured in the cerebellum and forebrain of rats belonging to the control or the experimental group, the cerebellar activity being significantly increased and the forebrain activity somewhat decreased, as compared to the previous stages ŽFig. 1.. The inhibitory action of L-NAME was a true competitive inhibition at the catalytic site of the enzyme, as predicted by the pharmacology of the compound, since we were unable to observe any significant alteration in the expression of the protein itself. Western blotting, carried out on protein extracts from the cerebellum Žnot shown. and the forebrain ŽFig. 2. of control and treated rats using a specific antibody against nNOS, did not reveal any clear decrease of the protein expression at the middle of the treatment Žnot shown. nor at the end of it ŽFig. 2. or 6 days after the end of the treatment ŽFig. 2.. The histochemical reaction for NADPH diaphorase gave a strongly reduced stain of positive brain structures at postnatal day 23, while the intensity of the reaction was completely recovered on day 29. This was true concerning
the reaction present in the molecular and the granular layer of the cerebellum Žnot shown. as well as the strong positivity of individual neurons localized in the striatum ŽFig. 3A,B,E,F. and the cerebral cortex ŽFig. 3C,D,G,H.. The lack of differences between control and treated animals was also conserved in adult animals Žnot shown.. In agreement with a previous report w30x based on a similar schedule of L-NAME administration, Nissl-stained sections did not reveal any gross abnormality in the cytoarchitecture in the various brain regions of young–adult rats Žnot shown.. To test whether the chronic neonatal blockade of NOS activity was detrimental to the neurochemical brain maturation, we assayed in the cerebellum, hippocampus and cortex of adult control and neonatally-treated rats, the activity of three neurotransmitter-related and one astrocytic marker. In all the three regions, a marker for cholinergic neurons ŽChAT., a marker for GABAergic neurons ŽGAD., a marker for glutamatergic neurons Ž3 H-D-aspartate high affinity uptake. and an astrocytic marker ŽGS. appeared not significantly affected by the neonatal treatment ŽTable 1.. Similar results were obtained in experiments carried on young–adult rats subjected to a short period of NOS inhibition during pregnancy, from gestational day 14 to gestational day 19 ŽTable 2.. 3.2. In Õitro experiments Chronic exposure of cerebellar granule cell cultures to Ž500 mM. had a moderately negative effect on
L-NAME
Table 2 Neurochemical markers after gestational NOS inhibition Brain regions
Cortex Hippocampus Cerebellum
ChAT Žmmol Žg prot.y1 hy1 .
GAD Žmmol Žg prot.y1 hy1 .
GLN SYNTH Žmmol Žg prot.y1 hy1 .
D-Asp
uptake Žpmol Žmg prot.y1 Ž3 min.y1 .
Control
NAME
Control
NAME
Control
NAME
Control
NAME
32.1 " 1.4 57.3 " 2.4 3.21 " 0.16
34.2 " 0.81 53.1 " 1.5 3.20 " 0.13
142 " 4.0 138 " 4.0 79 " 3.0
150 " 4.0 142 " 4.0 79 " 2.0
2.29 " 0.07 2.32 " 0.09 1.62 " 0.09
2.39 " 0.05 2.40 " 0.08 1.79 " 0.07
276.4 " 23.0 360.2 " 42.8 49.8 " 6.0
312.2 " 9.9 379.1 " 32.5 40.5 " 6.7
Samples were taken from brains of 40-day-old rats whose mothers had been treated with L-NAME or saline from gestational day 14 to gestational day 19. Values are the mean " S.E. of eight experiments.
M. Virgili et al.r Brain Research 839 (1999) 164–172
169
Fig. 4. Effect of exposure to L-NAME on survival of cerebellar granule cells in culture after 4 or 8 days in vitro ŽDIV.. Bars are the mean" S.E. of 18–24 cell counting performed on at least 8 dishes from four different cultures. UU p - 0.01, student’s t-test, as compared to control.
the survival of granule neurons since their density was slightly decreased, as compared to control cultures, after 8
days in vitro Žbut not after 4 days in vitro. ŽFig. 4.. However, these cultures did not show any obvious differ-
Fig. 5. Cerebellar granule cells after 8 days in vitro under control conditions Ž5A. or after chronic exposure to 500 mM L-NAME Ž5B. and effect of shifting the cultures for 16 h to a low Kq medium Ž5C, control; 5D, exposed to L-NAME.. Note the lower density of granule neurons after chronic exposure to q L-NAME and the reduced effect of shifting these cultures to a low K medium as compared to control cultures. Calibration bar, 60 mm.
170
M. Virgili et al.r Brain Research 839 (1999) 164–172
ence, as compared to control cultures, with respect to the morphology and progressive differentiation of granule neurons ŽFig. 5A,B.. Other features characterizing mature granule neurons in culture could have been modified by the treatment: for instance, shifting 8-day-old cultures from a medium containing 25 mM Kq to a medium containing 5 mM Kq for 16 h, resulted in substantial elimination of granule neurons in control cultures Ž5C., while the number of viable cells was much less affected in cultures exposed to L-NAME Ž5D., taking into account the fact that the total cell density was lower in these cultures.
4. Discussion The present study shows that the catalytic activity of NOS can be almost completely inhibited during the critical period of brain development without dramatic consequences on the synthesis of the enzymatic protein during the pharmacological treatment and the recovery of activity after its suspension. This prolonged blockade did not result in any appreciable alteration of the final neurochemical maturation of the brain, at least concerning some relevant markers previously studied by us during development w40x. This parallels the absence of gross alterations of brain morphology, previously noticed after a similar schedule of treatment w30x, and confirmed by us in the present study. This result adds uncertainty to the exact role that NO may play in synaptic plasticity during development. Since LNAME is a competitive inhibitor acting at the enzyme catalytic site w8x, and its action is unspecific with regards to the different NOS isoforms w21x, it is unlikely that a substantial production of NO could have occurred under a tight schedule of inhibitor administration such as the one used in the present study. This seems to exclude a mutual compensation through other NOS isoforms as it may occur in knockout mice lacking the expression of a single isoform w16x. Different timing of NOS inhibition could theoretically bring to different long-term effects, since the critical period may be not completely covered by the treatment or compensatory mechanisms may occur with prolonged inhibition. We have, therefore, performed additional experiments, in which NOS was inhibited during a short gestational period critical for the expression of cerebral nNOS w4x, from gestational day 14 to gestational day 19. The results obtained with these experiments, however, suggested that also this pulse treatment had no gross consequences on the neurochemical maturation of the brain in the long run. The critical processes mediated by NO production during development may be too subtle, even if physiologically relevant, to be detected by observations aimed at outlining gross alterations of brain structure w30x and neurochemistry Žpresent results. or of complex behaviours w43x. The most convincing results so far produced on NO role in brain
maturation based on NOS inhibition, are those demonstrating a derangement from the normal pattern of segregation of retinal input to the optic tectum in the chick and the lateral geniculate nucleus in the ferret w6,44x. When the focus was shifted to the thalamocortical connections, however, no evidence was found for an altered pattern of the visual or the somatosensory projections to the cortex, as a consequence of the chronic neonatal blockade of catalytic NO production w9x. This result, together with the fact that a similar lack of effect could be demonstrated in a cholinergic projection to the superior colliculus w25x, suggests that the functional interaction of NO system with synaptogenesis may be selective for some projections and not for others, probably in connection with the neurotransmitter– receptor combinations present at the synaptic levels. Our in vivo observations, demonstrate that also the diaphorase activity associated with NOS is strongly reduced by competitive NOS inhibitors and that full recovery occurs in concomitance with the recovery of the catalytic activity. It was shown before w28x that NOS inhibitors strongly inhibited the diaphorase activity associated with NOS in homogenates of chicken retina. Furthermore, the diaphorase activity measured in tissue from Drosophila was inhibited by excess of the natural substrate, arginine w27x. Concerning histochemical detection, pre-incubation of formaldehyde-fixed frozen sections with L-nitro arginine impaired the NADPH diaphorase reaction in spinal cord neurons w2x. In line with those previous data, our present results suggest that the diaphorase activity related to NOS is impaired when the catalytic site for NO production is occupied. Our present in vitro observations demonstrate that blockade of NO production is slightly detrimental to survival of cerebellar granule cells in culture, without apparently affecting their morphological differentiation. This modest effect, must take into account the fact that under the standard culture conditions Ži.e., high Kq concentration., nNOS may be downregulated in granule cells w1x. Tanaka et al. w35x previously demonstrated alterations in granule cell proliferation and migration in in vitro cultures of cerebellar slices exposed to L-nitro arginine. We also show here that NOS inhibition may protect granule cells when they are exposed to stressful conditions, such as the shifting from high to low Kq. Inhibition of NOS has been previously shown to partially protect cerebellar granule neurons from excitotoxic damage w5x as well as from spermine-mediated toxicity w34x. In conclusion, the present results suggest that relevant aspects of brain organization are not affected by an almost complete inhibition of NOS during the critical period of maturation. Therefore, the role of NO in developmental brain plasticity should be related to a fine tuning of neural circuits and subtle aspects of the stabilization and modification of synaptic connections. In in vitro conditions, the effects of NOS inhibition, may be more pronounced and affect basic aspects of neuronal viability and function.
M. Virgili et al.r Brain Research 839 (1999) 164–172
Acknowledgements The present work was supported by grants from the University of Bologna and from the targeted project ‘‘Biotechnology’’ of the Italian National Research Council. The skilful technical help of Luciana di Pietrangelo in preparing photographic illustrations is gratefully acknowledged.
References w1x S.L. Baader, K. Schilling, Glutamate receptors mediate dynamic regulation of nitric oxide synthase expression in cerebellar granule cells, J. Neurosci. 16 Ž1996. 1440–1449. w2x D. Blottner, H.G. Baumgarten, L-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons, Histochem. Cell Biol. 103 Ž1995. 379–385. w3x D.S. Bredt, S.H. Snyder, Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A. 86 Ž1990. 9030–9033. w4x D.S. Bredt, S.H. Snyder, Nitric oxide, a physiological messenger molecule, Annu. Rev. Biochem. 63 Ž1994. 175–195. w5x E. Ciani, L. Groneng, M. Voltattorni, R. Veslemoy, A. Contestabile, R.E. Paulsen, Inhibition of free radical production or free radical scavenging protects from excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons, Brain Res. 728 Ž1996. 1–6. w6x K.S. Cramer, A. Angelucci, J.O. Hahm, M.B. Bogdanov, M. Sur, A role for nitric oxide in the development of the ferret retinogeniculate projection, J. Neurosci. 15 Ž1996. 7995–8004. w7x T.M. Dawson, S.H. Snyder, Gases as biological messengers: nitric oxide and carbon monoxide in the brain, J. Neurosci. 14 Ž1994. 5147–5159. w8x V.L. Dawson, T.M. Dawson, Function of nitric oxide in neuronal cell death, in: M.D. Maines ŽEd.., Nitric Oxide Synthase: Characterization and Functional Analysis, Academic Press, San Diego, 1996, pp. 228–240. w9x E.M. Finney, C.J. Shatz, Establishment of patterned thalamocortical connections does not require nitric oxide synthase, J. Neurosci. 18 Ž1998. 8826–8838. w10x F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 Ž1975. 407–409. w11x F. Fonnum, I. Grofova, E. Rinvik, J. Storm-Mathiesen, F. Walberg, Origin and distribution of glutamate decarboxylase in the substantia nigra of the cat, Brain Res. 71 Ž1974. 77–92. w12x V. Gallo, A. Kingsbury, R. Balazs, O.S. Jorgensen, The role of depolarization in the survival and differentiation of cerebellar granule cells in culture, J. Neurosci. 7 Ž1987. 2203–2213. w13x J.A. Gally, R.P. Montague, G.N. Reeke, G.E. Edelman, The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 3547–3551. w14x J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 Ž1995. 683–706. w15x C. Holscher, Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity, Trends Neurosci. 20 Ž1997. 298–303. w16x P.L. Huang, M.C. Fishman, Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice, Mol. Med. 74 Ž1996. 415–421. w17x P.L. Huang, T.M. Dawson, D.S. Bredt, S.H. Snyder, M.C. Fischman, Targeted disruption of the neuronal nitric oxide synthase gene, Cell 75 Ž1993. 1268–1271. w18x G. Keilhoff, B. Seidel, H. Noak, W. Tischmeyer, D. Stanek, Patterns of nitric oxide synthase at the messenger RNA and protein levels during early brain development, Neuroscience 75 Ž1996. 1193–1201.
171
w19x B. Krekelberg, J.G. Taylor, Nitric oxide in cortical map formation, J. Chem. Neuroanat. 10 Ž1996. 191–196. w20x I. Kvale, V.M. Fosse, F. Fonnum, Development of neurotransmitter parameters in lateral geniculate body, superior colliculus and visual cortex of the albino rat, Dev. Brain Res. 7 Ž1983. 137–145. w21x C. Iadecola, Bright and dark sides of nitric oxide in ischemic brain injury, Trends Neurosci. 20 Ž1997. 132–139. w22x C.J. Lowenstein, S.H. Snyder, Nitric oxide, a novel biological messenger, Cell 70 Ž1992. 705–707. w23x O.H. Lowry, R.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. w24x N. Mitrovic, M. Schachner, Transient expression of NADPH diaphorase activity in the mouse whisker to barrel field pathway, J. Neurocytol. 25 Ž1996. 429–437. w25x R.R. Mize, C.A. Scheiner, M.F. Salvatore, R.J. Cork, Inhibition of nitric oxide synthase fails to disrupt the development of cholinergic fiber patches in the rat superior colliculus, Dev. Neurosci. 19 Ž1997. 260–273. w26x R.J. Nelson, G.E. Demas, P.L. Huang, M.C. Fishman, W.L. Dawson, T.M. Dawson, S.H. Snyder, Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase, Nature 378 Ž1995. 383–386. w27x T.I. Oparina, S.V. Myl’nikov, The effect of L-arginine on the NADPH-diaphorase activity in Drosophila melanogaster homogenates, Zh. Evol. Biokhim. Fiziol. 32 Ž1996. 217–219. w28x R. Paes de Carvalho, M.H. de Faria, J.L.M. do Nascimiento, J.N. Hokoc, Development of NADPH-diaphorase in the avian retina: regulation by calcium ions and relation to nitric oxide synthase, J. Neurochem. 67 Ž1996. 1063–1071. w29x A.J. Patel, A. Hunt, C.S.M. Tahourdin, Regional development of glutamine synthetase activity in rat brain and its association with the differentiation of astrocytes, Dev. Brain Res. 8 Ž1983. 31–37. w30x J. Prickaerts, J. De Vente, M. Markerink-Van Ittersum, H.W.M. Steinbusch, Behavioural, neurochemical and neuroanatomical effects of chronic postnatal N-nitro-L-arginine methyl ester treatment in neonatal and adult rats, Neuroscience 87 Ž1998. 181–195. w31x A. Resink, N. Hack, G.J. Boer, R. Balazs, Growth conditions differentially modulate the vulnerability of developing granule cells to excitatory amino acids, Brain Res. 655 Ž1994. 222–232. w32x N.L. Scholz, E.S. Chang, K. Graubard, J.W. Truman, The NOrcGMP pathway and the development of neural networks in postembryonic lobsters, J. Neurobiol. 34 Ž1998. 208–226. w33x E.M. Schuman, D.V. Madison, Nitric oxide and synaptic function, Annu. Rev. Neurosci. 17 Ž1994. 153–183. w34x M. Sparapani, R. Dall’Olio, O. Gandolfi, E. Ciani, A. Contestabile, Neurotoxicity of polyamines and pharmacological neuroprotection in cultures of rat cerebellar granule cells, Exp. Neurol. 148 Ž1997. 157–166. w35x M. Tanaka, S. Yoshida, M. Yano, F. Hanaoka, Roles of endogenous nitric oxide in cerebellar cortical development in slice cultures, NeuroReport 5 Ž1994. 2049–2052. w36x E. Thomas, A.G.E. Pearse, The solitary active cells: histochemical demonstration of damage-resistant nerve cells with a TNP-diaphorase reaction, Acta Neuropathol. 3 Ž1964. 238–249. w37x M. Tregnago, M. Virgili, B. Monti, T. Guarnieri, A. Contestabile, Alteration of neuronal nitric oxide synthase activity and expression in the cerebellum and the forebrain of microencephalic rats, Brain Res. 793 Ž1998. 54–60. w38x R.R. Vaid, B.K. Yee, U. Shalev, J.N. Rawlins, I. Weiner, J. Feldon, S. Totterdell, Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon’s horn of rats, J. Neurosci. 17 Ž1997. 5599–5609. w39x F.L. Van Muiswinkel, B. Drukarch, H.W. Steinbusch, J. De Vente, Sustained pharmacological inhibition of nitric oxide synthase does
172
M. Virgili et al.r Brain Research 839 (1999) 164–172
not affect the survival of intrastriatal rat fetal mesencephalic transplants, Brain Res. 792 Ž1998. 48–58. w40x M. Virgili, O. Barnabei, A. Contestabile, Regional maturation of neurotransmitter-related and glial markers during postnatal development in the rat, Int. J. Dev. Neurosci. 8 Ž1990. 156–159. w41x M. Virgili, F. Facchinetti, M. Sparapani, M. Tregnago, R. Lucchi, R. Dall’Olio, O. Gandolfi, A. Contestabile, Neuronal nitric oxide synthase is permanently decreased in the cerebellum of rats subjected to chronic neonatal blockade of NMDA receptors, Neurosci. Lett., in press.
w42x C.V. Williams, D. Nordquist, S.C. McLoon, Correlation of nitric oxide synthase expression with changing patterns of axonal projections in the developing visual system, J. Neurosci. 14 Ž1994. 1746– 1755. w43x G. Wortwein, B. Gustafson, K.L. Hansen, J. Mogensen, Behavioural symptoms in adult rats after postnatal L-nitro-arginine, Int. J. Dev. Neurosci. 15 Ž1997. 147–154. w44x H.H. Wu, C.V. Williams, S.C. McLoon, Involvement of nitric oxide in the elimination of a transient retinotectal projection in development, Science 265 Ž1994. 1593–1596.
Research report
Developmental effects of in vivo and in vitro inhibition of nitric oxide synthase in neurons Marco Virgili, Barbara Monti, Anna LoRusso, Monia Bentivogli, Antonio Contestabile
)
Department of Biology, UniÕersity of Bologna, Via Selmi 3, 40126 Bologna, Italy Accepted 15 June 1999
Abstract The diffusible chemical messenger nitric oxide ŽNO. is involved in neuronal plasticity and it is, therefore, supposed to play a role in brain development. A shortage of NO during the critical period of brain maturation may theoretically have long-lasting consequences on the organization of the adult brain. We have performed in neonatal rats a chronic inhibition of the enzyme responsible for NO production, nitric oxide synthase ŽNOS., from postnatal day 3 to postnatal day 23, through administration of the competitive antagonist N-nitro-L-arginine methylester ŽL-NAME.. The calcium-dependent catalytic activity resulted almost completely inhibited throughout the period of treatment and it took more than 4 days after its suspension to get a full recovery. The expression of the neuronal isoform of the enzyme ŽnNOS., revealed by immunoblotting, was unchanged during the treatment and after it. The histochemical reaction for NADPH diaphorase was reduced at the end of the treatment and recovered in concomitance with the recovery of the catalytic NOS activity. No gross structural alterations were detected in brain morphology. The levels of three neurotransmitter-related and one astrocytic marker were unchanged in the cerebellum, hippocampus and cortex of 60-day-old rats which had been neonatally treated. A similar lack of significant effects on neurochemical brain maturation was also noticed in a parallel series of experiments, in which a short pulse of NOS inhibition was performed at a critical prenatal time of brain development, from gestational day 14 to gestational day 19. In vitro, chronic exposure of cerebellar granule cells to L-NAME Ž500 mM. resulted in slight decrease of surviving neurons after 8 days in culture and in better resistance to the challenge of stressful culture conditions. The present results suggest that the basic plan of brain organization can be achieved despite an almost complete NOS inhibition during the maturation period. In vitro, NOS inhibition may bring to more pronounced consequences on neuronal viability and function. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Brain development; Nitric oxide; Nitric oxide synthase inhibitor; Cerebellar granule cell culture; Neurochemistry
1. Introduction The diffusible free radical nitric oxide ŽNO. is a cell-derived chemical messenger in several systems, including the immune, vascular and nervous ones. The role of NO as a novel biological messenger w4,22x and its involvement in brain development and function w13x is, at present, substantiated by a rather extensive evidence. NO-dependent processes in the nervous system include synaptogenesis, modulation of neurotransmitter release, synaptic plasticity and cell death, suggesting that this simple molecule plays important roles in the development, maintenance and functional modifications of brain circuits w7,13–15,33x. Experimental evidence for a role of NO in brain development is based on profiles of NO-related markers during normal ) C orresponding author. [email protected]
Fax:
q 39-51-251208;
E-m ail:
development w4,18,19,24,32,42x as well as in conditions of experimental manipulation of the development w37,38, 41,42x. Surprisingly enough, knockout mice for the neuronal isoform of the synthesizing enzyme, nitric oxide synthase ŽnNOS., do not show any patent alteration in brain structure and function w17x even if they are abnormal in aggressive and sexual behaviour w26x and their neurons are less sensitive to excitotoxic damage w21x. Contrasting results have also arisen from studies based on pharmacological manipulation of NO–NOS system during critical periods of brain development. In both mammals and in the chicken, some aspects of the normal pattern of retinal projections to the brain were altered upon chronic treatment with NOS inhibitors at critical times for synaptic refinement w6,44x. However, a recent study based on pharmacological NOS inhibition associated with knockout technology w9x concluded that NO was not essential for the patterning of either visual or somatosensory thalamocorti-
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 7 5 0 - 3
M. Virgili et al.r Brain Research 839 (1999) 164–172
cal connections. Furthermore, other reports were not able to demonstrate clear effects of chronic NOS blockade during development on projection topography w25x, learning and memory w43x and cell survival w39x. Very recently w30x chronic administration of L-NAME to rat pups from birth to 24 days of age, was reported to have no effects on brain morphology nor on animal behaviour in adults. In most cases, however, the actual extent of the inhibition of the catalytic NOS activity was not measured during the treatment and the recovery of it was not assessed upon suspension of the treatment. In the present study, we have performed a chronic long term inhibition of NOS during the critical period of structural, functional and neurochemical maturation of the brain and we have checked for the effective block of the catalytic activity as well as for the expression of the neuronal NOS isoform during the period of its functional inactivation. Both parameters, as well as NADPH histochemistry, were also determined after suspension of the treatment in order to assess the recovery of the enzyme function. Having established the extent of the inhibition of NOS catalytic activity, we have performed a regional survey of some neurochemical markers whose possible alteration in the adult could have been taken as a long-term effect of the neonatal treatment. The same neurochemical markers were evaluated in young–adult rats subjected to a short pulse of NOS inhibition during a critical period for brain gestational development. In parallel experiments, we have estimated the effect of the chronic exposure to a NOS inhibitor on the survival and differentiation of cerebellar granule cells in primary culture.
2. Materials and methods 2.1. In ÕiÕo experiments Wistar rat pups were treated from postnatal day 3 to postnatal day 23 Žday of birth being counted as postnatal day 1. with a total dosage of 60 mg kgy1 dayy1 of N-nitro-L-arginine methylesther ŽL-NAME., dissolved in saline and delivered subcutaneously divided into two daily administrations at 8 AM and 8 PM. Control pups received an equivalent volume of the saline only. At different intervals from the beginning of the treatment, at the end of it and at different times after the end of it, animals were killed by decapitation, the cerebellum and the whole forebrain were rapidly dissected out and stored in the deep freezer until assayed. During and at the end of the treatment, samples were always collected 10–12 h after the last administration of L-NAME. For the determination of the calcium-dependent NOS activity w3x, the tissue was homogenized in 50 mM Tris– HCl buffer at pH 7.4 containing 2 mM EDTA and centrifuged at 11,000 = g for 15 min. Aliquots of the supernatant, containing 10–20 mg of protein in 25 ml, were
165
added with a solution containing 3 H-arginine Žspecific activity 40 Cirmmol, 19 nM final concentration; NEN Dupont., b-NADPH Ž0.7 mM final concentration. and CaCl 2 Ž0.4 mM final concentration. in a total volume of 135 ml. Incubation was started by addition of CaCl 2 , lasted for 15 min at 258C and was blocked by addition of excess 20 mM HEPES buffer ŽpH 5.5. containing 2 mM EDTA. Blanks were incubated without adding calcium. The product of the enzymatic reaction, 3 H-citrulline was collected in scintillation vials by chromatography on Dowex 50 W = 8 resin Žsodium form. and counted in 5 ml of an appropriate scintillation cocktail. Protein content of the samples was determined colorimetrically w23x. For detection of nNOS protein through immunoblotting, the tissue was homogenized in 50 mM Tris–HCl buffer containing 1 mM EDTA, trypsin inhibitor, aprotinin and leupeptin Ž10 mgrml each. and centrifuged at 30,000 = g for 30 min at 48C. Aliquots of centrifugation supernatant, containing 100 mg of protein, were loaded in each lane of a 10% acrylamide gel in SDS for electrophoretic separation. The samples were then transferred to a Hybond-C-extra nitrocellulose membrane ŽAmersham. and incubated overnight at 48C with a polyclonal antibody raised in rabbits against rat nNOS ŽEuro-Diagnostica, B 220-1. diluted 1:2000. The immunoreaction was visualized by the ABC technique and intensified. The cerebellum, hippocampus and cerebral cortex, freshly dissected from the brain of young adult rats Ž60 days old., belonging to the neonatally L-NAME- or salinetreated groups, were homogenized in 0.32 M sucrose Ž10 strokes at 1000 rpm., in glass tubes with a teflon pestle. Aliquots were used to measure 3 H D-aspartate high affinity uptake w20x. The remaining homogenate was added with Triton X-100 Ž0.5% final concentration. and used to assay choline acetyltransferase ŽChAT. w10x, glutamate decarboxylase ŽGAD. w11x and glutamine synthetase ŽGS. w29x. In a parallel series of experiments, the same neurochemical markers were measured in the cerebellum, hippocampus and cerebral cortex of 40-day-old rat, whose mother had been subjected to NOS inhibition for a short period during gestation. To this aim, pregnant females were injected subcutaneously with 60 mg kgy1 dayy1 L-NAME, divided into two daily administrations, or saline, from gestational day 14 to gestational day 19. The beginning of pregnancy of these rats was ascertained from the presence of sperms in the vaginal smear after mating. For the histochemical localization of NADPH-diaphorase activity, L-NAME- and saline-treated rats at 23, 29 and 60 days of age were anaesthetized with ether and perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. Brains were removed, post-fixed overnight in the same fixative and washed in buffered 15% sucrose until sunk to the bottom. Parasagittal sections of the cerebellum and transverse sections of the forebrain were cut with a freezing microtome at 40-mm thickness. After permeabilization for 5 min with 0.1% Triton X-100
166
M. Virgili et al.r Brain Research 839 (1999) 164–172
in order to prevent proliferation of non-neuronal cells. While doing so, half of the cultures were added with a concentrated solution of L-NAME to bring it to a final concentration of 500 mM and the other cultures were added with the same volume of the solvent. A further addition of L-NAME was made at the end of the fourth day in culture and treated and control cultures were maintained up to the 8th day. At this stage, some of the cultures were shifted for 16 h to a fresh serum-free medium containing 5 mM KCl with L-NAME Žfinal concentration 500 mM. being added to those cultures which had been previously exposed chronically to the drug. At various stages of culture, viable cells were stained with fluorescein diacetate, observed and photographed with a fluorescence microscope. Survival of cells was evaluated by counting stained granule neurons in standard photographic prints.
3. Results 3.1. In ÕiÕo experiments
Fig. 1. Developmental pattern of calcium dependent NOS activity in the cerebellum and the forebrain of control rats and effect of the chronic treatment with L-NAME from postnatal day 3 to postnatal day 23. Each point is the mean of 6–10 experiments"S.E.
The chronic neonatal L-NAME treatment had a modest effect on body weight gain, treated animals showing a 10% decrease in body weight as compared to controls on day 23. This difference was completely recovered in young– adult rats Ž60 days old.. Furthermore, no patent alterations of behaviour or motor activity were noticed in the treated rats. The schedule of L-NAME administration adopted for the present study provided an almost complete inhibition of the calcium-dependent NOS activity in both the cerebellum and the forebrain throughout the entire period of treatment ŽFig. 1.. The inhibitory potency of the treatment was essentially the same, or even higher, as compared to
in PBS, NADPH-diaphorase activity was revealed by incubation for 45 min at 378C in PBS containing 0.1 mgrml nitro blue tetrazolium and 1 mgrml b-NADPH w36x. Some of the sections were Nissl-stained in order to appreciate the general architecture of the various brain regions. Experiments were performed according to the Italian law on the use of experimental animals after having been approved by a local bioethical committee, under the supervision of a veterinary committee to control animal care and comfort. 2.2. In Õitro experiments Cerebella were explanted from 8-day-old rat pups and cultures of dissociated cells were prepared as described w12,31x and plated at a density of 2.4 = 10 6rdish in polyL-lysine-coated 35 mm dish in basal modified Eagle’s medium ŽBME. containing 10% heat inactivated calf serum, 2 mM glutamine, gentamicin Ž100 mgrml. and 25 mM KCl. Cultures were placed in a humidified incubator at 378C under 95% airr5% CO 2 atmosphere. Cytosine arabino-furanoside Ž10 mM. was added 16 h after plating
Fig. 2. Immunoblotting for nNOS extracted from the forebrain of control ŽC. and L-NAME-treated ŽN. rats at postnatal days 23 Ž23d. and 29 Ž29d..
M. Virgili et al.r Brain Research 839 (1999) 164–172
167
Fig. 3. NADPH-diaphorase positive neurons in the striatum and the cortex of control and L-NAME-treated rats at postnatal days 23 and 29. ŽA. Striatum of a control rat at postnatal day 23; ŽB. striatum of a treated rat at postnatal day 23; ŽC. cortex of a control rat at postnatal day 23; ŽD. cortex of a treated rat at postnatal day 23; ŽE. striatum of a control rat at postnatal day 29; ŽF. striatum of a treated rat at postnatal day 29; ŽG. cortex of a control rat at postnatal day 29; ŽH. cortex of a treated rat at postnatal day 29. Calibration bar, 80 mm.
M. Virgili et al.r Brain Research 839 (1999) 164–172
168
Table 1 Neurochemical markers after neonatal NOS inhibition Brain regions
Cortex Hippocampus Cerebellum
ChAT Žmmol Žg prot.y1 hy1 .
GAD Žmmol Žg prot.y1 hy1 .
GLN SYNTH Žmmol Žg prot.y1 hy1 .
D-Asp
uptake Žpmol Žmg prot.y1 Ž3 min.y1 .
Control
NAME
Control
NAME
Control
NAME
Control
NAME
39.9 " 1.0 71.1 " 6.4 4.7 " 0.3
45.6 " 3.7 65.5 " 3.3 4.7 " 0.3
170.0 " 10.1 131.6 " 11.1 106.9 " 4.0
167.4 " 7.5 150.8 " 9.9 111.9 " 6.1
1.84 " 0.05 1.47 " 0.05 1.86 " 0.03
2.05 " 0.14 1.59 " 0.08 1.77 " 0.08
471.5 " 23.6 517.9 " 40.7 62.2 " 6.2
478.5 " 34.0 632.8 " 19.0 56.2 " 3.3
Samples were taken from brains of 60-day-old rats subjected to the neonatal ŽP3–P23. treatment with L-NAME or saline-injected controls. Values are the mean " S.E. of six experiments.
adult rats in which 6 days of administration of the same dosage of L-NAME resulted in comparable inhibition of calcium-dependent NOS activity Žcerebellum: controls, 91.5 " 1.8, treated, 7.2 " 0.5; forebrain: controls 24.5 " 3.4, treated, 1.3 " 0.1 pmol Žmg prot.y1 hy1 ; N s 6.. At the end of the pharmacological treatment, the recovery of the enzymatic activity was relatively slow and it took more than 4 days before that the values of control animals were reached by treated rats ŽFig. 1.. In young–adults, no differences were noticed in the calcium-dependent NOS activity measured in the cerebellum and forebrain of rats belonging to the control or the experimental group, the cerebellar activity being significantly increased and the forebrain activity somewhat decreased, as compared to the previous stages ŽFig. 1.. The inhibitory action of L-NAME was a true competitive inhibition at the catalytic site of the enzyme, as predicted by the pharmacology of the compound, since we were unable to observe any significant alteration in the expression of the protein itself. Western blotting, carried out on protein extracts from the cerebellum Žnot shown. and the forebrain ŽFig. 2. of control and treated rats using a specific antibody against nNOS, did not reveal any clear decrease of the protein expression at the middle of the treatment Žnot shown. nor at the end of it ŽFig. 2. or 6 days after the end of the treatment ŽFig. 2.. The histochemical reaction for NADPH diaphorase gave a strongly reduced stain of positive brain structures at postnatal day 23, while the intensity of the reaction was completely recovered on day 29. This was true concerning
the reaction present in the molecular and the granular layer of the cerebellum Žnot shown. as well as the strong positivity of individual neurons localized in the striatum ŽFig. 3A,B,E,F. and the cerebral cortex ŽFig. 3C,D,G,H.. The lack of differences between control and treated animals was also conserved in adult animals Žnot shown.. In agreement with a previous report w30x based on a similar schedule of L-NAME administration, Nissl-stained sections did not reveal any gross abnormality in the cytoarchitecture in the various brain regions of young–adult rats Žnot shown.. To test whether the chronic neonatal blockade of NOS activity was detrimental to the neurochemical brain maturation, we assayed in the cerebellum, hippocampus and cortex of adult control and neonatally-treated rats, the activity of three neurotransmitter-related and one astrocytic marker. In all the three regions, a marker for cholinergic neurons ŽChAT., a marker for GABAergic neurons ŽGAD., a marker for glutamatergic neurons Ž3 H-D-aspartate high affinity uptake. and an astrocytic marker ŽGS. appeared not significantly affected by the neonatal treatment ŽTable 1.. Similar results were obtained in experiments carried on young–adult rats subjected to a short period of NOS inhibition during pregnancy, from gestational day 14 to gestational day 19 ŽTable 2.. 3.2. In Õitro experiments Chronic exposure of cerebellar granule cell cultures to Ž500 mM. had a moderately negative effect on
L-NAME
Table 2 Neurochemical markers after gestational NOS inhibition Brain regions
Cortex Hippocampus Cerebellum
ChAT Žmmol Žg prot.y1 hy1 .
GAD Žmmol Žg prot.y1 hy1 .
GLN SYNTH Žmmol Žg prot.y1 hy1 .
D-Asp
uptake Žpmol Žmg prot.y1 Ž3 min.y1 .
Control
NAME
Control
NAME
Control
NAME
Control
NAME
32.1 " 1.4 57.3 " 2.4 3.21 " 0.16
34.2 " 0.81 53.1 " 1.5 3.20 " 0.13
142 " 4.0 138 " 4.0 79 " 3.0
150 " 4.0 142 " 4.0 79 " 2.0
2.29 " 0.07 2.32 " 0.09 1.62 " 0.09
2.39 " 0.05 2.40 " 0.08 1.79 " 0.07
276.4 " 23.0 360.2 " 42.8 49.8 " 6.0
312.2 " 9.9 379.1 " 32.5 40.5 " 6.7
Samples were taken from brains of 40-day-old rats whose mothers had been treated with L-NAME or saline from gestational day 14 to gestational day 19. Values are the mean " S.E. of eight experiments.
M. Virgili et al.r Brain Research 839 (1999) 164–172
169
Fig. 4. Effect of exposure to L-NAME on survival of cerebellar granule cells in culture after 4 or 8 days in vitro ŽDIV.. Bars are the mean" S.E. of 18–24 cell counting performed on at least 8 dishes from four different cultures. UU p - 0.01, student’s t-test, as compared to control.
the survival of granule neurons since their density was slightly decreased, as compared to control cultures, after 8
days in vitro Žbut not after 4 days in vitro. ŽFig. 4.. However, these cultures did not show any obvious differ-
Fig. 5. Cerebellar granule cells after 8 days in vitro under control conditions Ž5A. or after chronic exposure to 500 mM L-NAME Ž5B. and effect of shifting the cultures for 16 h to a low Kq medium Ž5C, control; 5D, exposed to L-NAME.. Note the lower density of granule neurons after chronic exposure to q L-NAME and the reduced effect of shifting these cultures to a low K medium as compared to control cultures. Calibration bar, 60 mm.
170
M. Virgili et al.r Brain Research 839 (1999) 164–172
ence, as compared to control cultures, with respect to the morphology and progressive differentiation of granule neurons ŽFig. 5A,B.. Other features characterizing mature granule neurons in culture could have been modified by the treatment: for instance, shifting 8-day-old cultures from a medium containing 25 mM Kq to a medium containing 5 mM Kq for 16 h, resulted in substantial elimination of granule neurons in control cultures Ž5C., while the number of viable cells was much less affected in cultures exposed to L-NAME Ž5D., taking into account the fact that the total cell density was lower in these cultures.
4. Discussion The present study shows that the catalytic activity of NOS can be almost completely inhibited during the critical period of brain development without dramatic consequences on the synthesis of the enzymatic protein during the pharmacological treatment and the recovery of activity after its suspension. This prolonged blockade did not result in any appreciable alteration of the final neurochemical maturation of the brain, at least concerning some relevant markers previously studied by us during development w40x. This parallels the absence of gross alterations of brain morphology, previously noticed after a similar schedule of treatment w30x, and confirmed by us in the present study. This result adds uncertainty to the exact role that NO may play in synaptic plasticity during development. Since LNAME is a competitive inhibitor acting at the enzyme catalytic site w8x, and its action is unspecific with regards to the different NOS isoforms w21x, it is unlikely that a substantial production of NO could have occurred under a tight schedule of inhibitor administration such as the one used in the present study. This seems to exclude a mutual compensation through other NOS isoforms as it may occur in knockout mice lacking the expression of a single isoform w16x. Different timing of NOS inhibition could theoretically bring to different long-term effects, since the critical period may be not completely covered by the treatment or compensatory mechanisms may occur with prolonged inhibition. We have, therefore, performed additional experiments, in which NOS was inhibited during a short gestational period critical for the expression of cerebral nNOS w4x, from gestational day 14 to gestational day 19. The results obtained with these experiments, however, suggested that also this pulse treatment had no gross consequences on the neurochemical maturation of the brain in the long run. The critical processes mediated by NO production during development may be too subtle, even if physiologically relevant, to be detected by observations aimed at outlining gross alterations of brain structure w30x and neurochemistry Žpresent results. or of complex behaviours w43x. The most convincing results so far produced on NO role in brain
maturation based on NOS inhibition, are those demonstrating a derangement from the normal pattern of segregation of retinal input to the optic tectum in the chick and the lateral geniculate nucleus in the ferret w6,44x. When the focus was shifted to the thalamocortical connections, however, no evidence was found for an altered pattern of the visual or the somatosensory projections to the cortex, as a consequence of the chronic neonatal blockade of catalytic NO production w9x. This result, together with the fact that a similar lack of effect could be demonstrated in a cholinergic projection to the superior colliculus w25x, suggests that the functional interaction of NO system with synaptogenesis may be selective for some projections and not for others, probably in connection with the neurotransmitter– receptor combinations present at the synaptic levels. Our in vivo observations, demonstrate that also the diaphorase activity associated with NOS is strongly reduced by competitive NOS inhibitors and that full recovery occurs in concomitance with the recovery of the catalytic activity. It was shown before w28x that NOS inhibitors strongly inhibited the diaphorase activity associated with NOS in homogenates of chicken retina. Furthermore, the diaphorase activity measured in tissue from Drosophila was inhibited by excess of the natural substrate, arginine w27x. Concerning histochemical detection, pre-incubation of formaldehyde-fixed frozen sections with L-nitro arginine impaired the NADPH diaphorase reaction in spinal cord neurons w2x. In line with those previous data, our present results suggest that the diaphorase activity related to NOS is impaired when the catalytic site for NO production is occupied. Our present in vitro observations demonstrate that blockade of NO production is slightly detrimental to survival of cerebellar granule cells in culture, without apparently affecting their morphological differentiation. This modest effect, must take into account the fact that under the standard culture conditions Ži.e., high Kq concentration., nNOS may be downregulated in granule cells w1x. Tanaka et al. w35x previously demonstrated alterations in granule cell proliferation and migration in in vitro cultures of cerebellar slices exposed to L-nitro arginine. We also show here that NOS inhibition may protect granule cells when they are exposed to stressful conditions, such as the shifting from high to low Kq. Inhibition of NOS has been previously shown to partially protect cerebellar granule neurons from excitotoxic damage w5x as well as from spermine-mediated toxicity w34x. In conclusion, the present results suggest that relevant aspects of brain organization are not affected by an almost complete inhibition of NOS during the critical period of maturation. Therefore, the role of NO in developmental brain plasticity should be related to a fine tuning of neural circuits and subtle aspects of the stabilization and modification of synaptic connections. In in vitro conditions, the effects of NOS inhibition, may be more pronounced and affect basic aspects of neuronal viability and function.
M. Virgili et al.r Brain Research 839 (1999) 164–172
Acknowledgements The present work was supported by grants from the University of Bologna and from the targeted project ‘‘Biotechnology’’ of the Italian National Research Council. The skilful technical help of Luciana di Pietrangelo in preparing photographic illustrations is gratefully acknowledged.
References w1x S.L. Baader, K. Schilling, Glutamate receptors mediate dynamic regulation of nitric oxide synthase expression in cerebellar granule cells, J. Neurosci. 16 Ž1996. 1440–1449. w2x D. Blottner, H.G. Baumgarten, L-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons, Histochem. Cell Biol. 103 Ž1995. 379–385. w3x D.S. Bredt, S.H. Snyder, Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A. 86 Ž1990. 9030–9033. w4x D.S. Bredt, S.H. Snyder, Nitric oxide, a physiological messenger molecule, Annu. Rev. Biochem. 63 Ž1994. 175–195. w5x E. Ciani, L. Groneng, M. Voltattorni, R. Veslemoy, A. Contestabile, R.E. Paulsen, Inhibition of free radical production or free radical scavenging protects from excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons, Brain Res. 728 Ž1996. 1–6. w6x K.S. Cramer, A. Angelucci, J.O. Hahm, M.B. Bogdanov, M. Sur, A role for nitric oxide in the development of the ferret retinogeniculate projection, J. Neurosci. 15 Ž1996. 7995–8004. w7x T.M. Dawson, S.H. Snyder, Gases as biological messengers: nitric oxide and carbon monoxide in the brain, J. Neurosci. 14 Ž1994. 5147–5159. w8x V.L. Dawson, T.M. Dawson, Function of nitric oxide in neuronal cell death, in: M.D. Maines ŽEd.., Nitric Oxide Synthase: Characterization and Functional Analysis, Academic Press, San Diego, 1996, pp. 228–240. w9x E.M. Finney, C.J. Shatz, Establishment of patterned thalamocortical connections does not require nitric oxide synthase, J. Neurosci. 18 Ž1998. 8826–8838. w10x F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 Ž1975. 407–409. w11x F. Fonnum, I. Grofova, E. Rinvik, J. Storm-Mathiesen, F. Walberg, Origin and distribution of glutamate decarboxylase in the substantia nigra of the cat, Brain Res. 71 Ž1974. 77–92. w12x V. Gallo, A. Kingsbury, R. Balazs, O.S. Jorgensen, The role of depolarization in the survival and differentiation of cerebellar granule cells in culture, J. Neurosci. 7 Ž1987. 2203–2213. w13x J.A. Gally, R.P. Montague, G.N. Reeke, G.E. Edelman, The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 3547–3551. w14x J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 Ž1995. 683–706. w15x C. Holscher, Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity, Trends Neurosci. 20 Ž1997. 298–303. w16x P.L. Huang, M.C. Fishman, Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice, Mol. Med. 74 Ž1996. 415–421. w17x P.L. Huang, T.M. Dawson, D.S. Bredt, S.H. Snyder, M.C. Fischman, Targeted disruption of the neuronal nitric oxide synthase gene, Cell 75 Ž1993. 1268–1271. w18x G. Keilhoff, B. Seidel, H. Noak, W. Tischmeyer, D. Stanek, Patterns of nitric oxide synthase at the messenger RNA and protein levels during early brain development, Neuroscience 75 Ž1996. 1193–1201.
171
w19x B. Krekelberg, J.G. Taylor, Nitric oxide in cortical map formation, J. Chem. Neuroanat. 10 Ž1996. 191–196. w20x I. Kvale, V.M. Fosse, F. Fonnum, Development of neurotransmitter parameters in lateral geniculate body, superior colliculus and visual cortex of the albino rat, Dev. Brain Res. 7 Ž1983. 137–145. w21x C. Iadecola, Bright and dark sides of nitric oxide in ischemic brain injury, Trends Neurosci. 20 Ž1997. 132–139. w22x C.J. Lowenstein, S.H. Snyder, Nitric oxide, a novel biological messenger, Cell 70 Ž1992. 705–707. w23x O.H. Lowry, R.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. w24x N. Mitrovic, M. Schachner, Transient expression of NADPH diaphorase activity in the mouse whisker to barrel field pathway, J. Neurocytol. 25 Ž1996. 429–437. w25x R.R. Mize, C.A. Scheiner, M.F. Salvatore, R.J. Cork, Inhibition of nitric oxide synthase fails to disrupt the development of cholinergic fiber patches in the rat superior colliculus, Dev. Neurosci. 19 Ž1997. 260–273. w26x R.J. Nelson, G.E. Demas, P.L. Huang, M.C. Fishman, W.L. Dawson, T.M. Dawson, S.H. Snyder, Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase, Nature 378 Ž1995. 383–386. w27x T.I. Oparina, S.V. Myl’nikov, The effect of L-arginine on the NADPH-diaphorase activity in Drosophila melanogaster homogenates, Zh. Evol. Biokhim. Fiziol. 32 Ž1996. 217–219. w28x R. Paes de Carvalho, M.H. de Faria, J.L.M. do Nascimiento, J.N. Hokoc, Development of NADPH-diaphorase in the avian retina: regulation by calcium ions and relation to nitric oxide synthase, J. Neurochem. 67 Ž1996. 1063–1071. w29x A.J. Patel, A. Hunt, C.S.M. Tahourdin, Regional development of glutamine synthetase activity in rat brain and its association with the differentiation of astrocytes, Dev. Brain Res. 8 Ž1983. 31–37. w30x J. Prickaerts, J. De Vente, M. Markerink-Van Ittersum, H.W.M. Steinbusch, Behavioural, neurochemical and neuroanatomical effects of chronic postnatal N-nitro-L-arginine methyl ester treatment in neonatal and adult rats, Neuroscience 87 Ž1998. 181–195. w31x A. Resink, N. Hack, G.J. Boer, R. Balazs, Growth conditions differentially modulate the vulnerability of developing granule cells to excitatory amino acids, Brain Res. 655 Ž1994. 222–232. w32x N.L. Scholz, E.S. Chang, K. Graubard, J.W. Truman, The NOrcGMP pathway and the development of neural networks in postembryonic lobsters, J. Neurobiol. 34 Ž1998. 208–226. w33x E.M. Schuman, D.V. Madison, Nitric oxide and synaptic function, Annu. Rev. Neurosci. 17 Ž1994. 153–183. w34x M. Sparapani, R. Dall’Olio, O. Gandolfi, E. Ciani, A. Contestabile, Neurotoxicity of polyamines and pharmacological neuroprotection in cultures of rat cerebellar granule cells, Exp. Neurol. 148 Ž1997. 157–166. w35x M. Tanaka, S. Yoshida, M. Yano, F. Hanaoka, Roles of endogenous nitric oxide in cerebellar cortical development in slice cultures, NeuroReport 5 Ž1994. 2049–2052. w36x E. Thomas, A.G.E. Pearse, The solitary active cells: histochemical demonstration of damage-resistant nerve cells with a TNP-diaphorase reaction, Acta Neuropathol. 3 Ž1964. 238–249. w37x M. Tregnago, M. Virgili, B. Monti, T. Guarnieri, A. Contestabile, Alteration of neuronal nitric oxide synthase activity and expression in the cerebellum and the forebrain of microencephalic rats, Brain Res. 793 Ž1998. 54–60. w38x R.R. Vaid, B.K. Yee, U. Shalev, J.N. Rawlins, I. Weiner, J. Feldon, S. Totterdell, Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon’s horn of rats, J. Neurosci. 17 Ž1997. 5599–5609. w39x F.L. Van Muiswinkel, B. Drukarch, H.W. Steinbusch, J. De Vente, Sustained pharmacological inhibition of nitric oxide synthase does
172
M. Virgili et al.r Brain Research 839 (1999) 164–172
not affect the survival of intrastriatal rat fetal mesencephalic transplants, Brain Res. 792 Ž1998. 48–58. w40x M. Virgili, O. Barnabei, A. Contestabile, Regional maturation of neurotransmitter-related and glial markers during postnatal development in the rat, Int. J. Dev. Neurosci. 8 Ž1990. 156–159. w41x M. Virgili, F. Facchinetti, M. Sparapani, M. Tregnago, R. Lucchi, R. Dall’Olio, O. Gandolfi, A. Contestabile, Neuronal nitric oxide synthase is permanently decreased in the cerebellum of rats subjected to chronic neonatal blockade of NMDA receptors, Neurosci. Lett., in press.
w42x C.V. Williams, D. Nordquist, S.C. McLoon, Correlation of nitric oxide synthase expression with changing patterns of axonal projections in the developing visual system, J. Neurosci. 14 Ž1994. 1746– 1755. w43x G. Wortwein, B. Gustafson, K.L. Hansen, J. Mogensen, Behavioural symptoms in adult rats after postnatal L-nitro-arginine, Int. J. Dev. Neurosci. 15 Ž1997. 147–154. w44x H.H. Wu, C.V. Williams, S.C. McLoon, Involvement of nitric oxide in the elimination of a transient retinotectal projection in development, Science 265 Ž1994. 1593–1596.