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Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization
Molecular Brain Research 53 Ž1998. 1–12
Research report
Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization Katsuro Iwase a , Ken-ichi Iyama b, Kiwamu Akagi a , Shigetoshi Yano c , Kohji Fukunaga c , Eishichi Miyamoto c , Masataka Mori a , Masaki Takiguchi a,) a
Department of Molecular Genetics, Kumamoto UniÕersity School of Medicine, Kuhonji 4-24-1, Kumamoto 862, Japan b Department of Surgical Pathology, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan c Department of Pharmacology, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan Accepted 25 March 1997
Abstract Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon’s horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase. q 1998 Elsevier Science B.V. Keywords: Neuronal nitric oxide synthase; mRNA; Hybridization; In situ; Digoxigenin; Rat brain; Hippocampus
1. Introduction Nitric oxide ŽNO. is a gaseous messenger that plays a role in neurotransmission as well as in cardiovascular and immune functions Žreviewed in w6,10,15x.. NO is formed by NO synthase ŽNOS. from arginine. Purification and molecular cloning led to identification of at least three NOS isoforms designated as neuronal NOS ŽnNOS., endothelial NOS ŽeNOS. and inducible NOS ŽiNOS. Žreviewed in w6,15x.. In the central nervous system, NO seems to be involved in plasticity and cytotoxicity. Biological roles of nNOS have been examined using targeted disruption of the nNOS gene w13x. Mutant mice deficient in nNOS displayed aggressive behavior and excess inappropriate sexual behavior w18x, in addition to the enlarged
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0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 1 3 9 - 3
stomach with hypertrophy of the pyloric sphincter w13x. nNOS-deficient mice exhibited significantly decreased areas of infarction after cerebral ischemia w14x. On the other hand, long-term potentiation in the CA1 region of hippocampal slices w19x and long-term depression in cultured Purkinje neurons w16x are normal in the mutant animals. It has just been reported that mice doubly disrupted in nNOS and eNOS exhibited a significant reduction of long-term potentiation in the stratum radiatum of the CA1 region, suggesting that nNOS and eNOS can compensate for each other w22x. To better understand mechanisms controlling NO production, we have studied the regulation of genes for enzymes involved in the synthesis and degradation of arginine in peripheral organs w11,17,23x Žsee w24x for review on genes for arginine metabolism.. To clarify precise functions and the regulation of the nNOS gene in the brain, a detailed localization of cells expressing the nNOS gene has to be defined. Here, we examined the distribution of nNOS
mRNA using non-radioisotopic in situ hybridization, a procedure which allows a highly-resolved detection of positive neurons. In addition to previously reported regions w1,8x, signals for nNOS mRNA were observed in a number of discrete areas.
2. Materials and methods 2.1. Hybridization probes nNOS cDNA segments each corresponding to positions 296–2150 or positions 3360–3707 of the published sequence ŽEMBLrGenBankrDDBJ database accession number X59949 w2x. were amplified by reverse transcription–polymerase chain reaction using rat total brain RNA, then were inserted into the HincII site of the plasmid pGEM-3ZfŽq. ŽPromega.. Digoxigenin-labeled RNA probes were prepared according to instructions of Boehringer Mannheim. Antisense and sense strand RNAs were synthesized with SP6 or T7 RNA polymerases after linearizing the plasmids with appropriate restriction enzymes. 2.2. RNA blot analysis Total RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform extraction procedure w4x. The RNA Ž5 mg per lane. was electrophoresed in denaturing formaldehyde–agarose Ž1%. gels. After visualizing 28S and 18S rRNAs by ethidium bromide staining to check integrity of the RNA samples and equal loading, RNA was transferred onto nylon membranes. Hybridization was performed overnight at 688C in a mixture of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% SDS, 0.1% N-lauroylsarcosine, 2% Blocking Reagent ŽBoehringer Mannheim. and 1 mgrml of the digoxigenin-labeled RNA probe. After hybridization, membranes were washed in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 708C. Colorimetric detection of the hybridized probe was made according to the protocol of Boehringer Mannheim using as chromogens nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.
2.3. Tissue preparation and in situ hybridization Five Wistar male rats were anaesthetized with ether followed by pentobarbital, and perfused through the heart with 4% paraformaldehyde made in Dulbecco’s phosphate-buffered saline ŽPBS. consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO4 ŽpH 7.4. and 1.5 mM KH 2 PO4 . The brains were removed, fixed at 48C overnight in the same paraformaldehyde solution, dehydrated in serially concentrated ethanol and in xylene, and embedded in paraffin. Tissue sections Ž6 mm thick. were cut and mounted on glass slides coated with 3-aminopropyltriethoxysilane. De-waxing was done by heating the slides in an oven at 608C for 1 h followed by immersions in xylene three times Ž30 min, 15 min, 15 min.. The slides were then rehydrated in serially diluted ethanol Ž95%, 70%, 30%. and in PBS. The sections were subjected to digestion in 1 mgrml proteinase K in PBS for 10 min, followed by washing in PBS. The sections were fixed with 4% paraformaldehyde in PBS, and thoroughly washed in PBS. The slides were incubated at 688C for 2 h in prehybridization solution consisting of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% SDS, 0.1% Nlauroylsarcosine, 2% Blocking Reagent. The digoxigeninlabeled RNA probe was heat-treated at 808C for 3 min, and diluted to the final concentration of 1 mgrml with the prehybridization solution supplemented with 0.1 mgrml yeast RNA. The resulting hybridization mixture Ž100 ml. was applied to each slide. The sections were covered with a high-density polyethylene membrane and incubated overnight at 688C in a moist chamber. After hybridization, slides were washed twice in a solution consisting of 300 mM NaCl, 30 mM sodium citrate and 0.1% SDS at room temperature for 5 min, and twice in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 688C for 20 min. Color detection of the hybridized probe was performed essentially according to the instruction of Boehringer Mannheim. The slides were serially soaked in buffer 1 Ž100 mM maleic acid, pH 7.5, 150 mM NaCl. for 1 min, in buffer 2 Ž1% Blocking Reagent in buffer 1. for 30 min, in alkaline phosphatase-conjugated anti-digoxigenin antibody solution Ždiluted 1r5000 in buffer 2. for 30 min, twice in buffer 1 for 15 min, and in buffer 3 Ž100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl 2 . for 2 min. Color development was allowed to proceed overnight in a mixture of 0.45 mgrml nitro blue tetra-
Fig. 1. Specific detection of nNOS mRNA using digoxigenin-labeled RNA probes. A: RNA blot analysis. Total RNA Ž5 mg. derived from rat brain regions and liver was probed with digoxigenin-labeled antisense-strand RNA corresponding to nucleotides 296–2150 of the published sequence w2x. The arrow indicates the specific band. The positions of 28S and 18S rRNA bands are shown. Below, ethidium bromide staining of 28S and 18S rRNAs is presented. B–G: in situ hybridization. Sagittal brain sections were probed with antisense-strand RNA ŽB. or sense-strand RNA ŽC.. At high magnification ŽD–G., densely stained cytoplasm and unstained nuclei are apparent for positive neurons in the cerebral cortex ŽD., the corpus callosum ŽE., the cerebellum ŽF. and the laterodorsal tegmental nucleus ŽG.. In E–G, sections were counterstained with hematoxylin. In D and E, sections were observed using Nomarski optics. gr, granular layer; mol, molecular layer; Pur, Purkinje cell layer.
zolium chloride and 0.175 mgrml 5-bromo-4-chloro-3-indolyl phosphate in buffer 3. The reaction was stopped by rinsing the sections in a solution consisting of 10 mM
Tris-HCl ŽpH 8.0. and 1 mM EDTA. When appropriate, sections were counterstained with hematoxylin, and the slides were mounted in Aquatex ŽMerck..
Fig. 2. The telencephalon examined for expression of nNOS mRNA. A–C: the olfactory bulb. The main ŽB. and accessory ŽC. olfactory bulbs are shown at higher magnification. D, E: the caudate–putamen and the ventral striatal complex. In E, the islands of Calleja are shown at higher magnification. F, G: the amygdala at a rostral ŽF. or caudal ŽG. level in coronal sections. H: the cerebral cortex. Arrows indicate intensely stained isolated neurons. I: the corpus callosum. J–L: the hippocampus. The inner blade of the dentate gyrus ŽK. and the CA1 field of the Ammon’s horn ŽL. are shown at higher magnification. In D, E, H, I, sections were counterstained with hematoxylin. In B, C, E, sections were observed using Nomarski optics. Acb, accumbens nucleus; ACo, anterior cortical nucleus; AOB, accessory olfactory bulb; APir, amygdalopiriform transition; BM, basomedial nucleus; CA1–3, Ammon’s horn CA1–3; CPu, caudate–putamen; EPl, external plexiform layer; Gl, glomerular cell layer; Gr, granule cell layer; GrA, granule cell layer of the accessory olfactory bulb; ICj, islands of Calleja; IPl, internal plexiform layer; Me, medial nucleus; Mi, mitral cell layer; MirTuA, mitralrtufted cell layer of the accessory olfactory bulb; MOB, main olfactory bulb; PMCo, posteromedial cortical nucleus; Tu, olfactory tubercle.
3. Results 3.1. Specific detection of nNOS mRNA To confirm the specificity for detection of nNOS mRNA by digoxigenin-labeled RNA probes, RNA blot analysis was performed ŽFig. 1A.. Using an antisense-strand probe corresponding to nucleotides 296–2150, a single mRNA
band was detected with RNAs from various regions of the rat brain, but not with liver RNA. nNOS mRNA levels were higher in the olfactory bulb and cerebellum than in other regions, concordant with results obtained using a radioisotopic DNA probe w2x. The following in situ hybridization studies were carried out under conditions essentially comparable to those of this filter hybridization. Sections derived from the paraformaldehyde-fixed
Fig. 3. The diencephalon examined for expression of nNOS mRNA. A: the bed nucleus of the stria terminalis. B: the supraoptic nucleus. C: the paraventricular nucleus of the hypothalamus. D: the posterior hypothalamus. E: the medial region of the thalamus. F, G: the lateral region of the caudal thalamus. In G, the ventral lateral geniculate nucleus in a different section is shown. The bracket indicates the most lateral layers enriched in strongly stained neurons. H: the medial habenular nucleus. In B, F, G, sections were counterstained with hematoxylin. 3V, third ventricle; ac, anterior commissure; BST, bed nucleus of the stria terminalis; CM, central medial nucleus; D3V, dorsal third ventricle; DLG, dorsal lateral geniculate nucleus; M, mammillary nucleus; MG, medial geniculate nucleus; opt, optic tract; MHb, medial habenular nucleus; PaV, paraventricular nucleus of the hypothalamus; PM, premammillary nucleus; PV, paraventricular nucleus of the thalamus; Re, reuniens nucleus; SO, supraoptic nucleus; SuM, supramammillary nucleus; VMH, ventromedial hypothalamic nucleus; VLG, ventral lateral geniculate nucleus; ZI, zona incerta. Fig. 4. The midbrain examined for expression of nNOS mRNA in a coronal section of the caudal region ŽA. and in a sagittal section of the dorsal region ŽB.. In A, the section was counterstained with hematoxylin. CG, central gray; IC, inferior colliculus; MCPC, magnocellular nucleus of the posterior commissure; SC, superior colliculus.
paraffin-embedded brain were subjected to in situ hybridization. As shown in Fig. 1B, region-specific hybridization signals were detected. A similar hybridization
pattern was observed with a probe corresponding to nucleotides 3360–3707 Ždata not shown.. No obvious signal was detected with sense-strand probes spanning nu-
Fig. 6. The cerebellum examined for expression of nNOS mRNA. A, B: the cerebellar cortex. C, D: the deep nuclei. In B and D, sections were counterstained with hematoxylin. gr, granular layer; mol, molecular layer; Pur, Purkinje cell layer.
cleotides 296–2150 ŽFig. 1C. and nucleotides 3360–3707 Ždata not shown.. Fig. 1D–G shows examples of positive neurons at high magnification. A strong staining was localized in the cytoplasm of the cell body and stem processes. On the other hand, the nucleus was unstained and translucent, confirming that nonspecific hybridization to nuclear materials was under a detectable level. 3.2. Distribution of nNOS mRNA in the rat brain Distribution of signals for nNOS mRNA was examined in sagittal and coronal sections.
3.2.1. Telencephalon In the main olfactory bulb ŽFig. 2A,B., strong signals were detected in the glomerular cell layer, the mitral cell layer and the granule cell layer. Less intense staining was observed in the internal plexiform layer. In the external plexiform layer only a small number of isolated positive neurons was detected. Intensely stained isolated neurons were also scattered in other layers. In the accessory olfactory bulb ŽFig. 2A,C., strong signals were detected in the granule cell layer. A small number of isolated positive neurons located in the mitralrtufted cell layer. As shown in Fig. 2D, in the caudate–putamen and the ventral striatal complex including the accumbens nucleus and the olfactory tubercle, strongly stained scattered neu-
Fig. 5. The pons and the medulla oblongata examined for expression of nNOS mRNA. A: overview of a sagittal section. B: the pedunculopontine tegmental nucleus. C: the pontine nucleus. D: the nucleus of the trapezoid body. E: the medial vestibular nucleus and the nucleus of the solitary tract. F: a coronal section of the rostral medulla. In C–F, sections were counterstained with hematoxylin. DMSp5, dorsomedial spinal trigeminal nucleus; DPGi, dorsal paragigantocellular field; IRt, intermediate reticular field; LDTg, laterodorsal tegmental nucleus; MVe, medial vestibular nucleus; PCRt, parvocellular reticular field; Pn, pontine nucleus; PPTg, pedunculopontine tegmental nucleus; PrH, prepositus hypoglossal nucleus; RF, reticular formation; Sol, nucleus of the solitary tract; Tz, nucleus of the trapezoid body.
rons were detected. Clustering of small positive cells was observed in the islands of Calleja ŽFig. 2D,E.. In the amygdala, the medial nucleus showed condensation of nNOS mRNA-expressing cells ŽFig. 2F.. The anterior cortical nucleus and the basomedial nucleus also exhibited clustering of positive cells. In the posteromedial cortical nucleus and the amygdalopiriform transition ŽFig. 2G., clustering of moderately stained neurons was observed. The cerebral cortex ŽFig. 2H, see also Fig. 1D. exhibited scattering of isolated neurons with highly dense staining. The corpus callosum ŽFig. 2I, see also Fig. 1E. harbored strongly stained isolated neurons. In the hippocampus ŽFig. 2J–L., moderately stained neurons were diffusely distributed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of CA1–CA3 fields of the Ammon’s horn. Distinguishably from these diffuse patterns, more intense signals were detected in scattered isolated neurons over the entire region of the hippocampus. 3.2.2. Diencephalon In the bed nucleus of the stria terminalis ŽFig. 3A., there was a clustering of moderately or weakly stained cells. Within the hypothalamic area, nNOS mRNA was detected in neurons of the supraoptic nucleus ŽFig. 3B.. Clustering of positive cells was observed in the paraventricular nucleus ŽFig. 3C. and the ventromedial nucleus of the hypothalamus ŽFig. 3D., and also in the premammillary nucleus, the mammillary nucleus and the supramammillary nucleus ŽFig. 3D.. The thalamus was generally free of stained cells. However, the paraventricular nucleus was rich in stained cells ŽFig. 3E.. The central medial nucleus and the reuniens nucleus also exhibited enrichment of scattered positive cells ŽFig. 3E.. The zona incerta of the subthalamic region showed enrichment of stained cells ŽFig. 3F.. In the dorsal and ventral lateral geniculate nucleus, clustering of well-stained isolated neurons was observed ŽFig. 3F.. In some section ŽFig. 3G., strongly stained neurons were arranged in the most lateral layers of the ventral lateral geniculate nucleus. This distribution is concordant with that of NADPH diaphorase-positive neurons w9x. In the epithalamic area, moderate staining was noted for the medial habenular nucleus ŽFig. 3H.. 3.2.3. Midbrain The magnocellular nucleus of the posterior commissure exhibited clustering of highly stained neurons ŽFig. 4A.. The central gray showed enrichment of positive cells ŽFig. 4A.. In the superior colliculus ŽFig. 4B., intense staining was noted, especially for neurons in the superficial gray layer. Almost all the area of the inferior colliculus was rich in strongly stained neurons ŽFig. 4B..
3.2.4. Pons and medulla oblongata The laterodorsal ŽFig. 5A, see also Fig. 1G. and pedunculopontine ŽFig. 5B. tegmental nuclei exhibited clustering of very strongly stained neurons. Cells in the pontine nucleus showed faint staining ŽFig. 5A,C.. In the nucleus of the trapezoid body ŽFig. 5A,D., clustering of intensely stained cells was evident. Scattered positive neurons were abundant in the medial vestibular nucleus ŽFig. 5A,E.. Intensely stained neurons clustered in the nucleus of the solitary tract ŽFig. 5A,E.. Clustering of positive cells was also observed in the prepositus hypoglossal nucleus and the dorsomedial spinal trigeminal nucleus ŽFig. 5F.. As for the reticular formation, scattered positive cells were more abundant in the medulla than in the pons ŽFig. 5A.. In the rostral portion of the medulla ŽFig. 5F., enrichment of positive cells was observed in the dorsal paragigantocellular field, the parvocellular reticular field and the intermediate reticular field. 3.2.5. Cerebellum Strong diffuse staining was observed in the granular layer ŽFig. 6A,B, see also Fig. 1F.. nNOS mRNA was also enriched in neurons of the molecular layer. Isolated positive neurons were scattered in the deep cerebellar nuclei ŽFig. 6C,D..
4. Discussion We performed in situ hybridization study to detect nNOS mRNA in the rat brain, using non-radioisotopic digoxigenin-labeled RNA probes and paraformaldehydefixed paraffin-embedded tissue sections. The combination of these probes and sections gave a high resolution sufficient to distinguish the densely stained cytoplasm and unstained nucleus in a neuron. We could clearly differentiate nonspecific signals derived from hybridization with nuclear materials, an event which often hampers the in situ hybridization study. Specificity of signals in this study was also ensured by using stringent conditions comparable with those for RNA blot analysis in which a single specific band was detected. The entire procedure is rapid and simple: pretreatments of sections such as acetylation and acidification prior to hybridization were omitted; detection of signals by color development was usually completed overnight. Highly resolved detection of nNOS mRNA enabled clarification of the size and shape of the positive cells to the extent comparable to that seen in immunocytochemistry using anti-nNOS antibodies w1,3,20x and histochemistry derived from NADPH diaphorase activity that reflects NOS activity w5,12,25x. This increases each other’s reliability for nNOS mRNA and protein localizations. In addition to previously stated areas such as the main and accessory olfactory bulbs, the amygdala, the supraoptic nucleus, the
Table 1 Classification of areas expressing nNOS mRNA Olfactoryrvomeronasal system Main olfactory bulb ŽMOB. Glomerular cell layer ŽGl. Mitral cell layer ŽMi. Internal plexiform layer ŽIP1. Granule cell layer ŽGr. ŽIsolated scattered neurons over the entire region. Accessory olfactory bulb ŽAOB. Mitralrtufted cell layer ŽMirTuA. Granule cell layer ŽGrA. Limbic and related system Amygdala Medial nucleus ŽMe. Anterior cortical nucleus ŽACo. Basomedial nucleus ŽBM. Posteromedial cortical nucleus ŽPMCo. Amygdalopiriform transition ŽAPir. Hippocampus Dentate gyrus ŽDG. Ammon’s horn CA1–3 ŽCA1–3. ŽIsolated scattered neurons over the entire region. Bed nucleus of the stria terminalis ŽBST. Epithalamus Medial habenular nucleus ŽMHb. Hypothalamus Supraoptic nucleus ŽSO. Paraventricular nucleus ŽPaV. Ventromedial nucleus ŽVMH. Premammillary nucleus ŽPM. Mammillary nucleus ŽM. Supramammillary nucleus ŽSuM. Cerebral cortex Corpus callosum Basal ganglia Caudate–putamen ŽCPu. Accumbens nucleus ŽAcb. Olfactory tubercle ŽTu. Islands of Calleja ŽICj.
Visual system Lateral geniculate nucleus Dorsal lateral geniculate nucleus ŽDLG. Ventral lateral geniculate nucleus ŽVLG. Superior colliculus ŽSC. Auditory system Nucleus of the trapezoid body ŽTz. Inferior colliculus ŽIC. Vestibular and related system Medial vestibular nucleus ŽMVe. Prepositus hypoglossal nucleus ŽPrH. Gustatory system Nucleus of the solitary tract ŽSol. Somatosensory system Dorsomedial spinal trigeminal nucleus ŽDMSp5. Thalamus Paraventricular nucleus ŽPV. Central medial nucleus ŽCM. Reuniens nucleus ŽRe. Subthalamic region Zona incerta ŽZI. Pontine nucleus ŽPn. Cerebellum Cortex Granular layer Žgr. Molecular layer Žmol. Deep nuclei Others Magnocellular nucleus of the posterior commissure ŽMCPC. Midbrain central gray ŽCG. Laterodorsal tegmental nucleus ŽLDTg. Pedunculopontine tegmental nucleus ŽPPTg. Medulla reticular formation Dorsal paragigantocellular field ŽDPGi. Parvocellular reticular field ŽPCRt. Intermediate reticular field ŽIRt.
Areas expressing nNOS mRNA are classified, based on anatomical location andror assigned major function of each area. Abbreviations used in the figures are shown in parentheses.
lateral geniculate nucleus, the superior and inferior colliculi, the pedunculopontine tegmental nucleus and the cerebellum w1,8x, clustering of neurons expressing nNOS mRNA was observed most abundantly in areas including the islands of Calleja, the paraventricular nucleus of the hypothalamus, the paraventricular nucleus of the thalamus, the magnocellular nucleus of the posterior commissure, the laterodorsal tegmental nucleus, the nucleus of the trapezoid body and the nucleus of the solitary tract. Most of these areas have been shown to contain neurons immunoreactive with nNOS antibodies w1,20,21x, except for the magnocellular nucleus of the posterior commissure, the immunohistochemical analysis of which remains to be done. Neurons in the molecular layer of the cerebellum, which were previously reported to be positive in immunohistochemistry w1,20,21x but almost negative with in situ hybridization w1x, were seen to express nNOS mRNA in the present study. The islands of Calleja, in which nNOS mRNA was
hardly detectable in a foregoing study w1x, also exhibited a strong hybridization signal, concordant with the expression of intense immunoreactivity w1,20x. Expression profiles of nNOS in the hippocampus have been controversial in the previous studies w1,7,8,20,21,26x. In the present study, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon’s horn, while more intensely stained isolated neurons were scattered over the entire region of the hippocampus. In Table 1, areas expressing nNOS mRNA are classified, taking into account anatomical location andror assigned major function of each area. Based on these distributions, it is safe to speculate that nNOS is involved in a number of brain functions related to sensory, motor, autonomic, limbic and higher systems, as has been repeatedly noted mainly based on immunohistochemical studies w1,20,21x. On the other hand, in each system, a high level
expression of nNOS mRNA is restricted to isolated individual neurons andror discrete cell groups, i.e. cell layers, nuclei, etc. This characteristic feature of localization of neurons expressing the nNOS gene was clearly demonstrated in our study using high-resolution in situ hybridization. The present data provide the basis for future investigations on roles of nNOS in each neural system, by clarifying the most remarkable areas in each system. Acknowledgements We thank S. Goto for advice in microscopic identification of neural structures, M. Ohara for helpful comments on the manuscript, and our colleagues for suggestions and discussions. This work was supported in part by grants-inaid from the Ministry of Education, Science, Sports and Culture of Japan. References w1x D.S. Bredt, C.E. Glatt, P.M. Hwang, M. Fotuhi, T.M. Dawson, S.H. Snyder, Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron 7 Ž1991. 615–624. w2x D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, S.H. Snyder, Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature 351 Ž1991. 714–718. w3x D.S. Bredt, P.M. Hwang, S.H. Snyder, Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347 Ž1990. 768–770. w4x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w5x T.M. Dawson, D.S. Bredt, M. Fotuhi, P.M. Hwang, S.H. Snyder, Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA 88 Ž1991. 7797–7801. w6x T.M. Dawson, S.H. Snyder, Gases as biological messengers: nitric oxide and carbon monoxide in the brain, J. Neurosci. 14 Ž1994. 5147–5159. w7x J.L. Dinerman, T.M. Dawson, M.J. Schell, A. Snowman, S.H. Snyder, Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity, Proc. Natl. Acad. Sci. USA 91 Ž1994. 4214–4218. w8x M. Endoh, K. Maiese, J.A. Wagner, Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons, Neuroscience 63 Ž1994. 679–689. w9x P.L. Gabbott, S.J. Bacon, An oriented framework of neuronal processes in the ventral lateral geniculate nucleus of the rat demonstrated by NADPH diaphorase histochemistry and GABA immunocytochemistry, Neuroscience 60 Ž1994. 417–440. w10x J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 Ž1995. 683–706.
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Research report
Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization Katsuro Iwase a , Ken-ichi Iyama b, Kiwamu Akagi a , Shigetoshi Yano c , Kohji Fukunaga c , Eishichi Miyamoto c , Masataka Mori a , Masaki Takiguchi a,) a
Department of Molecular Genetics, Kumamoto UniÕersity School of Medicine, Kuhonji 4-24-1, Kumamoto 862, Japan b Department of Surgical Pathology, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan c Department of Pharmacology, Kumamoto UniÕersity School of Medicine, Kumamoto 860, Japan Accepted 25 March 1997
Abstract Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon’s horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase. q 1998 Elsevier Science B.V. Keywords: Neuronal nitric oxide synthase; mRNA; Hybridization; In situ; Digoxigenin; Rat brain; Hippocampus
1. Introduction Nitric oxide ŽNO. is a gaseous messenger that plays a role in neurotransmission as well as in cardiovascular and immune functions Žreviewed in w6,10,15x.. NO is formed by NO synthase ŽNOS. from arginine. Purification and molecular cloning led to identification of at least three NOS isoforms designated as neuronal NOS ŽnNOS., endothelial NOS ŽeNOS. and inducible NOS ŽiNOS. Žreviewed in w6,15x.. In the central nervous system, NO seems to be involved in plasticity and cytotoxicity. Biological roles of nNOS have been examined using targeted disruption of the nNOS gene w13x. Mutant mice deficient in nNOS displayed aggressive behavior and excess inappropriate sexual behavior w18x, in addition to the enlarged
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0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 1 3 9 - 3
stomach with hypertrophy of the pyloric sphincter w13x. nNOS-deficient mice exhibited significantly decreased areas of infarction after cerebral ischemia w14x. On the other hand, long-term potentiation in the CA1 region of hippocampal slices w19x and long-term depression in cultured Purkinje neurons w16x are normal in the mutant animals. It has just been reported that mice doubly disrupted in nNOS and eNOS exhibited a significant reduction of long-term potentiation in the stratum radiatum of the CA1 region, suggesting that nNOS and eNOS can compensate for each other w22x. To better understand mechanisms controlling NO production, we have studied the regulation of genes for enzymes involved in the synthesis and degradation of arginine in peripheral organs w11,17,23x Žsee w24x for review on genes for arginine metabolism.. To clarify precise functions and the regulation of the nNOS gene in the brain, a detailed localization of cells expressing the nNOS gene has to be defined. Here, we examined the distribution of nNOS
mRNA using non-radioisotopic in situ hybridization, a procedure which allows a highly-resolved detection of positive neurons. In addition to previously reported regions w1,8x, signals for nNOS mRNA were observed in a number of discrete areas.
2. Materials and methods 2.1. Hybridization probes nNOS cDNA segments each corresponding to positions 296–2150 or positions 3360–3707 of the published sequence ŽEMBLrGenBankrDDBJ database accession number X59949 w2x. were amplified by reverse transcription–polymerase chain reaction using rat total brain RNA, then were inserted into the HincII site of the plasmid pGEM-3ZfŽq. ŽPromega.. Digoxigenin-labeled RNA probes were prepared according to instructions of Boehringer Mannheim. Antisense and sense strand RNAs were synthesized with SP6 or T7 RNA polymerases after linearizing the plasmids with appropriate restriction enzymes. 2.2. RNA blot analysis Total RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform extraction procedure w4x. The RNA Ž5 mg per lane. was electrophoresed in denaturing formaldehyde–agarose Ž1%. gels. After visualizing 28S and 18S rRNAs by ethidium bromide staining to check integrity of the RNA samples and equal loading, RNA was transferred onto nylon membranes. Hybridization was performed overnight at 688C in a mixture of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% SDS, 0.1% N-lauroylsarcosine, 2% Blocking Reagent ŽBoehringer Mannheim. and 1 mgrml of the digoxigenin-labeled RNA probe. After hybridization, membranes were washed in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 708C. Colorimetric detection of the hybridized probe was made according to the protocol of Boehringer Mannheim using as chromogens nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.
2.3. Tissue preparation and in situ hybridization Five Wistar male rats were anaesthetized with ether followed by pentobarbital, and perfused through the heart with 4% paraformaldehyde made in Dulbecco’s phosphate-buffered saline ŽPBS. consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO4 ŽpH 7.4. and 1.5 mM KH 2 PO4 . The brains were removed, fixed at 48C overnight in the same paraformaldehyde solution, dehydrated in serially concentrated ethanol and in xylene, and embedded in paraffin. Tissue sections Ž6 mm thick. were cut and mounted on glass slides coated with 3-aminopropyltriethoxysilane. De-waxing was done by heating the slides in an oven at 608C for 1 h followed by immersions in xylene three times Ž30 min, 15 min, 15 min.. The slides were then rehydrated in serially diluted ethanol Ž95%, 70%, 30%. and in PBS. The sections were subjected to digestion in 1 mgrml proteinase K in PBS for 10 min, followed by washing in PBS. The sections were fixed with 4% paraformaldehyde in PBS, and thoroughly washed in PBS. The slides were incubated at 688C for 2 h in prehybridization solution consisting of 0.75 M NaCl, 75 mM sodium citrate, 50% formamide, 0.02% SDS, 0.1% Nlauroylsarcosine, 2% Blocking Reagent. The digoxigeninlabeled RNA probe was heat-treated at 808C for 3 min, and diluted to the final concentration of 1 mgrml with the prehybridization solution supplemented with 0.1 mgrml yeast RNA. The resulting hybridization mixture Ž100 ml. was applied to each slide. The sections were covered with a high-density polyethylene membrane and incubated overnight at 688C in a moist chamber. After hybridization, slides were washed twice in a solution consisting of 300 mM NaCl, 30 mM sodium citrate and 0.1% SDS at room temperature for 5 min, and twice in a solution consisting of 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS at 688C for 20 min. Color detection of the hybridized probe was performed essentially according to the instruction of Boehringer Mannheim. The slides were serially soaked in buffer 1 Ž100 mM maleic acid, pH 7.5, 150 mM NaCl. for 1 min, in buffer 2 Ž1% Blocking Reagent in buffer 1. for 30 min, in alkaline phosphatase-conjugated anti-digoxigenin antibody solution Ždiluted 1r5000 in buffer 2. for 30 min, twice in buffer 1 for 15 min, and in buffer 3 Ž100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl 2 . for 2 min. Color development was allowed to proceed overnight in a mixture of 0.45 mgrml nitro blue tetra-
Fig. 1. Specific detection of nNOS mRNA using digoxigenin-labeled RNA probes. A: RNA blot analysis. Total RNA Ž5 mg. derived from rat brain regions and liver was probed with digoxigenin-labeled antisense-strand RNA corresponding to nucleotides 296–2150 of the published sequence w2x. The arrow indicates the specific band. The positions of 28S and 18S rRNA bands are shown. Below, ethidium bromide staining of 28S and 18S rRNAs is presented. B–G: in situ hybridization. Sagittal brain sections were probed with antisense-strand RNA ŽB. or sense-strand RNA ŽC.. At high magnification ŽD–G., densely stained cytoplasm and unstained nuclei are apparent for positive neurons in the cerebral cortex ŽD., the corpus callosum ŽE., the cerebellum ŽF. and the laterodorsal tegmental nucleus ŽG.. In E–G, sections were counterstained with hematoxylin. In D and E, sections were observed using Nomarski optics. gr, granular layer; mol, molecular layer; Pur, Purkinje cell layer.
zolium chloride and 0.175 mgrml 5-bromo-4-chloro-3-indolyl phosphate in buffer 3. The reaction was stopped by rinsing the sections in a solution consisting of 10 mM
Tris-HCl ŽpH 8.0. and 1 mM EDTA. When appropriate, sections were counterstained with hematoxylin, and the slides were mounted in Aquatex ŽMerck..
Fig. 2. The telencephalon examined for expression of nNOS mRNA. A–C: the olfactory bulb. The main ŽB. and accessory ŽC. olfactory bulbs are shown at higher magnification. D, E: the caudate–putamen and the ventral striatal complex. In E, the islands of Calleja are shown at higher magnification. F, G: the amygdala at a rostral ŽF. or caudal ŽG. level in coronal sections. H: the cerebral cortex. Arrows indicate intensely stained isolated neurons. I: the corpus callosum. J–L: the hippocampus. The inner blade of the dentate gyrus ŽK. and the CA1 field of the Ammon’s horn ŽL. are shown at higher magnification. In D, E, H, I, sections were counterstained with hematoxylin. In B, C, E, sections were observed using Nomarski optics. Acb, accumbens nucleus; ACo, anterior cortical nucleus; AOB, accessory olfactory bulb; APir, amygdalopiriform transition; BM, basomedial nucleus; CA1–3, Ammon’s horn CA1–3; CPu, caudate–putamen; EPl, external plexiform layer; Gl, glomerular cell layer; Gr, granule cell layer; GrA, granule cell layer of the accessory olfactory bulb; ICj, islands of Calleja; IPl, internal plexiform layer; Me, medial nucleus; Mi, mitral cell layer; MirTuA, mitralrtufted cell layer of the accessory olfactory bulb; MOB, main olfactory bulb; PMCo, posteromedial cortical nucleus; Tu, olfactory tubercle.
3. Results 3.1. Specific detection of nNOS mRNA To confirm the specificity for detection of nNOS mRNA by digoxigenin-labeled RNA probes, RNA blot analysis was performed ŽFig. 1A.. Using an antisense-strand probe corresponding to nucleotides 296–2150, a single mRNA
band was detected with RNAs from various regions of the rat brain, but not with liver RNA. nNOS mRNA levels were higher in the olfactory bulb and cerebellum than in other regions, concordant with results obtained using a radioisotopic DNA probe w2x. The following in situ hybridization studies were carried out under conditions essentially comparable to those of this filter hybridization. Sections derived from the paraformaldehyde-fixed
Fig. 3. The diencephalon examined for expression of nNOS mRNA. A: the bed nucleus of the stria terminalis. B: the supraoptic nucleus. C: the paraventricular nucleus of the hypothalamus. D: the posterior hypothalamus. E: the medial region of the thalamus. F, G: the lateral region of the caudal thalamus. In G, the ventral lateral geniculate nucleus in a different section is shown. The bracket indicates the most lateral layers enriched in strongly stained neurons. H: the medial habenular nucleus. In B, F, G, sections were counterstained with hematoxylin. 3V, third ventricle; ac, anterior commissure; BST, bed nucleus of the stria terminalis; CM, central medial nucleus; D3V, dorsal third ventricle; DLG, dorsal lateral geniculate nucleus; M, mammillary nucleus; MG, medial geniculate nucleus; opt, optic tract; MHb, medial habenular nucleus; PaV, paraventricular nucleus of the hypothalamus; PM, premammillary nucleus; PV, paraventricular nucleus of the thalamus; Re, reuniens nucleus; SO, supraoptic nucleus; SuM, supramammillary nucleus; VMH, ventromedial hypothalamic nucleus; VLG, ventral lateral geniculate nucleus; ZI, zona incerta. Fig. 4. The midbrain examined for expression of nNOS mRNA in a coronal section of the caudal region ŽA. and in a sagittal section of the dorsal region ŽB.. In A, the section was counterstained with hematoxylin. CG, central gray; IC, inferior colliculus; MCPC, magnocellular nucleus of the posterior commissure; SC, superior colliculus.
paraffin-embedded brain were subjected to in situ hybridization. As shown in Fig. 1B, region-specific hybridization signals were detected. A similar hybridization
pattern was observed with a probe corresponding to nucleotides 3360–3707 Ždata not shown.. No obvious signal was detected with sense-strand probes spanning nu-
Fig. 6. The cerebellum examined for expression of nNOS mRNA. A, B: the cerebellar cortex. C, D: the deep nuclei. In B and D, sections were counterstained with hematoxylin. gr, granular layer; mol, molecular layer; Pur, Purkinje cell layer.
cleotides 296–2150 ŽFig. 1C. and nucleotides 3360–3707 Ždata not shown.. Fig. 1D–G shows examples of positive neurons at high magnification. A strong staining was localized in the cytoplasm of the cell body and stem processes. On the other hand, the nucleus was unstained and translucent, confirming that nonspecific hybridization to nuclear materials was under a detectable level. 3.2. Distribution of nNOS mRNA in the rat brain Distribution of signals for nNOS mRNA was examined in sagittal and coronal sections.
3.2.1. Telencephalon In the main olfactory bulb ŽFig. 2A,B., strong signals were detected in the glomerular cell layer, the mitral cell layer and the granule cell layer. Less intense staining was observed in the internal plexiform layer. In the external plexiform layer only a small number of isolated positive neurons was detected. Intensely stained isolated neurons were also scattered in other layers. In the accessory olfactory bulb ŽFig. 2A,C., strong signals were detected in the granule cell layer. A small number of isolated positive neurons located in the mitralrtufted cell layer. As shown in Fig. 2D, in the caudate–putamen and the ventral striatal complex including the accumbens nucleus and the olfactory tubercle, strongly stained scattered neu-
Fig. 5. The pons and the medulla oblongata examined for expression of nNOS mRNA. A: overview of a sagittal section. B: the pedunculopontine tegmental nucleus. C: the pontine nucleus. D: the nucleus of the trapezoid body. E: the medial vestibular nucleus and the nucleus of the solitary tract. F: a coronal section of the rostral medulla. In C–F, sections were counterstained with hematoxylin. DMSp5, dorsomedial spinal trigeminal nucleus; DPGi, dorsal paragigantocellular field; IRt, intermediate reticular field; LDTg, laterodorsal tegmental nucleus; MVe, medial vestibular nucleus; PCRt, parvocellular reticular field; Pn, pontine nucleus; PPTg, pedunculopontine tegmental nucleus; PrH, prepositus hypoglossal nucleus; RF, reticular formation; Sol, nucleus of the solitary tract; Tz, nucleus of the trapezoid body.
rons were detected. Clustering of small positive cells was observed in the islands of Calleja ŽFig. 2D,E.. In the amygdala, the medial nucleus showed condensation of nNOS mRNA-expressing cells ŽFig. 2F.. The anterior cortical nucleus and the basomedial nucleus also exhibited clustering of positive cells. In the posteromedial cortical nucleus and the amygdalopiriform transition ŽFig. 2G., clustering of moderately stained neurons was observed. The cerebral cortex ŽFig. 2H, see also Fig. 1D. exhibited scattering of isolated neurons with highly dense staining. The corpus callosum ŽFig. 2I, see also Fig. 1E. harbored strongly stained isolated neurons. In the hippocampus ŽFig. 2J–L., moderately stained neurons were diffusely distributed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of CA1–CA3 fields of the Ammon’s horn. Distinguishably from these diffuse patterns, more intense signals were detected in scattered isolated neurons over the entire region of the hippocampus. 3.2.2. Diencephalon In the bed nucleus of the stria terminalis ŽFig. 3A., there was a clustering of moderately or weakly stained cells. Within the hypothalamic area, nNOS mRNA was detected in neurons of the supraoptic nucleus ŽFig. 3B.. Clustering of positive cells was observed in the paraventricular nucleus ŽFig. 3C. and the ventromedial nucleus of the hypothalamus ŽFig. 3D., and also in the premammillary nucleus, the mammillary nucleus and the supramammillary nucleus ŽFig. 3D.. The thalamus was generally free of stained cells. However, the paraventricular nucleus was rich in stained cells ŽFig. 3E.. The central medial nucleus and the reuniens nucleus also exhibited enrichment of scattered positive cells ŽFig. 3E.. The zona incerta of the subthalamic region showed enrichment of stained cells ŽFig. 3F.. In the dorsal and ventral lateral geniculate nucleus, clustering of well-stained isolated neurons was observed ŽFig. 3F.. In some section ŽFig. 3G., strongly stained neurons were arranged in the most lateral layers of the ventral lateral geniculate nucleus. This distribution is concordant with that of NADPH diaphorase-positive neurons w9x. In the epithalamic area, moderate staining was noted for the medial habenular nucleus ŽFig. 3H.. 3.2.3. Midbrain The magnocellular nucleus of the posterior commissure exhibited clustering of highly stained neurons ŽFig. 4A.. The central gray showed enrichment of positive cells ŽFig. 4A.. In the superior colliculus ŽFig. 4B., intense staining was noted, especially for neurons in the superficial gray layer. Almost all the area of the inferior colliculus was rich in strongly stained neurons ŽFig. 4B..
3.2.4. Pons and medulla oblongata The laterodorsal ŽFig. 5A, see also Fig. 1G. and pedunculopontine ŽFig. 5B. tegmental nuclei exhibited clustering of very strongly stained neurons. Cells in the pontine nucleus showed faint staining ŽFig. 5A,C.. In the nucleus of the trapezoid body ŽFig. 5A,D., clustering of intensely stained cells was evident. Scattered positive neurons were abundant in the medial vestibular nucleus ŽFig. 5A,E.. Intensely stained neurons clustered in the nucleus of the solitary tract ŽFig. 5A,E.. Clustering of positive cells was also observed in the prepositus hypoglossal nucleus and the dorsomedial spinal trigeminal nucleus ŽFig. 5F.. As for the reticular formation, scattered positive cells were more abundant in the medulla than in the pons ŽFig. 5A.. In the rostral portion of the medulla ŽFig. 5F., enrichment of positive cells was observed in the dorsal paragigantocellular field, the parvocellular reticular field and the intermediate reticular field. 3.2.5. Cerebellum Strong diffuse staining was observed in the granular layer ŽFig. 6A,B, see also Fig. 1F.. nNOS mRNA was also enriched in neurons of the molecular layer. Isolated positive neurons were scattered in the deep cerebellar nuclei ŽFig. 6C,D..
4. Discussion We performed in situ hybridization study to detect nNOS mRNA in the rat brain, using non-radioisotopic digoxigenin-labeled RNA probes and paraformaldehydefixed paraffin-embedded tissue sections. The combination of these probes and sections gave a high resolution sufficient to distinguish the densely stained cytoplasm and unstained nucleus in a neuron. We could clearly differentiate nonspecific signals derived from hybridization with nuclear materials, an event which often hampers the in situ hybridization study. Specificity of signals in this study was also ensured by using stringent conditions comparable with those for RNA blot analysis in which a single specific band was detected. The entire procedure is rapid and simple: pretreatments of sections such as acetylation and acidification prior to hybridization were omitted; detection of signals by color development was usually completed overnight. Highly resolved detection of nNOS mRNA enabled clarification of the size and shape of the positive cells to the extent comparable to that seen in immunocytochemistry using anti-nNOS antibodies w1,3,20x and histochemistry derived from NADPH diaphorase activity that reflects NOS activity w5,12,25x. This increases each other’s reliability for nNOS mRNA and protein localizations. In addition to previously stated areas such as the main and accessory olfactory bulbs, the amygdala, the supraoptic nucleus, the
Table 1 Classification of areas expressing nNOS mRNA Olfactoryrvomeronasal system Main olfactory bulb ŽMOB. Glomerular cell layer ŽGl. Mitral cell layer ŽMi. Internal plexiform layer ŽIP1. Granule cell layer ŽGr. ŽIsolated scattered neurons over the entire region. Accessory olfactory bulb ŽAOB. Mitralrtufted cell layer ŽMirTuA. Granule cell layer ŽGrA. Limbic and related system Amygdala Medial nucleus ŽMe. Anterior cortical nucleus ŽACo. Basomedial nucleus ŽBM. Posteromedial cortical nucleus ŽPMCo. Amygdalopiriform transition ŽAPir. Hippocampus Dentate gyrus ŽDG. Ammon’s horn CA1–3 ŽCA1–3. ŽIsolated scattered neurons over the entire region. Bed nucleus of the stria terminalis ŽBST. Epithalamus Medial habenular nucleus ŽMHb. Hypothalamus Supraoptic nucleus ŽSO. Paraventricular nucleus ŽPaV. Ventromedial nucleus ŽVMH. Premammillary nucleus ŽPM. Mammillary nucleus ŽM. Supramammillary nucleus ŽSuM. Cerebral cortex Corpus callosum Basal ganglia Caudate–putamen ŽCPu. Accumbens nucleus ŽAcb. Olfactory tubercle ŽTu. Islands of Calleja ŽICj.
Visual system Lateral geniculate nucleus Dorsal lateral geniculate nucleus ŽDLG. Ventral lateral geniculate nucleus ŽVLG. Superior colliculus ŽSC. Auditory system Nucleus of the trapezoid body ŽTz. Inferior colliculus ŽIC. Vestibular and related system Medial vestibular nucleus ŽMVe. Prepositus hypoglossal nucleus ŽPrH. Gustatory system Nucleus of the solitary tract ŽSol. Somatosensory system Dorsomedial spinal trigeminal nucleus ŽDMSp5. Thalamus Paraventricular nucleus ŽPV. Central medial nucleus ŽCM. Reuniens nucleus ŽRe. Subthalamic region Zona incerta ŽZI. Pontine nucleus ŽPn. Cerebellum Cortex Granular layer Žgr. Molecular layer Žmol. Deep nuclei Others Magnocellular nucleus of the posterior commissure ŽMCPC. Midbrain central gray ŽCG. Laterodorsal tegmental nucleus ŽLDTg. Pedunculopontine tegmental nucleus ŽPPTg. Medulla reticular formation Dorsal paragigantocellular field ŽDPGi. Parvocellular reticular field ŽPCRt. Intermediate reticular field ŽIRt.
Areas expressing nNOS mRNA are classified, based on anatomical location andror assigned major function of each area. Abbreviations used in the figures are shown in parentheses.
lateral geniculate nucleus, the superior and inferior colliculi, the pedunculopontine tegmental nucleus and the cerebellum w1,8x, clustering of neurons expressing nNOS mRNA was observed most abundantly in areas including the islands of Calleja, the paraventricular nucleus of the hypothalamus, the paraventricular nucleus of the thalamus, the magnocellular nucleus of the posterior commissure, the laterodorsal tegmental nucleus, the nucleus of the trapezoid body and the nucleus of the solitary tract. Most of these areas have been shown to contain neurons immunoreactive with nNOS antibodies w1,20,21x, except for the magnocellular nucleus of the posterior commissure, the immunohistochemical analysis of which remains to be done. Neurons in the molecular layer of the cerebellum, which were previously reported to be positive in immunohistochemistry w1,20,21x but almost negative with in situ hybridization w1x, were seen to express nNOS mRNA in the present study. The islands of Calleja, in which nNOS mRNA was
hardly detectable in a foregoing study w1x, also exhibited a strong hybridization signal, concordant with the expression of intense immunoreactivity w1,20x. Expression profiles of nNOS in the hippocampus have been controversial in the previous studies w1,7,8,20,21,26x. In the present study, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon’s horn, while more intensely stained isolated neurons were scattered over the entire region of the hippocampus. In Table 1, areas expressing nNOS mRNA are classified, taking into account anatomical location andror assigned major function of each area. Based on these distributions, it is safe to speculate that nNOS is involved in a number of brain functions related to sensory, motor, autonomic, limbic and higher systems, as has been repeatedly noted mainly based on immunohistochemical studies w1,20,21x. On the other hand, in each system, a high level
expression of nNOS mRNA is restricted to isolated individual neurons andror discrete cell groups, i.e. cell layers, nuclei, etc. This characteristic feature of localization of neurons expressing the nNOS gene was clearly demonstrated in our study using high-resolution in situ hybridization. The present data provide the basis for future investigations on roles of nNOS in each neural system, by clarifying the most remarkable areas in each system. Acknowledgements We thank S. Goto for advice in microscopic identification of neural structures, M. Ohara for helpful comments on the manuscript, and our colleagues for suggestions and discussions. This work was supported in part by grants-inaid from the Ministry of Education, Science, Sports and Culture of Japan. References w1x D.S. Bredt, C.E. Glatt, P.M. Hwang, M. Fotuhi, T.M. Dawson, S.H. Snyder, Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron 7 Ž1991. 615–624. w2x D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, S.H. Snyder, Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature 351 Ž1991. 714–718. w3x D.S. Bredt, P.M. Hwang, S.H. Snyder, Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347 Ž1990. 768–770. w4x P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w5x T.M. Dawson, D.S. Bredt, M. Fotuhi, P.M. Hwang, S.H. Snyder, Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA 88 Ž1991. 7797–7801. w6x T.M. Dawson, S.H. Snyder, Gases as biological messengers: nitric oxide and carbon monoxide in the brain, J. Neurosci. 14 Ž1994. 5147–5159. w7x J.L. Dinerman, T.M. Dawson, M.J. Schell, A. Snowman, S.H. Snyder, Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity, Proc. Natl. Acad. Sci. USA 91 Ž1994. 4214–4218. w8x M. Endoh, K. Maiese, J.A. Wagner, Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons, Neuroscience 63 Ž1994. 679–689. w9x P.L. Gabbott, S.J. Bacon, An oriented framework of neuronal processes in the ventral lateral geniculate nucleus of the rat demonstrated by NADPH diaphorase histochemistry and GABA immunocytochemistry, Neuroscience 60 Ž1994. 417–440. w10x J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 Ž1995. 683–706.
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