Nitric oxide synthase-expressing neurons are area-specifically distributed within the cerebral cortex of the rat

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Neuroscience Vol. 81, No. 2, pp. 321–330, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00131-0

NITRIC OXIDE SYNTHASE-EXPRESSING NEURONS ARE AREA-SPECIFICALLY DISTRIBUTED WITHIN THE CEREBRAL CORTEX OF THE RAT H.-J. BIDMON,*¶ J. WU,*§ A. GO } DECKE,† A. SCHLEICHER,* B. MAYER‡ and K. ZILLES* *Department of Neuroanatomy, Heinrich–Heine University, 40001 Du¨sseldorf, Germany †Department of Cardiovascular Physiology, Heinrich–Heine University, 40001 Du¨sseldorf, Germany ‡Department of Toxicology and Pharmacology, Karl Franzens University, Graz, Austria §Department of Anatomy, North China Coal Mining Medical College, Jianshelu, Thangshan 063000, Province Hebei, China Abstract––Neuronal nitric oxide synthase produces nitric oxide, a radical involved in neurotransmission as well as in cytotoxicity during stroke and neurodegenerative diseases. In the adult Wistar rat neuronal nitric oxide synthase-positive neurons are inhomogenously distributed along defined cortical areas, with highest densities (18 cells/mm2) in cingular area 1, piriform cortex, frontal motor area Fr 2 and in the medial visual association area Oc 2MM. A medium packing density of neuronal nitric oxide synthase neurons (10/mm2) characterizes primary sensory areas, whereas retrosplenial cortices contain lowest cell numbers (3–5/mm2). The data suggest that functions of certain cortical areas are more dependent on intracortically produced nitric oxide than others, and that cortical injury may cause more severe nitric oxide-related cytotoxicity in areas with higher numbers of neuronal nitric oxide synthase-positive neurons. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: nitric oxide, NADPH diaphorase, brain, cortical parcellation, non-phosphorylated neurofilament, in situ hybridization.

Nitric oxide (NO) was discovered as a retrograde messenger. It modulates synaptic plasticity,23,52 neuronal oscillations24,44 and cerebral blood flow.33,40,41,57 Three NO-producing synthases (NOS),40,46 one of which is neuronal NOS (nNOS), are under intensive investigation since nitric oxide may also be involved in the progression of neurodegenerative diseases11,40,41,51,52 and in injury-related cytotoxicity mainly by the formation of peroxynitrite.11,51 It is indicated that the latter actions are mainly influenced by the constitutive nNOS, since NOS inhibition during the first 2 h post cerebral injury reduces post-lesional neuronal damage51 and mice lacking the nNOS gene are less vulnerable to post-ischaemic neurotoxicity.12,32 In contrast to the former observations, deletion of the endothelial NOS-III gene results in a progession of post-lesional neurotoxicity.37 ¶To whom correspondence should be addressed. Abbreviations: DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetate; NADPH-d, NADPH-diaphorase; NHS, normal horse serum; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal NOS; PB, phosphate buffer; SMI 32, non-phosphorylated neurofilament H; SSC, saline sodium citrate; TB, Tris buffer; TBS, Trisbuffered saline; WFA, Wisteria floribunda lectin.

nNOS is co-localized with the enzyme NADPHdiaphorase (NADPH-d).2,3,27,39,50,52 NADPH-d enzyme histochemistry revealed that NADPH-d or nNOS-positive neurons are restricted to the septa of the somatosensory barrel cortex in rats18,50 and that they are lamina-specific in their distribution throughout the cortex of rats and humans.14,54–56 A more homogeneous and widespread distribution of nNOS containing neurons in the cerebral cortex was described for other species.13,16,27,29,35,36,47,53 However, in humans,16,17 monkeys29 and mice,13,27 the published figures indicated that such neurons were inhomogeneously distributed as quantitatively shown for NADPH-d-positive fibres in cortical areas 4, 17 and 24 of human brains17 as well as for NOS-positive cells in the developing cortex of sheep43 and prefrontal cortex of macaques.14 In order to study the distribution of NOS-positive neurons in rat brains, which often serve as models to study neuronal plasticity and post-lesional changes after cerebral infarcts,26 we used NADPH-d enzyme histochemistry, NOS immunohistochemistry and in situ hybridization in combination with the demonstration of area-specific markers such as nonphosphorylated neurofilament H (SMI 32) or proteoglycans.2,5–10,31,49

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Neuronal NO-synthase in cerebral cortex EXPERIMENTAL PROCEDURES

Subjects were male Wistar rats (n=7; 220–250 g body weight, Han: WIST, multiplication stock, TVA Du¨sseldorf), which were anaesthetized with nembutal and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were dissected, postfixed and serially-sectioned (50 µm) on a Vibratome. Brains of five additional male rats were dissected after decapitation, frozen on dry-ice and serially-sectioned (14 µm) in a cryostat. For NADPH-d histochemistry, Vibratome sections were rinsed in PB (4#15 min), transferred to 0.1 M Tris–HCl buffer (pH 8.0, containing 0.3% Triton X-100; 1 mg â-NADPH/ml, and 2.5 mg Nitro Blue Tetrazolium/ml [Boehringer]), incubated for 30 min at 37)C, rinsed twice in 50 mM Tris buffer containing 0.9% NaCl, pH 7.4 (Trisbuffered saline, TBS) and mounted onto glass slides. In the case of double-staining, endogenous peroxidase was blocked with 0.6% H2O2 in TBS. After several rinses in TBS, alternating sections were stained either with Wisteria floribunda lectin (WFA 1 µg/ml TBS; Sigma) following a standard protocol5 or for SMI 32. Immunohistochemistry For SMI 32 immunohistochemistry, non-specific staining was blocked by incubation of sections in TBS with 2% normal horse serum (NHS, Vectastain) and 10% fetal calf serum. Sections were then incubated at 4)C for 48 h in SMI 32 antibody (Sternberger monoclonals, Baltimore) diluted 1:1000 with TBS+2% NHS. After rinsing in TBS (4#15 min), they were incubated for 3 h in peroxidasecoupled antimouse antibody (Sigma/Deisenhofen) at a dilution of 1:50 in TBS. After two rinses in TBS and two in 50 mM Tris–HCl, pH 7.6 (Tris buffer, TB), sections were incubated in TB containing 0.75 mg/ml 3,3diaminobenzidine (Sigma) for 10 min, and 0.003% H2O2 was then added. Staining was terminated by several rinses in TB. Sections were mounted, air-dried and coverslipped with DePex (Fluka). Specificity of staining was controlled on sections incubated in NHS instead of SMI 32 antibody. Alternate frontal sections from 12 levels were stained with an antiserum against nNOS (Eurodiagnostica) at a dilution of 1:200. Non-specific staining was blocked with 2% normal goat serum. The secondary antibody was peroxidasecoupled anti-rabbit serum (Sigma) at a dilution of 1:200. The procedures were otherwise as described above for SMI 32. Additionally, another series of sections was similarily stained with nNOS antibody from B. Mayer at a final dilution of 1:1000.33 To prove if NADPH-d and nNOS were co-localized in all cortical neurons, an alternate series of sections was stained for NADPH-d followed by nNOS immunohistochemistry (Fig. 2f). In situ hybridization The probe for in situ hybridization was prepared as follows: 1 µg of total rat cerebellar RNA was reverse

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transcribed with MMLV-Reverse Transcriptase (Gibco BRL). A nNOS-specific fragment corresponding to nucleotides 1173–1632 was amplified by polymerase chain reaction using 35 cycles 1* 94)C, 2* 55)C, 2* 72)C with the 5* oligoprimer AGA GAA GGA ACA GTC CCC TAC C and the 3* oligoprimer ATC GAA CAC CTG CAG CTT G sequences. The resulting fragment was cloned into pGEM-T (Promega). The DNA was linearized and afterwards transcribed using T7- or SP6-RNA polymerases (Boehringer) and á-[35S]UTP (NEN Dupont) following a previously described protocol.4 Frozen sections were fixed in 4% paraformaldehyde, rinsed in TBS and alternate sections were stained for either NADPH-d or nNOS as described above, except that incubation time in primary antiserum was reduced to 14 h. Additional alternate paraformaldehyde-fixed sections were used for in situ hybridization with the antisense or sense nNOS-mRNA-probe according to a previously published protocol4 using a probe concentration of 1,000,000 c.p.m./ 20 µl hybridization buffer for each section. The hybridization buffer consisted of 50% formamide; 10 mM Tris–HCl, pH 7.5; 10 mM PB, pH 6.8; 5 mM EDTA; 2#saline sodium citrate (SSC); 150 µg/ml yeast tRNA (RNase-free, Boehringer); 0.1 mM UTP and 10% dextrane sulphate (all from Sigma). Shortly before use, 1/100 volume of 100 mM ADPßS; 1 mM ATPâS, 1 M dithiothreitol (DTT) and 1 M 2-mercaptoethanol (Boehringer) were added. In brief, sections were hybridized at 50)C for 24 h in a humid chamber, washed for 2 h at 37)C in wash-buffer (50% formamide; 2#SSC; 10 mM 2-mercaptoethanol), rinsed in TBS followed by incubation in RNase 20 µg/ml TBS (15 min), rinsed in TBS for 15 min, and rinsed overnight in wash-buffer+5 mM DTT. Sections were dehydrated in graded ethanols from 30%–100%. Dry slides were dipped in NTB 2 nuclear track emulsion (Eastman Kodak), exposed at 4)C for 21 days and developed and stained as previously described.1 Quantitative analysis For quantitative analyses we first tested if the changes in the distribution pattern of SMI 32 corresponded to areal borders within the cortex as is known for WFA.5 Sections stained with SMI 32 were drawn and sections stained with the area-specifically distributed WFA were superimposed and areal borders were marked. This comparison revealed that changes in the SMI 32 distribution corresponded to known areal borders. This was also shown in a study where paraffin-embedded sections stained for SMI 32 were compared with alternate sections stained for cytoarchitectonic parcellation with Ag–Nissl.2,58 The latter technique was not directly applicable to this study since the classical Nissl stain leads to a shrinkage of the free floating Vibratome sections, making it difficult to compare immuno- or WFA-stained sections directly with alternate sections stained with Ag– Nissl for classical cytoarchitectonic evaluation. Furthermore, the use of WFA or SMI 32 allowed the double

Fig. 1. Images of sections (a–d) and original micrographs (e–k). a–d) Four levels of the rat brain in rostrocaudal direction showing areal distribution patterns of the SMI 32 epitope (grey patches) upon which nNOS-positive neurons have been superimposed (each solid black dot represents one neuron). Note intense somatodendritic staining for SMI 32, but low number of nNOS-positive neurons in retrosplenial areas (RSG, RSA in b–d). Also sensory areas (Par1, HL) and motor area 3 (Fr3) show intense staining for SMI 32 in L II–III and V–VI (a–d), but nNOS-positive neurons are most numerous in areas with weak staining for SMI 32, such as in Cg1 (a), Fr2 (b–c), Oc2MM (d). e) Area Fr2 with neurons expressing nNOS mRNA (arrowheads). f) Area Oc2MM with multipolar neurons immunostained for nNOS (triangle marks cells enlarged in insets in e and f). g) Detail of NADPH-d-positive neurons in area Fr2 imaged in b, h and i) Area Pir in which both NADPH-d and nNOS mRNA detection techniques show strongly- and weakly-labelled nNOS-positive neurons. j and k) Original NADPH-d-positive neurons shown in RSA, Oc2MM/ML and part of Oc1M in d. k) Enlargement of the neurons marked by a black triangle in j; j is an adjacent section to f. For abbrevations see Fig. 3. Scale bars=2.0 mm (a–d), 100 µm (e–g, j), 50 µm (h,i,k and inset f).

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Neuronal NO-synthase in cerebral cortex staining with NADPH-d or nNOS on one and the same section, making it possible to examine the areaspecific borders as well as the area-specific amount of nNOS-positive neurons in the same section. For quantitative evaluation, photographs of each fifth section (magnification 16#), which was double-stained (NADPH-d/SMI 32 or NADPH-d/WFA) or NOS-I, were taken from six brains. In these photographs, areal borders were marked according to the WFA and SMI 32 pattern and in comparison to current atlases.44,57 These borders corresponded well and were easily defined by the changes in the distribution pattern of SMI 32 and WFA (Figs 1, 2). Each cortical area was then planimetrically determined using a graphics tablet (Jandel-Scan). To prevent double counting only the nNOS-positive neurons with a clearly visible nucleus were counted in each cortical area of each section. Additional sections on which small areas were represented had to be counted, since for statistical analysis each area had to be counted in at least seven sections from each brain. The data were statistically processed for cluster analysis and tested according to P-Stat Cluster analysis (P-Stat user’s manual; Vol. 2/3; P-Stat, Inc. Princeton, NJ, U.S.A. 1990), an analysis which groups areas into clusters which differ most significantly from each other (Fig. 3). Additionally, it was tested whether cortical areas differ significantly to each other by Student’s t-test (Fig. 4). RESULTS

With the combinations of NADPH-d and SMI 32 or WFA histochemistry, cortical parcellation was demonstrated in a reproducible manner for each brain (Figs 1a, 2f) and the areas identified with SMI 32 or WFA were similarily located to those identified by WFA histochemistry5 or other techniques.45,58 Staining for nNOS and NADPH-d revealed a 98–100% co-localization in most cortical areas. Slightly but not significantly more NADPH-d neurons were present in layer (L) VI compared to nNOS-immunoreactive neurons (Eurodiagnostika) whereas slightly more nNOS-immunoreactive neurons (antibody from B. Mayer; B.M.) existed in the piriform and entorhinal cortex (Fig. 2f). Similar to the nNOS antibody (B.M.), nNOS mRNA-positive perikarya (Figs 1, 2b,c) were more numerous in piriform and entorhinal regions, otherwise comparable values to those for NADPH-d were obtained. The nNOS-positive interneurons were laminaspecific distributed within most cortical areas with highest packing densities in layers VI and II/III as already described in detail.38,54 Only nonpyramidal

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bi- and multipolar neurons were nNOS-positive and no localization of nNOS or NADPH-d in neurons positively stained for SMI 32 or WFA was observed. The main purpose of this study was the evaluation of the area-specific packing density of nNOS-positive perikarya. The quantitative analysis of NADPH-dand nNOS-positive neurons for each cortical area revealed a clearly inhomogenuous distribution (Figs 1, 2). Sections showing the nNOS mRNA expression were not directly comparable to those sections stained for NADPH-d or nNOS, since they were from different animals and of different thickness. Especially the latter point makes it difficult to compare the number of cells counted in a 50-µm section with those seen in a 14-µm section. However, even without a direct quantitative comparison it was apparent that nNOS mRNA-expressing cortical cells showed the same inhomogenous, area-specific distribution pattern as the ones stained for NADPH-d or nNOS (Figs 1e,h, 2b,c). In order to demonstrate differences and/or similarities among cortical areas with regard to the amount of nNOS-positive cells we used cluster analysis. Cluster analysis revealed three clusters of cortical areas which differed with high significance from each other and which contained an average of 4.7 (Cluster 1), 9.8 (Cluster 2) or 17.8 (Cluster 3) nNOS-positive neurons/mm2 (Fig. 3). While cluster analysis groups cortical areas into groups which differ significantly from each other, it does not determine the level of significance by which individual areas differ from each other. Therefore, we determined cortical areas which differed, in their packing density for nNOS-neurons, showing significance using a Student’s t-test (Fig. 4). Cluster 1 consisted of areas with few positive neurons and was comprised of retrosplenial cortical areas, the parietal primary sensory, the occipital monocular, and the frontal primary motor cortex 3. The latter lies next to the primary somatosensory cortex and shares more similarites with sensory areas than with other frontal motor areas which clearly lack layer IV58 and which show less immunoreactivity for SMI 32. As shown in Figs 1b–d,i, 2e, and 3, cortical areas belonging to cluster 1 exhibited strongest WFA binding and highest amounts of SMI 32positive pyramidal neurons.

Fig. 2. Shows original sections (a, d–f) and digitized, colour-inverted images of sections hybridized with nNOS probe (b–c). a) Shows the distribution of dendritic SMI 32 (brown) in Fr2 and Cg1 compared to the distribution of NOS-positive interneurons (blue) with increased packing density of NOS in Cg1. b–c) Distribution of nNOS mRNA at two levels of the rat brain showing increased labelling in Cg1, Fr2/1 and Par 2 (Note the low amount of labelling in Cg2). d–e) Shows the area-specific distribution of WFA (brown) with strong binding in L IV–V in RSG and L II/III and weaker binding in L V in RSA (RSA lacks L IV). Fr2 show continuous WFA binding in L VI only, whereas Fr1 shows slight increase in L V compared to Fr2 (Areal borders marked by dashed lines). nNOS-positive neurons (blue) are more numerous in Fr2 where multi- and bipolar interneurons (d) lie next to single WFA-binding ones. f) Co-localization of NADPH-d and nNOS antibody (B.M.) in piriform cortex showing three clearly co-localized neurons (black arrowheads) and four neurons with weak but clear immunoreactivity for nNOS but with background staining for NADPH-d (white arrowheads). Corpus callosum, cc; striatum, St; lateral ventricle, v. Scale bars=500 µm (a,e); 50 µm (d,f); 1 mm (b,c).

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Fig. 3. Shows the packing density of nNOS-positive neurons (&S.D.; n=6) for each cortical area. The cortical areas are grouped according to P-Stat cluster analysis (Cluster 1, white; cluster 2, grey; and cluster 3, black columns). The roman numbers within each column indicate the staining intensity for SMI 32 within each area (I=stained single fibres and some single, mainly non-pyramidal somata; II=stained pyramidal neurons and fibres mainly in L V–VI; III=loosely packed SMI 32-positive pyramidal neurons and fibres in L II/III, V and VI; IV=densely-packed SMI 32-positive pyramidal neurons in L II/III, V–VI with dense fibre plexus in L I and some non-pyramidal neurons in L IV). AID, agranular dorsal insular cortex; AIP, agranular posterior insular cortex; Cg1–3, cingular cortex areas 1, 2 and 3; Ent, entorhinal cortex; FL, forelimb area; Fr1–3, frontal cortex areas 1, 2 and 3; Gu, gustatory cortex; HL, hindlimb area; IL, infralimbic area; Oc1–2, occipital cortex areas 1 and 2, B (binocular), M (monocular), L (lateral), ML (mediolateral), MM (mediomedial); Par1–2, parietal cortex areas 1 and 2; Pir, piriform cortex; PRh, perirhinal area; RS, retroslenial agranular (A) and granular (G) cortex; Te1–3, temporal cortex areas 1, 2 and 3.

Cluster 2 included mainly sensory and secondary sensory areas: the primary visual binocular and primary auditory cortices, the gustatory and agranular insular cortices as well as periallocortical and allocortical cortices (Figs 1–4). These areas express low or medium amounts of SMI 32-immunoreactive pyramidal neurons and WFA-binding and contain approximately nine to 10 nNOS-positive neurons/mm2. Cluster 3 consisted also of a heterogeneous group of areas with high densities of nNOS-positive neurons: the periallocortical infralimbic area, the more isocortical part of the cingular cortex area 1 (Cg1) (Fig. 2a–b), the laterally adjacent frontal cortex 2 (Fr2), the visual association cortex (Oc2MM) bordering it in the occipital direction (Figs 1–3) and the piriform cortex. These cortical areas showed

low amounts of SMI 32-immunoreactive neurons and low levels of WFA-binding, which was seen, when present, only in the deeper layers. In this regard, Fr2 and Oc2MM, which were clearly separated from the retrosplenial cortices by their low amounts of SMI 32 immunoreactivity and WFA binding in L I–VI, also contained the lowest amounts of both epitopes in comparison to the other adjacent frontal and/or occipital cortices (Figs 1b–d, 2d–e, 3). Also cingular cortex 1 and the piriform cortex contained low numbers of SMI 32-positive pyramidal neurons (Figs 1a, 2a, 3). With the exception of the cingular areas 2 and 3, the latter observations showed an almost reciprocal distribution of nNOS-positive neurons and the epitopes for SMI 32 or WFA among cortical areas.

Fig. 4. Graphical summary of the levels of significance for the differences in packing density of NOS-positive neurons among the different cortical areas as revealed by Student’s t-test. Black squares=P¦0.0009; dark grey squares=P¦0.009; light grey squares=P¦0.05; white=non significant. For abbreviations see Fig. 3.

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H.-J. Bidmon et al. DISCUSSION

Our study showed clearly an inhomogenous distribution of nNOS-positive interneurons among several cytoarchitectonically and functionally defined areas of the rat cerebral cortex. The observed areal distribution of nNOS-positive neurons was not related to the pattern of cerebral vascularization.48 For the rat these areas and their characterization have been reviewed by Zilles and Wree58 and their identification by the use of WFA binding has been shown for rats, gerbils and in part for humans.5,49 In our study we added the somatodendritic pyramidal cell marker SMI 32 to the list of markers which are useful for cortical parcellation in rats. SMI 32 has been identified as a marker for transhemispheric projection neurons in monkeys.6,7 Since SMI 32 is widely used in the parcellation of the primate cortex including man this marker offers the opportunity to compare areal staining patterns among species.6–8,30,31 With regard to nNOS-positive interneurons, our main goal was the area-specific distribution of these cells rather than a repetition of the results describing a lamina-specific distribution with highest numbers of nNOS-positive neurons in layers II/III and VI.34,38,39,50,54 Also the nNOS-positive cell types of bipolar, bitufted, and multipolar interneurons including Martinotti cells have been characterized in detail.34,38,39,50,54 In contrast to previous results our study showed some differences between NADPH-d staining and nNOS immunoreactivity. It is well accepted that only 98% of NADPH-d-positive neurons contain nNOS within the cortex.34 We obtained similar results. However, within the piriform cortex slightly more neurons were only immunostained for nNOS compared to other cortical areas. This was not due to the differences in staining intensities for NADPH-d among type-I (strongly stained) and type II (weakly stained) neurons according to Yan et al.,54–56 since both cell types were identified as NADPH-d-positive cells and counted for the determination of the total number of NADPH-d positive neurons/mm2. Similarily, many more cells contained nNOS mRNA within the piriform cortex. These cells were not quantified, since direct comparisons were not made among thick sections stained for nNOS and thin sections used for in situ hybridization. But this qualitative observation suggests differences in the sensitivity between the methods, or nNOS mRNA is not directly translated into protein in all neurons of the piriform cortex. About 2% of nNOS containing cortical interneurons contain GABA 14,19,20,50,56 and in rats many GABAergic neurons co-localize with parvalbumin which is a constituent of neurons positive for WFA.28 Since we observed no co-localization of NADPH-d and WFA binding the results suggest that the GABAergic nNOS-positive neurons belong to a different specialized subset of GABAergic interneurons. In rat and human cerebral cortex, NADPH-dpositive neurons partly co-localize somatostatin

and/or neuropeptide Y.15,42 Cortical somatostatinexpressing neurons outnumber those which were positive for nNOS. However, somatostatin-positive neurons are also area-specifically distributed throughout the rat cortex.21,22 The relative changes in the area-specific packing densities of somatostatinpositive neurons as well as their lamina-specific distribution pattern reflects those seen for nNOS and NADPH-d-positive neurons. Therefore, all cortical areas belonging to cluster 3 in our study also contain the highest packing densities of somatostatinexpressing neurons, whereas those of cluster 1 contain the lowest densities. This correspondence in the relative packing densities of nNOS and/ or somatostatin-containing neurons suggest areaspecific neurochemical functions which seem to rely on characteristic amounts of these substances. Our results as well as those described for somatostatin corroborate findings which show that Cg1, Fr2 and Oc2MM differ significantly in connectivity and function from the other cingular, frontal and occipital areas, respectively.58 Particularly, Fr2 of the rat has been considered to be homologous to the primate supplementary motor cortex (for discussion see Ref. 58). Additionally, the obtained results indicate the importance of detailed cortical parcellation, since inhomogeneity in the packing density of nNOSpositive neurons was not observed if the cingular, frontal, parietal or occipital cortices had not been parcellated into their subdivisions. In detail, no significant differences were found when Cg1–3, Fr1–3, Par1–2 or Oc1–2 were treated as single areas, as has been done in earlier studies.35,38,39,41,47,54 This becomes understandable, for example, in the frontal motor areas where all three areas belong to a different cluster (Fig. 3). Fr2 belongs to cluster 3 and is suggested to represent a supplementary motor area, Fr1 (cluster 2) represents a primary motor area and Fr3 (cluster 1) has many similarities with the parietal somatosensory area Par1 (Fig. 3). Therefore, nNOS neurons and SMI 32 distribution showed a clear parcellation which is comparable with that seen in classical cyto- and myeloarchitectonic studies, neurotransmitter receptor distribution58 and WFAbinding.5 In rats and humans, cortical areas and their subdivisions, which are characterized by classical techniques and certain types of neurotransmitters, show clear functional differences.25,58 Therefore, it is suggested that the observed area-specific levels of nNOS are also essential for area-specific functions. CONCLUSION

The present data indicate that intra-areal nNOS expression is more essential for certain cortical areas than for others, but they do not suggest that areas with low densities of nNOS-positive neurons lack functions modulated by NO. Extracortical nNOS and NO may be supplied via afferents from

Neuronal NO-synthase in cerebral cortex

subcortical and brainstem regions, as shown for the occipital cortex.38 It is known that retrosplenial areas, which show the lowest densities of nNOSpositive neurons, receive many projections from the bed nucleus of the stria terminalis, the presubiculum, Cg1 and Oc2MM. In the rat forebrain, these latter areas all contain great numbers of nNOS-positive perikarya. Concerning the cytotoxic effects of NO, local injuries which lead to a deregulated production of NO and related radicals11,51 may be more devastating during the initial phase of injury in areas containing more nNOS-positive neurons than in other cortical regions. In contrast deregulation of NO production in subcortical regions which are rich in nNOS, however, may affect cortical areas to which they are connected more severely. This suggestion

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may be also supported by the observation that mice lacking the nNOS gene are less vulnerable to cerebral infarcts.32 Furthermore, nNOS, NO and its metabolites are involved in neurodegenerative diseases in motor or motor-associated cortical areas51 which show high levels of nNOS in humans17 as well as in rats. Therefore, the observed inhomogenous distribution of nNOS within the rat cortex may offer one explanation for the differences found for NO-dependent post-lesional damage among different lesion models. Acknowledgements—The authors thank Dr J. Williams, Department of Physiology, for helpful discussions and corrections. The study was supported by the DFG; SFB 194/A6.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Bidmon H.-J. and Stumpf W. E. (1995) Localization of specific high-affinity binding sites (receptors) for ecdysteroids in invertebrates. In Autoradiography and Correlative Imaging (eds Stumpf W. E. and Solomon H. F.), pp. 83–110. Academic, San Diego. Bidmon H.-J., Wu J., Go¨decke A., Jancsik V. and Zilles K. (1995) Areal-spezifische Verteilung von nichtphosphoryliertem Neurofilament (SMI 32), Mikrotubuli assoziertem Protein (MAP 2) und NO-Synthase im Rattengehirn. Ann. Anat. 177, 56. Bredt D. S., Glatt C. E., Hwang P. M., Fohtui M., Dawson T. M. and Snyder S. H. (1991) Nitric oxide synthase protein and mRNA are discretly localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7, 615–624. Breier G., Albrecht U., Sterrer S. and Risau W. (1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114, 521–532. Bru¨ckner G., Seeger G., Brauer K., Ha¨rtig W., Kacza J. and Bigl V. (1994) Cortical areas are revealed by distribution patterns of proteoglycan components and parvalbumin in the mongolian gerbil and rat. Brain Res. 658, 67–86. Campbell M. J., Hof P. R. and Morrison J. H. (1991) A subpopulation of primate corticocortical neurons is distinguished by somatodendritic distribution of neurofilament protein. Brain Res. 539, 133–136. Campell M. J. and Morrison J. H. (1989) Monoclonal antibody to neurofilament protein (SMI 32) labels a subpopulation of pyramidal neurons in the human and monkey neocortex. J. comp. Neurol. 282, 191–205. Carmichael S. T. and Price J. L. (1994) Architectonic subdivision of the orbital and medial prefrontal cortex in the macaque monkey. J. comp. Neurol. 346, 366–402. Celio M. R. (1986) Parvalbumin in most ã-aminobutric acid-containing neurons of the rat cerebral cortex. Science 231, 995–997. Celio M. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475. Dawson V. L. and Dawson T. M. (1996) Free radicals and neuronal cell death. Cell Death Diff. 3, 71–78. Dawson V. L., Kizushi V. M., Huang P. L., Snyder S. H. and Dawson T. M. (1996) Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. J. Neurosci. 16, 2479–2487. Derer P. and Derer M. (1993) Ontogenesis of NADPH-diaphorase neurons in the mouse forebrain. Neurosci. Lett. 152, 21–24. Dombrowski S. M. and Barbas H. (1996) Differential expression of NADPH diaphorase in functionally distinct prefrontal cortices in the rhesus monkey. Neuroscience 72, 49–62. Dun N. J., Dun S. L., Wong R. K. and Fo¨rstermann U. (1994) Colocalization of nitric oxide synthase and somatostatin immunoreactivity in rat dentate hilar neurons. Proc. natn. Acad. Sci. U.S.A. 91, 2955–2958. Egberongbe Y. I., Gentleman S. M., Falkai P., Bogerts B., Polak J. M. and Roberts G. W. (1994) The distribution of nitric oxide synthase immunoreactivity in the human brain. Neuroscience 59, 561–578. Fisher H. C. and Kuljis R. O. (1994) Multiple types of nitrogen monoxide synthase-/NADPH diaphorase-containing neurons in the human cerebral neocortex. Brain Res. 654, 105–117. Franca J. G. and Volchan E. (1995) NADPH diaphorase histochemistry as a marker for barrels in rat somatosensory cortex. Braz. J. med. biol. Res. 28, 787–790. Gabbot P. L. A. and Bacon S. J. (1995) Co-localisation of NADPH diaphorase activity and GABA immunoreactivity in local circuit neurones in the medial prefrontal cortex (MPFC) of the rat. Brain Res. 699, 321–328. Gabbot P. L. A. and Bacon S. J. (1996) Local circuit neurons in the medial prefrontal cortex (areas 24A, B, C, and 32) in the monkey. 1. Cell morphology and morphometrics. J. comp. Neurol. 364, 567–608. Garrett B., Finsen B. and Wree A. (1994) Parcellation of cortical areas by in situ hybridization for somatostatin mRNA in the adult rat: frontal, parietal, occipital, and temporal regions. Anat. Embryol. 190, 389–398. Garrett B., Finsen B. and Wree A. (1996) In situ hybridization for somatostatin mRNA in the adult rat: cingulate, insular, prepiriform, perirhinal, entorhinal, and retrosplenial cortical regions. Anat. Embryol. 193, 387–395. Garthwaite J. (1991) Glutamate, nitric oxide and cell–cell signaling in the nervous system. Trends Neurosci. 14, 60–68. Gelperin A. (1994) Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc. Nature 369, 61–63. Geyer S., Ledberg A., Schleicher A., Kinomura S., Schormann T., Bu¨rgel U., Klingberg T., Larsson J., Zilles K. and Roland P. E. (1996) Two different areas within the primary motor cortex of man. Nature 382, 805–807.

330

H.-J. Bidmon et al.

26. Ginsberg M. D. (1996) The validity of rodent brain-ischemia models is self-evident. Arch. Neurol. 53, 1065–1067. 27. Giuili G., Luzi A., Poyard M. and Guellaen G. (1994) Expression of mouse brain soluble guanylyl cyclase and NO synthase during ontogeny. Devl Brain. Res. 81, 269–283. 28. Ha¨rtig W., Brauer K. and Bru¨ckner G. (1992) Wisteria floribunda agglutinin-labelled nets surround parvalbumincontaining neurons. NeuroReport 3, 869–872. 29. Hashikawa T., Leggio M. G., Hattori R. and Yui Y. (1994) Nitric oxide synthase immunoreactivity colocalized with NADPH-diaphorase histochemistry in monkey cerebral cortex. Brain Res. 641, 561–578. 30. Hayes T. L. and Lewis D. A. (1995) Anatomical specialization of the anterior motor speech area: hemispheric differences in magnopyramidal neurons. Brain and Language 49, 289–308. 31. Hof P. R. and Morrison J. H. (1995) Neurofilament protein defines regional patterns of cortical organisation in the macaque monkey visual system: a quantitative immunohistochemical analysis. J. comp. Neurol. 352, 161–186. 32. Huang Z., Huang P. L., Panahian N., Dalkara T., Fishman M. C. and Moskowitz M. A. (1994) Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265, 1883–1885. 33. Iadecola C., Beitz A. J., Renno W., Xu X., Mayer B. and Zhang F. (1993) Nitric oxide synthase-containing neuronal processes on large cerebral arteries and cerebral microvessels. Brain Res. 606, 148–155. 34. Kharazia V. N., Schmidt H. H. H. W. and Weinberg R. J. (1994) Type I nitric oxide synthase fully accounts for NADPH-diaphorase in rat striatum, but not in cortex. Neuroscience 62, 983–987. 35. Kidd E. J., Michel A. D. and Humphrey P. P. A. (1995) Autoradiographic distribution of [3H]-NG-nitro-arginine binding in rat brain. Neuropharmacology 34, 63–73. 36. Kuchiiwa S., Kuchiiwa T., Mori S. and Nakagawa S. (1994) NADPH diaphorase neurons are evenly distributed throughout cat neocortex irrespective of functional specialization of each region. NeuroReport 5, 1662–1664. 37. Lo E. H., Hara H., Rogowska J., Trocha M., Pierce A. R., Huang P. L., Fishman M. C., Wolf G. L. and Moskowitz M. A. (1996) Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression. Stroke 27, 1381–1385. 38. Lu¨th H.-J. (1995) The source of nitric oxide positive neurons in the visual cortex of the rat. In Learning and Memory (eds Elsner N. and Menzel R.), Vol. 2, p. 582. Thieme-Verl., Stuttgart. 39. Lu¨th H.-J., Hedlich A., Hilbig H., Winkelmann E. and Mayer B. (1995) Postnatal development of NADPHdiaphorase/nitric oxide synthase positive nerve cells in visual cortex of the rat. Brain Res. 36, 313–328. 40. Moncada S. and Higgs E. A. (1995) Molecular mechanisms and therapeutic strategies related to nitric oxide. Fedn Am. Socs exp. Biol. J. 9, 1319–1330. 41. Moro V., Badaut J., Springhetti V., Edvinsson L., Seylaz J. and Lasbennes F. (1995) Regional study of the co-localization of neuronal nitric oxide synthase with muscarinic receptors in the rat cerebral cortex. Neuroscience 69, 797–805. 42. Norris P. J., Waldvogel H. J., Faull R. L. M., Love D. R. and Emson P. C. (1996) Decreased neuronal nitric oxide synthase messenger RNA and somatostatin messenger RNA in the striatum of Huntington disease. Neuroscience 72, 1037–1047. 43. Northington F. J., Koehler R. C., Traystman R. J. and Martin L. J. (1996) Nitric oxide synthase 1 and nitric oxide synthase 3 protein expression is regionally and temporally regulated in fetal brain. Devl Brain Res. 95, 1–14. 44. Pape H.-C. (1995) Nitric oxide: an adequate modulatory link between biological oscillators and control systems in mammalian brain. Semin. Neurosci. 7, 329–340. 45. Paxinos G. and Watson C. (eds) (1986) The Rat Brain in Stereotactic Coordinates, Vol. 2. Academic, Sydney. 46. Pneunova N. and Eniklopov G. (1995) Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375, 68–73. 47. Rodrigo J., Springall D. R., Uttentahl O., Bentura M. L., Abadia-Molina F., Riveros-Moreno V., Martinez-Murillo R., Polak J. M. and Moncada S. (1994) Localization of nitric oxide synthase in adult rat brain. Phil. Trans. R. Soc. 345, 175–221. 48. Scremin O. U. (1995) Cerebral vascular system. In The Rat Central Nervous System (ed. Paxinos G.), pp. 3-35. Academic Press, San Diego.. 49. Seeger G., Lu¨th H.-J., Winkelmann E. and Brauer K. (1996) Distribution patterns of Wisteria floribunda agglutinin binding sites and parvalbumin-immunoreactivite neurons in the human visual cortex: a double-labelling study. Brain Res. 37, 351–366. 50. Valtschanoff J. G., Weinberg R. G., Kharazia V. N., Schmidt H. H. H. W., Nakane M. and Rustioni A. (1993) Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunohistochemistry, and colocalization with GABA. Neurosci. Lett. 157, 157–161. 51. Varner P. M. and Beckman J. S. (1995) Nitric oxide toxicity in neuronal injury and degeneration. In Nitric Oxide in the Nervous System (ed. Vincent S.), pp. 191–206. Academic, San Diego. 52. Vincent S. R. (1994) Nitric oxide: a radical neurotransmitter in the central nervous system. Prog. Neurobiol. 42, 129–160. 53. Vincent S. R. and Kimura H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755–784. 54. Yan X. X., Garey L. J. and Jen L. S. (1994) Development of NADPH-diaphorase activity in the rat neocortex. Devl Brain Res. 79, 29–38. 55. Yan X. X., Garey L. J. and Jen L. S. (1996) Prenatal development of NADPH-diaphorase-reactive neurons in human frontal cortex. Cerebr. Cortex 6, 737–745. 56. Yan X. X., Jen L. S. and Garey L. J. (1996) NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins. Cerebr. Cortex 6, 524–529. 57. Zhang F., Xu S. and Iadecola C. (1995) Role of nitric oxide and acetylcholine in neocortical hyperemia elicited by basal forebrain stimulation: evidence for an involvement of endothelial nitric oxide. Neuroscience 69, 1195–1204. 58. Zilles K. and Wree A. (1995) Cortex: Areal and laminar structure. In The Rat Nervous System (ed. Paxinos G.), pp. 649–685. Academic, San Diego. (Accepted 12 March 1997)