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Acta histochemica 107 (2005) 113—120

KEYWORDNOS I;Vestibularcomplex;Cat

0065-1281/$ - sdoi:10.1016/j.

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Light microscopical study of nitric oxide synthaseI-positive neurons, including fibres in the vestibularnuclear complex of the cat

Vassil Papantchev�, Adrian Paloff, Tatiana Christova, Dimka Hinova-Palova, Wladimir Ovtscharoff

Department of Anatomy and Histology, Medical University, 1431 Sofia, Bulgaria

Received 22 July 2004; received in revised form 21 January 2005; accepted 21 January 2005

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ee front matter & 200acthis.2005.01.004

ing author. Tel.: +359ess: vassil_papantche

SummaryNitric oxide is a gaseous neurotransmitter that is synthesized by the enzyme nitricoxide synthase I (NOS I). At present, little is known of NOS I-positive neurons in thevestibular nuclear complex of the cat (VNCc). The aim of the present study was toexamine the morphology, distribution patterns and interconnections of NOS I-positiveneurons, including fibres in the VNCc. Five adult cats were used as experimentalanimals. All cats were anaesthetized and perfused transcardially. Brains wereremoved, postfixed, cut on a freezing microtome and stained in three differentways. Every third section was treated with the Nissl method, other sections werestained either histochemically for NADPH diaphorase or immunohistochemically forNOS I. The atlas of Berman (1928) was used for orientation in the morphometric study.NOS I-positive neurons and fibres were found in all parts of VNCc: medial vestibularnucleus (MVN); lateral vestibular nucleus (LVN); superior vestibular nucleus (SVN);inferior vestibular nucleus (IVN); X, Y, Z groups and Cajal’s nucleus. The NOS I-positiveneurons were classified according to their size (small, medium-sized, large neuronstype I and type II) and their shape (oval, fusiform, triangular, pear-shaped, multipolarand irregular). In every nucleus, a specific neuronal population was observed. In SVN,a large number of interconnections between NOS I-positive neurons were identified. InMVN, chain-like rolls of small neurons were found. Tiny interconnections between MVNand mesencephalic reticular formation were present. Our data provide information onthe morphology, distribution patterns and interconnections of NOS I-positive neuronsin the VNCc and can be extrapolated to other mammals.& 2005 Elsevier GmbH. All rights reserved.

5 Elsevier GmbH. All rights reserved.

889 700 343.v@yahoo.com (V. Papantchev).

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V. Papantchev et al.114

Introduction

Nitric oxide (NO) is a neurotransmitter that hasbeen studied frequently over the last years(Mizukawa et al., 1989; Mizukawa, 1990; Dawsonet al., 1991; Hope et al., 1991; Rodrigo et al., 1994;Kullo et al., 1997; Iwase et al., 2000; Soares et al.,2003; Marino and Cudeiro, 2003; Sears et al., 2004;Seyidova et al., 2004). This interest is caused byand focused on the unique properties of NOdescribed below. It is not stored in vesicles, it isonly synthesized instantly by the enzyme NOsynthase (NOS; Martinelli et al., 2002). NO is nota ligand of postsynaptic receptors; it diffuses freelythrough cell membranes (Martinelli et al., 2002). Itis not inactivated by a specific enzyme, but itdissociates spontaneously (Martinelli et al., 2002)and there are no mechanisms of reuptake (Holsteinet al., 2001; Martinelli et al., 2002).

A number of methods exist to demonstrate NOS.Moreover, all three isoforms of NOS can bevisualized: NOS I or neuronal NOS (nNOS), which ismainly present in neurons (Dawson et al., 1991;Hope et al., 1991), skeletal muscular cells(Dahrmann and Gossrau, 1996) and heart musclecells (Sears et al., 2003); NOS II or inducible (iNOS),which can be found in macrophages (Sugiyama etal., 2003); NOS III or endothelial (eNOS), which ispresent in endothelial cells (Seidel et al., 1997).

Each isoform can be demonstrated by in situhybridization of mRNA (Seidel et al., 1997) or byimmunohistochemical methods, using antibodies

Figure 1. Schematic drawing of a cat’s vestibular nuclear commedial vestibular nucleus; LVN, lateral vestibular nucleus; SVNX; Zg, group Z; lg, group l; Cn, nucleus of Cajal (after Pomp

against the corresponding isoforms (Moreno-Lopezet al., 1998).

For localizing nNOS in the CNS, further twomethods have been developed. One of them is thehistochemical method based on demonstratingNADPH diaphorase (NADPHd) activity of nNOS(Dawson et al., 1991; Hope et al., 1991). The othermethod is an immunohistochemical method withthe use of an antibody against L-arginin (Holsteinet al., 2001; Martinelli et al., 2002). The lattermethod is based on the facts that L-arginin isgenerated during synthesis of NO in equivalentamounts as NO and L-arginin is not a metabolite inthe urea cycle in the CNS.

NADPHd-positive neurons and fibres are presentin many parts of the nervous system of variousspecies including the human (Mizukawa et al.,1989; Mizukawa, 1990; Vincent and Kimura, 1992;Hinova-Palova et al., 1997; Paloff and Hinova-Palova, 1998; Moreno-Lopez et al., 1998; Saxonand Beitz, 2000; Holstein et al., 2001; Martinelliet al., 2002; Lysakowski and Singer, 2000). Never-theless, as far as we know, there is no definitivelight microscopical study of nNOS-positive neurons,including fibres in the vestibular complex of a cat.In this respect, there are only studies concerningcertain nuclei (Mizukava et al., 1989; Vincent andKimura, 1992; Moreno-Lopez et al., 1996, 1998,2001). The aim of the present study was todemonstrate nNOS-positive neurons in VNCc andto describe their morphology, distribution patternsand interconnections.

plex. Major nuclei: IVN, inferior vestibular nucleus; MVN,, superior vestibular nucleus; additional nuclei: Xg, groupeano et al., 1966 with modifications).

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Light microscopical study of nitric oxide synthase I-positive neurons 115

Topographic verification of the various parts ofthe VNCc was based on the classification system ofPompeano and co-workers (Pompeano et al., 1966).The VNCc is divided into several major nuclei (MVN,medial vestibular nucleus; IVN, inferior vestibularnucleus – which contains the f group; LVN, lateralvestibular nucleus – which contains the l group; andSVN, superior vestibular nucleus) and additionalnuclei (group X, group Y, group Z, group l, group f,nucleus of Cajal; Fig. 1).

Figure 2. NADPH diaphorase-positive neurons in a majornucleus of the cVNC, the inferior vestibular nucleus. Bar,500 mm.

Material and methods

Five adult cats of both sexes (three female andtwo male) with an average weight of 2.5 kg wereused. All animals had free access to waterand food. Efforts were made to minimize thenumber of animals participating in the experimentand to ensure their humane treatment. All animalswere anaesthetized with Urethan (40mg/kg)and perfused transcardially with 500ml physiologi-cal solution and immediately afterwards with2500ml 4% paraformaldehyde. After perfusion, allbrains were removed and postfixed in the samesolution for the next 2 h. After 12 h of washing inPBS (pH, 7.4) at 4 1C, brains were cut using afreezing microtome 1260 (Reichert-Jung, Muench-en, Germany). The thickness of slices was 30 mm.Every third slice was stained using the Nisslmethod. All other slices were incubated with1mM NADPH, 1mM nitro BT and 0.1% Triton X100in TBS (pH 7.6) at 37 1C for 30–60min (Paloff andHinova-Palova, 1998; Papantchev et al., 2003). Allslices stained in this way were mounted on glassand air-dried for 24 h. All preparations werecovered using Enthalan and were examined usinga light microscope (Olympus, Tokyo, Japan) on thebasis of Berman’s atlas (Berman 1968). Later,explanatory marks were added to all images usingAdobe Photoshop 7.0.

Figure 3. NADPH diaphorase-positive neurons in a majornucleus of the cVNC, the medial vestibular nucleus. Bar,500 mm.

Morphometric study

For quantitative analysis, sections stained forNADPHd were used. It was performed using amicroanalysis system and a 40� objective. Thirtyslides from all major and accessory nuclei wereexamined. The maximum diameter of 450 neuronswas measured and the cells were divided intogroups. Data were recorded automatically using thecomputer program (CUE-2; Olympus).

Results

NADPHd-positive neurons and fibres were foundin each part of the VNCc (Figs. 2–11) from therostral to the caudal parts of every nucleus. Allneurons were similarly stained: the perikaryon washeavily stained, whereas the nucleus was free ofstaining (Fig. 6). With respect to their size, neuronswere classified as follows: small (10–18 mm), med-ium-sized (20–25 mm), large type I (30–35 mm),large type II (40–70 mm). According to the shapeof the perikaryon, the neurons were characterizedas oval, fusiform, triangular, multipolar, pear-shaped, and irregular. All small neurons were ovalor fusiform whereas all large type II neurons weremultipolar in shape. Medium-sized neurons andlarge type I neurons were represented by all of theshapes described. Every major nucleus had its ownspecific type of neurons (Figs. 2–5).

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Figure 4. NADPH diaphorase-positive neurons in a majornucleus of the cVNC, the lateral vestibular nucleus. Bar,500 mm.

Figure 5. NADPH diaphorase-positive neurons in a majornucleus of the cVNC, the superior vestibular nucleus. Bar,500 mm.

Figure 6. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a pear-shaped NADPHdiaphorase positive neuron from the X group (arrow). Bar,100 mm.

Figure 7. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a small NADPHdiaphorase positive neuron from Cajal’s nucleus (arrow).Bar, 100 mm.

Figure 8. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a triangular NADPHdiaphorase positive neuron from the Y group. Bar,100 mm.

Figure 9. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a small fusiform NADPNdiaphorase positive neuron from the Z group. Bar,100 mm.

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Figure 10. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a large type I neuronstained positive for NADPH diaphorase (in the centre) andsome small NADPH diaphorase positive neurons from the fgroup. Bar, 100 mm.

Figure 11. NADPH diaphorase-positive neurons in acces-sory nuclei of the cVNC, showing a multipolar NADPHdiaphorase positive neuron from the l group. Bar, 100 mm.

Figure 12. Clusters of NADPH diaphorase-positive neu-rons from the MVN. Bar, 100 mm.

Figure 13. Chain-like rows of small NADPH diaphorase-positive neurons (arrows). These rows were present inMVN of only one of the animals. Bar, 100 mm.

Light microscopical study of nitric oxide synthase I-positive neurons 117

IVN

In IVN, we observed neurons of all shapes andsizes. The main neuronal population consisted ofsmall and medium-sized neurons (Fig. 2). The largetype II neurons were exceptions in this nucleus. AllNADPHd positive fibres were distributed in aspecific pattern, which determined the typicalbackground of the nucleus. The neurons from groupf were also NADPHd-positive (Fig. 10).

MVN

In the MVN, we observed the largest populationof NADPHd-positive neurons and fibres of all nucleiin the VNCc. This population comprised small,medium and large type I neurons. Large type II

neurons were rarely found in this nucleus. NADPHd-positive fibres were intensely stained and formed adense network in the background of the nucleus asis shown in Fig. 3. Only in one of the animals, weobserved small NADPHd-positive neurons, forming aline. We called this pattern chain-like rows (Figs. 12and 13). In all animals, we found tiny interconnec-tions between the MVN and the reticular formationof the pons. According to our as yet unpublisheddata, these interconnections are afferent (Fig. 14)and efferent (Fig. 15) in both structures. In someintersections, we observed that neurons werelinked (Fig. 16).

LVN

The main neuronal population in the LVN con-sisted of large type II neurons (Fig. 4). SmallNADPHd-positive neurons were also observed. All

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Figure 14. Large numbers of interconnections betweenMVN and reticular formation of the pons. White arrows,dendrites; black arrows, axons. Bar, 200 mm.

Figure 15. Interconnections between MVN and reticularformation of the pons. White arrows, dendrites; blackarrows, axons. Bar, 200 mm.

Figure 16. Interconnections between MVN and reticularformation of the pons. Arrows, two NADPH diaphorase-positive neurons in contact. Bar, 200 mm.

Figure 17. Large type II neurons with dendrites (arrows).Bar, 200 mm.

Figure 18. Large type II neurons with dendrites (arrows).Bar, 200 mm.

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other types of neurons were rare in this nucleus.The neurons from group l were also NADPHd-positive (Fig. 11). The amount of NADPHd-positive

fibres was fewer here than in other parts of theVNCc. This is the reason for the pale background ofthis nucleus.

SVN

In the SVN, neurons of all shapes and sizes wereobserved (Fig. 5). We found large type II neuronswith many dendrites in the section (Figs. 17 and18). All NADPHd-positive fibres were perpendicularto the wall of the fourth brain ventricle (Fig. 5).

Groups X, Y, Z, and Cajal nucleus

In all additional nuclei, NADPHd-positive neuronsand fibres were found (Figs. 6–11). The mainpopulation of neurons consisted of small andmedium-sized neurons. NADPHd positive fibresformed the greater part of the background stainingof each nucleus.

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Table 1. Morphological classification of NADPH diaphorase-positive neurons according to size and shape

Neuronal type Size (mm) Shape Function

Small 10–18 Oval, fusiform InterneuronsMedium-sized 20–25 Oval, fusiform, triangular, multipolar, pear-shaped, and irregular Different functionsLarge type I 30–35 Oval, fusiform, triangular, multipolar, pear-shaped, and irregular Different functionsLarge type II 40–70 Multipolar Projection neurons

Light microscopical study of nitric oxide synthase I-positive neurons 119

Discussion

Our results are compared here with all publisheddata on cats (Moreno-Lopez et al., 1996, 1998) anddata on rats (Vincent and Kimura, 1992; Saxon andBeitz, 2000; Holstein et al., 2001; Martinelli et al.,2002). None of the above-mentioned studiesprovide a strict morphological classification ofnNOS-positive neurons in the vestibular complex.A classification of this kind is useful in practicebecause the specific morphology is linked with thefunction of the neuron. For example, smallNADPHd-positive neurons have the morphologyand size of interneurons, i.e. in such a neuronalpopulation NO will be active only within theboundaries of the nucleus. On the other hand, thelarge type II neurons have the size and morphologyof projective neurons, i.e. NO could affect the cellthat has produced it as a feedback mechanism.Table 1 shows the suggested classification.

Moreno-Lopez et al. (1998) proved that NOfunctions within boundaries of the MVN and IVN.This conclusion can be also be drawn by using ourclassification described above. The connections ofthe MVN with the reticular formation of the ponswere described as early as in 1966 (Pompeano etal., 1966). However, no data are available in theliterature about the fact illustrated by us thatNADPHd-positive neurons of the MVN receive andsend information from the reticular formation andNADPHd-positive neurons from the reticular forma-tion get and transmit information towards the MVN.According to our as yet unpublished data, theseinterconnections are afferent and efferent in bothstructures.

As far as we know, chain-like rows have not beenmentioned in the literature before. The fact thatthey were observed in only one of the animals andtheir specific pattern of arrangement indicates thatthey are artificially stained glial cells. A possiblereason for this pattern of arrangement is prolongedincubation (60min).

The LVN is known to be the beginning of thevestibospinal tract, the axons of which end onalpha-motor neurons from all parts of the spinalcord. It is also known that the large type II neurons

are the morphological substratum of this tract,which means that they are projective. At the sametime, the half-life of NO is very short, for example,it cannot build up sufficient concentrations insacral segments. It should be noted that NO is partof a feedback mechanism involving the neuron thatsynthesize it.

The large number of dendrites in the plane ofsection in the SVN shows that there are numerousinterconnections within the nucleus. Another inter-estina observation is that the typical backgroundstaining of each nucleus is the result of staining ofNADPHd-positive fibres. The possible origin of thesefibres is described in detail by Saxon and Beitz(2000) and therefore it is not discussed here.

In conclusion, we hope that our study contributesto a better understanding of the functioning of theVNCc and that the classification suggested by uswill help to identity NADPHd-positive neurons infuture studies.

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