Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain

Brain Research Bulletin 66 (2005) 555–559

Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain Jes´us M. L´opez, Nerea Moreno, Ruth Morona, Margarita Mu˜noz, Agust´ın Gonz´alez ∗ Department of Cell Biology, Faculty of Biology, University Complutense of Madrid, 28040 Madrid, Spain Available online 6 June 2005

Abstract By means of double immunohistofluorescence techniques, we have investigated the colocalization of nitric oxide synthase and tyrosine hydroxylase (TH) or serotonin (5-HT) in the central nervous system of the anurans Rana perezi and Xenopus laevis and the urodele Pleurodeles waltl. A wide codistribution of neuronal populations, expressing these markers, was found throughout the brain and spinal cord. In contrast, colocalization of these markers was rather restricted. Only in the caudal portion of the brainstem raphe column in anurans, approximately 80% of the 5-HT-positive cells were also NOS-immunoreactive, whereas in the urodele brain, about 40% of the serotonergic cells at the level of the glossopharyngeal motor nucleus were simultaneously NOS-positive. In various brain regions, a wide codistribution of NOSand TH-containing neurons was observed, but real colocalization of nitrergic and catecholaminergic cells was only found in a small neuron population in the posterior tubercle of anuran amphibians. Therefore, in amphibians, only a distinct and small cell population within the serotonergic raphe column (anurans and urodele) and in the catecholaminergic posterior tubercle (anurans) seem to produce simultaneously nitric oxide. © 2005 Elsevier Inc. All rights reserved. Keywords: Catecholamines; Serotonin; Nitric oxide; Immunohistochemistry; Amphibians

1. Introduction In the brain of all vertebrates, a system of neuronal centers exists that produces nitric oxide (NO) by means of the enzyme nitric oxide synthase (NOS). The anatomy of the nitrergic system has been extensively studied in the brain of almost all vertebrate classes by means of the NADPH-diaphorase (NADPHd) histochemical technique or NOS immunocytochemistry [1]. Given the wide distribution of nitrergic cells in the brain and their patterns of organization, it was suggested that they do not correspond with any other neurotransmitter system [1]. However, partial overlaps between NOS- and various other neurotransmitter-containing centers have been described, suggesting a multiple action of NO in relation with other systems in the brain [24]. Among those substances that have been investigated for colocalization with NOS are the monoamines. This research was prompted by the early observation that both catecholamines and serotonin (5-HT) showed ∗

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a distribution pattern in the brain that extensively overlapped with that observed for NOS-immunoreactive (NOSir) structures [11]. The results of a series of studies aiming to clarify the actual colocalization of monoamines and NOS in the same cells showed that only a small portion of the widely codistributed cells were double labeled with both substances [6,10,18–20,22,23]. Most of these studies were carried out in mammalian species. In particular, only a restricted number of catecholaminergic cells in the mesencephalon was observed to contain NOS in rats [10] and other mammals, including cats and guinea pigs [2,13,14]. In mammals, the serotonergic dorsal raphe nucleus colocalizes NOS [6,10,12,27,28]. Studies in birds and reptiles are very sparse and the results about colocalization of TH and NOS demonstrated that many neurons, simultaneously expressing both substances, are located in the mesencephalic dopaminergic groups [17,20,23]. Data about colocalization of NOS and 5-HT are not available for non-mammalian species. In amphibians, the extensive distribution of nitrergic cells suggests the possible colocalization of NOS with catecholamines and 5-HT in many brain areas [5,8,9,18]. Preliminary results suggested

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that NADPHd/TH colocalization is only found in the posterior tubercle and the locus coeruleus of anurans [18] or even absent in the urodele Pleurodeles waltl [9]. Therefore, in the present study, we analyzed the degree of colocalization between catecholamines or 5-HT with NOS by means of a sensitive double labeling technique using antibodies against TH, 5-HT and NOS. Two anurans (Rana perezi, Xenopus laevis) and one urodele (P. waltl) were used.

2. Materials and methods In the present study, a total of nine green frogs (R. perezi), five South African clawed frogs (X. laevis) and six Iberian ribbed newts (P. waltl) were used. The animals were obtained from the laboratory stock of the Department of Cell Biology, University Complutense of Madrid. The original research reported herein was performed under the guidelines established by the Spanish Royal Decree 223/1988. All animals were deeply anesthetized in a 0.3% solution of tricaine methanesulfonate (MS222, Sandoz; pH 7.3) and perfused transcardially with saline followed by 200 ml of 4% paraformaldehyde in a 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed and kept in the same fixative for 2 h. They were then immersed in a solution of 30% sucrose in PB for 5 h at 4 ◦ C, embedded in a solution of 20% gelatine with 30% sucrose in PB, and then stored for 6 h in a 4% formaldehyde solution at 4 ◦ C. The brains were cut into transverse sections on a freezing microtome of 40 ␮m and collected in PB. For the simultaneous detection of NOS and monoamines, the following procedure was used: (1) incubation for 60 h at 4 ◦ C in a mixture of sheep anti-NOS (K205 antibody, kindly donated by Dr. P.C. Emson; diluted 1:20,000), and either rabbit anti-5-HT (diluted 1:1000; Incstar) or rabbit antiTH (diluted 1:100; Chemicon) in PB containing 0.5% Triton X-100; (2) second incubation for 2 h at room temperature in a mixture of antisera rhodamine-conjugated donkey antisheep (red fluorescence, diluted 1:100, Chemicon) and FITCconjugated chicken anti-rabbit (green fluorescence, diluted 1:100, Chemicon) in PB containing 0.5% Triton X-100. After rinsing three times in PB, the sections were mounted on glass slides and coverslipped with Vectashield (Vector). Alternating the appropriate filter combinations in a Zeiss fluorescence microscope allowed the identification of NOS and 5-HT or TH labeled structures.

3. Results In all double labeled sections of the anuran and urodele brain, a distinct distribution for NOSir cells and fibers was obtained. In addition, either THir or 5-HTir structures were simultaneously detected. Differences were observed among the three species used but, in general, the results in the two anurans were largely similar and will be described together.

3.1. Colocalization of NOS and TH Double immunohistochemistry for NOS and TH resulted in the total lack of colocalization of these substances in neurons of the urodele brain, in spite of the high degree of codistribution of neurons expressing them in regions such as the olfactory bulbs, the hypothalamus, the mesencephalon, the locus coeruleus and the caudal rhombencephalon. In the two anuran species, a small contingent of double labeled neurons was found in the posterior tubercle (Fig. 1a–c). In this region, among the ventrolateral portion of the dopaminergic cell group [8], scattered cells were NOSir and up to 70% of the latter cells were simultaneously labeled for TH. No colocalization of TH and NOS was found elsewhere in the anuran brain, although extensive codistribution of both substances was observed, as in the case of the urodele. 3.2. Colocalization of NOS and 5-HT The individual analysis of the distribution of NOSir neurons and 5-HTir cells in the brain of anurans and urodeles suggested that only the caudal portion of the long serotoninergic cell column of the raphe could contain colocalized these two substances. Double labeled sections in anurans demonstrated that at levels just rostral to the obex (coincident with the vagal motor nucleus), a relatively large population of the 5-HTir cells (70–80%) was simultaneously labeled for NOS (Fig. 1d–f). Most of the cells were located close to the midline and only a few double labeled neurons were located more ventrolaterally in the inferior reticular zone. This cell population extended caudally to the obex into the rostral spinal segments (Fig. 1A and B). Double labeled cells were also found in the brain of the urodele within the caudal portion of the raphe column (Fig. 1C and D). This cell population was very scarce and was made up by a few cells at the level of the glossopharyngeal motor nucleus, located exclusively close to the midline beneath the ventricular lining. No more than 40% of the 5-HTir cells in this region were simultaneously labeled with the NOS antibody.

4. Discussion The present study has revealed that the amphibian brain shows a limited and selective expression of NOSimmunoreactivity within subpopulations of monoaminergic neurons, suggesting a functional correlation between the two systems. Although scarce, the colocalization of TH/NOS and 5-HT/NOS in the amphibian brain may give clues to the degree of conservation of this characteristic across vertebrates. 4.1. NOS in catecholaminergic neurons In most vertebrates studied, on the basis of distribution of TH immunocytochemistry and NADPHd/NOS-positive

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Fig. 1. (a–c) Photomicrographs of a transverse section through the ventrolateral part of the posterior tubercle (TPvl) of R. perezi showing NOSir cells (red), THir neurons (green) and double labeled cells (yellow in c). (d–f) Photomicrographs of a transverse section through the caudal raphe column (Ra) of R. perezi showing NOSir cells (red), 5-HTir neurons (green) and double labeled cells (yellow in f). The panel below the photographs illustrates two transverse sections through the caudal rhombencephalon showing the localization of the double labeled cells for NOS/5-HT in the anuran and urodele brain. A and C are rostral to B and D, respectively. Abbreviations: DCN, dorsal column nucleus; Nsol, nucleus of the solitary tract; sol, solitary tract; Rai, inferior raphe nucleus; Ri, inferior reticular nucleus; IXm, glossopharyngeal motor nucleus; Xm, vagal motor nucleus.

cell bodies, cellular localization of these enzymes could be expected in many places of the CNS. However, double staining methods revealed that in most of these places, the two kinds of cells lie closely intermingled, but constitute separate populations of cells. Numerous studies attempted to clarify the possible colocalization of catecholamines and NOS by combining NADPHd-histochemistry and THimmunocytochemistry [3,10,13,14,17,18,20,23]. With this technique, the blue NADPHd reaction product of NOS cells and the brown DAB reaction product of TH cells are distinguished in separate populations of neurons. However, in some cases, the two labelings are difficult to discern mainly in areas where dark-blue neurons could mask the putative brown staining. The actual codistribution/colocalization is more reliably observed with double NOS/TH immunofluorescence [2,6,9,21,23]. In mammals, discrepancies have been reported among the different species studied. Thus, in rats, with the NADPHd/TH technique, only a very small number of double labeled cells were found in the periaqueductal gray, the rostral linear nucleus and the rostrodorsal aspect of the ventral tegmental area [10]. Subsequently, with the same technique, colocalization was described in the magnocellular neurosecretory nuclei of the hypothalamus [3]. The more sensitive NOS/TH technique revealed in addition dou-

ble labeled cells in the rhombencephalic reticular formation and the nucleus of the solitary tract [2,6,21]. Colocalization within the isthmic region and in particular in the locus coeruleus was not observed [6,10,19,29]. In other mammals as the guinea pig and the cat, no double labeled cells were found in the brainstem, with the exception of the nucleus of the solitary tract [2,13]. Also in humans, considering the distribution of NADPHd and NOS in the midbrain [7], a general lack of colocalization in the midbrain is to be expected. In birds, two studies using the NADPHd/TH technique in chicken and quail yielded very different results. A total lack of colocalization was reported in the chicken brain [17], whereas about 40% of the THir cell bodies in the ventral tegmental area and substantia nigra of the quail also contained NADPHd [20]. Studies with double NOS/TH immunofluorescence are lacking in birds. Interestingly, in the lizard Gekko gecko, both NADPHd/TH and NOS/TH techniques revealed extensive colocalization in the dopaminergic cell groups of the mesencephalon [23]. Therefore, NO might play an important role in the regulation of the activity of the dopaminergic system in the mesencephalon in amniotes, albeit exceptions may exist. Of note, in amphibians, differences have also been detected between the two orders studied. Thus, only in anurans, NOS/TH colocalization was found in the posterior

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tubercle. This observation was possible only with the double immunofluorescence technique, given the weak NADPHd histochemical reaction showed by the neurons in this region [5,18]. The posterior tubercle harbors a dopaminergic cell population that has been compared with the ventral tegmental area/substantia nigra complex of amniotes [16]. Therefore, the results in anurans are in line with the observations in at least in some amniotes [10,20,23]. The lack of colocalization in the urodele posterior tubercle points to interspecific differences between amphibians. As in amniotes, the possible colocalization of NOS in the noradrenergic cells of the anuran locus coeruleus could not be confirmed with the NOS/TH immunohistochemistry. 4.2. NOS in serotoninergic neurons Using double labeling techniques, a considerable colocalization of 5-HT and NOS/NADPHd has been reported in the dorsal raphe nucleus of the rat [6,10,22,27,28] with a high degree of coexistence in the medial areas and a low one laterally. Only with the NOS/5-HT method, a few cells were detected in the caudal raphe nuclei [6]. In contrast to the data in rats, in double labeling studies in mice, guinea pigs and cats, no NOS was found in 5-HT cells [12,13]. Therefore, the colocalization of NOS/5-HT in the dorsal raphe nucleus of the rat is the only positive datum available of such a chemically characterized 5-HT subpopulation. Furthermore, this specific group of cells has been correlated with raphecortical projection cells [10,22]. The possible colocalization of NOS with 5-HT in birds (quail) was excluded on the basis of the different topography of the neurons containing either one or the other labeling [20]. In reptiles, double labeling experiments have not been made, but differences may exist since in the lizard Gekko gecko many NOSir cells were described in the caudal raphe nuclei coincident with the localization of 5HT cells [23], whereas in turtles, both substances seem to be present in separate populations of neurons [4,26]. The finding in amphibians of a subpopulation of 5-HTir cells in the caudal raphe column that also contains NOS suggests that, as in rats, this chemical content may characterize a subpopulation of the serotoninergic raphe cells. However, the situation may be totally different from rats since the position of these double labeled cells mainly account for raphespinal cells [25] more than raphetelencephalic cells [15]. In summary, from the available data, it can be concluded that colocalization of NOS and catecholamines and serotonin occurs in many vertebrates, not only in amniotes but also in amphibians. However, there appears to be considerable variation between species, not only across vertebrate classes but also within the same class, as seen in amphibians.

Acknowledgements Grant sponsor: Spanish Ministry of Science and Technology. Grant number: BFI2003-03756.

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