Developmental Brain Research 101Ž. 1997 177±186 Research report Transient expression of NADPH-diaphorasernitric oxide synthase in the paratenial nucleus of the rat thalamus E. Garcõa-Ojeda,ÂÄ J.R. Alonso ), C. Crespo, E. Weruaga, J.G. Brinon, R. Arevalo, J. Aijon Departamento de Biologõa Celular y Patologõa, UniÕersidad de Salamanca, AÕenida Campo Charro 1, 37007 Salamanca, Spain Accepted 18 March 1997 Abstract The distribution pattern of nitric oxide synthesizing neurons was studied in the paratenial nucleus throughout the rat development using the NADPH-diaphoraseŽ. ND histochemical method and nitric oxide synthase Ž NOS . immunocytochemistry. The onset of NDrNOS activity in the paratenial nucleus was detected in the postnatal life day 1. Until the postnatal stage 4, a quick increase in the number and staining intensity of the NDrNOS positive neurons was observed. From postnatal day 4 to postnatal day 6, these variations continued slowly, whereas an increase in the neuronal size was evident. In these stages, densely packed NDrNOS-labeled neurons were observed. From stages 6 to 10, the NDrNOS-positive elements demonstrated similar number, size, and staining intensity. These cells had medium size, variable morphology and showed reaction product in the cell bodies and, at most, their proximal dendrites. After postnatal day 10, a quick decrease in the staining intensity and in the number of NDrNOS-labeled elements was detected, although no changes were observed in their morphological characteristics. Postnatal day 15 was the last developmental stage studied in which NDrNOS-posi- tive elements were observed. Finally, the paratenial nucleus did not present NDrNOS-positive elements in adult animals. This transient expression of the NDrNOS-activity suggests a role of nitric oxide in the reorganization of the paratenial nucleus during the first postnatal fortnight. q 1997 Elsevier Science B.V. Keywords: Development; Nitric oxide; Ontogeny; Plasticity 1. Introduction reaction has been used to locate neurons producing nitric oxideŽ. NO throughout the brain Ž NDrNOS neurons . NADPH-diaphoraseŽ. ND activity can be easily de- wx6,8,23 . NO seems to be involved in particular processes tected in fixed tissues using a histochemical reaction during brain developmentwx 13,25 including the establish- wx2,22,30 . It has been reported that ND activity is produced ment of synapseswx 12,18,26 , changes occurring in the last by neuronal nitric oxide synthaseŽ. NOSwx 20 , and this developmental stages, such as apoptosis and reorganization of cell populationswx 9 , functional modulation of hypothala- mic neuronswx 35 , and in the maturation of motor neurons Abbreviations: AD, anterodorsal thalamic nucleus; AM, anteromedial 21,38 . The diversity of suggested roles for NO during thalamic nucleus; AT, anterior thalamus; AV, anteroventral thalamic wx nucleus; B, basal nucleus of Meynert; BST, bed nucleus of the stria brain development is probably correlated to the variations terminalis; CM, centromedial thalamic nucleus; f, fornix; GP, globus in the distribution pattern of NDrNOS neurons during pallidus; Hb, habenular nucleus; IAM, interanteromedial thalamic nu- ontogeny. cleus; ic, internal capsule; LD, laterodorsal thalamic nucleus; mt, mam- The paratenial nucleusŽ. PT in the rat is a paired millothalamic tract; ND, NADPH-diaphorase; NO, nitric oxide; NOS, nitric oxide synthase; P0-P30, postnatal day from 0 to 30; PC, paracentral structure located in the rostral region of the thalamus. All thalamic nucleus; PT, paratenial thalamic nucleus; PV, paraventricular midline thalamic nuclei, including PT, are easily identifi- thalamic nucleus; Pva, paraventricular hypothalamic nucleus; Re, re- able in perinatal periodswx 4 . The nuclei of the midline uniens thalamic nucleus; Rh, rhomboid thalamic nucleus; Rt, reticular thalamic region project to the ipsilateral forebrain, demon- thalamic nucleus; sm, stria medullaris; st, stria terminalis; VL, ventrolat- strating a bilateral symmetry. PT receives afferents from eral thalamic nucleus; VM, ventromedial thalamic nucleus; VP, ventro- posterior thalamic nucleus; ZI, zona incerta the neocortex, and projects to the orbital cortex, nucleus ) Corresponding author. Fax: q34Ž. 23 294549. E-mail: accumbens, amygdala and hippocampuswx 5,34 . In the rat, [email protected] the midline thalamic nuclei including the PT are involved 0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S0165-3806Ž. 97 00062-X 178 E. Garcõa-Ojeda et al.rDeÕelopmental Brain Research 101() 1997 177±186 in the cortico-thalamic limbic pathway, and seem to be mogenŽ. nitroblue tetrazolium . In both cases, no reaction related with learning, storage and memory mechanismswx 4 . product was observed. In the adult nervous system, NO has been related with these same functionswx 34 . In a general study throughout 2.3. NOS-immunocytochemistry the adult rat brain, Vincent and Kimurawx 37 described a scarce number of ND-labeled elements in the thalamic In order to check the coincidence of ND-activity and region and, specifically, the absence of ND-positive fibers NOS-immunoreactivity, two animals of the more critical or neurons in the PT. However, in a preliminary study on ages: P0, P1, P5, P10, P15, P20 and P30, were processed the ontogeny of ND-activity in the brain, we observed as described above, and consecutive sections were stained ND-stained elements in the PT of perinatal animals, sug- for ND and NOS. gesting time-related changes in the ND-activity in this For NOS immunostaining, sections were successively brain nucleus during the development. incubated inŽ. a normal goat serum diluted 1 : 10 in PB for The aim of this study is to analyze the onset of 30 min,Ž. b primary antibody Ž K205 sheep anti-rat neu- NDrNOS-stained elements in the rat PT during the devel- ronal NOS antibodywx 19. diluted 1 : 20 000 in PB overnight, opment of the nervous system and to correlate these Ž.c biotinylated anti-sheep immuno-gammaglobulin Ž Vec- findings to morphological changes in this thalamic nu- tor Laboratories, Burlingame, USA. diluted 1 : 250 in PB cleus. The differential expression of this neuronal marker for 90 min, andŽ. d avidin-peroxidase complex Ž Vector, may help to understand its role in the nervous system both Elite kit. diluted 1 : 500 in PB for 60 min. The reaction during the development and in the adult animal. was revealed incubating the sections with 0.07% 3,3X-di- aminobenzidine and 0.003% hydrogen peroxide in 0.1 M Tris-HCl buffer, pH 7.6. All steps were carried out at room 2. Materials and methods temperature. The K205 neuronal NOS antibody has been fully characterizedwx 11 . Specificity of the antibody was 2.1. Animals and tissue preparation assessed using Western blot analysis and liquid phase pre-adsorption experiments with purified recombinant neu- Fetuses, postnatal pups and adult Wistar rats were used ronal NOSwx 11 . in this study. The brains of adults, of fetuses from embry- Controls of the specificity for the immunocytochemical onic day 16Ž. E16 to 21 Ž. E21 , of pups from birth day Ž. P0 procedure were carried out as previously describedwx 1 . No to postnatal day 15Ž. P1±P15 , postnatal days 20 Ž. P20 , 25 residual reaction was observed. Ž.P25 and 30 Ž. P30 were analyzed. Six animals of each age were used for P0, P1, P5, P10, P15, P20 and P30, four 2.4. Quantification animals were used for the remaining stages. Male and female adult rats were housed together for approximately 4 For the measurement of cell sizes, in the perinatal hr. The following 24 h after the detection of sperm were animals only those neurons which exhibit, at least, the considered as embryonic day 0Ž. E0 . The fetuses from initial portion of one cellular process were used. In the E16±E19 were decapitated, their brains were dissected out remaining animals, only those neurons which presented and fixed for 20±24 h in a mixture containing 4% para- two or more cellular processes were considered. In each formaldehyde and 15% saturated picric acid in 0.1 M age, 100 ND-positive neurons of two different animals sodium phosphate bufferŽ. PB . Fetuses from E20 and E21, were measured. The cells were plotted using a 40= and postnatal pups were deeply anesthetized with ether or planapochromatic objective connected through a digitizer ketamine and perfused through the ascending aorta with tablet and optic pen to a semiautomatic image analysis saline followed by the fixative described above. Thirty-mm systemŽ. MOP-Videoplan 2000, Kontron . The mean and coronal sections were cut using a cryostat. S.E.M. were calculated using the corrected average for each group. The result were statistically analyzed using 2.2. NADPH-diaphorase histochemistry ANOVA. Values of P-0.01 for Fisher PLSD and Scheffe F-tests jointly were considered statistically significant. The sections were processed for NADPH-diaphorase as The labeling distribution was drawn using a Zeiss cam- described previouslywx 2 . Briefly, brain sections from four era lucida and Canvase 3.0.6. software. animals for each development stage were incubated for 60±90 min at 378C in a solution made up of 1 mM b-NADPH, 0.8 mM nitroblue tetrazolium, and 0.08% Tri- 3. Results ton X-100 in 0.1 M Tris-HCl buffer, pH 8.0. All reagents were purchased from Sigma. The course of the reaction 3.1. General characteristics was controlled by observation under the microscope. Con- trols for the specificity of the histochemical procedure The onset and evolution of the NDrNOS staining in the included incubation without substrateŽ. NADPH or chro- elements of the PT of the rat have been studied throughout E. Garcõa-Ojeda et al.rDeÕelopmental Brain Research 101() 1997 177±186 179 the development. In this study, the parcellation and some differences between both stainings were observed. nomenclature proposed by Paxinos et al.wx 29 for the Employing the ND-technique, in addition to the ND-stained developing brain, and Paxinos and Watsonwx 28 for the neuronal population, blood vessels were labeled.