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Noradrenaline-like terminals in the cat nucleus ventralis posterior of the thalamus
Brain Research Bulierin. Vol. 26, pp. 533-537. * Pergamon Press
plc, 1991. Printed in the U.S.A
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Noradrenaline-Like Terminals in the Cat Nucleus Ventralis Posterior of the Thalamus PHILIPPE DELAGRANGE, * MARIE CONRATH ,I. MICHEL GEFFARD, $ DJAMILA TADJER,* JEAN-JACQUES BOUYER*’ AND ARLETTE ROUGEUL* *lnstitut des Neurosciences, CNRS-UniversitP Pierre et Marie Curie De’partement de Neurophysiologie Cornparke, 9 quai St. Bernard, 75005, Paris, France l’lnstitut des Neurosciences, CNRS-Universite’ Pierre et Marie Curie Dtfpartement de Cytologie, 7 quai St. Bernard, 75005, Paris, France $L_aboratoire d’immuno~ogie et Pathologic (CJF 88-13 INSERM] BP 66 University Bordeaw II, 146 rue Lko Saignat, 33076 Bordeaux Cedex, France Received
16 April 1990
DELAGRANGE, P., M. CONRATH, M. GEFFARD, D. TADJER, J.-J. BOUYER AND A. ROUGEUL. Noradrenaline-like rerminulJ in rhe car nucleus venrrulis posterior of the thalamus. BRAIN RES BULL 26(4) 533-537, 1991. -Noradrenaline-like immunoreactivity in the cat nucleus ventralis posterior of the thalamus was investigated using an indirect immunocytochemical technique. Specific antinoradrenaline antibodies, raised in rabbits, were used. It was first verified that these antibodies recognize noradrenaline cells bodies of the locus coeruleus and their ascending axons in the ascending noradrenergic tract. In the nucleus ventralis posterior itself, noradrenaline-like fibers were observed. They were either randomly distributed or grouped around nonlabeled cell bodies. These neurons were generally oblong and measured 60-80 pm. With electron microscopy, preliminary results showed immunoreactive fibers in close apposition to unlabeled celi bodies or dendrites. The precise nature of these profiles was sometimes difficult to ascertain, since experiments were done in presence of detergent. In some cases symmetric synapses might be observed between i~unoreactive axon terminals and unlabeled dendrites. The specificity of the reaction is discussed in the light of several control experiments. I~unocytochemist~ Cat Nucleus ventralis posterior
Locus coetuleus
Noradrenaline
SEVERAL techniques have been used to study the catecholaminergic systems in the mammalian brain. Histofluorescence methods provided the first global description of the noradrenergic cells, terminals and paths (5, 14, 15, 18). Further methods based upon the specific uptake of 3H-noradrenaline (NA) have brought more specific results (6,26). At the same time, ascending projections from the locus coeruleus (LC) were studied with various tracing techniques (1223). Immunohist~h~mist~ using first antityrosine hydroxylase or antidop~ine-~-hydroxylase (DPH) antibodies lead to the discovery of new terminal fields and new neuronal groups, respectively catecholaminergic (11,20) and noradrenergic (24.28). Antisera specifically directed against conjugated catecholamines were produced by overcoming two major difficulties: chemical coupling and evaluation of antibody affinity and specificity (7). With such specific antibodies, NA-like (NA-LI) immunoreactivity was investigated in the rat hypothalamus (3,9) and thalamus
iCharge! de Recherches
specific antiserum
Thalamus
(22). But, no previous data existed concerning the cat. Our purpose here was to test these antibodies in the cat LC and thalamus, particularly in the nucleus ventralis posterior (VP). This was performed on adult cats at the light microscopic level. Preliminary results with electron microscopy are also presented. METHOD
The conjugated NA antiserum was raised after adminis~ation of NA conjugated to proteins by glut~aldehyde followed by a saturation of the double bonds by 0.1 M sodium borohydride solution (7,8). The experimental protocol used for immunocytochemistry was that described by Geffard et al. (8). Nine adult cats were perfused under ketamine anaesthesia (35 mg/kg) through the ascending aorta with 500 ml of 0.9% N&l containing 1% sodium metabisulfite (MBS) to wash out the circulatory system,
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1Fr 7.5 FIG. 1. Schematic diagram of three levels of the cat pons and thalamus redrawn from Snider and Niemer’s atlas (27). Boxes A, B, C represent the levels illustrated in immunohistochemistry in Fig. 2. CM: nucleus centralis medialis; GL: lateral geniculate body; GM: medial geniculate body; LC: locus coeruleus; LME: lam-
ina medulla& extema; SN: substantia nigra; VPM: nucleus ventralis posterior, pars medialis.
followed by 3 1 of 2.5% glutaraldehyde in 0.15 M Sorensen MBS buffer (pH = 7.6). After 3 h postfixation in the same fixative and washing overnight at 4°C in 0.15 M Sorensen MBS buffer, 50-pm vibratome frontal sections were made from the level of the hypothalamus to that of the LC. The sections were then washed in 0.15 M Tris buffer (pH=7.4). Double bonds were saturated with 1% sodium borohydride in 0.15 M Tris buffer (5 min). After extensive washing in 0.15 M Sorensen MBS buffer, they were preincubated during 30 min in Sorensen MBS buffer containing 1% normal sheep serum to prevent nonspecific binding. Triton X-100 (0.1% to 0.3%) was added to improve penetration of the antiserum. Sections were then incubated at 4°C in rabbit conjugated NA antiserum at l/2,000-l/5,000. After 48 h incubation, sections were washed in Sorensen buffer, incubated in goat antirabbit immunoglobuhn coupled to horseradish peroxidase (l/250) for 3 h and then washed again in Siirensen buffer. After 15 min rinsing in Tris buffer (pH 7.4; 0.1 M) the peroxidase activity was revealed with the 3-3’-diaminobenzidine reaction (15 mg/50 ml Tris buffer containing 0.03% hydrogen peroxide) and washed. Sections were mounted on glass slides with Sorensen-Glycerol
(1: 1). In some cases, the peroxidase reaction was intensified by the Silver-Gold method (16). After light microscopic observation, the VP nucleus was dissected out from sections between frontal planes 6.5 and 9.5 and prepared for electron microscopy. Briefly, blocks were postfixed during 1% h in 2% osmium tetroxide and dehydrated in graded alcohols before being embedded in araldite. Ultrathin sections were made, stained with lead citrate, and examined with a Philips EM 300 electron microscope. To assess the specificity of the reaction, the following controls were performed. 1) Regions known to be rich in NA-LI neurons were observed. 2) The diluted antiserum (l/2,000) was preabsorbed with l-10 mg/ml NA or 5-10 mg/ml dopamine (DA) or GABA during three hours at room temperature before addition of the sections. 3) The nonspecific absorption of the goat anti-rabbit IgG antibodies coupled to peroxidase was determined by omitting the anti-NA antiserum in the first incubation step. This control was made with and without sodium borohydride. 4) A monoclonal anti-DA antibody was tested in the cat thalamus using the same experimental protocol.
FACING PAGE FIG. 2. Noradrenaline-like immunoreactivity in the cat at levels indicated in Fig. 1. A: Fr-3, locus coeruleus; B: Fr 6, ascending noradrenergic-like tract; C: Fr 7.5, nucleus ventralis posterior of the thalamus. Al: Numerous immunoreactive cell bodies are observed in all the subdivisions of the nucleus locus coeruleus: principal LC, LCu, subcoeruleus (SC). Some immunoreactive fibers are also observed. BC: bra&mm conjunctivum; nV: tract of the mesencephalic trigeminal nucleus. A2: The antiserum (l/2,000) has been preincubated with 5 mg/ml NA during 3 h at room temperature before addition of the section. Note the almost total absence of reaction in the LC compared to Al. B: Punctate immunoreactivity in the dorsal ascending NAergic tract. The points most likely correspond to transversally cut axons. Cl: Immunoreactive fibers in nucleus ventralis posterior of the thahunus. They are grouped around probably nonlabeled cell bodies (arrows). Note the reaction extending along cellular processes. C2: Higher magnification of immunoreactive fibers in the nucleus ventralis posterior surrounding cell bodies. The presence of detergent in the incubation medium led to a diffusion into the cell bodies which are considered to be unlabeled. These cell bodies are oblong and measure 60-80 Fm. C3: Silver-gold intensification of the reaction in the nucleus ventralis posterior. In addition to clustered immunoteactive fibers around cell bodies (thick arrows), numerous isolated immunoreactive fibers are observed (thin arrows). A, B, C scale bars: 100 p,m; D, E scale bars: 50 pm.
NA-LIKE I~MUNOREACTIVITY
IN CAT VP
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some cases, the immunoreactivity extended along proximal dendrites. When the reaction was intensified with the silver-gold method, numerous immunoreactive isolated fibers could be seen (Fig. 2-C3). These pictures were obtained when 0.1 to 0.3% Triton X-100 was added in the preincubation medium and after 48 lr incubation in the specific antiserum. This may explain the diffuse staining in some cell bodies (Fig. 2-C) which did not present the punctate-like feature of the staining observed around the cell bodies. When the specific antiserum was omitted from the first mcubation step, no immunolabeling was observed in the VP or in the LC. Preincubation of the diluted anti-NA (112,000) with I mg/ml NA totally prevented the reaction in the VP. However, preincubation of the antiserum with DA or GABA did not modify the immunostaining. It was also verified that no immunoreactive fibers were observed in the VP after incubation in a monoclonal anti-DA antibody. This antibody labeled numerous cell bodies in the substantia nigra. In electron microscopy, the morphology of the tissue was poorly preserved because of the technical conditions used (addition of a detergent in the incubation media and long period of incubation). Our preliminary results have, however, permitted to observe numerous immunoreactive fibers in close apposition to unlabeled cell bodies (Fig. 3-l) or proximal dendrites (Fig. 3-2). In Fig. 3-2, the immunoreactive profile was presumably an axon terminal and established a symmetric synaptic contact, identified by the regular space between plasma membranes, with a proximal dendrite. DISCUSSION FIG. 3. Noradrenaline-like immunoreactivity in electron microscopy in nucleus ventralis posterior of the thalamus. 1: An immunoreactive profile is in close apposition with an unlabeled neuron. Cy: cytoplasm, N: nucleus. 2: An immunoreactive axon (A) establishes a symmetric synaptic contact with a proximal dendrite (D). Note the regular space between the two plasma membranes (arrow heads). Scale bars: 0.5 km.
RESULTS
The different regions of the cat brain observed in this study are schematically represented in the diagrams of Fig. 1. The diagrams, redrawn from Snider and Niemer’s atlas (27), represent frontal planes. Fr-3 corresponds to the locus coeruleus, Fr 6 to the ascending noradrenergic tract and Fr 7.5 to the nucleus ventralis posterior of the thalamus. Boxes A, B, C, correspond to the micrographs presented in Fig. 2. After incubation of the sections in the anti-NA antiserum, numerous immunoreactive cell bodies were observed in the region surrounding the brachium conjunctivum and the tract of the mesencephalic trigeminal nucleus (Fig. 2-Al). The different subdivisions of the LC were immunoreactive: the principal LC, the LCa and the subcoeruleus. Immunoreactive fibers were also observed. When the diluted antiserum was preincubated in 5 mg/ml NA, the immunolabeling was absent (Fig. 2-A2). At the frontal level Fr 6, a punctate NA-LI immunoreactivity was observed in the region of the ventral noradmnergic ascending bundle (Fig. 2-B). In the nucleus ventralis posterior, the immunoreactivity was localized between frontal planes 7 and 8.5, mainly in the ventral part of the VP medialis (VPM), just above the lamina medullaris extema and particularly on its lateral part. Neighbouring regions were devoid of immunoreactivity, particularly the lateral posterior nucleus located above the VP. In the VPM, the immunoreactivity was localized in fibers generally surrounding nonimmunoreactive cell bodies (Fig. 2-Cl-C3). These cell bodies were generally oblong and measured 60-80 Wm. In
The data presented here are to our knowledge the first description of NA-LI immunoreactivity in the cat obtained with a specific conjugated NA antiserum. NA-LI cell bodies were observed in the LC and NA-LI fibers in the noradrenergic ascending tract and in the VP of the thalamus. With the same antiserum several studies have been performed in the rat, in the LC (8). thalamus (22) and hypothalamus (3,9). NA-LI cell bodies of the cat LC were observed with an antiserum concentration of l/5,000, a concentration generally used for studies performed on the rat (see the references above). However, NA-LI fibers in the cat VP were observed only at antiserum concentration of l/2,000 and when Triton X-100 (0.1-0.3%) was added in the preincubation medium. This could result from the low concentration of NA-LI material in this species. This hypothesis is reinforced by the fact that we and others (17) were unable to detect tyrosine hydroxylase (TH)-positive axon terminals in the cat VP, suggesting that this enzyme also has a low concentration in this region. The latter authors could detect TH-LI axon terminals in the monkey thalamus (17) and, as already mentioned, NA-LI axon terminals are present in the rat thalamus. The specificity of the NA antiserum used here has been biochemically investigated (21). Immunocytochemical controls gave the expected results: in the LC, the immunoreaction decreased markedly when the antiserum (115,000) was preincubated in 1 mg/ml NA and disappeared totally after preincubation in 5 mg/ml NA. The same results were obtained in the VP with an antiserum concentration of l/2,000. In contrast with the biochemical controls (21), the free immunogen (nonconjugated to bovine serum albumine) is well recognized in immunocytochemistry. In addition, at both levels (LC and VP), when the antiserum was preincubated in other compounds, the labeling was never affected whatever were the concentrations tested. Moreover, we have seen that intraperitoneal injections of 10 mg/kg DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine), the drug reparted to destroy specifically the noradrenergic endings in rodents (10,13), resulted,
NA-LIKE IMMUNOREACTIVITY
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537
15-90 days later, in a great decrease in the number of TH-LI cell bodies in the LC (4) and NA-LI fibers in the VP. This argument is in favor of the specificity of the noradrenergic nature of the immunoreactive terminals observed here in the VP. At the light microscopic level, NA-LI fibers of the VP were either isolated or grouped around nonimmunoreactive cell bodies. In the latter case. punctate immunostaining was observed around the cell body and often extended along proximal dendrites. This configuration is confirmed in our preliminary results at the electron microscopic level. Immunoreactive neurites. sometimes clearly identified as axon terminals were observed in close apposition with unlabeled cell bodies or proximal dendrites. Synapses were seldomly encountered, perhaps due to the poor preservation of tissues which results from the necessity of adding Triton X-100 to the preincubation medium. However, in some cases, these synapses were suspected to be symmetrical since the intermembranous space is large and regular. Data obtained in the rat nucleus ventralis basahs with the same antiserum have
shown that synaptic differentiations are never observed (22). In the monkey VP, it seems that only 5-107~ of TH-LI terminals form conventional synapses. It has been suggested that NA is released nonsynaptically (at least partly) in the VP of these two species. Concerning the cat, more detailed data at the electron microscopic level would be necessary to make the same claim. The origin of NA-LI axon terminals of the VP remains to be demonstrated with double labeling studies. However, histofluorescence studies have indeed shown a light catecholaminergic innervation of different thalamic nuclei including the VP (25,29). This hypothesis was reinforced by the demonstration with horseradish peroxidase (HRP) or wheat-germ-agglutinin peroxidase (WGA-HRP) of a direct projection from the locus coeruleus to the VP in the cat (1.2) and in the rat (19.33). ACKNOWLEDGEMENTS
This study was supported by DRET (Grant 86-101 pour la Recherche Medicale.
I and La Fondation
REFERENCES 1. Barbaresi, P.; Conti, F.; Manzoni. T. Periaqueductal grey projection to the ventrobasal complex in the cat: an horseradish peroxidase study. Neurosci. Lett. 30:205-209; 1982. 2. Bouyer, J. J.; Tilquin, C.; Delagrange, P.; Rougeul, A.; Buser. P. Voies de regulation du comportement d’attente chez le chat: etude histologique. C. R. Acad. Sci. [III] 309:637-646; 1989. 3. Decavel, C.: Geffard. M.; Calas, A. Comparative study of dopamine- and noradrenaline immunoreactive terminals in the paraventricular and supraoptic nuclei of the rat. Neurosci. Lett. 77:149-154; 1987. 4. Delagrange, P.; Tadjer, D.; Bouyer, J. J.; Rougeul, A.; Conrath. M. Effect of DSP4, a neurotoxic agent, on attentive behaviour and related electrocortical activity in cat. Behav. Brain Res. 33:3343; 1989. 5. Falck. 9.; Hillarp, N. A.: Thieme, Cl.; Tarp. A. Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10:348-354; 1962. 6. Fujiwara. H.; Fujii, Y.: Saijoh, K.; Tanaka, C. Evidence for the neurotransmitter role of norepinephrine in the ventral thalamic nucleus of the guinea pig: localization, uptake and release. Brain Res. 301:131-137; 1984. 7. Geffard, M.: Henrich-Rock. A. M.; Dulluc, J. Antisera against small neurotransmitter-like molecules. Neurochem. Int. 7:403-413; 1985. 8. Geffard, M.; Patel, M.; Dulluc. J.; Rock, A. M. Specific detection of noradrenaline in the rat brain by using antibodies. Brain Res. 363: 395400; 1986. 9. Grzanna, R.: Berger, U.: Fritschy, J.-M.; Geffard, M. Acute action of DSP-4 on central norepinephrine axons: Biochemical and immunohistochemical evidence for differential effects. J. Histochem. Cytochem. 37:1435-1442; 1989. 10. Jaim-Etcheverry. G.; Zieher, L. M. DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of the adult and developing rats. Brain Res. 188:513-523: 1980. 11. Jones, 9. E.; Beaudet, A. Distribution of acetylcholine and catecholamine neurons in the cat brainstem: a choline acetyltransferase and tyrosine hydroxylase immunohistochemical study. J. Comp. Neurol. 261:15-32; 1987. 12. Jones. 9. E.; Moore, R. Y, Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study. Brain Res. 127:23-53; 1977. 13. Jonsson. G.: Hallman, H.; Ponzio, F.; Ross, S. DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine)-a useful denervation tool for the central and peripheral noradrenaline neurons. Eur. J. Pharmacol. 72:173-188: 1981. 14. Levitt, P.: Moore, R. Y. Origin and organization of brainstem catecholamine innervation in the rat. J. Comp. Neurol. 186:505-528; 1979. 15. Lindvall. 0.; Bjorklund, A. The organization of the ascending catecholamine neuron systems in the rat brain as revealed by glyoxylic
acid fluorescence method. Acta Physiol. Stand. 154:3 17-348; 1974. 16. Liposits. Z. S.; Setalo. G. Y.; Flerko. 9. Application of silver-gold intensified 3-3’.diaminobenzidine chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain. Neuroscience 13:5 13-525; 1984. 17. Liu, X. 9.; Jones, E. G. Electron microscopy of 5-HT and TH immunoreactive axon terminals in the VP nucleus of cat and monkey thalamus. Sot. Neurosci. Abstr. 16:255: 1990. 18. Maeda. T.: Pin, C.; Salvert, D.: Ligier. M.; Jouvet. M. Les neurones contenant des catecholamines du tegmentum pontique et leurs voies de projection chez le chat. Brain Res. 57:119-152; 1973. 19. Mason, S. T.; Fibiger, H. C. Regional topography within noradrenergic locus coeruleus as revealed by retrograde transport of horseradish peroxidase. J. Comp. Neurol. 187:703-724; 1979. 20. Milner, T. A.; Joh, T. H.; Pickel, V. M. Tyrosine hydroxylase m the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity. J. Neurosci. 6:2585-2603; 1986. 21. Mons, N.; Geffard, M. Specific antisera against the catecholamines: L-3.4-dihydroxyphenylalanine, dopamine, noradrenaline, and octopamine tested by an enzyme-linked immunosorbent assay. J. Neurothem. 481826-1833; 1987. 22. Nothias. F.; Onteniente, B.; Roudier. F.: Peschanski, M. Immunocytochemical study of serotoninergic and noradrenergic innervation of the ventrobasal complex of the rat tkalamus. Neurosci. Lett. 95: 59-63; 1988. 23. Peschanski, M.; Besson, J. M. Coerulear projections to the lateral diencephalon in the rat. An anatomical study using wheat-germ agglutinin conjugated to HRP. Neurosci. Lett. 46:329-334; 1984. 24. Pretel, S.; Ruda, M. A. Immunocytochemical analysis of noradrenaline, substance P and enkephalin axonal contacts on serotonin neurons in the caudal raphe nuclei of the cat. Neurosci, Lett. 89: 19-24; 1988. 25. Rainbow, T. C.; Biegon, A. Quantitative autoradiography of [3H]prazosin binding sites in rat forebrain. Neurosci. Lett. 40:221226; 1983. 26. Silverman. A. J.; Oldfield, 9.; Hou-Yu, A.; Zimmerman, E. A. The noradrenergic innervation of vasopressin neurons in the paraventricular nucleus of the hypothalamus: an ultrastructural study using radioautography and immunocytochemistry. Brain Res. 325:215-229; 1985. 27. Snider, R. S.; Niemer. W. T. A stereotaxic atlas of the cat brain. Chicago: The University of Chicago Press; 1961. 28. Swanson, L. W.; Hartman, 9. K. The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-B-hydroxylase as a marker. J. Comp. Neurol. 163:467-506; 1975. 29. Versteeg, D. H. G.; Van der Gugten, J.; De Jong, W.; Palkovits, M. Regional concentrations of noradrenaline and dopamine in rat brain. Brain Res. 113563-574; 1976.
plc, 1991. Printed in the U.S.A
Q3hl-9230191 $3.00 + .IXl
Noradrenaline-Like Terminals in the Cat Nucleus Ventralis Posterior of the Thalamus PHILIPPE DELAGRANGE, * MARIE CONRATH ,I. MICHEL GEFFARD, $ DJAMILA TADJER,* JEAN-JACQUES BOUYER*’ AND ARLETTE ROUGEUL* *lnstitut des Neurosciences, CNRS-UniversitP Pierre et Marie Curie De’partement de Neurophysiologie Cornparke, 9 quai St. Bernard, 75005, Paris, France l’lnstitut des Neurosciences, CNRS-Universite’ Pierre et Marie Curie Dtfpartement de Cytologie, 7 quai St. Bernard, 75005, Paris, France $L_aboratoire d’immuno~ogie et Pathologic (CJF 88-13 INSERM] BP 66 University Bordeaw II, 146 rue Lko Saignat, 33076 Bordeaux Cedex, France Received
16 April 1990
DELAGRANGE, P., M. CONRATH, M. GEFFARD, D. TADJER, J.-J. BOUYER AND A. ROUGEUL. Noradrenaline-like rerminulJ in rhe car nucleus venrrulis posterior of the thalamus. BRAIN RES BULL 26(4) 533-537, 1991. -Noradrenaline-like immunoreactivity in the cat nucleus ventralis posterior of the thalamus was investigated using an indirect immunocytochemical technique. Specific antinoradrenaline antibodies, raised in rabbits, were used. It was first verified that these antibodies recognize noradrenaline cells bodies of the locus coeruleus and their ascending axons in the ascending noradrenergic tract. In the nucleus ventralis posterior itself, noradrenaline-like fibers were observed. They were either randomly distributed or grouped around nonlabeled cell bodies. These neurons were generally oblong and measured 60-80 pm. With electron microscopy, preliminary results showed immunoreactive fibers in close apposition to unlabeled celi bodies or dendrites. The precise nature of these profiles was sometimes difficult to ascertain, since experiments were done in presence of detergent. In some cases symmetric synapses might be observed between i~unoreactive axon terminals and unlabeled dendrites. The specificity of the reaction is discussed in the light of several control experiments. I~unocytochemist~ Cat Nucleus ventralis posterior
Locus coetuleus
Noradrenaline
SEVERAL techniques have been used to study the catecholaminergic systems in the mammalian brain. Histofluorescence methods provided the first global description of the noradrenergic cells, terminals and paths (5, 14, 15, 18). Further methods based upon the specific uptake of 3H-noradrenaline (NA) have brought more specific results (6,26). At the same time, ascending projections from the locus coeruleus (LC) were studied with various tracing techniques (1223). Immunohist~h~mist~ using first antityrosine hydroxylase or antidop~ine-~-hydroxylase (DPH) antibodies lead to the discovery of new terminal fields and new neuronal groups, respectively catecholaminergic (11,20) and noradrenergic (24.28). Antisera specifically directed against conjugated catecholamines were produced by overcoming two major difficulties: chemical coupling and evaluation of antibody affinity and specificity (7). With such specific antibodies, NA-like (NA-LI) immunoreactivity was investigated in the rat hypothalamus (3,9) and thalamus
iCharge! de Recherches
specific antiserum
Thalamus
(22). But, no previous data existed concerning the cat. Our purpose here was to test these antibodies in the cat LC and thalamus, particularly in the nucleus ventralis posterior (VP). This was performed on adult cats at the light microscopic level. Preliminary results with electron microscopy are also presented. METHOD
The conjugated NA antiserum was raised after adminis~ation of NA conjugated to proteins by glut~aldehyde followed by a saturation of the double bonds by 0.1 M sodium borohydride solution (7,8). The experimental protocol used for immunocytochemistry was that described by Geffard et al. (8). Nine adult cats were perfused under ketamine anaesthesia (35 mg/kg) through the ascending aorta with 500 ml of 0.9% N&l containing 1% sodium metabisulfite (MBS) to wash out the circulatory system,
INSERM.
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1Fr 7.5 FIG. 1. Schematic diagram of three levels of the cat pons and thalamus redrawn from Snider and Niemer’s atlas (27). Boxes A, B, C represent the levels illustrated in immunohistochemistry in Fig. 2. CM: nucleus centralis medialis; GL: lateral geniculate body; GM: medial geniculate body; LC: locus coeruleus; LME: lam-
ina medulla& extema; SN: substantia nigra; VPM: nucleus ventralis posterior, pars medialis.
followed by 3 1 of 2.5% glutaraldehyde in 0.15 M Sorensen MBS buffer (pH = 7.6). After 3 h postfixation in the same fixative and washing overnight at 4°C in 0.15 M Sorensen MBS buffer, 50-pm vibratome frontal sections were made from the level of the hypothalamus to that of the LC. The sections were then washed in 0.15 M Tris buffer (pH=7.4). Double bonds were saturated with 1% sodium borohydride in 0.15 M Tris buffer (5 min). After extensive washing in 0.15 M Sorensen MBS buffer, they were preincubated during 30 min in Sorensen MBS buffer containing 1% normal sheep serum to prevent nonspecific binding. Triton X-100 (0.1% to 0.3%) was added to improve penetration of the antiserum. Sections were then incubated at 4°C in rabbit conjugated NA antiserum at l/2,000-l/5,000. After 48 h incubation, sections were washed in Sorensen buffer, incubated in goat antirabbit immunoglobuhn coupled to horseradish peroxidase (l/250) for 3 h and then washed again in Siirensen buffer. After 15 min rinsing in Tris buffer (pH 7.4; 0.1 M) the peroxidase activity was revealed with the 3-3’-diaminobenzidine reaction (15 mg/50 ml Tris buffer containing 0.03% hydrogen peroxide) and washed. Sections were mounted on glass slides with Sorensen-Glycerol
(1: 1). In some cases, the peroxidase reaction was intensified by the Silver-Gold method (16). After light microscopic observation, the VP nucleus was dissected out from sections between frontal planes 6.5 and 9.5 and prepared for electron microscopy. Briefly, blocks were postfixed during 1% h in 2% osmium tetroxide and dehydrated in graded alcohols before being embedded in araldite. Ultrathin sections were made, stained with lead citrate, and examined with a Philips EM 300 electron microscope. To assess the specificity of the reaction, the following controls were performed. 1) Regions known to be rich in NA-LI neurons were observed. 2) The diluted antiserum (l/2,000) was preabsorbed with l-10 mg/ml NA or 5-10 mg/ml dopamine (DA) or GABA during three hours at room temperature before addition of the sections. 3) The nonspecific absorption of the goat anti-rabbit IgG antibodies coupled to peroxidase was determined by omitting the anti-NA antiserum in the first incubation step. This control was made with and without sodium borohydride. 4) A monoclonal anti-DA antibody was tested in the cat thalamus using the same experimental protocol.
FACING PAGE FIG. 2. Noradrenaline-like immunoreactivity in the cat at levels indicated in Fig. 1. A: Fr-3, locus coeruleus; B: Fr 6, ascending noradrenergic-like tract; C: Fr 7.5, nucleus ventralis posterior of the thalamus. Al: Numerous immunoreactive cell bodies are observed in all the subdivisions of the nucleus locus coeruleus: principal LC, LCu, subcoeruleus (SC). Some immunoreactive fibers are also observed. BC: bra&mm conjunctivum; nV: tract of the mesencephalic trigeminal nucleus. A2: The antiserum (l/2,000) has been preincubated with 5 mg/ml NA during 3 h at room temperature before addition of the section. Note the almost total absence of reaction in the LC compared to Al. B: Punctate immunoreactivity in the dorsal ascending NAergic tract. The points most likely correspond to transversally cut axons. Cl: Immunoreactive fibers in nucleus ventralis posterior of the thahunus. They are grouped around probably nonlabeled cell bodies (arrows). Note the reaction extending along cellular processes. C2: Higher magnification of immunoreactive fibers in the nucleus ventralis posterior surrounding cell bodies. The presence of detergent in the incubation medium led to a diffusion into the cell bodies which are considered to be unlabeled. These cell bodies are oblong and measure 60-80 Fm. C3: Silver-gold intensification of the reaction in the nucleus ventralis posterior. In addition to clustered immunoteactive fibers around cell bodies (thick arrows), numerous isolated immunoreactive fibers are observed (thin arrows). A, B, C scale bars: 100 p,m; D, E scale bars: 50 pm.
NA-LIKE I~MUNOREACTIVITY
IN CAT VP
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DELAGRANGE
ET AL
some cases, the immunoreactivity extended along proximal dendrites. When the reaction was intensified with the silver-gold method, numerous immunoreactive isolated fibers could be seen (Fig. 2-C3). These pictures were obtained when 0.1 to 0.3% Triton X-100 was added in the preincubation medium and after 48 lr incubation in the specific antiserum. This may explain the diffuse staining in some cell bodies (Fig. 2-C) which did not present the punctate-like feature of the staining observed around the cell bodies. When the specific antiserum was omitted from the first mcubation step, no immunolabeling was observed in the VP or in the LC. Preincubation of the diluted anti-NA (112,000) with I mg/ml NA totally prevented the reaction in the VP. However, preincubation of the antiserum with DA or GABA did not modify the immunostaining. It was also verified that no immunoreactive fibers were observed in the VP after incubation in a monoclonal anti-DA antibody. This antibody labeled numerous cell bodies in the substantia nigra. In electron microscopy, the morphology of the tissue was poorly preserved because of the technical conditions used (addition of a detergent in the incubation media and long period of incubation). Our preliminary results have, however, permitted to observe numerous immunoreactive fibers in close apposition to unlabeled cell bodies (Fig. 3-l) or proximal dendrites (Fig. 3-2). In Fig. 3-2, the immunoreactive profile was presumably an axon terminal and established a symmetric synaptic contact, identified by the regular space between plasma membranes, with a proximal dendrite. DISCUSSION FIG. 3. Noradrenaline-like immunoreactivity in electron microscopy in nucleus ventralis posterior of the thalamus. 1: An immunoreactive profile is in close apposition with an unlabeled neuron. Cy: cytoplasm, N: nucleus. 2: An immunoreactive axon (A) establishes a symmetric synaptic contact with a proximal dendrite (D). Note the regular space between the two plasma membranes (arrow heads). Scale bars: 0.5 km.
RESULTS
The different regions of the cat brain observed in this study are schematically represented in the diagrams of Fig. 1. The diagrams, redrawn from Snider and Niemer’s atlas (27), represent frontal planes. Fr-3 corresponds to the locus coeruleus, Fr 6 to the ascending noradrenergic tract and Fr 7.5 to the nucleus ventralis posterior of the thalamus. Boxes A, B, C, correspond to the micrographs presented in Fig. 2. After incubation of the sections in the anti-NA antiserum, numerous immunoreactive cell bodies were observed in the region surrounding the brachium conjunctivum and the tract of the mesencephalic trigeminal nucleus (Fig. 2-Al). The different subdivisions of the LC were immunoreactive: the principal LC, the LCa and the subcoeruleus. Immunoreactive fibers were also observed. When the diluted antiserum was preincubated in 5 mg/ml NA, the immunolabeling was absent (Fig. 2-A2). At the frontal level Fr 6, a punctate NA-LI immunoreactivity was observed in the region of the ventral noradmnergic ascending bundle (Fig. 2-B). In the nucleus ventralis posterior, the immunoreactivity was localized between frontal planes 7 and 8.5, mainly in the ventral part of the VP medialis (VPM), just above the lamina medullaris extema and particularly on its lateral part. Neighbouring regions were devoid of immunoreactivity, particularly the lateral posterior nucleus located above the VP. In the VPM, the immunoreactivity was localized in fibers generally surrounding nonimmunoreactive cell bodies (Fig. 2-Cl-C3). These cell bodies were generally oblong and measured 60-80 Wm. In
The data presented here are to our knowledge the first description of NA-LI immunoreactivity in the cat obtained with a specific conjugated NA antiserum. NA-LI cell bodies were observed in the LC and NA-LI fibers in the noradrenergic ascending tract and in the VP of the thalamus. With the same antiserum several studies have been performed in the rat, in the LC (8). thalamus (22) and hypothalamus (3,9). NA-LI cell bodies of the cat LC were observed with an antiserum concentration of l/5,000, a concentration generally used for studies performed on the rat (see the references above). However, NA-LI fibers in the cat VP were observed only at antiserum concentration of l/2,000 and when Triton X-100 (0.1-0.3%) was added in the preincubation medium. This could result from the low concentration of NA-LI material in this species. This hypothesis is reinforced by the fact that we and others (17) were unable to detect tyrosine hydroxylase (TH)-positive axon terminals in the cat VP, suggesting that this enzyme also has a low concentration in this region. The latter authors could detect TH-LI axon terminals in the monkey thalamus (17) and, as already mentioned, NA-LI axon terminals are present in the rat thalamus. The specificity of the NA antiserum used here has been biochemically investigated (21). Immunocytochemical controls gave the expected results: in the LC, the immunoreaction decreased markedly when the antiserum (115,000) was preincubated in 1 mg/ml NA and disappeared totally after preincubation in 5 mg/ml NA. The same results were obtained in the VP with an antiserum concentration of l/2,000. In contrast with the biochemical controls (21), the free immunogen (nonconjugated to bovine serum albumine) is well recognized in immunocytochemistry. In addition, at both levels (LC and VP), when the antiserum was preincubated in other compounds, the labeling was never affected whatever were the concentrations tested. Moreover, we have seen that intraperitoneal injections of 10 mg/kg DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine), the drug reparted to destroy specifically the noradrenergic endings in rodents (10,13), resulted,
NA-LIKE IMMUNOREACTIVITY
IN CAT VP
537
15-90 days later, in a great decrease in the number of TH-LI cell bodies in the LC (4) and NA-LI fibers in the VP. This argument is in favor of the specificity of the noradrenergic nature of the immunoreactive terminals observed here in the VP. At the light microscopic level, NA-LI fibers of the VP were either isolated or grouped around nonimmunoreactive cell bodies. In the latter case. punctate immunostaining was observed around the cell body and often extended along proximal dendrites. This configuration is confirmed in our preliminary results at the electron microscopic level. Immunoreactive neurites. sometimes clearly identified as axon terminals were observed in close apposition with unlabeled cell bodies or proximal dendrites. Synapses were seldomly encountered, perhaps due to the poor preservation of tissues which results from the necessity of adding Triton X-100 to the preincubation medium. However, in some cases, these synapses were suspected to be symmetrical since the intermembranous space is large and regular. Data obtained in the rat nucleus ventralis basahs with the same antiserum have
shown that synaptic differentiations are never observed (22). In the monkey VP, it seems that only 5-107~ of TH-LI terminals form conventional synapses. It has been suggested that NA is released nonsynaptically (at least partly) in the VP of these two species. Concerning the cat, more detailed data at the electron microscopic level would be necessary to make the same claim. The origin of NA-LI axon terminals of the VP remains to be demonstrated with double labeling studies. However, histofluorescence studies have indeed shown a light catecholaminergic innervation of different thalamic nuclei including the VP (25,29). This hypothesis was reinforced by the demonstration with horseradish peroxidase (HRP) or wheat-germ-agglutinin peroxidase (WGA-HRP) of a direct projection from the locus coeruleus to the VP in the cat (1.2) and in the rat (19.33). ACKNOWLEDGEMENTS
This study was supported by DRET (Grant 86-101 pour la Recherche Medicale.
I and La Fondation
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