- Email: [email protected]
Multiple sources of the pituitary pars intermedia innervation in amphibians: A DiI retrograde tract-tracing study
ELSEVIER
Neuroscience Letters 169 (1994) 163-166
H[UROSCIENCE LETTERS
Multiple sources of the pituitary pars intermedia innervation in amphibians: a DiI retrograde tract-tracing study C. Artero, A. Fasolo, M.F. Franzoni* Laboratorio di Anatomia Comparata, Dipartimento di Biologia Animale, Universit~ di Torino, 10123 Torino, Italy Received 20 August 1993; Revised version received 18 November 1993; Accepted 7 January 1994
Abstract Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.
Key words: Tract-tracing; DiI; Pars intermedia; Adenohypophysis; Rana esculenta; Triturus carnifex
A number of studies has been conducted to determine the neurochemical nature of the nerve terminals in the pars intermedia (p.i.) of anurans [2,4,9,16,17,18,20, 22,23] and urodeles [5,11,12,16] since the classic histofluorescence studies that revealed the presence of monoamines in such innervation in Rana temporaria [9], Bufo arenarum [10], and Xenopus laevis [27]. More recently, several ultrastructural immunocytochemical studies have demonstrated that dopamine (DA), neuropeptide Y (NPY) and y-aminobutyric acid (GABA) co-exist in nerve terminals of the p.i. in both Xenopus laevis [7] and Rana ridibunda [28]. These data together with results from pharmacological and superfusion studies carried out with p.i. kept in vitro [1,4,29,33] have provided new insight into the involvement of these molecules as melanotropic regulators• Conversely, tracttracing investigations on the sources of p.i. innervation have been restricted to Xenopus laevis [30] and Bufo japonicus [21]. Such results differ in that either the suprachiasmatic nucleus (Xenopus) or the preoptic recess (Bufo) appears to control the p.i. Therefore, in the present study, we traced the afferents to the p.i. in two other amphibian species, the anuran Rana esculenta and the
*Corresponding author. Dipartimento di Biologia Animale, Universitfi di Torino, via Accademia Albertina 17, 20123 Torino. Italy. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)0006 I-E
urodelan Triturus carnifex, using the DiI tract-tracing method, in order to evaluate possible species-specific differences in amphibians. Male and female adult specimens of Rana esculenta (9 specimens) and Triturus carnifex (6 specimens), deeply anaesthetized with 1:1000 tricaine methanesulphonate (MS222, Sandoz Ltd., Basel, Switzerland), were transcardially perfused with 0.1 M phosphate buffer (PB), pH 7.4 and subsequentely with 4% paraformaldehyde (PFA) in PB. The skulls were opened and brains were immersed in situ overnight in 4% PFA. A minute crystal of DiI (1,1 'dioctadecyl-3,3,3',3' tetramethylindocarbocyanine, Molecular Probes, Eugene, OR, USA) was unilaterally inserted into the p.i. and kept in place with liquid 8% agar-agar. The brains were then incubated at 37°C in 4% PFA for 2 weeks to allow the retrograde axonal diffusion of the dye. After incubation, the brains were dissected out and cut with a vibratome in 50/lm coronal sections. The tissue sections were observed with a Leitz microscope equipped for epifluorescence (Leitz 1 2 filter). An overall similarity of labeling was observed in both species, although some differences regarding the extension rather than the localization of labeling between Rana and Triturus, were seen. Therefore, the locations of the DiI labeled cell bodies have been reconstructed and presented in a schematic longitudinal section of the frog brain (Fig. 1).
164
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
*
P
~
v
PVO
Fig. 1. Schematic drawing of a longitudinal section through the brain of Rana esculenta showing the distribution of DiI labeled perikarya (asterisks), after insertion of the fluorescent dye DiI within the pituitary intermediate lobe (stippled area), dp, dorsal pallium; inf, infundibulum; OCH, optic chiasm; OT, optic tectum; PA, preoptic area; pd, pars distalis of the pituitary; pn, pars nervosa of the pituitary; PVO, paraventricular organ; th, thalamus.
Labeled neurons in the anterior hypothalamus and the brain stem appeared more numerous in frog than in the newt. In both species, labeled neurons and nerve processes were generally concentrated in the preoptic area, where a substantial number of DiI fluorescent cell bodies, variable in size (12 22/~m), were localized mainly in the preoptic nucleus (nucleus preopticus of Herrick [19]) (Fig. 2a). Such neurons, generally bipolar (in some cases, unipolar) in shape, were endowed with laterally directed, tufted, dendritic arborization and with cerebrospinal fluid (CSF)-contacting processes (Fig. 2a). Frequently, a subependymal plexus of fluorescent thin processes running parallel to the ventricular surface, particularly developed in Rana (Fig. 2a), was observed. Sometimes, scattered ependymal tanycytes were also labeled. Several DiI-positive pear-shaped neurons were found in the supra- and retro-chiasmatic hypothalamus (Fig. 2b,c) and also the post-optic commissure was heavily stained in all specimens. In the tuberal lobes of the hypothalamus, rare cells appeared labeled in the ventromedial perinfundibular gray. Labeled neurons, bipolar or multipolar in shape together with varicose nerve processes were scattered from the dorso-lateral isthmic tegmentum to the area adjacent to the solitary tract nucleus. A number of labeled cells was observed in the area indicated as locus coeruleus (Fig. 2d,e). The DiI tract-tracing method applied to fixed brains [15] is considered far more sensitive than the classical HRP method [34]. It was successfully used in poikilothermic vertebrates to study different neural systems (e.g. the retinofugal pathway in Xenopus [8] and the bulbospinal serotoninergic pathway in Rana pipiens [26]). In the present study, after application of a minute DiI crystal into the intermediate lobe, patterns of labeling distribution appeared to be similar in both species investigated with some differences related to the extent of labeling rather than its location. In the newt the number of cell bodies containing the fluorescent dye was generally lower than in frog, both in the preoptic area and in
the brain stem, likely due to the overall simpler brain of urodeles compared to anurans [25]. In both species, a number of retrogradelly labeled neurons was found in the preoptic nucleus (although a possible retrograde labeling coming from the neural lobe could not be excluded for the animals which have received a large DiI crystal in the p.i.) and in supra-chiasmatic hypothalamus and, to a lesser extent, in the tuberal hypothalamus. Since our findings partially agree with those reported in the toad Xenopus laevis [30,31], they strengthen the involvement of the suprachiasmatic hypothalamus in the regulation of the intermediate lobe of amphibians. On the other hand, some species-specific variations may occur in anurans in the hypothalamic control of melanotrope cells as indicated by us (present data) and Kato et al. [21] in Bufo japonicus where, after DiI and lesion techniques, the preoptic recess organ was found to play a pivotal role in white-adaptation in background response. In this respect Triturus and Rana differ from Xenopus in that the latter species does not show significant DiI labeling in the preoptic nucleus [31]. A number of immunohistochemical studies has reported the occurrence in nerve terminals of amphibian p.i. of DA [16,17], GABA [1,14] and neuropeptides such as thyrotropin-releasing hormone (TRH) and NPY [5]. Moreover, GABA, DA and NPY were found to co-exist within the same nerve terminals in Xenopus [7] as well as in Rana [28] intermediate lobes, and the suprachiasmatic hypothalamus was proposed as a possible source of such nerve terminals [30,31]. Our results support, at least partly, the immunohistochemical data cited above, because the suprachiasmatic hypothalamus, which in our experiments was heavily labeled by DiI, also contains cell bodies immunoreactive with antibodies against TH (in Rana [32] and Triturus [13]), NPY (in Rana [2]) and GABA (in Rana and Triturus [14]). Furthermore, by means of confocal microscopic analysis, NPY and TH have been found to co-exist in the supra-chiasmatic hypothalamus of Rana esculenta (Fasolo and Feuilloley, unpublished data). Using an anti-noradrenaline (NA) antiserum, Gonzalez and Smeets [18] have demonstrated a rich NA innervation in the p.i. of Xenopus laevis. This is in agreement with previous studies indicating the presence of NAergic and adrenergic receptors on melanotropic cells [6,33] in the same species. In addition, Gonzalez and Smeets [16] have postulated, using anti-TH and anti-DA antibodies in Rana and the urodele Pleurodeles waltlii, the NA-ergic nature of the isthmic catecholaminergic cell bodies. They have also demonstrated [18] the presence of NA-immunopositive cell bodies in Xenopus both in the paraventricular organ (PVO) and in the isthmus (locus coeruleus) and, more caudally, close to the solitary tract nucleus. Some histochemical observations carried in Xenopus [27] have suggested the PVO as a possible source of afferents to the p.i. On the contrary, we did not
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
165
Fig. 2. DiI labelings in coronal sections of the brain of (a) Rana esculenta: a number of labeled unipolar and bipolar cells within the proptic nucleus (IIIV, 3rd ventricle), 125 x; (b) Rana esculenta: labeled neurons in the suprachiasmatic hypothalamus, 400 ×; (c) Triturus carnifex: a group of labeled neurons in the suprachiasmatic hypothalamus. Photomontage, 410 x; (d) Rana esculenta and (e) Triturus carnifex: DiI-containing neurons in the locus coeruleus, (d) 300 ×, (e) 400 ×. find a n y significant labeling in the PVO. Since in the species investigated labeled p e r i k a r y a were identified in the d0rso-lateral isthmus, we can infer that the principal source of the NA-ergic i n n e r v a t i o n of the intermediate lobe is located in the b r a i n stem.
[1] Adjeroud, S., Tonon, M.C., Lamacz, M., Leneveu, E., Stockel, M.E., Tappaz, M.L., Cazin, L., Danger, J.M., Bernard, C. and Vaudry, H, GABA-ergic control of ~-melanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro, Brain Res. Bull., 17 (1986) 717-724. [2] Andersen, A.C., Tonon M.C., Pelletier G., Conlon M., Fasolo A.
166
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
and Vaudry, H., Neuropeptides in the amphibian brain, Int. Rev. Cytol., 138 (1992) 89 188. [3] Caillez, D., Danger, J.M., Andersen, A.C., Polak, J.M., Pelletier, G., Kawamura, K., Kikuyama, S. and Vaudry, H., Neuropeptide Y (NPY)-like immunoreactive neurons in the brain and pituitary of the amphibian Rana catesbeiana, Zool. Sci., 4 (1987) 123 134. [4] Danger, J.M., Leboulenger, F., Guy, J., Tonon, M.C., Benyamina, M., Martel, J.C., Saint-Pierre, S., Pelletier, G. and Vaudry, H., Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an -MSH-release inhibiting factor, Life Sci., 39 (1986) 1183 1192. [5] Danger, J.M., Perroteau, I., Franzoni, M.F., Saint-Pierre, S., Fasolo, A. and Vaudry, H., Innervation of the pars intermedia and control of alpha-melanotropin secretion in the newt, Neuroendocrinology, 50 (1989) 543-549. [6] de Koning, H.P., Jenks, B.G. and Roubos, E.W., Evidence for three different cathecolamine receptors on pituitary melanotrope cells of Xenopus laevis, Abstr. 7th Int. Catechol. Symp., Amsterdam, 1992, p. 68. [7] de Rijk, E.P.C.T., van Strien, F.J.C. and Roubos, E.W., Demonstration of coexisting catecholamine (dopamine), aminoacid (GABA), and peptide (NPY) involved in inhibition ofmelanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze substitution immunocytochemical study, J. Neurosci., 12 (1992) 864-87 I. [8] Easter, S.S.J. and Taylor, J.S.H., The development of the Xenopus retinofugal pathway: optic fibers join a preexisting tract, Development, 107 (1989) 553-573. [9] Enemar, A. and Falk, B., On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria, Gen.; Comp. Endocrinol., 5 (1965) 577 583. [10] Enemar, A., Falk, B. and Iturriza, F.C., Adrenergic nerves in the pars intermedia of the pituitary in the toad, BuJb arenarum, Z. Zellforsch., 77 (1967) 325 330. [11] Franzoni, M.F., Fasolo, A. and Mazzi, V., Observations on the pars intermedia of the pituitary in the crested newt under various light conditions, Monitore Zool. Ital., 6 (1972) 113 128. [12] Franzoni, M.F, and Fasolo, A., Fine structural changes induced by 6-hydroxydopamine in the pars intermedia of the newt, J. Submicrosc. Cytol., 7 (1975) 107-119. [13] Franzoni, M.F., Thibault, J., Fasolo, A., Martinoli, M.G., Scaranari, F. and Calas, A., Organization of tyrosine-hydroxylase immunopositive neurons in the brain of the crested newt, Triturus cristatus carnifex, J. Comp. Neurol., 251 (1986) 121 134. [14] Franzoni, M.F, and Morino, P., The distribution of GABA-immunoreactive neurons in the brain of the newt (Triturus cruitatus carnifex) and the green frog (Rana eseulenta), Cell Tissue Res., 255 (1989) 155 166. [15] Godement, R, Vanselow, J., Thanos, S. and Bonhoffer, F., A study in developing visual system with a new method of staining neurons and their processes in fixed tissue, Development, 101 (1987) 697 713. [16] Gonzalez, A. and Smeets, W.J.A.J., Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the brain of two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii, J. Comp. Neurol., 303 (1991) 457-477. [17] Gonzalez, A., Tuinhof, R. and Smeets, W.J.A.J., Distribution of tyrosine hydroxylase- and dopamine-immunoreactivities in the brain of the south african clawed frog Xenopus laevis, Anat. Embryol., 187 (1993) 193 201. [18] Gonzalez, A. and Smeets, W.J.A.J., Noradrenaline in the brain of the south african clawed frog Xenopus laevis: a study with antibodies against noradrenaline and dopamine-fl-hydroxylase, J. Comp. Neurol., 331 (1993) 363 374. [19] Herrick, C.J., The Brain of the Tiger Salamander Ambystoma tigrinum, University of Cichago Press, Cichago, IL, 1948, p. 241.
[20] Hopkins, C.R., Localization of adrenergic fibers in the amphibian pars intermedia by electron microscope autoradiography and their selective removal by 6-hydroxydopamine, Gen. Comp. Endocrinol., 16 (1970) 112-120. [21] Kato, Z., Kawamura, K. and Kikuyama, S., Role of preoptic recess organ in background response of the toad, Bufo japonicus, Abstr. Proc. Int. Symp. Amphibian Endocrinol., Tokyo 1992, p. 65. [22] Lamacz, M., Hindelang, C., Tonon, M.C., Vaudry, H. and Stoeckel, M.E., Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for co-localization of thyrotropin-releasing hormone and mesotocin in fibers innervating pars intermedia cells, Neuroscience, 32 (1989) 451~462. [23] Lamacz, M., Tonon, M.C., Leboulanger, F., H6ry, F., Idres, S., Verhofstad, A.J., Pelletier, G. and Vaudry, H., Effect of serotonin on alfa-melanocyte-stimulating hormone secretion from perifused frog neurointermediate lobe: evidence for the presence of serotonin-containing cells in the frog pars intermedia, J. Endocrinol., 122 (1989) 135-146. [24] Laz~tr, G., Maderdrut, J.L., Trasti, S.L., Liposits, Z., T6th, P., Kozicz, T. and Merchenthaler, I., Distribution ofproneuropeptide Y-derived peptides in the brain of Rana eseulenta and Xenopus laevis, J. Comp. Neurol., 327 (1993) 551-571. [25] Schmidt, A. and Roth, G., Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltlii, Cell Tissue Res., 272 (1993) 273-287. [26] Tan, H. and Miletic, V., Bulbospinal serotoninergic pathways in the frog Rana pipiens, J. Comp. Neurol., 292 (1990) 291 302. [27] Terlou, M. and Ploemacher, R.E., The distribution of monoamines in the tel-, di- and mesencephalon of Xenopus laevis tadpoles with special reference to the hypothalamo-hypophysial system, Z. Zellforsh., 137 (1973) 521-540. [28] Tonon, M.C., Bosler, O., Stoeckel, M.E., Pelletier, G., Tappaz, M. and Vaudry H., Co-localization of tyrosine hydroxylase, GABA and neuropeptide Y within axon terminals innervating the intermediate lobe of the frog Rana ridibunda, J. Comp: Neurol., 319 (1992) 599 605. [29] Tonon, M.C., Danger, J.M., Lamacz, M., Leroux, P., Adjeroud, S., Andersen A.C., Verburg-van Kemenade, L., Jenks, B.G. Pelletier, G., Stoeckel, L., Burlet, A., Kupryszewski, G. and Vaudry, H., Multihormonal control of melanotropin secretion in cold blooded vertebrates. In M.E. Hadley (Eds.), The Melanotropic Peptides, CRC, New York, 1988, pp. 127 171. [30] Tuinhof, R., Laurent, F., Brandt, J.W., Michielsen, C. and Roubos, E.W., Role of the hypothalamo-hypophysial system in control of background adaptation by the amphibian Xenopus laevis, Netherlands J. Zool., 41 (1991) 311-312. [31] Tuinhof, R., Artero, C., Fasolo, A., Franzoni, M.F., Ten Donkelaar, H.J., Wismans, P.G.P. and Roubos, E.W., Involvement of retinohypothalamic input, suprachiasmatic nucleus and locus coeruleus in neural control of melanotrope cells of Xenopus laevis, in preparation. [32] Tuninetti, P., Mulatero, B., Franzoni, M.F., Thibault, J., Vaudry, H. and Fasolo, A., Immunohistochemical analysis of tyrosine hydroxylase positive systems in the frog brain, Eur. J. Neurosci., Suppl. 2 (1989) p. 31. [33] Verburg van Kemenade, B.M.L., Tonon, M.C., Jenks, B.G. and Vaudry, H., Characteristics of receptors for dopamine in the pars intermedia of amphibian Xenopus laevis, Neuroendocrinology, 44 (1986) 446456. [34] Wicht, H. and Northcutt, R.G., Retinofugal and retinopetal projections in the pacific hagfish Eptatretus stanti (myxinoidea), Brain Behav. Evol., 36 (1990) 315 328.
Neuroscience Letters 169 (1994) 163-166
H[UROSCIENCE LETTERS
Multiple sources of the pituitary pars intermedia innervation in amphibians: a DiI retrograde tract-tracing study C. Artero, A. Fasolo, M.F. Franzoni* Laboratorio di Anatomia Comparata, Dipartimento di Biologia Animale, Universit~ di Torino, 10123 Torino, Italy Received 20 August 1993; Revised version received 18 November 1993; Accepted 7 January 1994
Abstract Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.
Key words: Tract-tracing; DiI; Pars intermedia; Adenohypophysis; Rana esculenta; Triturus carnifex
A number of studies has been conducted to determine the neurochemical nature of the nerve terminals in the pars intermedia (p.i.) of anurans [2,4,9,16,17,18,20, 22,23] and urodeles [5,11,12,16] since the classic histofluorescence studies that revealed the presence of monoamines in such innervation in Rana temporaria [9], Bufo arenarum [10], and Xenopus laevis [27]. More recently, several ultrastructural immunocytochemical studies have demonstrated that dopamine (DA), neuropeptide Y (NPY) and y-aminobutyric acid (GABA) co-exist in nerve terminals of the p.i. in both Xenopus laevis [7] and Rana ridibunda [28]. These data together with results from pharmacological and superfusion studies carried out with p.i. kept in vitro [1,4,29,33] have provided new insight into the involvement of these molecules as melanotropic regulators• Conversely, tracttracing investigations on the sources of p.i. innervation have been restricted to Xenopus laevis [30] and Bufo japonicus [21]. Such results differ in that either the suprachiasmatic nucleus (Xenopus) or the preoptic recess (Bufo) appears to control the p.i. Therefore, in the present study, we traced the afferents to the p.i. in two other amphibian species, the anuran Rana esculenta and the
*Corresponding author. Dipartimento di Biologia Animale, Universitfi di Torino, via Accademia Albertina 17, 20123 Torino. Italy. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)0006 I-E
urodelan Triturus carnifex, using the DiI tract-tracing method, in order to evaluate possible species-specific differences in amphibians. Male and female adult specimens of Rana esculenta (9 specimens) and Triturus carnifex (6 specimens), deeply anaesthetized with 1:1000 tricaine methanesulphonate (MS222, Sandoz Ltd., Basel, Switzerland), were transcardially perfused with 0.1 M phosphate buffer (PB), pH 7.4 and subsequentely with 4% paraformaldehyde (PFA) in PB. The skulls were opened and brains were immersed in situ overnight in 4% PFA. A minute crystal of DiI (1,1 'dioctadecyl-3,3,3',3' tetramethylindocarbocyanine, Molecular Probes, Eugene, OR, USA) was unilaterally inserted into the p.i. and kept in place with liquid 8% agar-agar. The brains were then incubated at 37°C in 4% PFA for 2 weeks to allow the retrograde axonal diffusion of the dye. After incubation, the brains were dissected out and cut with a vibratome in 50/lm coronal sections. The tissue sections were observed with a Leitz microscope equipped for epifluorescence (Leitz 1 2 filter). An overall similarity of labeling was observed in both species, although some differences regarding the extension rather than the localization of labeling between Rana and Triturus, were seen. Therefore, the locations of the DiI labeled cell bodies have been reconstructed and presented in a schematic longitudinal section of the frog brain (Fig. 1).
164
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
*
P
~
v
PVO
Fig. 1. Schematic drawing of a longitudinal section through the brain of Rana esculenta showing the distribution of DiI labeled perikarya (asterisks), after insertion of the fluorescent dye DiI within the pituitary intermediate lobe (stippled area), dp, dorsal pallium; inf, infundibulum; OCH, optic chiasm; OT, optic tectum; PA, preoptic area; pd, pars distalis of the pituitary; pn, pars nervosa of the pituitary; PVO, paraventricular organ; th, thalamus.
Labeled neurons in the anterior hypothalamus and the brain stem appeared more numerous in frog than in the newt. In both species, labeled neurons and nerve processes were generally concentrated in the preoptic area, where a substantial number of DiI fluorescent cell bodies, variable in size (12 22/~m), were localized mainly in the preoptic nucleus (nucleus preopticus of Herrick [19]) (Fig. 2a). Such neurons, generally bipolar (in some cases, unipolar) in shape, were endowed with laterally directed, tufted, dendritic arborization and with cerebrospinal fluid (CSF)-contacting processes (Fig. 2a). Frequently, a subependymal plexus of fluorescent thin processes running parallel to the ventricular surface, particularly developed in Rana (Fig. 2a), was observed. Sometimes, scattered ependymal tanycytes were also labeled. Several DiI-positive pear-shaped neurons were found in the supra- and retro-chiasmatic hypothalamus (Fig. 2b,c) and also the post-optic commissure was heavily stained in all specimens. In the tuberal lobes of the hypothalamus, rare cells appeared labeled in the ventromedial perinfundibular gray. Labeled neurons, bipolar or multipolar in shape together with varicose nerve processes were scattered from the dorso-lateral isthmic tegmentum to the area adjacent to the solitary tract nucleus. A number of labeled cells was observed in the area indicated as locus coeruleus (Fig. 2d,e). The DiI tract-tracing method applied to fixed brains [15] is considered far more sensitive than the classical HRP method [34]. It was successfully used in poikilothermic vertebrates to study different neural systems (e.g. the retinofugal pathway in Xenopus [8] and the bulbospinal serotoninergic pathway in Rana pipiens [26]). In the present study, after application of a minute DiI crystal into the intermediate lobe, patterns of labeling distribution appeared to be similar in both species investigated with some differences related to the extent of labeling rather than its location. In the newt the number of cell bodies containing the fluorescent dye was generally lower than in frog, both in the preoptic area and in
the brain stem, likely due to the overall simpler brain of urodeles compared to anurans [25]. In both species, a number of retrogradelly labeled neurons was found in the preoptic nucleus (although a possible retrograde labeling coming from the neural lobe could not be excluded for the animals which have received a large DiI crystal in the p.i.) and in supra-chiasmatic hypothalamus and, to a lesser extent, in the tuberal hypothalamus. Since our findings partially agree with those reported in the toad Xenopus laevis [30,31], they strengthen the involvement of the suprachiasmatic hypothalamus in the regulation of the intermediate lobe of amphibians. On the other hand, some species-specific variations may occur in anurans in the hypothalamic control of melanotrope cells as indicated by us (present data) and Kato et al. [21] in Bufo japonicus where, after DiI and lesion techniques, the preoptic recess organ was found to play a pivotal role in white-adaptation in background response. In this respect Triturus and Rana differ from Xenopus in that the latter species does not show significant DiI labeling in the preoptic nucleus [31]. A number of immunohistochemical studies has reported the occurrence in nerve terminals of amphibian p.i. of DA [16,17], GABA [1,14] and neuropeptides such as thyrotropin-releasing hormone (TRH) and NPY [5]. Moreover, GABA, DA and NPY were found to co-exist within the same nerve terminals in Xenopus [7] as well as in Rana [28] intermediate lobes, and the suprachiasmatic hypothalamus was proposed as a possible source of such nerve terminals [30,31]. Our results support, at least partly, the immunohistochemical data cited above, because the suprachiasmatic hypothalamus, which in our experiments was heavily labeled by DiI, also contains cell bodies immunoreactive with antibodies against TH (in Rana [32] and Triturus [13]), NPY (in Rana [2]) and GABA (in Rana and Triturus [14]). Furthermore, by means of confocal microscopic analysis, NPY and TH have been found to co-exist in the supra-chiasmatic hypothalamus of Rana esculenta (Fasolo and Feuilloley, unpublished data). Using an anti-noradrenaline (NA) antiserum, Gonzalez and Smeets [18] have demonstrated a rich NA innervation in the p.i. of Xenopus laevis. This is in agreement with previous studies indicating the presence of NAergic and adrenergic receptors on melanotropic cells [6,33] in the same species. In addition, Gonzalez and Smeets [16] have postulated, using anti-TH and anti-DA antibodies in Rana and the urodele Pleurodeles waltlii, the NA-ergic nature of the isthmic catecholaminergic cell bodies. They have also demonstrated [18] the presence of NA-immunopositive cell bodies in Xenopus both in the paraventricular organ (PVO) and in the isthmus (locus coeruleus) and, more caudally, close to the solitary tract nucleus. Some histochemical observations carried in Xenopus [27] have suggested the PVO as a possible source of afferents to the p.i. On the contrary, we did not
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
165
Fig. 2. DiI labelings in coronal sections of the brain of (a) Rana esculenta: a number of labeled unipolar and bipolar cells within the proptic nucleus (IIIV, 3rd ventricle), 125 x; (b) Rana esculenta: labeled neurons in the suprachiasmatic hypothalamus, 400 ×; (c) Triturus carnifex: a group of labeled neurons in the suprachiasmatic hypothalamus. Photomontage, 410 x; (d) Rana esculenta and (e) Triturus carnifex: DiI-containing neurons in the locus coeruleus, (d) 300 ×, (e) 400 ×. find a n y significant labeling in the PVO. Since in the species investigated labeled p e r i k a r y a were identified in the d0rso-lateral isthmus, we can infer that the principal source of the NA-ergic i n n e r v a t i o n of the intermediate lobe is located in the b r a i n stem.
[1] Adjeroud, S., Tonon, M.C., Lamacz, M., Leneveu, E., Stockel, M.E., Tappaz, M.L., Cazin, L., Danger, J.M., Bernard, C. and Vaudry, H, GABA-ergic control of ~-melanocyte-stimulating hormone (a-MSH) release by frog neurointermediate lobe in vitro, Brain Res. Bull., 17 (1986) 717-724. [2] Andersen, A.C., Tonon M.C., Pelletier G., Conlon M., Fasolo A.
166
C. Artero et al./Neuroscience Letters 169 (1994) 163 166
and Vaudry, H., Neuropeptides in the amphibian brain, Int. Rev. Cytol., 138 (1992) 89 188. [3] Caillez, D., Danger, J.M., Andersen, A.C., Polak, J.M., Pelletier, G., Kawamura, K., Kikuyama, S. and Vaudry, H., Neuropeptide Y (NPY)-like immunoreactive neurons in the brain and pituitary of the amphibian Rana catesbeiana, Zool. Sci., 4 (1987) 123 134. [4] Danger, J.M., Leboulenger, F., Guy, J., Tonon, M.C., Benyamina, M., Martel, J.C., Saint-Pierre, S., Pelletier, G. and Vaudry, H., Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an -MSH-release inhibiting factor, Life Sci., 39 (1986) 1183 1192. [5] Danger, J.M., Perroteau, I., Franzoni, M.F., Saint-Pierre, S., Fasolo, A. and Vaudry, H., Innervation of the pars intermedia and control of alpha-melanotropin secretion in the newt, Neuroendocrinology, 50 (1989) 543-549. [6] de Koning, H.P., Jenks, B.G. and Roubos, E.W., Evidence for three different cathecolamine receptors on pituitary melanotrope cells of Xenopus laevis, Abstr. 7th Int. Catechol. Symp., Amsterdam, 1992, p. 68. [7] de Rijk, E.P.C.T., van Strien, F.J.C. and Roubos, E.W., Demonstration of coexisting catecholamine (dopamine), aminoacid (GABA), and peptide (NPY) involved in inhibition ofmelanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze substitution immunocytochemical study, J. Neurosci., 12 (1992) 864-87 I. [8] Easter, S.S.J. and Taylor, J.S.H., The development of the Xenopus retinofugal pathway: optic fibers join a preexisting tract, Development, 107 (1989) 553-573. [9] Enemar, A. and Falk, B., On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria, Gen.; Comp. Endocrinol., 5 (1965) 577 583. [10] Enemar, A., Falk, B. and Iturriza, F.C., Adrenergic nerves in the pars intermedia of the pituitary in the toad, BuJb arenarum, Z. Zellforsch., 77 (1967) 325 330. [11] Franzoni, M.F., Fasolo, A. and Mazzi, V., Observations on the pars intermedia of the pituitary in the crested newt under various light conditions, Monitore Zool. Ital., 6 (1972) 113 128. [12] Franzoni, M.F, and Fasolo, A., Fine structural changes induced by 6-hydroxydopamine in the pars intermedia of the newt, J. Submicrosc. Cytol., 7 (1975) 107-119. [13] Franzoni, M.F., Thibault, J., Fasolo, A., Martinoli, M.G., Scaranari, F. and Calas, A., Organization of tyrosine-hydroxylase immunopositive neurons in the brain of the crested newt, Triturus cristatus carnifex, J. Comp. Neurol., 251 (1986) 121 134. [14] Franzoni, M.F, and Morino, P., The distribution of GABA-immunoreactive neurons in the brain of the newt (Triturus cruitatus carnifex) and the green frog (Rana eseulenta), Cell Tissue Res., 255 (1989) 155 166. [15] Godement, R, Vanselow, J., Thanos, S. and Bonhoffer, F., A study in developing visual system with a new method of staining neurons and their processes in fixed tissue, Development, 101 (1987) 697 713. [16] Gonzalez, A. and Smeets, W.J.A.J., Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the brain of two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii, J. Comp. Neurol., 303 (1991) 457-477. [17] Gonzalez, A., Tuinhof, R. and Smeets, W.J.A.J., Distribution of tyrosine hydroxylase- and dopamine-immunoreactivities in the brain of the south african clawed frog Xenopus laevis, Anat. Embryol., 187 (1993) 193 201. [18] Gonzalez, A. and Smeets, W.J.A.J., Noradrenaline in the brain of the south african clawed frog Xenopus laevis: a study with antibodies against noradrenaline and dopamine-fl-hydroxylase, J. Comp. Neurol., 331 (1993) 363 374. [19] Herrick, C.J., The Brain of the Tiger Salamander Ambystoma tigrinum, University of Cichago Press, Cichago, IL, 1948, p. 241.
[20] Hopkins, C.R., Localization of adrenergic fibers in the amphibian pars intermedia by electron microscope autoradiography and their selective removal by 6-hydroxydopamine, Gen. Comp. Endocrinol., 16 (1970) 112-120. [21] Kato, Z., Kawamura, K. and Kikuyama, S., Role of preoptic recess organ in background response of the toad, Bufo japonicus, Abstr. Proc. Int. Symp. Amphibian Endocrinol., Tokyo 1992, p. 65. [22] Lamacz, M., Hindelang, C., Tonon, M.C., Vaudry, H. and Stoeckel, M.E., Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for co-localization of thyrotropin-releasing hormone and mesotocin in fibers innervating pars intermedia cells, Neuroscience, 32 (1989) 451~462. [23] Lamacz, M., Tonon, M.C., Leboulanger, F., H6ry, F., Idres, S., Verhofstad, A.J., Pelletier, G. and Vaudry, H., Effect of serotonin on alfa-melanocyte-stimulating hormone secretion from perifused frog neurointermediate lobe: evidence for the presence of serotonin-containing cells in the frog pars intermedia, J. Endocrinol., 122 (1989) 135-146. [24] Laz~tr, G., Maderdrut, J.L., Trasti, S.L., Liposits, Z., T6th, P., Kozicz, T. and Merchenthaler, I., Distribution ofproneuropeptide Y-derived peptides in the brain of Rana eseulenta and Xenopus laevis, J. Comp. Neurol., 327 (1993) 551-571. [25] Schmidt, A. and Roth, G., Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltlii, Cell Tissue Res., 272 (1993) 273-287. [26] Tan, H. and Miletic, V., Bulbospinal serotoninergic pathways in the frog Rana pipiens, J. Comp. Neurol., 292 (1990) 291 302. [27] Terlou, M. and Ploemacher, R.E., The distribution of monoamines in the tel-, di- and mesencephalon of Xenopus laevis tadpoles with special reference to the hypothalamo-hypophysial system, Z. Zellforsh., 137 (1973) 521-540. [28] Tonon, M.C., Bosler, O., Stoeckel, M.E., Pelletier, G., Tappaz, M. and Vaudry H., Co-localization of tyrosine hydroxylase, GABA and neuropeptide Y within axon terminals innervating the intermediate lobe of the frog Rana ridibunda, J. Comp: Neurol., 319 (1992) 599 605. [29] Tonon, M.C., Danger, J.M., Lamacz, M., Leroux, P., Adjeroud, S., Andersen A.C., Verburg-van Kemenade, L., Jenks, B.G. Pelletier, G., Stoeckel, L., Burlet, A., Kupryszewski, G. and Vaudry, H., Multihormonal control of melanotropin secretion in cold blooded vertebrates. In M.E. Hadley (Eds.), The Melanotropic Peptides, CRC, New York, 1988, pp. 127 171. [30] Tuinhof, R., Laurent, F., Brandt, J.W., Michielsen, C. and Roubos, E.W., Role of the hypothalamo-hypophysial system in control of background adaptation by the amphibian Xenopus laevis, Netherlands J. Zool., 41 (1991) 311-312. [31] Tuinhof, R., Artero, C., Fasolo, A., Franzoni, M.F., Ten Donkelaar, H.J., Wismans, P.G.P. and Roubos, E.W., Involvement of retinohypothalamic input, suprachiasmatic nucleus and locus coeruleus in neural control of melanotrope cells of Xenopus laevis, in preparation. [32] Tuninetti, P., Mulatero, B., Franzoni, M.F., Thibault, J., Vaudry, H. and Fasolo, A., Immunohistochemical analysis of tyrosine hydroxylase positive systems in the frog brain, Eur. J. Neurosci., Suppl. 2 (1989) p. 31. [33] Verburg van Kemenade, B.M.L., Tonon, M.C., Jenks, B.G. and Vaudry, H., Characteristics of receptors for dopamine in the pars intermedia of amphibian Xenopus laevis, Neuroendocrinology, 44 (1986) 446456. [34] Wicht, H. and Northcutt, R.G., Retinofugal and retinopetal projections in the pacific hagfish Eptatretus stanti (myxinoidea), Brain Behav. Evol., 36 (1990) 315 328.