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Neuronal localization of immunoreactive adrenocorticotropin-like substance in the hypothalamus of elasmobranch fishes
Neuroscience Letters, 80 (1987) l~i Elsevier Scientific Publishers Ireland Ltd.
1
NSL 04783
Neuronal localization of immunoreactive adrenocorticotropin-like substance in the hypothalamus of elasmobranch fishes Mauro Vallarino and Irene Ottonello Istituto di Anatomia Comparata, Universit~ di Genova, Genova (Italy)
(Received 30 March 1987; Revised version received 23 April 1987; Accepted 8 May 1987) Key words: Adrenocorticotropin (ACTH); Hypothalamus; Elasmobranch fish; Immunocytochemistry
The occurrence and localization of adrenocorticotropin (ACTH)-Iike material in the brain of two species of elasmobranch fishes, Scyliorhinus canicula and Squalus acanthias, were studied by immunohistochemical techniques using an antiserum generated against human ACTH~_24.In both species immunopositive neurons and fibers were recognized in the basal hypothalamus mainly distributed in the nucleus lateralis tuberis, in which many of these elements were of the liquor-contacting type. A few ACTH-Iike positive cells were also found in the nucleus lobi lateralis hypothalami. The features of this peptidergic system, its topographic distribution to form intrinsic circuits within the posterior hypothalamic area and probably connections with the pituitary, suggest implications in brain neuromodulatory activities and hypophyseal regulation for this peptide.
A d r e n o c o r t i c o t r o p i n ( A C T H ) is one o f a n u m b e r o f n e u r o a c t i v e p e p t i d e s which have been identified in the b r a i n o f several m a m m a l i a n species [1, 13, 18]. R a d i o i m m u n o l o g i c a l a n d i m m u n o c y t o c h e m i c a l studies have s h o w n its presence in the neurons o f the a r c u a t e nucleus a n d within a rich fiber n e t w o r k e x t e n d i n g to m a n y h y p o t h a l a m i c a n d e x t r a h y p o t h a l a m i c regions [12], where it seems to p l a y a role in n e u r o e n d o c r i n e a n d b e h a v i o r a l functions [8]. In lower vertebrates, b r a i n A C T H i m m u n o r e a c t i v i t y has o n l y been r e c o r d e d in reptiles, in two s e p a r a t e centers o f the h y p o t h a l a m u s a n d m i d b r a i n t h a t are also i m m u n o r e a c t i v e for o t h e r o p i o m e l a n o c o r t i n p e p t i d e s [4]. In c y c l o s t o m e s positive b i o a s s a y results o b t a i n e d by E a s t m a n a n d P o r t a n o v a [5] were n o t c o n f i r m e d by i m m u n o h i s t o c h e m i c a l p r o c e d u r e s [ 10]. In this s t u d y the presence o f A C T H was i m m u n o c y t o c h e m i c a l l y investigated in the b r a i n o f e l a s m o b r a n c h fishes utilizing an a n t i s e r u m g e n e r a t e d a g a i n s t h u m a n A C T H I 24. Twelve male a n d female Scyliorhinus canicula a n d Squalus acanthias c a p t u r e d in Correspondence." M. Vallarino, Istituto di Anatomia Comparata, Universit~ di Genova, Via Balbi 5, 1-16126 Genova, Italy.
0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd.
the Ligurian Sea (Western Mediterranean) were anesthetized by immersion in tricaine methasulphonate (MS 222, Sandoz, 100 mg/liter seawater) and perfused through the ventral aorta with cold 0.01 M phosphate-buffered saline (PBS), pH 7.4, and then with a solution of 4% paraformaldehyde in PBS and alternatively with Bouin's fluid. The brains, together with attached pituitaries, were dissected out and postfixed overnight in the respective fixation solution. Bouin-fixed brains were dehydrated through a graded ethanol series and embedded in paraffin. Serial 5/~m thick sections were cut in either the sagittal or transverse plane and mounted on glass slides. Paraformaldehyde-fixed brains were instead transferred to 20% sucrose in 0.01 M phosphate buffer (4°C) for 24-48 h, and then sectioned frozen at 20/~m. The sections were treated according to the indirect immunofluorescent method [3] or with the peroxidase-anti-peroxidase (PAP) method [14], as described in detail elsewhere [ 15]. The sections were incubated with (1) normal swine serum (0.7 mg/ml) at room temperature for 20 min, (2) anti-ACTH serum (UCB, Brussels, Belgium) diluted 1:400 with Triton X-100/PBS for 24 h at 4°C, (3) fluorescein isothiocyanate conjugated swine anti-rabbit g a m m a globulin (Dakopatts, Denmark) diluted 1:500, at room temperature for 1 h, and then examined through a Zeiss epifluorescent microscope. For the PAP method the sections reacted with (1) primary antiserum, (2) swine antiserum to rabbit g a m m a globulin (Dakopatts, Denmark) diluted 1:500 for 1 h, (3) peroxidase-rabbit anti-peroxidase complex solution (Dakopatts, Denmark) diluted 1:800, for 1 h. This reaction was accomplished with the free base of 3,Y-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, U.S.A.) containing 0.01% H202. Some sections were also counterstained with Cresyl violet. An absorption test, performed by incubating alternate sections in anti-ACTH serum that had been absorbed with excess ACTH1 24 (50 nmol/ml), prevented all staining for A C T H , while absorption with ct-MSH (UCB, Brussels, Belgium), fl-MSH (Sigma, St. Louis, U.S.A.) and fl-LPH (Sigma, St. Louis, U.S.A.) did not produce any decrease in the intensity of the staining. Both immunocytochemical procedures revealed ACTH-reactive neurons in the ventral hypothalamus of all the specimens examined. Stained cell bodies were confined in the nucleus lateralis tuberis and in the pars ventralis of the nucleus lobi lateralis hypothalami. The nucleus lateralis tuberis exhibited a great deal of immunopositive cells and fibers surrounding the ventral wall of the infundibular recess (Fig. la,
Fig. 1. a-c: ACTH-likePAP-immunoreactiveneurons and fibers in the nucleus lateralis tuberis of Scyliorhinus canicula. Positiveelementsare distributed, among non-reactivecells, within the subependymallayers surrounding the infundibular (I) cavity of the third ventricle. Arrows indicate positive processes coursing ventrally, a: sagittal section. In the ventral side are also present some immunopositivecells of the rostral lobe of the pituitary. × 150. b: high magnification of reactive neurons. × 375. c: positive neurons of the liquor-contacting type showing a swelling of ACTH-like material on the wall of the infundibular recess. × 375. d: ACTH-like immunofluorescent neurons in the nucleus lobi lateralis hypothalami of Squalus acanthias. Cross-section. x 375. e: rostral lobe of the pituitary gland of Scyliorhinus with ACTH-positive cells mainly distributed in the anterior dorsal side. Sagittal section, x 150.
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b), with more rostral elements distributed in the subependymal layer and protruding a cerebrospinal fluid (CSF)-contacting process into the recess, with a swelling at its end (Fig. lc). Some immunoreactive processes were seen running ventrally towards the median eminence, but in this structure we found very little positive material. At a more caudal level immunoreactive cells appeared to be round in shape and more densely packed. In the nucleus lobi lateralis hypothalami ACTH-immunoreactive neurons were less numerous and scattered in its caudal part, within the subependymal layers (Fig. ld). In additional experiments, both the ACTH-immunoreactive nuclei were investigated for the presence of ~-MSH, using an antiserum against pure ~-MSH (UCB, Brussels, Belgium) previously employed to identify this peptide in the brain of reptiles [15] and amphibians [16]. In this case no immunoreaction was obtained. ACTH-immunopositive cells were also detected in the rostral lobe of the pituitary pars distalis (Fig. 1e), in agreement with previous cytological data [7] and biochemical studies revealing a corticotropin peptide with a high degree of sequence homology to human pituitary ACTH in these cells [9]. Our findings first establish that a substance, with immunological properties closely related to the pituitary ACTH, may be recognized in the hypothalamus of both Scyliorhinus and Squalus. Its distribution in neurons of the nucleus lateralis tuberis is in good agreement with that reported in the hypothalamus of reptiles [4] and mammals [13, 18], in which ACTH neurons were found in regions anatomically corresponding to this immunoreactive center, while a different pattern may be traced for positive fibers and terminals [12]. Its localization in the nucleus lobi lateralis hypothalami, instead, appears without any equivalent representation in tetrapods. In this study, ACTH-Iike material is confined within a well-defined neuronal system, located ventrally in the posterior hypothalamus to form intrinsic circuits and local connections with the median eminence, while in higher vertebrates ACTH is also involved in a wide extrahypothalamic fiber network with a role in adaptive behavior and cognitive function [8]. Concerning the presence of particular histological features of many ACTH-Iike reactive cells of the liquor-contacting type, it should be emphasized that these elements might be involved in intraventricular neurosecretory activities or in other functions related to the reception of ventricular information. In the first condition these cells might supply this peptide to different brain areas as well as to the neurointermediate lobe of the pituitary, while in the latter they act merely as sensory elements according to Vigh-Teichmann et al. [17]. A further question that arises from our data is the possibility of different biochemical processes for ACTH in the brain of these species, in comparison with related available data from reptiles and mammals [4, 11]. In fact, in the hypothalamus of these last groups ACTH is strongly thought to be derived from a 31,000 dalton proopiomelanocortin precursor, together with such peptides as ~-MSH, CLIP and pendorphin, by processes similar to those of the melanotrophic cells of the intermediate lobe. In our results, the lack of ~-MSH-like immunoreactive material within ACTH-positive centers suggests a resemblance to the biosynthetic pathway described
for this p e p t i d e in the c o r t i c o t r o p h cells o f the a n t e r i o r lobe o f the p i t u i t a r y o f b o t h fish a n d t e t r a p o d s [2, 6]. H o w e v e r , o u r results d o n o t c o m p l e t e l y clarify this interesting point. A d d i t i o n a l b i o c h e m i c a l investigations are n e e d e d to define the presence o f A C T H p r e c u r s o r m o l e c u l e s a n d related p r o c e s s i n g within these i m m u n o r e a c t i v e centers. In s u m m a r y , the i m m u n o h i s t o c h e m i c a l results p r e s e n t e d in this r e p o r t indicate that A C T H - I i k e m a t e r i a l m a y be d e t e c t e d in the b a s a l p o s t e r i o r h y p o t h a l a m u s in b o t h species o f e l a s m o b r a n c h e x a m i n e d . Its f u n c t i o n a l significance is u n k n o w n . W e s u p p o s e t h a t this p e p t i d e m a y exercise a role as n e u r o m o d u l a t o r a g e n t o r n e u r o t r a n s m i t t e r within the h y p o t h a l a m u s . Its l o c a l i z a t i o n in the nucleus lateralis tuberis, a m a s s o f cells a n a t o m i c a l l y closely related to the p i t u i t a r y , also suggests a p o s s i b l e i n v o l v e m e n t in h y p o p h y s e a l r e g u l a t i o n b y p e p t i d e r g i c fibers s u p p l y i n g b o t h the n e u r o i n t e r m e d i a t e lobe a n d the m e d i a n eminence. T h e finding o f A C T H - l i k e m a t e r i a l in this last structure s t r o n g l y s u p p o r t s this hypothesis. F i n a l l y , o u r d a t a p r o v i d e f u r t h e r f o u n d a t i o n to the i d e a t h a t substances related to p r o - o p i o m e l a n o c o r t i n - d e r i v e d peptides a r e p r e s e n t in the b r a i n o f all v e r t e b r a t e g r o u p s with specific b i o c h e m i c a l a n d t o p o g r a p h i c patterns. This w o r k was s u p p o r t e d b y g r a n t s f r o m M . P . I . (40 a n d 60%). The a u t h o r s w o u l d like to t h a n k Mr. R. M a r i n i for the p h o t o g r a p h y , a n d Mr. C. G r a t t a r o l a for e x p e r t technical assistance.
I Akil, H. and Watson, S.J., Beta-endorphin and biosynthetically related peptides in the central nervous system. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 16, Plenum, New York, 1983, pp. 209-253. 2 Chretien, M. and Seidah, N.G., Chemistry and biosynthesis of pro-opiomelanocortin, Mol. Cell. Biochem., 34 (1981) 101-127. 3 Coons, A.H., Leduc, E.H. and Conolly, J.M., Studies on antibody production. I. Method for the histochemical demonstration of specific antibody and its application to the study of the hyperimmune rabbit, J. Exp. Med., 102 (1955)49-59. 4 Dores, R.M., Khachaturian, H., Watson, S.J. and Akil, H., Localization of neurons containing proopiomelanocortin-relatedpeptides in the hypothalamus and midbrain of the lizard, Anolis carolinensis: evidence for region-specific offl-endorphin, Brain Res., 324 (1984) 384-389. 5 Eastman, J.T. and Portanova, R., ACTH activity in the pituitary and brain of the least brook lamprey, Lampetra aepyptera, Gen. Comp. Endocrinol., 47 (1982) 346-350. 6 Estivariz, F.E. and Iturizza, F.C., An investigation on pro-opiomelanocortin and processed peptides from the teleost fish Prochilodus platensis, Peptides, 6 (1985) 817-824. 7 Knowles, F., Volrath, L. and Meurling, P., Cytological and neuroendocrine relations of the pituitary of the dogfish, Scyliorhinus canicula, Proc. R. Soc. London Ser., B., 19l (1975) 507-525. 8 Krieger, D.T., Brain peptides: what, where, and why?, Science, 222 (1983) 975-985. 9 Lowry, P.J., Bennett, H.P.J., McMartin, C. and Scott, A.P., The isolation and amino acid sequence of adrenocorticotrophin from the pars distalis and an adrenocorticotrophin-likeintermediate lobe peptide from the neuro-intermediate lobe of the dogfish Squalus acanthias, Biochem. J., 141 (1974) 427 437. I0 Nozaki, M. and Gorbman, A., Distribution of immunoreactive sites for several components of proopiocortin in the pituitary and brain of adult lampreys, Petromyzon marinus and Entosphenus tridentatus, Gen. Comp. Endocrinol., 53 (1984) 335-352.
11 O'Donohue, T.L., The opiomelanotropinergic neuronal and endocrine system, Peptides, 3 (1981) 333344. 12 Pa|kovits, M., Topography of chemical identified neurons in the central nervous system: progress in 1981-1983. In E.E. Muller and M.R. MacLeod (Eds.), Neuroendocrine, Perspectives, Vol. 3, Elsevier, Amsterdam, 1984, pp. I ~ 9 . 13 Pelletier, G. and D6sy, L., Localization of ACTH in the human hypothalamus, Cell Tissue Res., 196 (1979) 525 530. 14 Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979, pp. 104-169. 15 Vallarino, M., Immunocytochemical localization of ~-melanocyte-stimulating hormone in the brain of the lizard, Lacerta muralis, Cell Tissue Res., 237 (1984) 521 524. 16 Vallarino, M., Immunocytochemical evidence for a-melanocyte-stimulating hormone in the hypothalamus of the frog Rana esculenta, Cell Tissue Res., in press. 17 Vigh-Teichmann, 1., Vigh, B., Rohlich, P. and Olsson, R., Phylogenetic aspects of the sensory neurons of the wall of the diencephalon. In L. Rakic and B.B. Mrsulja (Eds.), Circulatory and Developmental Aspects of Brain Metabolism, Plenum, New York, 1980, pp. 415~,28. 18 Watson, S.J., Richard, C.W. and Barkas, J.D., Adrenocorticotropin in rat brain: immunocytochemical localization in cells and axon, Science, 200 (1978) 1118-1182.
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NSL 04783
Neuronal localization of immunoreactive adrenocorticotropin-like substance in the hypothalamus of elasmobranch fishes Mauro Vallarino and Irene Ottonello Istituto di Anatomia Comparata, Universit~ di Genova, Genova (Italy)
(Received 30 March 1987; Revised version received 23 April 1987; Accepted 8 May 1987) Key words: Adrenocorticotropin (ACTH); Hypothalamus; Elasmobranch fish; Immunocytochemistry
The occurrence and localization of adrenocorticotropin (ACTH)-Iike material in the brain of two species of elasmobranch fishes, Scyliorhinus canicula and Squalus acanthias, were studied by immunohistochemical techniques using an antiserum generated against human ACTH~_24.In both species immunopositive neurons and fibers were recognized in the basal hypothalamus mainly distributed in the nucleus lateralis tuberis, in which many of these elements were of the liquor-contacting type. A few ACTH-Iike positive cells were also found in the nucleus lobi lateralis hypothalami. The features of this peptidergic system, its topographic distribution to form intrinsic circuits within the posterior hypothalamic area and probably connections with the pituitary, suggest implications in brain neuromodulatory activities and hypophyseal regulation for this peptide.
A d r e n o c o r t i c o t r o p i n ( A C T H ) is one o f a n u m b e r o f n e u r o a c t i v e p e p t i d e s which have been identified in the b r a i n o f several m a m m a l i a n species [1, 13, 18]. R a d i o i m m u n o l o g i c a l a n d i m m u n o c y t o c h e m i c a l studies have s h o w n its presence in the neurons o f the a r c u a t e nucleus a n d within a rich fiber n e t w o r k e x t e n d i n g to m a n y h y p o t h a l a m i c a n d e x t r a h y p o t h a l a m i c regions [12], where it seems to p l a y a role in n e u r o e n d o c r i n e a n d b e h a v i o r a l functions [8]. In lower vertebrates, b r a i n A C T H i m m u n o r e a c t i v i t y has o n l y been r e c o r d e d in reptiles, in two s e p a r a t e centers o f the h y p o t h a l a m u s a n d m i d b r a i n t h a t are also i m m u n o r e a c t i v e for o t h e r o p i o m e l a n o c o r t i n p e p t i d e s [4]. In c y c l o s t o m e s positive b i o a s s a y results o b t a i n e d by E a s t m a n a n d P o r t a n o v a [5] were n o t c o n f i r m e d by i m m u n o h i s t o c h e m i c a l p r o c e d u r e s [ 10]. In this s t u d y the presence o f A C T H was i m m u n o c y t o c h e m i c a l l y investigated in the b r a i n o f e l a s m o b r a n c h fishes utilizing an a n t i s e r u m g e n e r a t e d a g a i n s t h u m a n A C T H I 24. Twelve male a n d female Scyliorhinus canicula a n d Squalus acanthias c a p t u r e d in Correspondence." M. Vallarino, Istituto di Anatomia Comparata, Universit~ di Genova, Via Balbi 5, 1-16126 Genova, Italy.
0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd.
the Ligurian Sea (Western Mediterranean) were anesthetized by immersion in tricaine methasulphonate (MS 222, Sandoz, 100 mg/liter seawater) and perfused through the ventral aorta with cold 0.01 M phosphate-buffered saline (PBS), pH 7.4, and then with a solution of 4% paraformaldehyde in PBS and alternatively with Bouin's fluid. The brains, together with attached pituitaries, were dissected out and postfixed overnight in the respective fixation solution. Bouin-fixed brains were dehydrated through a graded ethanol series and embedded in paraffin. Serial 5/~m thick sections were cut in either the sagittal or transverse plane and mounted on glass slides. Paraformaldehyde-fixed brains were instead transferred to 20% sucrose in 0.01 M phosphate buffer (4°C) for 24-48 h, and then sectioned frozen at 20/~m. The sections were treated according to the indirect immunofluorescent method [3] or with the peroxidase-anti-peroxidase (PAP) method [14], as described in detail elsewhere [ 15]. The sections were incubated with (1) normal swine serum (0.7 mg/ml) at room temperature for 20 min, (2) anti-ACTH serum (UCB, Brussels, Belgium) diluted 1:400 with Triton X-100/PBS for 24 h at 4°C, (3) fluorescein isothiocyanate conjugated swine anti-rabbit g a m m a globulin (Dakopatts, Denmark) diluted 1:500, at room temperature for 1 h, and then examined through a Zeiss epifluorescent microscope. For the PAP method the sections reacted with (1) primary antiserum, (2) swine antiserum to rabbit g a m m a globulin (Dakopatts, Denmark) diluted 1:500 for 1 h, (3) peroxidase-rabbit anti-peroxidase complex solution (Dakopatts, Denmark) diluted 1:800, for 1 h. This reaction was accomplished with the free base of 3,Y-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, U.S.A.) containing 0.01% H202. Some sections were also counterstained with Cresyl violet. An absorption test, performed by incubating alternate sections in anti-ACTH serum that had been absorbed with excess ACTH1 24 (50 nmol/ml), prevented all staining for A C T H , while absorption with ct-MSH (UCB, Brussels, Belgium), fl-MSH (Sigma, St. Louis, U.S.A.) and fl-LPH (Sigma, St. Louis, U.S.A.) did not produce any decrease in the intensity of the staining. Both immunocytochemical procedures revealed ACTH-reactive neurons in the ventral hypothalamus of all the specimens examined. Stained cell bodies were confined in the nucleus lateralis tuberis and in the pars ventralis of the nucleus lobi lateralis hypothalami. The nucleus lateralis tuberis exhibited a great deal of immunopositive cells and fibers surrounding the ventral wall of the infundibular recess (Fig. la,
Fig. 1. a-c: ACTH-likePAP-immunoreactiveneurons and fibers in the nucleus lateralis tuberis of Scyliorhinus canicula. Positiveelementsare distributed, among non-reactivecells, within the subependymallayers surrounding the infundibular (I) cavity of the third ventricle. Arrows indicate positive processes coursing ventrally, a: sagittal section. In the ventral side are also present some immunopositivecells of the rostral lobe of the pituitary. × 150. b: high magnification of reactive neurons. × 375. c: positive neurons of the liquor-contacting type showing a swelling of ACTH-like material on the wall of the infundibular recess. × 375. d: ACTH-like immunofluorescent neurons in the nucleus lobi lateralis hypothalami of Squalus acanthias. Cross-section. x 375. e: rostral lobe of the pituitary gland of Scyliorhinus with ACTH-positive cells mainly distributed in the anterior dorsal side. Sagittal section, x 150.
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b), with more rostral elements distributed in the subependymal layer and protruding a cerebrospinal fluid (CSF)-contacting process into the recess, with a swelling at its end (Fig. lc). Some immunoreactive processes were seen running ventrally towards the median eminence, but in this structure we found very little positive material. At a more caudal level immunoreactive cells appeared to be round in shape and more densely packed. In the nucleus lobi lateralis hypothalami ACTH-immunoreactive neurons were less numerous and scattered in its caudal part, within the subependymal layers (Fig. ld). In additional experiments, both the ACTH-immunoreactive nuclei were investigated for the presence of ~-MSH, using an antiserum against pure ~-MSH (UCB, Brussels, Belgium) previously employed to identify this peptide in the brain of reptiles [15] and amphibians [16]. In this case no immunoreaction was obtained. ACTH-immunopositive cells were also detected in the rostral lobe of the pituitary pars distalis (Fig. 1e), in agreement with previous cytological data [7] and biochemical studies revealing a corticotropin peptide with a high degree of sequence homology to human pituitary ACTH in these cells [9]. Our findings first establish that a substance, with immunological properties closely related to the pituitary ACTH, may be recognized in the hypothalamus of both Scyliorhinus and Squalus. Its distribution in neurons of the nucleus lateralis tuberis is in good agreement with that reported in the hypothalamus of reptiles [4] and mammals [13, 18], in which ACTH neurons were found in regions anatomically corresponding to this immunoreactive center, while a different pattern may be traced for positive fibers and terminals [12]. Its localization in the nucleus lobi lateralis hypothalami, instead, appears without any equivalent representation in tetrapods. In this study, ACTH-Iike material is confined within a well-defined neuronal system, located ventrally in the posterior hypothalamus to form intrinsic circuits and local connections with the median eminence, while in higher vertebrates ACTH is also involved in a wide extrahypothalamic fiber network with a role in adaptive behavior and cognitive function [8]. Concerning the presence of particular histological features of many ACTH-Iike reactive cells of the liquor-contacting type, it should be emphasized that these elements might be involved in intraventricular neurosecretory activities or in other functions related to the reception of ventricular information. In the first condition these cells might supply this peptide to different brain areas as well as to the neurointermediate lobe of the pituitary, while in the latter they act merely as sensory elements according to Vigh-Teichmann et al. [17]. A further question that arises from our data is the possibility of different biochemical processes for ACTH in the brain of these species, in comparison with related available data from reptiles and mammals [4, 11]. In fact, in the hypothalamus of these last groups ACTH is strongly thought to be derived from a 31,000 dalton proopiomelanocortin precursor, together with such peptides as ~-MSH, CLIP and pendorphin, by processes similar to those of the melanotrophic cells of the intermediate lobe. In our results, the lack of ~-MSH-like immunoreactive material within ACTH-positive centers suggests a resemblance to the biosynthetic pathway described
for this p e p t i d e in the c o r t i c o t r o p h cells o f the a n t e r i o r lobe o f the p i t u i t a r y o f b o t h fish a n d t e t r a p o d s [2, 6]. H o w e v e r , o u r results d o n o t c o m p l e t e l y clarify this interesting point. A d d i t i o n a l b i o c h e m i c a l investigations are n e e d e d to define the presence o f A C T H p r e c u r s o r m o l e c u l e s a n d related p r o c e s s i n g within these i m m u n o r e a c t i v e centers. In s u m m a r y , the i m m u n o h i s t o c h e m i c a l results p r e s e n t e d in this r e p o r t indicate that A C T H - I i k e m a t e r i a l m a y be d e t e c t e d in the b a s a l p o s t e r i o r h y p o t h a l a m u s in b o t h species o f e l a s m o b r a n c h e x a m i n e d . Its f u n c t i o n a l significance is u n k n o w n . W e s u p p o s e t h a t this p e p t i d e m a y exercise a role as n e u r o m o d u l a t o r a g e n t o r n e u r o t r a n s m i t t e r within the h y p o t h a l a m u s . Its l o c a l i z a t i o n in the nucleus lateralis tuberis, a m a s s o f cells a n a t o m i c a l l y closely related to the p i t u i t a r y , also suggests a p o s s i b l e i n v o l v e m e n t in h y p o p h y s e a l r e g u l a t i o n b y p e p t i d e r g i c fibers s u p p l y i n g b o t h the n e u r o i n t e r m e d i a t e lobe a n d the m e d i a n eminence. T h e finding o f A C T H - l i k e m a t e r i a l in this last structure s t r o n g l y s u p p o r t s this hypothesis. F i n a l l y , o u r d a t a p r o v i d e f u r t h e r f o u n d a t i o n to the i d e a t h a t substances related to p r o - o p i o m e l a n o c o r t i n - d e r i v e d peptides a r e p r e s e n t in the b r a i n o f all v e r t e b r a t e g r o u p s with specific b i o c h e m i c a l a n d t o p o g r a p h i c patterns. This w o r k was s u p p o r t e d b y g r a n t s f r o m M . P . I . (40 a n d 60%). The a u t h o r s w o u l d like to t h a n k Mr. R. M a r i n i for the p h o t o g r a p h y , a n d Mr. C. G r a t t a r o l a for e x p e r t technical assistance.
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