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Co-localization of enkephalin and TRH in perifornical neurons of the rat hypothalamus that project to the lateral septum
Brain Research, 544 (1991) 177-180 ADONIS 000689939124573J
177
BRES 24573
Co-localization of enkephalin and TRH in perifornical neurons of the rat hypothalamus that project to the lateral septum Istvan Merchenthaler Functional Morphology Section, Laboratory of Molecular and Integrattve Neuroscience, NIEHS, NIH, Research Trtangle Park, NC 27709 (U.S.A.)
(Accepted 11 December 1990) Key words: Retrograde tracer; Double labeling; Immunocytochemlstry; Neuropeptide; Neuroanatomy; Opioid; Telencephalon
Neurons of the lateral septal nucleus are surrounded by terminals lmmunoreactwe for thyrotropin-releasing hormone (TRH) and enkephalin (Enk). Retrograde labeling from the lateral septum in combination with lmmunocytochemical analyses for Enk and TRH in colchicine-treated rats has revealed that nearly all Enk- (and TRH-) containing periformcal neurons project to the lateral septum. Immunostaining of adjacent, thin paraffin sections for either TRH or Enk and double staining of thick vibratome sections for the two pepndes have shown that TRH and Enk ]mmunoreactivlties co-exist within the same neurons in the periformcal region of the hypothalamus. Recent immunocytochemical studies have indicated that thyrotropin-releasing hormone- (TRH) ~1 and enkephalin- (Enk) 2° containing nerve terminals in the lateral septum arise from perikarya just below the fornix at the level of the paraventricular nucleus (PVN). The distribution of Enk-immunoreactive (Enk-i) and T R H immunoreactive (TRH-i) nerve terminals in the lateral septum and the distribution and the morphology of Enk-i and TRH-i perikarya around the fornix at the level of the PVN are similar 15-~7. The purpose of the present study was to determine whether T R H and Enk are co-localized in perifornical neurons. By using a combination of retrograde tracing from the lateral septum with wheat germ agglutinin-enzymatically inactivated horseradish peroxidase-protein colloidal gold ( W G A - H R P - A U ) and Enk and/or T R H immunocytochemistry or adjacent, thin paraffin sections, we report a nearly complete colocalization of the two peptides in the perifornical neurons nearly all of which project to the lateral septum. Six male Sprague-Dawley rats (C.D., Charles River, Raleigh, NC) were anesthetized with 2.5% tribromoethanol intraperitoneally (1.0 ml/100 g b.wt.). The heads of the animals were fixed in a stereotaxic instrument (David Koppf Instruments, Tujunga, CA), and 2 ~1 of 1% W G A - H R P - A U - c o m p l e x was injected into the lateral septum through a micropipette with tip diameter of 30-50/~m. The retrograde tracer was prepared according to the protocols of Men6trey 14 and Basbaum and Men6trey ].
Forty-eight hours later the animals were reanesthetized, and 5/A of 2% colchicine in saline was injected into the lateral ventricle as described 15. Twenty-four hours after the colchicine-treatment, the animals were perfused transcardially under deep anesthesia with 1% paraformaldehyde in 0.05 M phosphate buffer followed by 4% paraformaldehyde in the same buffer. The brains were removed, cut into smaller pieces and further immersed in the same fixative overnight. Two brains were embedded in paraffin, and 4 were cut on a vibratome. In order to visualize the retrograde tracer, each section at first was subjected to a silver-intensification procedure, using Intense BL (Janssen Life Science Products, Olen, Belgium). Following development, the sections were washed in 0.01 M phosphate buffered saline (PBS), and every 4th section was mounted on slides for the evaluation of retrograde labeling. Two adjacent 30 /~m vibratome sections were immunostained for Enk 19 or T R H ~6 with antisera raised in rabbits (anti-Leu-Enk no. 206 and anti-TRH no. 4/6). The antigens were detected by a modification of the peroxidase-antiperoxidase (PAP) technique is with 3,3'-diaminobenzidine (DAB) as chromogen. The 3rd section was simultaneously immunostained for T R H and Enk according to the double labeling technique of Joseph and Sternberger 12. Briefly, following blocking of nonspecific background staining with 2% normal sheep serum (NSS), the sections were incubated with the following immunoreagents: (i) primary antiserum (anti-TRH no. 4/6), dilution 1:4000, 4 °C
Correspondence" I Merchenthaler, MD C4-07, LMIN, NIEHS, NIH, P O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
178 for 24 h. (ii) Sheep antt-rabbit IgG (Arnel Products, Co., New York, NY), dilution 1:500, room temperature, 30 min. Off) Rabbit PAP (Arnel), dilution 1:1000, room temperature, 30 min. (iv) DAB (40 mg/100 ml 0.05 M Tris), room temperature, 10 min. Each step was followed by an appropriate wash in phosphate-buffered saline (PBS). The first immunostaining, representing TRH, was followed by a second immunostaining for Enk using the PAP technique for the second time. However, instead of the brown DAB reaction product, the blue chromogen, 4-chloro-l-naphthol (50 mg/100 ml Tris) was used. For details, see refs. 12 and 18. Co-localization of the two antigens was also examined on adjacent 5/~m paraffin sections using a monoclonal Enk antibody 6 and a polyclonal antiserum against TRH ~6, respectively. For technical details and critiques of different co-localization techniques see ref. 10. All sections were examined with a Zeiss Axiophot photomicroscope.
Each of the antlsera had already been tested for specificity6"~6'w. In addition, preabsorption of the TRH antiserum with Met- or Leu-Enk (up to 20/~g/ml working dilution of the primary antiserum) or preabsorption of the Enk antiserum with TRH (up to 20 gg/ml working dilution of the antiserum) did not affect the intensity or the distribution of the immunostainings. On the contrary, preabsorption of the primary antisera with 1 gg of the corresponding anttgens completely abolished the tmmunostaining. Within the hypothalamus, TRH-1 perikarya were seen in the preoptlc area, the sexually dimorphic area, the PVN, the lateral basal hypothalamus and the dorsal hypothalamlc area. The most intensely stained cell bodies were present in the parvocellular PVN (cf. refs. 11 and 16). These neurons merged with another population of lmmunoreactive cells encircling the fornix and arched through the anterior and lateral-basal hypothalamus.
• ,o
,~
,
0
Fig. 1 Immunocytochemlcailocalization of TRH (A,C) and enkephahn (Enk) (B,D) m two adjacent paraffin sections of cell bodies of the penfornical region of the lateral hypothalamus Identical labels indicate penkarya lmmunoreactwe for both peptides. Stars in Figs A and B label the same blood vessel. TRH- (E) and Enk-lmmunoreacuve (F) perikarya m the periformcal region of the hypothalamus accumulating the retrograde tracer WGA-HRP-AU rejected previously m the lateral septum The endogenous peptides are represented by DAB, the retrograde tracer by small salver grains Almost all of the TRH and/or Enk penkarya accumulate the retrograde tracer Arrowheads in E and F indicate a TRH and an Enk penkaryon that does not contain silver grains. Arrow in E labels two neurons that accumulate the retrograde tracer but are not immunoreactive for TRH. Magnifications (A-D) × 170, (C-D) × 240.
179 Perikarya immunoreactive for Enk were observed in the preoptic area, and the PVN, the ventromedial, dorsomedial, and arcuate nuclei. The most intensely stained cell bodies could be detected just below the fornix (cf. ref. 23). Both T R H and Enk were present in nerve terminals of, among other regions, the lateral septum apparently surrounding unlabeled perikarya. Using adjacent, thin paraffin sections or doublelabeling immunocytochemistry, we have found an almost complete overlap between T R H - and Enk-immunoreactive perikarya around the fornix (Fig. 1A-D). Following injection of the retrograde tracer WGAHRP-AU into the lateral septal nucleus, retrogradely labeled perikarya were present in the bed nucleus of the stria terminalis, the anterior commissural nucleus, the lateral basal hypothalamus, and the around the fornix at the level of the PVN (Fig. 1E,F). Double-labeled perikarya, however, were present only in the perifornical region (data not shown). In the present study we provided further support for the existence of a 'hypothalamo-septal' connection 11'21'22'24 by detecting W G A - H R P - A U in the perifornical neurons following injection of the retograde tracer into the lateral septum. Furthermore, we have supplied evidence for the co-expression of T R H and Enk in these perifornical neurons. These observations strongly suggest that nerve terminals in the lateral septum also contain at least two peptidergic transmitters, i.e., T R H and Enk. Whether Enk and T R H are packaged into the same secretory granules is not known. Electron microscopic immunocytochemical investigations will be necessary to resolve this question. Co-localization of T R H and Enk in perifornical neurons of the hypothalamus has been achieved by two immunocytochemical techniques. Staining of adjacent, thin paraffin sections for T R H and Enk provided clearcut evidence for the co-existence of the two peptides in the same neurons. The strength of this technique is that no interference between antisera can occur; thus, no problems of crossreactivity between the two target antigens are encountered.
Because of the fact that T R H - and Enk-containing perikarya are not densely packed in this region, this approach becomes impractical when trying to evaluate systematically the co-localization of these peptides. The double-labeling technique of Joseph and Sternberger in vibratome sections 12 has made possible the systematic search for co-localization throughout the hypothalamus. Although the physiological significance of co-localization of T R H and Enk in perifornical hypothalamic neurons is not known, the massive connections of septal neurons with the hippocampus, the hypothalamus and other regions of the limbic system suggest that Enk and T R H , by acting in a synergistic or an antagonistic manner, modify the activity of septal neurons which, in turn, can modulate the activity of neurons in the limbic system. However, the identity of the neurotransmitter(s) used by the majority of these septal neurons has not been determined. The septum as an important component of the limbic system is involved in a variety of physiological functions, such as circulation, respiration, emotional expression, water intake, ovulation, and A C T H release 7. When T R H was injected into the septum, it produced a prompt rise in body temperature 3 and reversed pentobarbital-induced sleep 4' 13,20 Enkephalin is considered to play a role in the integration and regulation of autonomic responses and several aspects of adenohypophysial hormone secretion 8'9. In addition, Enk, as a neurotransmitter or neuromodulator, plays an important role in the central modulation of nociception 2"5. It might also be of importance that the immunostaining for both T R H and Enk, but particularly for Enk, in the perifornical cell group is very intense, perhaps the most intense in the central nervous system. Whether this phenomenon is due to increased synthesis or decreased release needs further investigations. It also remains to be determined if and how the 'hypothalamo-septal Enk and T R H pathway' participates in some of the above physiological processes.
1 Basbaum, A.I. and Menttrey, D , Wheat germ agglutmlnapoHRP gold. A new retrograde tracer for light- and electronmicroscopic single- and double-label studies, J. Comp. Neurol, 261 (1987) 306-318. 2 Belluzl, J D., Grant, N., Garsky, V, Sarantakls, D , Wise, C.D. and Stein, L., Analgesia induced m wvo by central administration of enkephahn in rat, Nature, 260 (1976) 625-626 3 Bosch1, G. and Rips, R., Effects of thyrotropin releasing hormone inJection into different loci of rat brain on core temperature, Neursct Lett., 23 (1974) 267-269 4 Brunello, N and Cheney, D.L., The septal-hlppocampal cholinerglc pathway: role in antagonism of pentobarbital anesthesm and regulation of various afferents, J. Pharmacol Exp. Ther, 219 (1981) 489-495. 5 Buscher, H.H, Hill, R.C., Romer, D., Cardinaux, E, Closse,
A., Hauser, D. and Pless, J., Evidence for analgesic activity of enkephalin m the mouse, Nature, 261 (1976) 423-425 Cuello, A.C., Milstein, C, Couture, R., Wright, B., Prietsley, J V. and Jarvis, J., Characterization and immunocytochemical apphcation of monoclonal antibodies agamst enkephalins, J. Htstochem. Cytochem., 32 (1984) 947-957. DeFrance, J.E, The Septal Nuclet, Plenum, New York, 1976. Denavit-Sauble, M., Champagnat, J. and Zleglgansberger, W., Effects of opiates and methlonin-enkephalin on pontine and bulbar respiratory neurons in the cat, Brain Research, 155 (1978) 55-67 Dupont, A., Cusan, L., Labrie, E, Coy, D.H. and 1.,1, C.H., Stimulation of prolactin release in the rat by intraventricnlar Injection of beta-endorphin and methionme-enkephahn, Btochem. Btophys. Res. Commun., 75 (1977) 76-82.
The author is grateful to Drs. Peter Petrusz, Paul Ordronneau, and Jerome L Maderdrut for cntlcally evaluating the manuscript.
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180 10 Hokfelt, T , Holets, V.R., Staines, W. et ai, Coexistence of neuronal messengers - - an overview. In T. Hokfelt, K. Fuxe and B. Pernow (Eds), Co Extstence of Neuronal Messengers New Prmctples m Chemical Transmission, Progress m Brain Research, Vol. 68, 1986, pp. 33-70 11 Ishlkawa, K., Tamguchi Y., Kurosuml, K. and Suzuki, M , Ongm of septal thyrotropin-releasmg hormone on the rat, Neuroendocrmology, 44 (1986) 54-58 12 Joseph, S.A. and Sternberger, L.A., The unlabeled anUbody method. Contrasting color staining of beta-hpotropin and ACTH-associated hypothalamic peptldes without antibody removal, J Histochem. Cytochem., 27 (1979) 1430-1437 13 Kalivas, P.W. and Horita, A., Thyrotropm releasing hormone. neurogenesls of actions in the pentobarbltal narcotized rat, J Pharmacol Exp Ther, 212 (1980) 203-210 14 Men6trey, D , Retrograde tracing of neuronal pathways with a protein-gold complex I" Light microscopic detection after sdver intensification, Hlstochemistry, 83 (1985) 391-395. 15 Merchenthaler, I , Maderdrut, J.L., Altshuler, R.A and Petrusz, P., Immunocytochemlcal locahzaUon of proenkephalinderived peptides in the central nervous system of the rat, Neuroscwnce, 17 (1986) 325-348. 16 Merchenthaler, I., Csernus, V , Csontos, C., Petrusz, P and Mess, B., New data on the lmmunocytochemlcal locahzatlon of thyrotropm-releasing hormone in the rat central nervous system, A m J A n a t , 181 (1988) 359-376. 17 Merchenthaler, I., Meeker, M., Petrusz, E and Klzer, J . S , Identification and ~mmunocytochemlcal localization of a new TRH precursor in rat brain, Endocrinology, 124 (1989) 18881897 18 Merchenthaler, I , S~thl~), G , Csontos, C , Petrusz, P , Flerk6,
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B. and Negro-Vd~r, A., Combined retrograde tracing and ~mmunocytochemlcal identification of lutemtzmg hormone-releasing hormone- and somatostatm-containing neurons projecting to the median eminence of the rat, Endocrinology, 125 (1989) 2812-2821 Mdler, R J , Chang, K J., Cooper, B and Cuatrecacas, P., Rad~o~mmunoassay and characterization of enkephahn in rat tissues, J Btol Chem , 253 (1978) 531-538 Mlyamoto, M., Nagal, Y , Naruml, S. and Nagava, Y , TRH and ~st novel analog (DN-1417): Antlpentobarbital action and involvement of chohnerglc mechamsms, Pharmacol Btochem. Behav , 17 (1982) 797-806 OntEmente, B., Men6trey, D., Arai, R and Calas A , Origin of the met-enkephahnerglc mnervauon of the lateral septum in the rat, Cell Ttssue Res., 256 (1989) 585-592. Poulaln, P., Martm-Bouyer, L., Beauvllhan, J C and Tramu, G , Study of the efferent connections of the enkephalinerglc magnocellular dorsal nucleus m the guinea pig hypothalamus using lesions, retrograde tracing and immunocytochem~stry: evidence for a projection to the lateral septum, Neuroscwnce, 11 (1984) 331-343 Petrusz, P., Merchenthaler, I and Maderdrut, J L., DlstnbuUon of enkephahn-containmg neurons in the central nervous system In B. Bjorklund and T Hokfelt (Eds.), Handbook of Chemical Neuroanatomy Vol 4 G A B A and Neuropeptides in the CNS, Part 1, Elsevier, Amsterdam, 1985, pp 273-332 Sakanaka, M. and Magan, S , Reassessment of enkephahn (ENK)-contammg afferents to the rat lateral septum with reference to the fine structures of septal ENK fibers, Bram Research, 479 (1989) 205-216
177
BRES 24573
Co-localization of enkephalin and TRH in perifornical neurons of the rat hypothalamus that project to the lateral septum Istvan Merchenthaler Functional Morphology Section, Laboratory of Molecular and Integrattve Neuroscience, NIEHS, NIH, Research Trtangle Park, NC 27709 (U.S.A.)
(Accepted 11 December 1990) Key words: Retrograde tracer; Double labeling; Immunocytochemlstry; Neuropeptide; Neuroanatomy; Opioid; Telencephalon
Neurons of the lateral septal nucleus are surrounded by terminals lmmunoreactwe for thyrotropin-releasing hormone (TRH) and enkephalin (Enk). Retrograde labeling from the lateral septum in combination with lmmunocytochemical analyses for Enk and TRH in colchicine-treated rats has revealed that nearly all Enk- (and TRH-) containing periformcal neurons project to the lateral septum. Immunostaining of adjacent, thin paraffin sections for either TRH or Enk and double staining of thick vibratome sections for the two pepndes have shown that TRH and Enk ]mmunoreactivlties co-exist within the same neurons in the periformcal region of the hypothalamus. Recent immunocytochemical studies have indicated that thyrotropin-releasing hormone- (TRH) ~1 and enkephalin- (Enk) 2° containing nerve terminals in the lateral septum arise from perikarya just below the fornix at the level of the paraventricular nucleus (PVN). The distribution of Enk-immunoreactive (Enk-i) and T R H immunoreactive (TRH-i) nerve terminals in the lateral septum and the distribution and the morphology of Enk-i and TRH-i perikarya around the fornix at the level of the PVN are similar 15-~7. The purpose of the present study was to determine whether T R H and Enk are co-localized in perifornical neurons. By using a combination of retrograde tracing from the lateral septum with wheat germ agglutinin-enzymatically inactivated horseradish peroxidase-protein colloidal gold ( W G A - H R P - A U ) and Enk and/or T R H immunocytochemistry or adjacent, thin paraffin sections, we report a nearly complete colocalization of the two peptides in the perifornical neurons nearly all of which project to the lateral septum. Six male Sprague-Dawley rats (C.D., Charles River, Raleigh, NC) were anesthetized with 2.5% tribromoethanol intraperitoneally (1.0 ml/100 g b.wt.). The heads of the animals were fixed in a stereotaxic instrument (David Koppf Instruments, Tujunga, CA), and 2 ~1 of 1% W G A - H R P - A U - c o m p l e x was injected into the lateral septum through a micropipette with tip diameter of 30-50/~m. The retrograde tracer was prepared according to the protocols of Men6trey 14 and Basbaum and Men6trey ].
Forty-eight hours later the animals were reanesthetized, and 5/A of 2% colchicine in saline was injected into the lateral ventricle as described 15. Twenty-four hours after the colchicine-treatment, the animals were perfused transcardially under deep anesthesia with 1% paraformaldehyde in 0.05 M phosphate buffer followed by 4% paraformaldehyde in the same buffer. The brains were removed, cut into smaller pieces and further immersed in the same fixative overnight. Two brains were embedded in paraffin, and 4 were cut on a vibratome. In order to visualize the retrograde tracer, each section at first was subjected to a silver-intensification procedure, using Intense BL (Janssen Life Science Products, Olen, Belgium). Following development, the sections were washed in 0.01 M phosphate buffered saline (PBS), and every 4th section was mounted on slides for the evaluation of retrograde labeling. Two adjacent 30 /~m vibratome sections were immunostained for Enk 19 or T R H ~6 with antisera raised in rabbits (anti-Leu-Enk no. 206 and anti-TRH no. 4/6). The antigens were detected by a modification of the peroxidase-antiperoxidase (PAP) technique is with 3,3'-diaminobenzidine (DAB) as chromogen. The 3rd section was simultaneously immunostained for T R H and Enk according to the double labeling technique of Joseph and Sternberger 12. Briefly, following blocking of nonspecific background staining with 2% normal sheep serum (NSS), the sections were incubated with the following immunoreagents: (i) primary antiserum (anti-TRH no. 4/6), dilution 1:4000, 4 °C
Correspondence" I Merchenthaler, MD C4-07, LMIN, NIEHS, NIH, P O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
178 for 24 h. (ii) Sheep antt-rabbit IgG (Arnel Products, Co., New York, NY), dilution 1:500, room temperature, 30 min. Off) Rabbit PAP (Arnel), dilution 1:1000, room temperature, 30 min. (iv) DAB (40 mg/100 ml 0.05 M Tris), room temperature, 10 min. Each step was followed by an appropriate wash in phosphate-buffered saline (PBS). The first immunostaining, representing TRH, was followed by a second immunostaining for Enk using the PAP technique for the second time. However, instead of the brown DAB reaction product, the blue chromogen, 4-chloro-l-naphthol (50 mg/100 ml Tris) was used. For details, see refs. 12 and 18. Co-localization of the two antigens was also examined on adjacent 5/~m paraffin sections using a monoclonal Enk antibody 6 and a polyclonal antiserum against TRH ~6, respectively. For technical details and critiques of different co-localization techniques see ref. 10. All sections were examined with a Zeiss Axiophot photomicroscope.
Each of the antlsera had already been tested for specificity6"~6'w. In addition, preabsorption of the TRH antiserum with Met- or Leu-Enk (up to 20/~g/ml working dilution of the primary antiserum) or preabsorption of the Enk antiserum with TRH (up to 20 gg/ml working dilution of the antiserum) did not affect the intensity or the distribution of the immunostainings. On the contrary, preabsorption of the primary antisera with 1 gg of the corresponding anttgens completely abolished the tmmunostaining. Within the hypothalamus, TRH-1 perikarya were seen in the preoptlc area, the sexually dimorphic area, the PVN, the lateral basal hypothalamus and the dorsal hypothalamlc area. The most intensely stained cell bodies were present in the parvocellular PVN (cf. refs. 11 and 16). These neurons merged with another population of lmmunoreactive cells encircling the fornix and arched through the anterior and lateral-basal hypothalamus.
• ,o
,~
,
0
Fig. 1 Immunocytochemlcailocalization of TRH (A,C) and enkephahn (Enk) (B,D) m two adjacent paraffin sections of cell bodies of the penfornical region of the lateral hypothalamus Identical labels indicate penkarya lmmunoreactwe for both peptides. Stars in Figs A and B label the same blood vessel. TRH- (E) and Enk-lmmunoreacuve (F) perikarya m the periformcal region of the hypothalamus accumulating the retrograde tracer WGA-HRP-AU rejected previously m the lateral septum The endogenous peptides are represented by DAB, the retrograde tracer by small salver grains Almost all of the TRH and/or Enk penkarya accumulate the retrograde tracer Arrowheads in E and F indicate a TRH and an Enk penkaryon that does not contain silver grains. Arrow in E labels two neurons that accumulate the retrograde tracer but are not immunoreactive for TRH. Magnifications (A-D) × 170, (C-D) × 240.
179 Perikarya immunoreactive for Enk were observed in the preoptic area, and the PVN, the ventromedial, dorsomedial, and arcuate nuclei. The most intensely stained cell bodies could be detected just below the fornix (cf. ref. 23). Both T R H and Enk were present in nerve terminals of, among other regions, the lateral septum apparently surrounding unlabeled perikarya. Using adjacent, thin paraffin sections or doublelabeling immunocytochemistry, we have found an almost complete overlap between T R H - and Enk-immunoreactive perikarya around the fornix (Fig. 1A-D). Following injection of the retrograde tracer WGAHRP-AU into the lateral septal nucleus, retrogradely labeled perikarya were present in the bed nucleus of the stria terminalis, the anterior commissural nucleus, the lateral basal hypothalamus, and the around the fornix at the level of the PVN (Fig. 1E,F). Double-labeled perikarya, however, were present only in the perifornical region (data not shown). In the present study we provided further support for the existence of a 'hypothalamo-septal' connection 11'21'22'24 by detecting W G A - H R P - A U in the perifornical neurons following injection of the retograde tracer into the lateral septum. Furthermore, we have supplied evidence for the co-expression of T R H and Enk in these perifornical neurons. These observations strongly suggest that nerve terminals in the lateral septum also contain at least two peptidergic transmitters, i.e., T R H and Enk. Whether Enk and T R H are packaged into the same secretory granules is not known. Electron microscopic immunocytochemical investigations will be necessary to resolve this question. Co-localization of T R H and Enk in perifornical neurons of the hypothalamus has been achieved by two immunocytochemical techniques. Staining of adjacent, thin paraffin sections for T R H and Enk provided clearcut evidence for the co-existence of the two peptides in the same neurons. The strength of this technique is that no interference between antisera can occur; thus, no problems of crossreactivity between the two target antigens are encountered.
Because of the fact that T R H - and Enk-containing perikarya are not densely packed in this region, this approach becomes impractical when trying to evaluate systematically the co-localization of these peptides. The double-labeling technique of Joseph and Sternberger in vibratome sections 12 has made possible the systematic search for co-localization throughout the hypothalamus. Although the physiological significance of co-localization of T R H and Enk in perifornical hypothalamic neurons is not known, the massive connections of septal neurons with the hippocampus, the hypothalamus and other regions of the limbic system suggest that Enk and T R H , by acting in a synergistic or an antagonistic manner, modify the activity of septal neurons which, in turn, can modulate the activity of neurons in the limbic system. However, the identity of the neurotransmitter(s) used by the majority of these septal neurons has not been determined. The septum as an important component of the limbic system is involved in a variety of physiological functions, such as circulation, respiration, emotional expression, water intake, ovulation, and A C T H release 7. When T R H was injected into the septum, it produced a prompt rise in body temperature 3 and reversed pentobarbital-induced sleep 4' 13,20 Enkephalin is considered to play a role in the integration and regulation of autonomic responses and several aspects of adenohypophysial hormone secretion 8'9. In addition, Enk, as a neurotransmitter or neuromodulator, plays an important role in the central modulation of nociception 2"5. It might also be of importance that the immunostaining for both T R H and Enk, but particularly for Enk, in the perifornical cell group is very intense, perhaps the most intense in the central nervous system. Whether this phenomenon is due to increased synthesis or decreased release needs further investigations. It also remains to be determined if and how the 'hypothalamo-septal Enk and T R H pathway' participates in some of the above physiological processes.
1 Basbaum, A.I. and Menttrey, D , Wheat germ agglutmlnapoHRP gold. A new retrograde tracer for light- and electronmicroscopic single- and double-label studies, J. Comp. Neurol, 261 (1987) 306-318. 2 Belluzl, J D., Grant, N., Garsky, V, Sarantakls, D , Wise, C.D. and Stein, L., Analgesia induced m wvo by central administration of enkephahn in rat, Nature, 260 (1976) 625-626 3 Bosch1, G. and Rips, R., Effects of thyrotropin releasing hormone inJection into different loci of rat brain on core temperature, Neursct Lett., 23 (1974) 267-269 4 Brunello, N and Cheney, D.L., The septal-hlppocampal cholinerglc pathway: role in antagonism of pentobarbital anesthesm and regulation of various afferents, J. Pharmacol Exp. Ther, 219 (1981) 489-495. 5 Buscher, H.H, Hill, R.C., Romer, D., Cardinaux, E, Closse,
A., Hauser, D. and Pless, J., Evidence for analgesic activity of enkephalin m the mouse, Nature, 261 (1976) 423-425 Cuello, A.C., Milstein, C, Couture, R., Wright, B., Prietsley, J V. and Jarvis, J., Characterization and immunocytochemical apphcation of monoclonal antibodies agamst enkephalins, J. Htstochem. Cytochem., 32 (1984) 947-957. DeFrance, J.E, The Septal Nuclet, Plenum, New York, 1976. Denavit-Sauble, M., Champagnat, J. and Zleglgansberger, W., Effects of opiates and methlonin-enkephalin on pontine and bulbar respiratory neurons in the cat, Brain Research, 155 (1978) 55-67 Dupont, A., Cusan, L., Labrie, E, Coy, D.H. and 1.,1, C.H., Stimulation of prolactin release in the rat by intraventricnlar Injection of beta-endorphin and methionme-enkephahn, Btochem. Btophys. Res. Commun., 75 (1977) 76-82.
The author is grateful to Drs. Peter Petrusz, Paul Ordronneau, and Jerome L Maderdrut for cntlcally evaluating the manuscript.
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