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Corticotropin-releasing factor mRNA is expressed in the inferior olives of rodents and primates
189
Molecular Brain Research, 1(1986) 189-192 Elsevier BRM 80004
Corticotropin-releasing factor mRNA is expressed in the inferior olives of rodents and primates W. SCOTT YOUNG
III’, LARY C. WALKER3,
RICHARD
E. POWERS3,
ERROL
B. DE SOUZA’ and DONALD L. PRICE3mS
‘National Institute of Drug Abuse, ‘Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.), Addiction Research Center, Neuroscience Branch, Baltimore, MD 21205 (U.S.A.) and Departments of ‘Pathology, “Neurology, and ‘Neuroscience, Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Accepted 1 July 1986) Key words: Corticotropin-releasing
factor-Inferior
olive -Cerebellum
- mRNA - In situ hybridization
Immunohistochemical studies have suggested that corticotropin-releasing factor (CRF) was a transmitter of the olivocerebellar projection. We used in situ hybridization histochemistry with a 3SS-labeled oligodeoxyribonucleotide probe for CRF mRNA to show that inferior olivary neurons of rats, baboons, and humans synthesize CRF.
The inferior olive (IO), a precerebellar relay nucleus, is the principal source of climbing fibers in cerebellar cortex6, an input with profound influence on the physiological activity of Purkinje cells5. The IO appears to play a key role in influencing movements, and dysfunction of the olivocerebellar pathway is implicated in a variety of movement disorderslO. Despite extensive information concerning the connections of the IO’, relatively little is known about the transmitters used by its neurons, particularly in primates. Complicating attempts to identify putative peptide transmitters in neurons of the IO is the fact that it is difficult to visualize some neuropeptides in neuronal perikarya immunohistochemically without pretreatment with colchicine. In situ hybridization histochemistry (ISHH) circumvents this difficulty by using radiolabeled probes to detect neurons which transcribe mRNA coding for particular neuropeptides. Since corticotropin-releasing factor (CRF), a 41 amino acid peptide 16,20,is present in rat IO neurons4”’ and has been identified recently as a possible neurotransmitter in olivocerebellar projections of the cat4 we used a “S-labeled synthetic oligodeoxyribonucleotide probe and ISHH to demonstrate CRF mRNA in the 10s of rats, baboons, and humans.
Five male Sprague-Dawley rats (NIH, 175-225 g) were given water ad libitum and maintained on a 12 h on, 12 h off lighting schedule. They were anesthetized with ether and their brains were perfused with formaldehyde as previously described23. Three baboons (genus Pupio), aged 174 gestational days (near term), 2.3 years, and approximately 2.5 years, were sacrificed by an overdose of sodium pentobarbital, and the brains were immediately removed. The rostral medullas of the 2.3-year-old and near-term baboons were immediately frozen on dry ice; tissues from the 2.5year-old baboon were placed in 25% sucrose at 4 “C for 3 days and then frozen. For the human studies, brains were obtained from 3 individuals without evidence of neurological disease (ages 41, 56, and 66 years with postmortem intervals of < 10 h); the medullas were dissected and frozen on dry ice. Frozen sections (12 pm) were cut and primate sections were postfixed in 4% formaldehyde for 5 min at room temperature. All sections were then hybridized with the CRF probe as described previously23, except that the temperature of the hybridization was 37 “C. After hybridization, sections were rinsed in 2 x SSC/50% formamide 4 times for 15 min at 40 “C
Correspondence: W.S. Young, III, Building 36, Room 3A-17, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD20892, U.S.A.
Fig. 1. CRF mRNA-containing neurons in rat (A), baboon (B. E), and human (C, F) inferior olives. A is a dark-field photomicrograph of an autoradiogram over rat inferior olive after hybridization with 35S-labeled CRF probe and 2 week exposure of emulsion-coated coverslip. Label (white grains) is found over the principal (p) and dorsal (d) and medial (m) accessory divisions of the IO. The dotted line denotes the midline and py is the pyramid. B and E show bright-field and dark-field photomicrographs, respectively. of baboon IO neurons containing CRF mRNA. C and Fare of human IO. Arrowheads indicate 4 of the labeled neurons. D shows only background labeling over hypoglossal neurons from same section as shown in C and F. Baboon and human autoradiographic exposures were 1 month. Bars equal 20Opm (A, E, F) or 50pm (B-D).
191 (20 “C below the theoretical melting temperature in the presence of formamide for rat, 14 “C below for human2s1’). Slides were rinsed twice for 1 h each at room temperature in 2 x SSC, dipped twice briefly into water to remove salts, and dried before they were dipped into NTB3 nuclear emulsion (1:l with water; Kodak) or apposed to emulsion-coated coverslips 22. After exposure, slides were developed, sections were stained with cresyl violet.
and
A 48 base synthetic oligodeoxyribonucleotide probe, complementary to the coding region for amino acids 22-37 of rat CRF9, was made on an Applied Biosystems DNA synthesizer (courtesy, Dr. M. Brownstein, NIMH). The probe had 94% base sequence homology with the corresponding human sequence18; the homology with baboon CRF is unknown. The probe was purified on a preparative sequencing gel apparatus with 8% polyacrylamide and 8 M urea and labeled on the 3’ end using terminal deoxynucleotidyl transferase (Bethesda Research Labs) and [35S]deoxyadenosine triphosphate (NEN, > 1000 Ci/mmol). In the rats, CRF mRNA was demonstrated in neurons in the principal and medial and dorsal accessory divisions of the IO* using the 35S-CRF probe (Fig. 1A). Similarly, in the tissues from 3 baboons and 3 humans, all the large neurons studied within the principal and medial and dorsal accessory IO’ were labeled by in situ hybridization (Fig. lB, C, E and F). Non-olivary neurons in these sections were not labeled with the probe (Fig. 1D). Specific labeling above background was not observed when a vasopressin probe of identical length and comparable base composition was used (data not shown). Although Several previous immunocytochemical studies did not demonstrate CRF-like immunoreactivity in IO neurons3.12.14.‘9, several lines of evidence suggest that CRF is a neurotransmitter synthesized in IO neurons and released from cerebellar climbing fibers. Retrograde tracers injected into the cerebellum appeared in IO neurons which were stained with CRF antisera4. Moreover, our recent immunocytochemical investigations of humans have identified CRF immunoreactivity in perikarya and fibers of IO
neurons15. In addition, CRF receptors have been demonstrated in the cerebellar cortex of primates13,15 and rodents7,21. The present study indicates that CRF mRNA is transcribed in IO neurons for translation to preproCRF. The results of this ISHH study depend on the specificity of our probe for CRF mRNA. Three lines of evidence suggest that this probe labeled CRF neurons. First, the ISHH23 and immunocytochemica13,‘2,14,17,19 studies showed parallel distributions of CRF mRNA and CRF, respectively, in the IO and hypothalamic paraventricular and supraoptic nuclei. Second, a control probe (vasopressin) did not label IO neurons, an observation which argues against non-specific labeling by the CRF probe. Third, using the CRF probe, Northern blot analysis of total RNA from rat paraventricular and supraoptic nuclei demonstrated only a single band of 1.3 kb9,23. The present investigation has implications for future studies of neurological diseases. Several degenerative diseases associated with disorders of movement, e.g., the olivopontocerebellar atrophies” involve the olivocerebellar system. Investigations of the biology of CRF neurons may serve to clarify aspects of these disorders, and studies of CRF receptors in the cerebellum may provide new information about the regulation of peptidergic receptors in these diseases. Finally, agents designed to act at CRF receptor sites may ameliorate some of the symptoms occurring in patients with disorders of the olivocerebellar system.
The authors thank Drs. Michael J. Brownstein and Miklos Palkovits for thoughtful advice, Mrs. M. Warden and Ms. J. Park for technical help, Mrs. P. Thurston for secretarial assistance, and Mr. Larry Ostby for photographic aid. This work was supported by grants from the U.S. Public Health Service (NIH NS 20471, AG 05146, AG 03359, and NS 07179) as well as funds from the Robert L. and Clara G. Patterson Trust. W.S.Y. was a Medical Staff Fellow of the Pharmacology Associates Training Program, NIGMS.
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8
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Brodal. A., Neurological Anatomy in Relation IO Clinical Medicine, 3rd edn., Oxford University Press. New York, 1981, pp. 329-343. Casey, J. and Davidson, N., Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res., 4 (1977) 1539-1552. Cummings, S., Elde, R., Ells, J. and Lindall, A.. Corticotropin-releasing factor immunoreactivity is widely distributed within the central nervous system of the rat: an imstudy. J. Neurosci., 3 (1983) munohistochemical 1355-1368. Cummings, S.. Elde, R. and Sharp, B., CRF-immunoreactive neurons within the inferior olive project to the flocculus and dorsal and ventral paraflocculi, Sot. Neurosci. Abstr., 11 (1985) 683. Demer. J.L., Echelman, D.A. and Robinson, D.A.. Effects of electrical stimulation and reversible lesions of the olivocerebellar pathway on Purkinje cell activity in the flocculus of the cat, Brain Res., 346 (1985) 22-3 I. Desclin, J.C., Histological evidence supporting the inferior olive as the major source of cerebellar climbing fibers in rat, Brain Res., 77 (1974) 365-384. DeSouza, E.B.. Insel, T.R., Perrin, M.H., Rivier. J., Vale, W.W. and Kuhar, M.J., Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study, J. Neurosci., 5 (1985) 3189-3203. Gwyn. D.G., Nicholson, G.P. and Flumerfelt, B.A.. The inferior olivary nucleus of the rat: a light and electron microscopic study, J. Comp. Neural., 174 (1977) 489-520. Jingami, H., Mizuno, N.. Takahashi. H.. Shibahara. S.. Furutani. Y., Imura. H. and Numa, S., Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor. FEBS Left., 192 (1985) 63-66. Koeppen. A.H. and Barron, K.D.. The neurophathology of olivopontocerebellar atrophy. In R.C. Duvoisin and A. Plaitakis (Eds.), The Olivopontocerebellar Atrophies, Raven Press, New York. 1984, pp. 13-38. Lathe. R.. Synaptic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J. Mol. Biol., 183 (1985) I - 12. Merchenthaler. I.. Vigh, S.. Petrusz, P. and Schally, A.V.. Immunochemical localization of corticotropin releasing factor (CRF) in the rat brain, Am. J. Anat.. 165 (1982) 385396.
I3 Millan, M.A., Jacobowitz, D.M., Hauger, R.L., Catt, K.J. and Aguilera, G., Distribution of corticotropin-releasing factor receptors in primate brain, Proc. Natl. Acad. Sci. L’.S.A., 83 (1986) 1921-1925. I4 Olschowka, J.A.. O’Donohue, T.L.. Mueller, G.P. and Jacobowitz, D.M.. The distribution of corticotropin-releasing factor-like immunoreactive neurons in rat brain, Peptides, 3 (1982) 995-1015. I5 Powers, R.E., DeSouza. E.B.. Walker. L.C.. Vale. W.W.. Price, D.L. and Young, W.S.. III, Corticotropin-releasing factor as a transmitter of inferior olivary neurons, Sot. Neurosci. Abstr., 12 (1986) 568. 16 Rivier, J., Spiess. J. and Vale. W., Characterization of rat hypothalamic corticotropin-releasing factor, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4851-4855. I7 Schipper, J., Steinbusch, H.W.M., Vermes, I. and Tilders, F.J.H.. Mapping of CRF-immunoreactive nerve fibers in the medulla oblongata and spinal cord of the rat, Brain Res., 267 (1983) 145-150. 18 Shibahara, S., Morimoto. Y., Furutani, Y.. Notake. M., Takahashi. H., Shimizu, H., Horikawa, S. and Numa. S., Isolation and sequence analysis of the human corticotropinreleasing factor precursor gene. EMBO J., 2 (1983) 775-779. IY Swanson. L.W., Sawchenko, P.E.. Rivier, J. and Vale. W. W., Organization of ovine cortocotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology, 36 (1983) 165-186. 20 Vale. W.. Spiess. J.. Rivier. C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and P-endorphin. Science, 213 (1981) 1394-1397. 21 Wynn, P.C., Hauger. R.L., Holmes. M.C.. Millan. M.A.. Catt. K.J. and Aquilcra. G.. Brain and pituitary receptors for corticotropin releasing factor: localization and differcntial regulation after adrenalectomy. Peptides, 5 (1984) 1077- 1084. 22 Young. W.S. III and Kuhar. M.J.. A new method for receptor autoradiography: (jH) opioid receptors in rat brain. Brain Res.. 179 (1979) 255-270. 23 Young, W.S. 111, Mezey. E. and Siegel, R.E.. Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats, Neurosci. Lrtt., in press.
Molecular Brain Research, 1(1986) 189-192 Elsevier BRM 80004
Corticotropin-releasing factor mRNA is expressed in the inferior olives of rodents and primates W. SCOTT YOUNG
III’, LARY C. WALKER3,
RICHARD
E. POWERS3,
ERROL
B. DE SOUZA’ and DONALD L. PRICE3mS
‘National Institute of Drug Abuse, ‘Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.), Addiction Research Center, Neuroscience Branch, Baltimore, MD 21205 (U.S.A.) and Departments of ‘Pathology, “Neurology, and ‘Neuroscience, Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Accepted 1 July 1986) Key words: Corticotropin-releasing
factor-Inferior
olive -Cerebellum
- mRNA - In situ hybridization
Immunohistochemical studies have suggested that corticotropin-releasing factor (CRF) was a transmitter of the olivocerebellar projection. We used in situ hybridization histochemistry with a 3SS-labeled oligodeoxyribonucleotide probe for CRF mRNA to show that inferior olivary neurons of rats, baboons, and humans synthesize CRF.
The inferior olive (IO), a precerebellar relay nucleus, is the principal source of climbing fibers in cerebellar cortex6, an input with profound influence on the physiological activity of Purkinje cells5. The IO appears to play a key role in influencing movements, and dysfunction of the olivocerebellar pathway is implicated in a variety of movement disorderslO. Despite extensive information concerning the connections of the IO’, relatively little is known about the transmitters used by its neurons, particularly in primates. Complicating attempts to identify putative peptide transmitters in neurons of the IO is the fact that it is difficult to visualize some neuropeptides in neuronal perikarya immunohistochemically without pretreatment with colchicine. In situ hybridization histochemistry (ISHH) circumvents this difficulty by using radiolabeled probes to detect neurons which transcribe mRNA coding for particular neuropeptides. Since corticotropin-releasing factor (CRF), a 41 amino acid peptide 16,20,is present in rat IO neurons4”’ and has been identified recently as a possible neurotransmitter in olivocerebellar projections of the cat4 we used a “S-labeled synthetic oligodeoxyribonucleotide probe and ISHH to demonstrate CRF mRNA in the 10s of rats, baboons, and humans.
Five male Sprague-Dawley rats (NIH, 175-225 g) were given water ad libitum and maintained on a 12 h on, 12 h off lighting schedule. They were anesthetized with ether and their brains were perfused with formaldehyde as previously described23. Three baboons (genus Pupio), aged 174 gestational days (near term), 2.3 years, and approximately 2.5 years, were sacrificed by an overdose of sodium pentobarbital, and the brains were immediately removed. The rostral medullas of the 2.3-year-old and near-term baboons were immediately frozen on dry ice; tissues from the 2.5year-old baboon were placed in 25% sucrose at 4 “C for 3 days and then frozen. For the human studies, brains were obtained from 3 individuals without evidence of neurological disease (ages 41, 56, and 66 years with postmortem intervals of < 10 h); the medullas were dissected and frozen on dry ice. Frozen sections (12 pm) were cut and primate sections were postfixed in 4% formaldehyde for 5 min at room temperature. All sections were then hybridized with the CRF probe as described previously23, except that the temperature of the hybridization was 37 “C. After hybridization, sections were rinsed in 2 x SSC/50% formamide 4 times for 15 min at 40 “C
Correspondence: W.S. Young, III, Building 36, Room 3A-17, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD20892, U.S.A.
Fig. 1. CRF mRNA-containing neurons in rat (A), baboon (B. E), and human (C, F) inferior olives. A is a dark-field photomicrograph of an autoradiogram over rat inferior olive after hybridization with 35S-labeled CRF probe and 2 week exposure of emulsion-coated coverslip. Label (white grains) is found over the principal (p) and dorsal (d) and medial (m) accessory divisions of the IO. The dotted line denotes the midline and py is the pyramid. B and E show bright-field and dark-field photomicrographs, respectively. of baboon IO neurons containing CRF mRNA. C and Fare of human IO. Arrowheads indicate 4 of the labeled neurons. D shows only background labeling over hypoglossal neurons from same section as shown in C and F. Baboon and human autoradiographic exposures were 1 month. Bars equal 20Opm (A, E, F) or 50pm (B-D).
191 (20 “C below the theoretical melting temperature in the presence of formamide for rat, 14 “C below for human2s1’). Slides were rinsed twice for 1 h each at room temperature in 2 x SSC, dipped twice briefly into water to remove salts, and dried before they were dipped into NTB3 nuclear emulsion (1:l with water; Kodak) or apposed to emulsion-coated coverslips 22. After exposure, slides were developed, sections were stained with cresyl violet.
and
A 48 base synthetic oligodeoxyribonucleotide probe, complementary to the coding region for amino acids 22-37 of rat CRF9, was made on an Applied Biosystems DNA synthesizer (courtesy, Dr. M. Brownstein, NIMH). The probe had 94% base sequence homology with the corresponding human sequence18; the homology with baboon CRF is unknown. The probe was purified on a preparative sequencing gel apparatus with 8% polyacrylamide and 8 M urea and labeled on the 3’ end using terminal deoxynucleotidyl transferase (Bethesda Research Labs) and [35S]deoxyadenosine triphosphate (NEN, > 1000 Ci/mmol). In the rats, CRF mRNA was demonstrated in neurons in the principal and medial and dorsal accessory divisions of the IO* using the 35S-CRF probe (Fig. 1A). Similarly, in the tissues from 3 baboons and 3 humans, all the large neurons studied within the principal and medial and dorsal accessory IO’ were labeled by in situ hybridization (Fig. lB, C, E and F). Non-olivary neurons in these sections were not labeled with the probe (Fig. 1D). Specific labeling above background was not observed when a vasopressin probe of identical length and comparable base composition was used (data not shown). Although Several previous immunocytochemical studies did not demonstrate CRF-like immunoreactivity in IO neurons3.12.14.‘9, several lines of evidence suggest that CRF is a neurotransmitter synthesized in IO neurons and released from cerebellar climbing fibers. Retrograde tracers injected into the cerebellum appeared in IO neurons which were stained with CRF antisera4. Moreover, our recent immunocytochemical investigations of humans have identified CRF immunoreactivity in perikarya and fibers of IO
neurons15. In addition, CRF receptors have been demonstrated in the cerebellar cortex of primates13,15 and rodents7,21. The present study indicates that CRF mRNA is transcribed in IO neurons for translation to preproCRF. The results of this ISHH study depend on the specificity of our probe for CRF mRNA. Three lines of evidence suggest that this probe labeled CRF neurons. First, the ISHH23 and immunocytochemica13,‘2,14,17,19 studies showed parallel distributions of CRF mRNA and CRF, respectively, in the IO and hypothalamic paraventricular and supraoptic nuclei. Second, a control probe (vasopressin) did not label IO neurons, an observation which argues against non-specific labeling by the CRF probe. Third, using the CRF probe, Northern blot analysis of total RNA from rat paraventricular and supraoptic nuclei demonstrated only a single band of 1.3 kb9,23. The present investigation has implications for future studies of neurological diseases. Several degenerative diseases associated with disorders of movement, e.g., the olivopontocerebellar atrophies” involve the olivocerebellar system. Investigations of the biology of CRF neurons may serve to clarify aspects of these disorders, and studies of CRF receptors in the cerebellum may provide new information about the regulation of peptidergic receptors in these diseases. Finally, agents designed to act at CRF receptor sites may ameliorate some of the symptoms occurring in patients with disorders of the olivocerebellar system.
The authors thank Drs. Michael J. Brownstein and Miklos Palkovits for thoughtful advice, Mrs. M. Warden and Ms. J. Park for technical help, Mrs. P. Thurston for secretarial assistance, and Mr. Larry Ostby for photographic aid. This work was supported by grants from the U.S. Public Health Service (NIH NS 20471, AG 05146, AG 03359, and NS 07179) as well as funds from the Robert L. and Clara G. Patterson Trust. W.S.Y. was a Medical Staff Fellow of the Pharmacology Associates Training Program, NIGMS.
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Brodal. A., Neurological Anatomy in Relation IO Clinical Medicine, 3rd edn., Oxford University Press. New York, 1981, pp. 329-343. Casey, J. and Davidson, N., Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res., 4 (1977) 1539-1552. Cummings, S., Elde, R., Ells, J. and Lindall, A.. Corticotropin-releasing factor immunoreactivity is widely distributed within the central nervous system of the rat: an imstudy. J. Neurosci., 3 (1983) munohistochemical 1355-1368. Cummings, S.. Elde, R. and Sharp, B., CRF-immunoreactive neurons within the inferior olive project to the flocculus and dorsal and ventral paraflocculi, Sot. Neurosci. Abstr., 11 (1985) 683. Demer. J.L., Echelman, D.A. and Robinson, D.A.. Effects of electrical stimulation and reversible lesions of the olivocerebellar pathway on Purkinje cell activity in the flocculus of the cat, Brain Res., 346 (1985) 22-3 I. Desclin, J.C., Histological evidence supporting the inferior olive as the major source of cerebellar climbing fibers in rat, Brain Res., 77 (1974) 365-384. DeSouza, E.B.. Insel, T.R., Perrin, M.H., Rivier. J., Vale, W.W. and Kuhar, M.J., Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study, J. Neurosci., 5 (1985) 3189-3203. Gwyn. D.G., Nicholson, G.P. and Flumerfelt, B.A.. The inferior olivary nucleus of the rat: a light and electron microscopic study, J. Comp. Neural., 174 (1977) 489-520. Jingami, H., Mizuno, N.. Takahashi. H.. Shibahara. S.. Furutani. Y., Imura. H. and Numa, S., Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor. FEBS Left., 192 (1985) 63-66. Koeppen. A.H. and Barron, K.D.. The neurophathology of olivopontocerebellar atrophy. In R.C. Duvoisin and A. Plaitakis (Eds.), The Olivopontocerebellar Atrophies, Raven Press, New York. 1984, pp. 13-38. Lathe. R.. Synaptic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J. Mol. Biol., 183 (1985) I - 12. Merchenthaler. I.. Vigh, S.. Petrusz, P. and Schally, A.V.. Immunochemical localization of corticotropin releasing factor (CRF) in the rat brain, Am. J. Anat.. 165 (1982) 385396.
I3 Millan, M.A., Jacobowitz, D.M., Hauger, R.L., Catt, K.J. and Aguilera, G., Distribution of corticotropin-releasing factor receptors in primate brain, Proc. Natl. Acad. Sci. L’.S.A., 83 (1986) 1921-1925. I4 Olschowka, J.A.. O’Donohue, T.L.. Mueller, G.P. and Jacobowitz, D.M.. The distribution of corticotropin-releasing factor-like immunoreactive neurons in rat brain, Peptides, 3 (1982) 995-1015. I5 Powers, R.E., DeSouza. E.B.. Walker. L.C.. Vale. W.W.. Price, D.L. and Young, W.S.. III, Corticotropin-releasing factor as a transmitter of inferior olivary neurons, Sot. Neurosci. Abstr., 12 (1986) 568. 16 Rivier, J., Spiess. J. and Vale. W., Characterization of rat hypothalamic corticotropin-releasing factor, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4851-4855. I7 Schipper, J., Steinbusch, H.W.M., Vermes, I. and Tilders, F.J.H.. Mapping of CRF-immunoreactive nerve fibers in the medulla oblongata and spinal cord of the rat, Brain Res., 267 (1983) 145-150. 18 Shibahara, S., Morimoto. Y., Furutani, Y.. Notake. M., Takahashi. H., Shimizu, H., Horikawa, S. and Numa. S., Isolation and sequence analysis of the human corticotropinreleasing factor precursor gene. EMBO J., 2 (1983) 775-779. IY Swanson. L.W., Sawchenko, P.E.. Rivier, J. and Vale. W. W., Organization of ovine cortocotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology, 36 (1983) 165-186. 20 Vale. W.. Spiess. J.. Rivier. C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and P-endorphin. Science, 213 (1981) 1394-1397. 21 Wynn, P.C., Hauger. R.L., Holmes. M.C.. Millan. M.A.. Catt. K.J. and Aquilcra. G.. Brain and pituitary receptors for corticotropin releasing factor: localization and differcntial regulation after adrenalectomy. Peptides, 5 (1984) 1077- 1084. 22 Young. W.S. III and Kuhar. M.J.. A new method for receptor autoradiography: (jH) opioid receptors in rat brain. Brain Res.. 179 (1979) 255-270. 23 Young, W.S. 111, Mezey. E. and Siegel, R.E.. Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats, Neurosci. Lrtt., in press.