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Corticotropin-releasing factor-containing afferents to the inferior colliculus of the rat brain
Brain Research, 414 (1987) 68-76
68
Elsevier
BRE 12644
Corticotropin-releasing factor-containing afferents to the inferior colliculus of the rat brain Masahiro Sakanaka 1, Tamotsu Shibasaki 2 and Karl Lederis 1 1Department of Pharmacology and Therapeutics, The Universityof Calgary, Calgary, Alta. (Canada) and eDepartment of Medicine, Tokyo Women's Medical College, Tokyo (Japan) (Accepted 11 November 1986)
Key words: Corticotropin-releasing factor; Lateral hypothalamic area; Zona incerta; Afferents to the inferior colliculus; Combination of HRP and immunohistochemistry
Using a modified cobalt-glucose oxidase-diaminobenzidine (Co-GOD) method in a combination of horseradish peroxidase (HRP) retrograde tracing and immunohistochemistry, a widespread localization of corticotropin releasing factor-like immunoreactive (CRFI) structures in the rat inferior colliculus (IC), and a CRFI-containing pathway from the subthalamus and the hypothalamus to the IC have been observed. By means of the modified Co-GOD method, CRFI cells were detected in almost all the subdivisions of the IC. including the dorsomedial part of the central nucleus, the ventrolateral part of the central nucleus, the pericentral nucleus and the external nucleus. Neural processes with CRFI were observed in all of the above areas. Following HRP injection into the IC, doublelabeled cells which contained a homogeneous brown immunoreaction product of CRF and a granular black reaction product of retrogradely transported HRP were identified in the lateral hypothalamic area (LH), zona incerta (ZI) and perifomical hypothalamic area (PeF). These double-labeled cells provide direct evidence for CRFI projections from the LH, ZI and PeF to the IC. Thus, the present study supports the view that CRF may act as a neurotransmitter or neuromodulator in the brain. INTRODUCTION Recent immunohistochemical studies have shown that the lateral hypothalamic area (LH) contains corticotropin releasing factor-like immunoreactive (CRFI) cells3'5'9'10'13"21"23,some of which are also immunoreactive to a-melanocyte-stimulating h o r m o n e (a-MSH) 5. In addition, there is evidence for the presence of a-MSH-containing projections from the LH to the inferior colliculus (IC) 2°. These findings raise a question of the location of the C R F I cells from which the axons to the IC originate. The present study was intended, first, to demonstrate the localization of CRFI structures in the IC, of which little is known and, second, to examine the existence of CRF-conraining pathway from the LH to the IC with the simultaneous use of retrograde transport of horseradish peroxidase (HRP) and C R F immunohistochemistry.
MATERIALS AND METHODS Twenty
Sprague-Dawley
male
rats
weighing
60-100 g were used in this study. All animals were housed under constant temperature (20 °C) with food and water ad libitum and 12:12 h l i g h t - d a r k cycle.
CRF-like immunoreactivity in the rat Preparation of tissue. Three animals were prepared to determine the localization of C R F I structures. Each animal was anesthetized with Nembutal (40 mg/kg) and perfused transcardially, first with 50 ml of heparinized saline, then with 30 ml of Bouin's solution (pH 2.0) 1. Perfusion of the fixative was followed by a further phosphate-buffered saline (PBS) wash to clear out the Bouin's solution, then the animal was finally perfused with 150 ml of Z a m b o n i ' s solution (pH 7.4) 28. The brain was quickly removed,
Correspondence: K. Lederis, Department of Pharmacology and Therapeutics, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada, T2N 4N1. 0006-8993/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
69 cut into 3 parts and sectioned at room temperature in the frontal plane at 40 pm with a Lancer vibratome. Serial sections were collected, kept in glass vials containing PBS for 2 h, then treated with ethanol (50% for 10 min, 70% for 30 rain, 50% for 10 min) to facilitate the penetration of antibodies, and rinsed again in PBS for 30 min. Four aminals received i.v. colchicine injections (25 pg/100 g b. wt.) 24 h before sacrifice, then they were treated as described above. Immunohistochemical procedures. The sections were kept in a PBS solution containing 0.1% gelatin and 0.005% hydrogen peroxide for 20 min to suppress endogenous peroxidase activity of the tissue. Following a brief rinse in phosphate-buffered saline (PBS), the sections were exposed to 10% normal goat serum (NGS) in PBS for 30 min, then rinsed in PBS containing 1% NGS and 0.25% carrageenan. Subsequently, they were stained while floating in glass vials according to the unlabeled antibody peroxidase-antiperoxidase (PAP) method 22 and cobaltglucose oxidase-diaminobenzidine (Co-GOD) intensification procedure 7'12. (1) The sections were incubated with the primary antiserum, produced in rabbits against synthetic rat CRF (see reference 4 for characterization of the antiserum), diluted 1:2000 with PBS containing 1% NGS and 0.1% Triton X-100 (36-48 h at room temperature). (2) Incubated with the bridge antibody (goat antirabbit IgG, Sigma) diluted 1:50 with PBS containing 1% NGS and 0.1% Triton X-100 (2 h at room temperature). Each incubation was followed by two washes in PBS containing 1% NGS and further wash in PBS containing 1% NGS and 0.25% carrageenan (10 rain in each at 4 °C). (3) The sections were incubated with peroxidaseantiperoxidase (PAP; DAKO) solution diluted 1:80 with PBS containing 0.1% Triton X-100 (1 h at room temperature). (4) Rinsed twice in PBS, once in 0.05 M Tris-HC1 buffer (pH 7.6) (15 min in each at 4 °C), then subjected to a modified version of the Co-GOD method of Itoh et al. 7 and Oldfield et al. 12. (5) The sections were washed briefly in 0.1 M TrisHCI buffer (pH 7.6). (6) Placed in a 0.5% solution of cobalt acetate in
0.1 M Tris-HC1 buffer (pH 7.6) for 10 rain at room temperature. (7) Washed 3 times in 0.1 M Tris-HCl buffer (pH 7.6), twice in 0.1 M phosphate buffer (pH 7.3) (10 min in each at room temperature). (8) Incubated for 12-16 h at 4 °C in a freshly prepared medium composed of 40 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB), 200 mg of fl-Dglucose, 40 mg of NH4C1, 0.5 U of glucose oxidase (GOD) (Sigma, Type V), and 100 ml of 0.1 M phosphate buffer (pH 7.3). (9) Washed 3 times in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C). Control sections were first incubated with antiserum absorbed with an excess of synthetic rat CRF (15 pg/ml), after which they were processed as described above.
Combined HRP and immunohistochemical study The method developed by Wainer and Rye 27 was applied in 13 animals to obtain direct evidence for CRF-containing projections from the LH to the IC, though some modifications were made. Each animal was anaesthetized with Nembutal before the experiment. A single injection of 0.04-0.3 pl of 20% HRP dissolved in 0.2 M Tris-HCl buffer (pH 8.6) was delivered into the unilateral IC over a 15-min period using a glass micropipette (i.d. 100 ~m) mounted in a stereotaxic apparatus and connected to a 1-pl Hamilton syringe. Each animal, kept alive for 24-48 h after the injection, was deeply reanaesthetized and then perfused transcardially with 50 ml of heparinized saline, followed by 100 ml of 0.075% glutaraldehyde-2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and finally with 40 ml of 0.1 M phosphate buffer (pH 7.4). Colchicine treatment was not carried out before the peffusion, because it appeared to suppress the retrograde transport of HRP. Moreover, it increased only the intensity but not the number of CRFI cells in areas containing 'double-labeled cells' (see Discussion). Following the perfusion, the brain was quickly removed and cut into sections of 60 pm thickness on a Lancer vibratome. The sections were collected serially, kept in glass vials containing 0.1 M phosphate buffer (pH 7.4) and subjected to the Co-GOD method described earlier for detection of retrogradely transported HRP 7,12. Subsequently, the sections were processed for immunohistochemistry
70 without cobalt intensification, using the coupled oxidation method 7'8. Briefly the following procedures were carried out after an incubation of the sections with PAP; the sections were washed twice in PBS, once in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C), and incubated for 12-16 h at 4 °C in a freshly prepared medium composed of 40 mg of DAB, 200 mg of fl-D-glucose, 40 mg of NHaC1, 0.5 U of glucose oxidase (GOD) (Sigma, Type V), and 100 ml of 0.1 M phosphate buffer (pH 7.3). Following the incubation, the sections were washed 3 times in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C). As a control, HRP was injected into the striate cortex or into areas caudal to the IC, and the animals were processed as described above.
Terminology and stereotaxic coordinates Terminology used was based upon the atlas of Paxinos and Watson ~5. The stereotaxic coordinates were obtained from the atlases of Sherwood and Timiras 19,
Paxinos and Watson 15, and Palkovits and Jacobowitz TM, though slight modifications of these atlases were made on the basis of pilot experiments. RESULTS
CRF-like immunoreactivity in the inferior colliculus In the most rostral part of the IC (rIC), CRFI cells were observed in the area just lateral to the superior colliculus (Figs. 1A and 2A). Caudally, the dorsomedial part of the central nucleus of the IC (CICDM) contained a small number of CRFI cells (Figs. 1BD) which extended dorsocaudally into the pericentral nucleus of the IC (PCIC) (Figs. 1C and 2C). Colchicine treatment resulted in the visualization of CRFI cells in the ventrolateral part of the central nucleus of the IC (CICVL) and in the external nucleus of the IC (EIC) (Figs. 1D and 2C, D) while increasing the number and intensity of stained cells in the above mentioned areas. Scattered CRFI cells were
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lo Fig. 1. A, B, and C: bright-field photomicrographsshowing CRFI cells and fibres in the rlC (A), CICDM (B, C) and PCIC (C) of a normal rat. D: bright-field photomicrographshowingCRFI cells and fibres in the CICVL and EIC of the colchicine-treated rat. Broken lines show nuclear boundaries. Frontal sections × 67.5.
71
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Fig. 2. Schematic representation showing CRFI structures in the rat IC. Larger black spheres indicate CRFI ceils (a sphere represents one to two CRFI cells per section), and small dots, CRFI fibres. The dots do not represent quantitative distribution of CRFI fibres. Modified frontal planes from the atlas of Paxinos and Watson 15. A: bregma -7.8 mm, B: bregma -8.3 mm, C: bregma-8.8 mm, D: bregma -9.3 mm.
seen in the brachium of the IC (bic) and in the commissure of the IC (cic) (Fig. 2 A , B). C R F I fibres were d e t e c t e d in almost all the subdivisions of the IC. A m o n g them, the P C I C and the E I C contained considerable n u m b e r s of C R F I fibres (Figs. 1C and 2C). Some of the C R F I fibres in the central nucleus of the IC were traced for a short distance from intrinsic C R F I cells, whereas others app e a r e d to run multidirectionally without any association with the intrinsic C R F I cells (Figs. 1 and 2). The immunoreactive structures which were stained with the antiserum against rat C R F were not seen in the sections incubated with the adsorption control serum.
Combined H R P and immunohistochemical Method Following H R P injection into the IC (Fig. 3), 3 Fig. 3. Bright-field photomicrograph showing an injection site of 0.095/~1 of HRP in the IC. Frontal section, x12.
3
72 kinds of cells (double-labeled cells, CRFI cells, HRP-labeled cells) were bilaterally identified in the LH, zona incerta (ZI) and perifornical hypothalamic area (PeF) with ipsilateral predominance. General picture of the 3 kinds of cells. Many of the double-labeled cells demonstrated a homogeneous brown reaction product throughout the cytoplasm and proximal dendrites, indicating the presence of CRFI, and a granular black reaction product over the cytoplasm, indicating the presence of retrogradely transported H R P (Fig. 4Aa, Ba). The CRFI cells showed the diffuse brown reaction product (Fig. 4Ab, Bb), occasionally with a granular brown reaction product within the cytoplasm (Fig. 4Ac, Bc). In many cases, this granular brown immunoreaction product was larger in diameter and coarser in surface than the granular black reaction product of retrogradely transported HRP. Nevertheless, it was sometimes difficult to distinguish a CRFI cell from a double-labeled cell on a black and white print, espe-
cially in the case where the double-labeled cell contained the intensely stained granular immunoreaction product and scattered retrogradely transported HRP. The HRP-labeled cells showed only the granular black reaction product with a faintly grey cytoplasm (Fig. 4Ad, Bd). The differences between the 3 kinds of cells were discernable at high magnification (Fig. 4B). Control sections stained with absorption control serum demonstrated only HRP-labeled cells. Distribution of double-labeled cells. After 0.05 ¢tl of HRP was injected into the IC, double-labeled cells were detected mainly in the caudal LH corresponding to the levels of bregma -2.8 mm to -3.3 mm in the atlas of Paxinos and Watson 15 (Figs. 4Aa, e, f, Ba, Ce, Df, and 5). Many of the double-labeled cells in the LH had, like other projecting neurons, multipolar processes with large perikarya (Fig. 4Ba, Ce and Of). In the ZI, double-labeled cells were seen through-
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Fig. 4. Bright-field photomicrographs showing 3 kinds of cells in the LH at low magnification (A) and at high magnification (B, C, D,) after HRP injection into the IC. Double-labeled cells demonstrate a homogeneous brown reaction product and a granular black reaction product over the cytoplasm (Aa, e, f, Ba, Ce, and Df). CRFI cells show the diffuse brown reaction product (Ab, Bb), occasionally with a granular brown reaction product within the cytoplasm (Ac and Bc). HRP-labeled cells contained only the granular black reaction product with a faintly grey cytoplasm (Ad and Bd). Frontal section, A, x72; B, C, and D, x464.
73
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was injected into the striate cortex or into areas caudal to the IC. These findings suggest that the double-labeled cells located in the LH, ZI and PeF project to the IC.
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Fig. 5. Schematic representation showing, on the right, doublelabeled cells (asterisks), CRFI cells (black spheres) and HRPlabeled cells (closed triangles) after HRP injection into the IC. Modified frontal planes from the atlas of Pa×inos and Watsonis. A: bregma -2.3 mm. B: bregma-2.8 ram. C: bregma -3.3 ram. Corresponding brain areas are indicated on the left side.
out its rostrocaudal extent (Figs. 5 and 6Aa, Ba, Cb, Db) with medial extension into the dorsal hypothalamic area (DA) (Fig. 5). The double-labeled cells in the ZI were mostly medium in size, though doublelabeled cells with a large fusiform cytoplasm were occasionally identified in the lateral Z I (Fig. 6Cb, Db). The PeF also contained double-labeled cells in its caudal part (Fig. 5B, C and 7). They were medium in size and possessed a variety of processes such as unipolar (Fig. 7Aa, Ba), bipolar and multipolar ones (Fig. 7Cb, Db). An increase in number of double-labeled cells in the LH, ZI and PeF was apparent when larger amounts (0.1-0.3 pl of 20% solution) of H R P were injected into the IC. Occasionally, a few doublelabeled cells were found in the central amygdaloid nucleus after the larger amounts of H R P had been injected, suggestive of H R P diffusion into the parabrachial nuclei and/or into the central gray matter (see 'Discussion')6.11,16. Double-labeled cells were not detected when H R P
DISCUSSION Using a homologous antiserum and modified cobalt intensification developed in our previous study 16, a more widespread localization of CRFI in the rostrocaudal IC could be seen as compared with a previous study 9 which used an antiserum against ovine CRF, and a silver-gold intensification. The immunohistochemical discrepancy between the present investigation and previous study 9 on the localization of CRFI in the IC may be attributable to the difference of antisera employed, as suggested by Skofitsch and Jacobowitz 21. The CRFI in the IC, like other neuropeptides 2°,24, might be involved in the auditory transmission system. In the present study, several modifications of the combined H R P and immunohistochemical method developed by Wainer and Rye 27 have been introduced to increase the sensitivity of H R P histochemistry and to retain antigen•city on the sections: first, the brain was cut into 60-/~m sections on a vibratome immediately after the perfusion instead of immersing it in phosphate buffer containing 30% sucrose for 24 h 27. This immediate cutting of the brain seemed to attenuate the suppressive effects of the fixative on H R P activity and on CRF antigen•city. Furthermore, the application of the Co-GOD method to H R P histochemistry allowed us to obtain heavily labeled cells with H R P on sections with low background staining 7. These sections with low background staining made the subsequent immunohistochemical visualization of CRFI easy. The coupled oxidation method 7'8 carried out at the end of the immunohistochemical procedures was also useful in achieving stable staining of CRFI. Since colchicine treatment was not carried out in the combined H R P and immunohistochemical study, a question may arise that some HRP-labeled cells identified in the LH, ZI and PeF following H R P injection into IC might contain small amounts of QRFI which would be immunohistochemically enhanced by colchicine treatment. It is not always possible to exclude the possibility of H R P labeled cells with 'false'
74
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Fig. 6. Bright-fieldphotomicrographsshowingdouble-labeled cells in the ZI at low (Aa and Cb) and high (Ba and Db) magnifications. Frontal sections, A and C, x80; B and D, x520.
negative CRFI. Although colchicine treatment generally increases the number and intensity of immunostained cells, our immunohistochemical results (unpublished observations) have shown that CRFI cells in the LH, ZI and PeF increase only in intensity, but hardly or not at all in number after colchicine treatment. Therefore, the possibility of HRP-labeled cells with 'false' negative CRFI can be excluded in the present combination study. Further experiments may have to be designed to determine why colchicine treatment can affect the number of CRFI cells in the IC but not in the LH, ZI and PeF. When a relatively small amount (0.05/~1) of HRP is injected into the IC without any apparent HRP diffusion to adjacent areas, double-labeled cells were
seen in the LH, ZI and PeF. Such projections are supported by previous neuroanatomical studies carried out with use of HRP and autoradiographic tracings ]7'2°. It may be worth noting that double-labeled cells were occasionally identified in the central amygdaloid nucleus (Ce) in addition to the above areas after the larger amounts (C. 1-0.3/A) of HRP had been injected into the IC. This occurrence of the doublelabeled cells in the Ce could be due to HRP diffusion into adjacent areas including the parabrachial nucleus (PB) and central gray (CG), since CRFI-containing projections from the Ce to the PB and CG have been reported previously 6'11'16. However, the increase in double-labeled cell number in the LH, ZI and PeF following the larger HRP injections into the
75 bination m e t h o d of r e t r o g r a d e tracer and i m m u n o histochemistry m a y be required. The comparison of H R P and fluorescent reagents m a y be of value in immunohistochemical studies c o m b i n e d with retrograde tracing 2a8,26. The functional significance of the C R F p a t h w a y shown in the present study has not been investigated. Nevertheless, the present study suggests that C R F m a y play a role as a neurotransmitter-like substance in the extrahypothalamic circuits from the subthalamic (ZI) and hypothalamic ( L H , PeF) regions to the IC as well as participating in n e u r o e n d o c r i n e regulation 25.
ACKNOWLEDGEMENTS Fig. 7. Bright-field photomicrographs showing double-labeled cells in the PeF at low (Aa and Cb) and high (Ba and Db) magnifications. Frontal sections, A and C, ×90 and D, x580. IC leaves two possibilities open: one is that the H R P diffusion into the PB and/or into the C G resulted in the increase in d o u b l e - l a b e l e d cells, and the other is that the large amounts of H R P injected into the IC caused m o r e labeling in C R F I cells within the L H , Z I and P e F than the small a m o u n t of H R P injection. To resolve this issue, a m o r e reliable and sensitive com-
ABBREVIATIONS Aq Arc bic Cb CG cic CICDM CICVL Cnf DA DM EIC
cerebral aqueduct (Sylvius) arcuate hypothalamic nucleus brachium of the IC cerebellum central (periaqueductal) gray commissure of the IC central nucleus of the IC, dorsomedial part central nucleus of the IC, ventrolateral part cuneiform nucleus dorsal hypothalamic area dorsomedial hypothalamic nucleus external nucleus of the IC
REFERENCES 1 Baker, J.R., Cytological Technique, Methuen, London, 1946. 2 Carlsen, J., Zaborszky, L. and Heimer, L., Cholinergic projections from the basal forebrain to the basolateral amygdaloid complex: a combined retrograde fluorescent and immunohistochemical study, J. Comp. Neurol., 234
This work was s u p p o r t e d by grants from the M R C ( C a n a d a ) to K.L. ( C a r e e r Investigator of the M R C ) , and from the A l b e r t a H e r i t a g e F o u n d a t i o n for Medical Research to M.S. ( A H F M R fellow) who is on leave of absence from the D e p a r t m e n t of A n a t o m y at O s a k a Medical College. The authors wish to t h a n k Prof. S. Magari for her interest and e n c o u r a g e m e n t throughout this work. W e are grateful to Mrs. M. Devlin for typing the manuscript, and to B a c h e m ( C A ) for their generous provision of synthetic rat CRF.
f IC ic LH mt opt PB PCIC PeF rlC SC VMH ZI
fornix inferior coUiculus internal capsule lateral hypothalamic area mammillothalamic tract optic tract parabrachial nuclei pericentral nucleus of the IC perifornical hypothalamic area most rostral part of the IC superior colliculus ventromedial hypothalamic nucleus zona incerta
(1985) 155-167. 3 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 immunohistochemical study, J. Neurosci., 3 (1983) 1355-1368. 4 Daikoku, S., Okamura, Y., Kawano, H., Tsuruo, Y., Maegawa, M. and Shibasaki, T., Immunohistochemical study on the development of CRF-containing neurons in the hy-
76 pothalamus of the rat, Cell Tissue Res., 238 (1984) 539-544. 5 Daikoku, S., Okamura, Y., Kawano, H., Tsuruo, Y., Maegawa, M. and Shibasaki, T., CRF-containing neurons of the rat hypothalamus, Cell Tissue Res., 240 (1985) 575-584. 6 Gray, T.S., Neuropeptide neuronal efferents from the amygdala to the central gray in the rat, Anat. Rec., 214 (1986) 44A. 7 Itoh, K. Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. and Sugimoto, T., Application of coupled oxidation reaction to electronmicroscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method, Brain Research, 175 (1979) 341-346. 8 Lundquist, I. and Josefsson, J.-O., Sensitive method for determination of peroxidase activity in tissue by means of coupled oxidation reaction, Anal. Biochem., 41 (1971) 567-577. 9 Merchenthaler, I., Corticotropin releasing factor (CRF)like immunoreactivity in the rat central nervous system. Extrahypothalamic distribution, Peptides, 5 Suppl. 1 (1984) 53-69. 10 Merchenthaler, I., Hynes, M.A., Vigh, S., Schally, A.V. and Petrusz, P., Corticotropin releasing factor (CRF): origin and course of afferent pathways to the median eminence (ME) of the rat hypothalamus, Neuroendocrinology, 39 (1984) 296-306. 11 Moga, M.M. and Gray, T.S., Evidence for corticotropinreleasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus, J. Comp. Neurol., 241 (1985) 275-284. 12 Oldfield, B.J., Hou-Yu, A, and Silverman, A.-J., Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunohistochemically identified neuropeptide, J. Histochem. Cytochem., 31 (1983) 1145-1150. 13 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. 14 Palkovits, M. and Jacobowitz, D.M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain, J. Comp. NeuroL, 157 (1974) 29-42. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. 16 Sakanaka, M., Shibasaki, T. and Lederis, K., Distribution and efferent projections of corticotropin releasing factorlike immunoreactivity in the rat amygdaloid complex,
Brain Research, 382 (1986) 213-238. 17 Saper, C.B., Swanson, L.W. and Cowan, W.M., An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat, J. Comp Neurol., 183 (1979) 689-706. 18 Sawchenko, P.E. and Swanson, L.W., A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Research, 210 (1981) 31-51. 19 Sherwood, N.M. and Timiras, P.S., A Stereotaxic Atlas of the Developing Rat Brain, University of California Press, Berkeley, 1970. 20 Shiosaka, S., Kawai, Y., Shibasaki, T. and Tohyama, M., The descending a-MSHergic (a-melanocyte-stimulating hormone-ergic) projections from the zona incerta and lateral hypothalamic area to the inferior colliculus and spinal cord in the rat, Brain Research, 338 (1985) 371-375. 21 Skofitsch, G. and Jacobowitz, D.M., Distribution and corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay: comparison and characterization of ovine and rat/human CRF antisera, Peptides, 6 (1985) 319-336. 22 Sternberger, L.A. lmmunohistochemistry, 2nd edn., Wiley, New York, 1979. 23 Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibres in the rat brain: an immunohistochemical study, Neuroendocrinology, 36 (1983) 165-186. 24 Takatsuki, K., Shiosaka, S., Sakanaka, M., Inagaki, S., Senba, E., Takagi, H. and Tohyama, M., Somatostatin in the auditory system of the rat, Brain Research, 213 (1981) 211-216. 25 Vale, W., Spiess, J., Rivier, C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and fl-endorphin, Science, 213 (1981) 1394-1397. 26 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., Substance P in the ascending cholinergic reticular system, Nature (London), 306 (1983) 688-691. 27 Wainer, B.H. and Rye, D.B., Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity, J. Histochem. Cytochem., 32 (1984) 439-443. 28 Zamboni, L. and DeMartino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electronmicroscopy, J. Cell Biol., 35 (1967) 148A.
68
Elsevier
BRE 12644
Corticotropin-releasing factor-containing afferents to the inferior colliculus of the rat brain Masahiro Sakanaka 1, Tamotsu Shibasaki 2 and Karl Lederis 1 1Department of Pharmacology and Therapeutics, The Universityof Calgary, Calgary, Alta. (Canada) and eDepartment of Medicine, Tokyo Women's Medical College, Tokyo (Japan) (Accepted 11 November 1986)
Key words: Corticotropin-releasing factor; Lateral hypothalamic area; Zona incerta; Afferents to the inferior colliculus; Combination of HRP and immunohistochemistry
Using a modified cobalt-glucose oxidase-diaminobenzidine (Co-GOD) method in a combination of horseradish peroxidase (HRP) retrograde tracing and immunohistochemistry, a widespread localization of corticotropin releasing factor-like immunoreactive (CRFI) structures in the rat inferior colliculus (IC), and a CRFI-containing pathway from the subthalamus and the hypothalamus to the IC have been observed. By means of the modified Co-GOD method, CRFI cells were detected in almost all the subdivisions of the IC. including the dorsomedial part of the central nucleus, the ventrolateral part of the central nucleus, the pericentral nucleus and the external nucleus. Neural processes with CRFI were observed in all of the above areas. Following HRP injection into the IC, doublelabeled cells which contained a homogeneous brown immunoreaction product of CRF and a granular black reaction product of retrogradely transported HRP were identified in the lateral hypothalamic area (LH), zona incerta (ZI) and perifomical hypothalamic area (PeF). These double-labeled cells provide direct evidence for CRFI projections from the LH, ZI and PeF to the IC. Thus, the present study supports the view that CRF may act as a neurotransmitter or neuromodulator in the brain. INTRODUCTION Recent immunohistochemical studies have shown that the lateral hypothalamic area (LH) contains corticotropin releasing factor-like immunoreactive (CRFI) cells3'5'9'10'13"21"23,some of which are also immunoreactive to a-melanocyte-stimulating h o r m o n e (a-MSH) 5. In addition, there is evidence for the presence of a-MSH-containing projections from the LH to the inferior colliculus (IC) 2°. These findings raise a question of the location of the C R F I cells from which the axons to the IC originate. The present study was intended, first, to demonstrate the localization of CRFI structures in the IC, of which little is known and, second, to examine the existence of CRF-conraining pathway from the LH to the IC with the simultaneous use of retrograde transport of horseradish peroxidase (HRP) and C R F immunohistochemistry.
MATERIALS AND METHODS Twenty
Sprague-Dawley
male
rats
weighing
60-100 g were used in this study. All animals were housed under constant temperature (20 °C) with food and water ad libitum and 12:12 h l i g h t - d a r k cycle.
CRF-like immunoreactivity in the rat Preparation of tissue. Three animals were prepared to determine the localization of C R F I structures. Each animal was anesthetized with Nembutal (40 mg/kg) and perfused transcardially, first with 50 ml of heparinized saline, then with 30 ml of Bouin's solution (pH 2.0) 1. Perfusion of the fixative was followed by a further phosphate-buffered saline (PBS) wash to clear out the Bouin's solution, then the animal was finally perfused with 150 ml of Z a m b o n i ' s solution (pH 7.4) 28. The brain was quickly removed,
Correspondence: K. Lederis, Department of Pharmacology and Therapeutics, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada, T2N 4N1. 0006-8993/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
69 cut into 3 parts and sectioned at room temperature in the frontal plane at 40 pm with a Lancer vibratome. Serial sections were collected, kept in glass vials containing PBS for 2 h, then treated with ethanol (50% for 10 min, 70% for 30 rain, 50% for 10 min) to facilitate the penetration of antibodies, and rinsed again in PBS for 30 min. Four aminals received i.v. colchicine injections (25 pg/100 g b. wt.) 24 h before sacrifice, then they were treated as described above. Immunohistochemical procedures. The sections were kept in a PBS solution containing 0.1% gelatin and 0.005% hydrogen peroxide for 20 min to suppress endogenous peroxidase activity of the tissue. Following a brief rinse in phosphate-buffered saline (PBS), the sections were exposed to 10% normal goat serum (NGS) in PBS for 30 min, then rinsed in PBS containing 1% NGS and 0.25% carrageenan. Subsequently, they were stained while floating in glass vials according to the unlabeled antibody peroxidase-antiperoxidase (PAP) method 22 and cobaltglucose oxidase-diaminobenzidine (Co-GOD) intensification procedure 7'12. (1) The sections were incubated with the primary antiserum, produced in rabbits against synthetic rat CRF (see reference 4 for characterization of the antiserum), diluted 1:2000 with PBS containing 1% NGS and 0.1% Triton X-100 (36-48 h at room temperature). (2) Incubated with the bridge antibody (goat antirabbit IgG, Sigma) diluted 1:50 with PBS containing 1% NGS and 0.1% Triton X-100 (2 h at room temperature). Each incubation was followed by two washes in PBS containing 1% NGS and further wash in PBS containing 1% NGS and 0.25% carrageenan (10 rain in each at 4 °C). (3) The sections were incubated with peroxidaseantiperoxidase (PAP; DAKO) solution diluted 1:80 with PBS containing 0.1% Triton X-100 (1 h at room temperature). (4) Rinsed twice in PBS, once in 0.05 M Tris-HC1 buffer (pH 7.6) (15 min in each at 4 °C), then subjected to a modified version of the Co-GOD method of Itoh et al. 7 and Oldfield et al. 12. (5) The sections were washed briefly in 0.1 M TrisHCI buffer (pH 7.6). (6) Placed in a 0.5% solution of cobalt acetate in
0.1 M Tris-HC1 buffer (pH 7.6) for 10 rain at room temperature. (7) Washed 3 times in 0.1 M Tris-HCl buffer (pH 7.6), twice in 0.1 M phosphate buffer (pH 7.3) (10 min in each at room temperature). (8) Incubated for 12-16 h at 4 °C in a freshly prepared medium composed of 40 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB), 200 mg of fl-Dglucose, 40 mg of NH4C1, 0.5 U of glucose oxidase (GOD) (Sigma, Type V), and 100 ml of 0.1 M phosphate buffer (pH 7.3). (9) Washed 3 times in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C). Control sections were first incubated with antiserum absorbed with an excess of synthetic rat CRF (15 pg/ml), after which they were processed as described above.
Combined HRP and immunohistochemical study The method developed by Wainer and Rye 27 was applied in 13 animals to obtain direct evidence for CRF-containing projections from the LH to the IC, though some modifications were made. Each animal was anaesthetized with Nembutal before the experiment. A single injection of 0.04-0.3 pl of 20% HRP dissolved in 0.2 M Tris-HCl buffer (pH 8.6) was delivered into the unilateral IC over a 15-min period using a glass micropipette (i.d. 100 ~m) mounted in a stereotaxic apparatus and connected to a 1-pl Hamilton syringe. Each animal, kept alive for 24-48 h after the injection, was deeply reanaesthetized and then perfused transcardially with 50 ml of heparinized saline, followed by 100 ml of 0.075% glutaraldehyde-2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and finally with 40 ml of 0.1 M phosphate buffer (pH 7.4). Colchicine treatment was not carried out before the peffusion, because it appeared to suppress the retrograde transport of HRP. Moreover, it increased only the intensity but not the number of CRFI cells in areas containing 'double-labeled cells' (see Discussion). Following the perfusion, the brain was quickly removed and cut into sections of 60 pm thickness on a Lancer vibratome. The sections were collected serially, kept in glass vials containing 0.1 M phosphate buffer (pH 7.4) and subjected to the Co-GOD method described earlier for detection of retrogradely transported HRP 7,12. Subsequently, the sections were processed for immunohistochemistry
70 without cobalt intensification, using the coupled oxidation method 7'8. Briefly the following procedures were carried out after an incubation of the sections with PAP; the sections were washed twice in PBS, once in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C), and incubated for 12-16 h at 4 °C in a freshly prepared medium composed of 40 mg of DAB, 200 mg of fl-D-glucose, 40 mg of NHaC1, 0.5 U of glucose oxidase (GOD) (Sigma, Type V), and 100 ml of 0.1 M phosphate buffer (pH 7.3). Following the incubation, the sections were washed 3 times in 0.1 M phosphate buffer (pH 7.3) (10 min in each at 4 °C). As a control, HRP was injected into the striate cortex or into areas caudal to the IC, and the animals were processed as described above.
Terminology and stereotaxic coordinates Terminology used was based upon the atlas of Paxinos and Watson ~5. The stereotaxic coordinates were obtained from the atlases of Sherwood and Timiras 19,
Paxinos and Watson 15, and Palkovits and Jacobowitz TM, though slight modifications of these atlases were made on the basis of pilot experiments. RESULTS
CRF-like immunoreactivity in the inferior colliculus In the most rostral part of the IC (rIC), CRFI cells were observed in the area just lateral to the superior colliculus (Figs. 1A and 2A). Caudally, the dorsomedial part of the central nucleus of the IC (CICDM) contained a small number of CRFI cells (Figs. 1BD) which extended dorsocaudally into the pericentral nucleus of the IC (PCIC) (Figs. 1C and 2C). Colchicine treatment resulted in the visualization of CRFI cells in the ventrolateral part of the central nucleus of the IC (CICVL) and in the external nucleus of the IC (EIC) (Figs. 1D and 2C, D) while increasing the number and intensity of stained cells in the above mentioned areas. Scattered CRFI cells were
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71
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Fig. 2. Schematic representation showing CRFI structures in the rat IC. Larger black spheres indicate CRFI ceils (a sphere represents one to two CRFI cells per section), and small dots, CRFI fibres. The dots do not represent quantitative distribution of CRFI fibres. Modified frontal planes from the atlas of Paxinos and Watson 15. A: bregma -7.8 mm, B: bregma -8.3 mm, C: bregma-8.8 mm, D: bregma -9.3 mm.
seen in the brachium of the IC (bic) and in the commissure of the IC (cic) (Fig. 2 A , B). C R F I fibres were d e t e c t e d in almost all the subdivisions of the IC. A m o n g them, the P C I C and the E I C contained considerable n u m b e r s of C R F I fibres (Figs. 1C and 2C). Some of the C R F I fibres in the central nucleus of the IC were traced for a short distance from intrinsic C R F I cells, whereas others app e a r e d to run multidirectionally without any association with the intrinsic C R F I cells (Figs. 1 and 2). The immunoreactive structures which were stained with the antiserum against rat C R F were not seen in the sections incubated with the adsorption control serum.
Combined H R P and immunohistochemical Method Following H R P injection into the IC (Fig. 3), 3 Fig. 3. Bright-field photomicrograph showing an injection site of 0.095/~1 of HRP in the IC. Frontal section, x12.
3
72 kinds of cells (double-labeled cells, CRFI cells, HRP-labeled cells) were bilaterally identified in the LH, zona incerta (ZI) and perifornical hypothalamic area (PeF) with ipsilateral predominance. General picture of the 3 kinds of cells. Many of the double-labeled cells demonstrated a homogeneous brown reaction product throughout the cytoplasm and proximal dendrites, indicating the presence of CRFI, and a granular black reaction product over the cytoplasm, indicating the presence of retrogradely transported H R P (Fig. 4Aa, Ba). The CRFI cells showed the diffuse brown reaction product (Fig. 4Ab, Bb), occasionally with a granular brown reaction product within the cytoplasm (Fig. 4Ac, Bc). In many cases, this granular brown immunoreaction product was larger in diameter and coarser in surface than the granular black reaction product of retrogradely transported HRP. Nevertheless, it was sometimes difficult to distinguish a CRFI cell from a double-labeled cell on a black and white print, espe-
cially in the case where the double-labeled cell contained the intensely stained granular immunoreaction product and scattered retrogradely transported HRP. The HRP-labeled cells showed only the granular black reaction product with a faintly grey cytoplasm (Fig. 4Ad, Bd). The differences between the 3 kinds of cells were discernable at high magnification (Fig. 4B). Control sections stained with absorption control serum demonstrated only HRP-labeled cells. Distribution of double-labeled cells. After 0.05 ¢tl of HRP was injected into the IC, double-labeled cells were detected mainly in the caudal LH corresponding to the levels of bregma -2.8 mm to -3.3 mm in the atlas of Paxinos and Watson 15 (Figs. 4Aa, e, f, Ba, Ce, Df, and 5). Many of the double-labeled cells in the LH had, like other projecting neurons, multipolar processes with large perikarya (Fig. 4Ba, Ce and Of). In the ZI, double-labeled cells were seen through-
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Fig. 4. Bright-field photomicrographs showing 3 kinds of cells in the LH at low magnification (A) and at high magnification (B, C, D,) after HRP injection into the IC. Double-labeled cells demonstrate a homogeneous brown reaction product and a granular black reaction product over the cytoplasm (Aa, e, f, Ba, Ce, and Df). CRFI cells show the diffuse brown reaction product (Ab, Bb), occasionally with a granular brown reaction product within the cytoplasm (Ac and Bc). HRP-labeled cells contained only the granular black reaction product with a faintly grey cytoplasm (Ad and Bd). Frontal section, A, x72; B, C, and D, x464.
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was injected into the striate cortex or into areas caudal to the IC. These findings suggest that the double-labeled cells located in the LH, ZI and PeF project to the IC.
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Fig. 5. Schematic representation showing, on the right, doublelabeled cells (asterisks), CRFI cells (black spheres) and HRPlabeled cells (closed triangles) after HRP injection into the IC. Modified frontal planes from the atlas of Pa×inos and Watsonis. A: bregma -2.3 mm. B: bregma-2.8 ram. C: bregma -3.3 ram. Corresponding brain areas are indicated on the left side.
out its rostrocaudal extent (Figs. 5 and 6Aa, Ba, Cb, Db) with medial extension into the dorsal hypothalamic area (DA) (Fig. 5). The double-labeled cells in the ZI were mostly medium in size, though doublelabeled cells with a large fusiform cytoplasm were occasionally identified in the lateral Z I (Fig. 6Cb, Db). The PeF also contained double-labeled cells in its caudal part (Fig. 5B, C and 7). They were medium in size and possessed a variety of processes such as unipolar (Fig. 7Aa, Ba), bipolar and multipolar ones (Fig. 7Cb, Db). An increase in number of double-labeled cells in the LH, ZI and PeF was apparent when larger amounts (0.1-0.3 pl of 20% solution) of H R P were injected into the IC. Occasionally, a few doublelabeled cells were found in the central amygdaloid nucleus after the larger amounts of H R P had been injected, suggestive of H R P diffusion into the parabrachial nuclei and/or into the central gray matter (see 'Discussion')6.11,16. Double-labeled cells were not detected when H R P
DISCUSSION Using a homologous antiserum and modified cobalt intensification developed in our previous study 16, a more widespread localization of CRFI in the rostrocaudal IC could be seen as compared with a previous study 9 which used an antiserum against ovine CRF, and a silver-gold intensification. The immunohistochemical discrepancy between the present investigation and previous study 9 on the localization of CRFI in the IC may be attributable to the difference of antisera employed, as suggested by Skofitsch and Jacobowitz 21. The CRFI in the IC, like other neuropeptides 2°,24, might be involved in the auditory transmission system. In the present study, several modifications of the combined H R P and immunohistochemical method developed by Wainer and Rye 27 have been introduced to increase the sensitivity of H R P histochemistry and to retain antigen•city on the sections: first, the brain was cut into 60-/~m sections on a vibratome immediately after the perfusion instead of immersing it in phosphate buffer containing 30% sucrose for 24 h 27. This immediate cutting of the brain seemed to attenuate the suppressive effects of the fixative on H R P activity and on CRF antigen•city. Furthermore, the application of the Co-GOD method to H R P histochemistry allowed us to obtain heavily labeled cells with H R P on sections with low background staining 7. These sections with low background staining made the subsequent immunohistochemical visualization of CRFI easy. The coupled oxidation method 7'8 carried out at the end of the immunohistochemical procedures was also useful in achieving stable staining of CRFI. Since colchicine treatment was not carried out in the combined H R P and immunohistochemical study, a question may arise that some HRP-labeled cells identified in the LH, ZI and PeF following H R P injection into IC might contain small amounts of QRFI which would be immunohistochemically enhanced by colchicine treatment. It is not always possible to exclude the possibility of H R P labeled cells with 'false'
74
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Fig. 6. Bright-fieldphotomicrographsshowingdouble-labeled cells in the ZI at low (Aa and Cb) and high (Ba and Db) magnifications. Frontal sections, A and C, x80; B and D, x520.
negative CRFI. Although colchicine treatment generally increases the number and intensity of immunostained cells, our immunohistochemical results (unpublished observations) have shown that CRFI cells in the LH, ZI and PeF increase only in intensity, but hardly or not at all in number after colchicine treatment. Therefore, the possibility of HRP-labeled cells with 'false' negative CRFI can be excluded in the present combination study. Further experiments may have to be designed to determine why colchicine treatment can affect the number of CRFI cells in the IC but not in the LH, ZI and PeF. When a relatively small amount (0.05/~1) of HRP is injected into the IC without any apparent HRP diffusion to adjacent areas, double-labeled cells were
seen in the LH, ZI and PeF. Such projections are supported by previous neuroanatomical studies carried out with use of HRP and autoradiographic tracings ]7'2°. It may be worth noting that double-labeled cells were occasionally identified in the central amygdaloid nucleus (Ce) in addition to the above areas after the larger amounts (C. 1-0.3/A) of HRP had been injected into the IC. This occurrence of the doublelabeled cells in the Ce could be due to HRP diffusion into adjacent areas including the parabrachial nucleus (PB) and central gray (CG), since CRFI-containing projections from the Ce to the PB and CG have been reported previously 6'11'16. However, the increase in double-labeled cell number in the LH, ZI and PeF following the larger HRP injections into the
75 bination m e t h o d of r e t r o g r a d e tracer and i m m u n o histochemistry m a y be required. The comparison of H R P and fluorescent reagents m a y be of value in immunohistochemical studies c o m b i n e d with retrograde tracing 2a8,26. The functional significance of the C R F p a t h w a y shown in the present study has not been investigated. Nevertheless, the present study suggests that C R F m a y play a role as a neurotransmitter-like substance in the extrahypothalamic circuits from the subthalamic (ZI) and hypothalamic ( L H , PeF) regions to the IC as well as participating in n e u r o e n d o c r i n e regulation 25.
ACKNOWLEDGEMENTS Fig. 7. Bright-field photomicrographs showing double-labeled cells in the PeF at low (Aa and Cb) and high (Ba and Db) magnifications. Frontal sections, A and C, ×90 and D, x580. IC leaves two possibilities open: one is that the H R P diffusion into the PB and/or into the C G resulted in the increase in d o u b l e - l a b e l e d cells, and the other is that the large amounts of H R P injected into the IC caused m o r e labeling in C R F I cells within the L H , Z I and P e F than the small a m o u n t of H R P injection. To resolve this issue, a m o r e reliable and sensitive com-
ABBREVIATIONS Aq Arc bic Cb CG cic CICDM CICVL Cnf DA DM EIC
cerebral aqueduct (Sylvius) arcuate hypothalamic nucleus brachium of the IC cerebellum central (periaqueductal) gray commissure of the IC central nucleus of the IC, dorsomedial part central nucleus of the IC, ventrolateral part cuneiform nucleus dorsal hypothalamic area dorsomedial hypothalamic nucleus external nucleus of the IC
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This work was s u p p o r t e d by grants from the M R C ( C a n a d a ) to K.L. ( C a r e e r Investigator of the M R C ) , and from the A l b e r t a H e r i t a g e F o u n d a t i o n for Medical Research to M.S. ( A H F M R fellow) who is on leave of absence from the D e p a r t m e n t of A n a t o m y at O s a k a Medical College. The authors wish to t h a n k Prof. S. Magari for her interest and e n c o u r a g e m e n t throughout this work. W e are grateful to Mrs. M. Devlin for typing the manuscript, and to B a c h e m ( C A ) for their generous provision of synthetic rat CRF.
f IC ic LH mt opt PB PCIC PeF rlC SC VMH ZI
fornix inferior coUiculus internal capsule lateral hypothalamic area mammillothalamic tract optic tract parabrachial nuclei pericentral nucleus of the IC perifornical hypothalamic area most rostral part of the IC superior colliculus ventromedial hypothalamic nucleus zona incerta
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