Immunohistochemistry and in Situ Hybridization Histochemistry

Home , Inferior olivary nucleus, Solitary tract

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3911-3915, June 1987 Neurobiology

Corticotropin-releasing factor in the olivocerebellar tract of rats: Demonstration by light- and electron-microscopic immunohistochemistry and in situ hybridization histochemistry (inferior olive/cerebelium/climbing fibers/messenger RNA) MIKLOS PALKOVITS*, CSABA LMRANTHt, TAMAS GtRCSt, AND W. SCOTT YOUNG III* *Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892; tSection of Neuroanatomy and Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510; and *U.S.-Japan Biomedical Research Laboratories, Laboratory for Molecular Neuroendocrinology and Diabetes, Belle Chasse, LA 70037 Communicated by Jdnos Szentdgothai, February 3, 1987

ABSTRACT The presence of corticotropin-releasing fac- rats and cats pretreated with colchicine (16, 17). Indeed, the tor (CRF) in the olivocerebellar system was demonstrated in inferior olive is rich in CRF: the second highest level of this rats by light- and electron-microscopic immunohistochemistry, peptide in the rat (18) and sheep (19) central nervous system as well as by in situ hybridization histochemistry. CRF-like was measured there by radioimmunoassay. In the cerebel- immunoreactivity was present in each portion of the olivocer- lum, fine (presumably climbing) fibers were immunostained ebellar system: (i) In colchicine-treated rats, CRF-im- for CRF, and CRF-like immunoreactive fibers were observed munostained cells were found in each nuclei and subunit of the in the vicinity of the cerebellar peduncles (17, 20). inferior olive. After surgical transection of the medullary In the present study, we performed various morphological portion of the olivocerebellar tract, CRF-immunostaining techniques on untreated, colchicine-treated, and brainstem- accumulated in inferior olive neurons contralateral to the operated rats to provide evidence for the presence of CRF in lesion. In situ hybridization histochemistry indicated that CRF each particular portion of the olivocerebellar tract including mRNA is transcribed in inferior olive neurons for translation neuronal cell bodies in the inferior olive, fibers in the medulla to CRF precursor, whereas CRF mRNA was depleted contra- oblongata and the cerebellar cortex, and presynaptic vari- lateral to the lesion. (ii) CRF-immunopositive fibers of the cosities of climbing fibers. Furthermore, we used in situ olivocerebellar tract were demonstrated ipsilateral to the hybridization histochemistry with a synthetic oligonucleotide transection due to an accumulation in the proximal portion of probe for CRF to demonstrate that CRF-immunoreactivity in transected axons. (iii) CRF-immunopositivity was demonstrat- the olivocerebellar system is indeed translated from CRF ed in climbing fibers in the molecular layer of the cerebellum mRNA there. at both light- and electron-microscopic levels. CRF-immunostained nerve terminals (climbing fiber varicosities) established synaptic contact with dendritic spines of the Purkinje cells. MATERIALS AND METHODS These three observations provide morphological evidence sug- Adult (200-250 g) male Sprague-Dawley (CD strain) rats gesting that CRF might serve as a neurotransmitter in the were used. The animals were kept on standard laboratory olivocerebellar system. conditions-24°C ± 1C with a dark-light cycle at 12/12 hr (light period from 6:00 AM) and dry laboratory food and tap The anatomical and functional connections between the water given ad libitum. In one group of rats, to visualize inferior olive and the cerebellum have been extensively immunostaining in neuronal perikarya, colchicine (120 ,ug/15 investigated during the past four decades. A great variety of ,u1 of physiological saline solution) was injected under ether techniques have revealed a crossed olivocerebellar projec- anesthesia into the lateral ventricle 48 hr before decapitation. tion in which every individual cerebellar cortical lobule Surgical Transection of the Olivocerebellar Tract. Under receives input from different and discrete nuclei of the ether anesthesia the head of the animal was stereotactically inferior olive complex (1-5). The olivocerebellar fibers enter fixed in a maximal nose-down position. Unilateral 1.5-mm the cerebellum through the inferior cerebellar peduncle, then sagittal knife incisions were made 1 mm lateral to the midline fan out as climbing fibers in the cerebellar white matter and at the level of the obex. A "glass knife" made from a provide terminals in the molecular layer synapsing there with histological coverslip was used for these transections. The dendrites of the Purkinje cells. It has been clearly demon- knife incisions reached the ventral surface of the medulla strated by nerve degeneration techniques following lesioning oblongatajust lateral to the inferior olive (Fig. 4a). Animals ofthe inferior olive (6-8), by autoradiography (9-12), as well were decapitated on the third postoperative day. as by an electrophysiological technique (2) that the inferior Light Microscopic Immunohistochemistry. Under Nembu- olive is the major, probably the sole significant, source of tal anesthesia (50 mg/kg) animals were perfused transaorti- cerebellar climbing fibers. Recent tract-tracing studies (13, cally with a fixative containing 4% paraformaldehyde and 1% 14) indicate that the olivocerebellar tract is exclusively a picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Each contralateral projection. brain was removed, and the medulla oblongata and cerebel- Little is known about the neurotransmitters in the olivo- lum were excised and placed in ice-cold fixative for an cerebellar system. Some studies have already implicated additional 90 min before being transferred to phosphate aspartate as a climbing fiber transmitter (10, 15). Recently, buffer and stored at 4°C overnight. Coronal sections of40-,um corticotropin-releasing factor (CRF)-like immunoreactivity thickness were cut from the caudal half of the medulla has been reported in neuronal perikarya of inferior olive in oblongata with a Vibratome (Oxford, England). Prior to the immunostaining, sections were rinsed for at least 6 hr in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: CRF, corticotropin-releasing factor or CRF-like- in accordance with 18 U.S.C. §1734 solely to indicate this fact. releasing factor.

Downloaded by guest on September 29, 2021 3911 3912 Neurobiology: Palkovits et al. Proc. Natl. Acad. Sci. USA 84 (1987) several changes of phosphate buffer and freeze-thaw treated examination of the sections. Selected sections were reem- in 0.5 ml of 10o sucrose in phosphate buffer to enhance the bedded in Araldite for electron microscopy. Ultrathin sec- antibody penetration. The avidin-biotin-peroxidase (ABC) tions, collected on Formwar-coated single-slot grids were technique (28) was applied on free floating sections. Sections examined in a Hitachi HU-12 electron microscope. were incubated in rabbit anti-ovine CRF in 1:10,000 dilution Antibody Specificity and Reactivity. The reactivity and titer with phosphate buffer containing 1% normal sheep serum and of antiserum (TG 84/951) raised against ovine hypothalamic 0.01% sodium azide for 48 hr at 40C, and then in avidin- CRF were determined by the microplate ELISA method: (i) biotin-peroxidase complex diluted with phosphate buffer Individual microtiter plate wells were coated with 0.1 ,g of (1:250) at 40C. Following each incubation step sections were one of the following peptides-oxytocin, Arg-vasopressin, washed for 4 x 15 min in phosphate buffer. The reagents were urotensin II, angiotensin II, cholecystokinin-8, bradykinin, purchased from Vector Laboratories (Burlingame, CA). The leu- and met-enkephalin, adrenocorticotrophic hormone (1- tissue-bound peroxidase was visualized with a diaminoben- 24) and (18-39), a-melanocyte-stimulating hormone, sub- zidine method (14.5 mg of 3,3'-diaminobenzidine, 165 1Ul of stance P, neurotensin, somatostatin-14 and -28 (1-14), neu- 0.3% H202 in 50 ml of phosphate buffer for 7-10 min). The ropeptide Y, vasoactive intestinal peptide, PHI-27 (a peptide sections were placed on gelatin-coated slides, dried, dehy- with NH2-terminal histidine and COOH-terminal isoleucine, drated, and mounted with plastic mounting medium. Some of 27 amino acids in length), luteinizing hormone-releasing the sections were counterstained with 0.05% toluidine blue hormone, thyrotropin-releasing hormone, hpGRF (human for a few sec to visualize Purkinje dendrites. pancreatic growth hormone-releasing factor), and ovine Electron-Microscopic Immunocytochemistry. Anesthetized CRF. (ii) Serial dilutions (0.5-128 x 1000) of CRF antiserum rats were perfused with 50 ml ofphysiological saline that was were applied to the plate wells with 0.1 ,ug ofovine CRF. The followed by a fixative of4% paraformaldehyde/15% saturat- antiserum was highly specific for ovine CRF and no crossed picric acid/0.1 M phosphate buffer, pH 7.35. After 30 min reactivity was observed with the other peptides investigated. of perfusion each brain was removed from the skull, and the The specificity of the immunostaining was tested by a cerebellum was dissected away and postfixed in the same liquid-phase preabsorption of the primary antibody with fixative without glutaraldehyde overnight. Sections were cut ovine CRF (10 ,ug of ovine CRF dissolved in 0.05 M acetic and immunostained as for light microscopy until their incu- acid was added to 1 ml of primary antibody diluted 1:10,000 bation in the diaminobenzidine/H202/phosphate buffer so- as before for 24 hr at 4°C). The preabsorption resulted in a lution. Thereafter, sections were osmicated (1% OS02 in complete loss of immunostaining as did omitting the second phosphate buffer for 30 min), dehydrated in graded ethanols or third antibodies from the procedure. (1% uranyl acetate was added only to the 70% ethanol step for In Situ Hybridization Histochemistry. Three male rats were 30 min), and flat-embedded (21) allowing light microscopic lesioned three days before sacrifice. The tissues were pre- a b .. _

*: F. .... w w _ * M fl sis s s g '. w t Sx - , *.k...... t

.s_ .. > 3'

.: .: $, E. 9, ... #.#u- ... -- 5K <= '&:. s. . e.

#: ,: .K::. 4,}i '. .. b^.'s ie- :t.-k'r e. - 2F 4 .,. a: t t : .. 31iLl ^s _ ;,s5 ]. Ffy_ _ 5s4 .. ..

:.;. w aX,*-^jap XFP wFer '.x ..... Y .... w l w l a ^*. : ..Bi .sT ": :! '':'3;R -' '' ''''' t ,,;Z x: ., g c d e

. .I. / teji.. -, .z j.>t

Al, .A-' 'r .' .. gS*.:ssT /-s# \' S . t .,.,X! tb*.*gv rw * X .... *W..a; ' mz ^qef.rtF;ffi;a *Wps: e.F;s SeFSr eS. | _,, b.. FIG. 2. CRF-immunostained Vibratome sections from the vermis (a and b) and from hemispheres (c-e) with toluidine blue counter- staining (a-c) or flat-embedded (d and e). Close topographical FIG. 1. (a) CRF-like immunoreactive neuronal cell bodies in the relationships are demonstrated between immunostained varicose principal olive nucleus in colchicine-treated rats. (b) Detection of climbing fibers (fine line of dots indicated by arrows) and counter- CRF mRNA over cells from the same part ofthe nucleus. (Bar = 100 stained Purkinje dendrites. M, molecular layer; P, Purkinje cell layer. am.) (Bar = lm.)100 Downloaded by guest on September 29, 2021 Neurobiology: Palkovits et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3913 pared for in situ hybridization histochemistry as described then dried and apposed to emulsion-coated coverslips. The (22). Briefly, the animals were anesthetized with ether and autoradiograms were exposed for 4 days and then developed perfused with 4% formaldehyde within 1 min of sacrifice. The as previously described (24). Some alternate sections were brains were frozen onto brass chucks, and 12-tkm thick not processed for in situ hybridization but were, instead, used sections were cut and thaw-mounted onto gelatin-coated to visualize CRF-immunostained cell bodies in the inferior slides. The sections were prepared for hybridization by olive and also to confirm the knife incision. The specificity of treatment with acetic anhydride and a series of ethanols and the hybridization has been described (22, 25). chloroform for delipidation. The hybridization buffer con- tained 1.3 x 106 dpm of the 48-base oligodeoxynucleotide RESULTS probe that was complementary to amino acids 22-37 of rat CRF proper (23) and was labeled to a specific activity of8500 The inferior olive is composed of the principal olive, the Ci/mmol (1 Ci = 37 GBq) with [35S]dAMP (22). This buffer dorsal and medial accessory olivary nuclei, as well as was applied to each section and incubated at room temper- additional minor subdivisions-the dorsal cap, the nucleus ature for 22 hr; the sections were then washed with 50% beta, the ventrolateral outgrowth, and the dorsomedial cell formaldehyde/2 x SSC (lx SSC = 0.15 M sodium chloride/ column. Each of these nuclei and subdivisions contain 0.015 M sodium citrate, pH 7.2) for 60 min at 40'C and then CRF-immunostained perikarya in colchicine-treated rats. with 2 x SSC for 3 hr at room temperature. The sections were The strongest accumulation of immunostained material was

-U

At * . A t F i As' s';s

V -.

i,; iA

i d ::,~ ._ ,ea

- *: ~~;7 .I"'mVI

FIG. 3. Electron micrographs from the middle third of the molecular layer of the vermis (a and b) and hemispheres (c-e) immunostained for CRF. Immunoreactive boutons are located along Purkinje dendrites (D) and synapse with dendritic spines (*). Note that e demonstrates the connections (arrow) of a postsynaptic spine (star) to the shaft of a Purkinje dendrite. Frequently, immunolabeled boutons establish synaptic connections with two spines (a and c). (Bar = 1 tLm.) Downloaded by guest on September 29, 2021 3914 Neurobiology: Palkovits et al. Proc. Natl. Acad. Sci. USA 84 (1987) observed in the principal olivary nucleus (Fig. la). CRF cell Three days after a sagittal transection of the medulla body immunostaining was seen in the solitary tract nucleus oblongata at the lateral tip of the inferior olive complex (Fig. but not in the cerebellum. The inferior olivary complex was 4a), CRF-immunopositive material accumulated in fibers readily visualized by in situ hybridization histochemistry medial to the knife incision (Fig. 4b). Topographically, fibers using the CRF oligodeoxynucleotide probe. Accumulation of that appear subsequent to the knife incision corresponded grains was detected over perikarya both in the principal (Fig. exactly to those that constitute the ventral medullary portion lb) and accessory olivary nuclei. Labeled neurons were also ofthe olivocerebellar tract. Heavy immunostaining appeared detected in the nucleus of the solitary tract and occasionally in inferior olive cells contralateral to the lesion (Fig. 4c). in the catecholaminergic cell group region. Immunolabeled perikarya were present in each major nuclei Fine dotted lines of CRF immunoreactivity indicated the and subunits of the inferior olive except the nucleus beta. No climbing fibers along the Purkinje cell dendrites in the immunostaining appeared in the ipsilateral inferior olive nor molecular layer of the cerebellar cortex (Fig. 2). Immunore- in cells elsewhere in the medulla oblongata. The unilateral active axons and varicosities could be followed up to the knife incision resulted in a contralateral decrease in CRF external third of the molecular layer. No differences were mRNA copies (Fig. 4d). This effect was more pronounced recognized between cerebellar cortical folia from the vermis over the principal olivary nucleus and in the dorsomedial cell or hemispheres in staining intensity, distribution pattern, or column on the side opposite the lesion than ipsilaterally. density of immunoreactive axons. Neither cortical nor medullary elements other than climbing fibers were immuno- DISCUSSION stained for CRF. In ultrathin sections, several section profiles of immunoreactive boutons of 0.6- to 1.5-gum diameter were Light and electron-microscopic immunohistochemistry, as observed in the molecular layer. Immunostained presynaptic well as in situ hybridization histochemistry, both demon- profiles were characteristic of climbing fiber varicosities. strated CRF in the olivocerebellar system in rats. Although They were densely packed with spheric agranular synaptic CRF immunoreactivity in neuronal perikarya has been dem- vesicles and established synaptic connections exclusively onstrated in the inferior olivary nuclei in rat (16, 25), cat (17), with unstained dendritic spines (Fig. 3). Frequently, im- baboon (25), and human (25) brains, and in presumptive munostained varicosities were in contact with more than one climbing fibers in the rat cerebellum (17, 20), the presence of spine (Fig. 3 a and c). These spines had short stalks with this neuropeptide in the olivocerebellar system had not been tubules of the endoplasmic reticulum and synaptic contact proven. In the present study, CRF has been demonstrated in with climbing axons on the side but not on the tip oftheir head each portion of this system: in neuronal perikarya of the (Fig. 3e). No synaptic connections of immunoreactive bou- inferior olive, in nerve fibers ofthe olivocerebellar tract in the tons were found on dendritic shafts. medulla oblongata, in climbing fibers in the molecular layer C

FIG. 4. (a) Coronal section of the medulla oblongata indicating the location of the knife incision. Al, catecholaminergic cell group; c, parvicellular reticular nucleus; 10, inferior olive; nts, nucleus of the solitary tract; P, pyramidal tract; ro, raphe obscurus; th, nucleus prepositus hypoglossi; tsV, spinal tract ofthe trigeminal nerve; V, vermis. (b) Accumulation of CRF-immunostained material in fibers of the olivocerebellar tract proximal to the knife incision (*). The size and topography of this enlarged region are indicated on a by the small rectangle. (Bar, 100 g.m.) (c) CRF-immunostained neurons in the inferior olive contralateral to the knife incision. (d) The level of CRF mRNA is decreased on the contralateral (right) side, especially over the dorsomedial cell column and the principal olive nucleus. c and d correspond to an area indicated by the large rectangle on a. Arrows indicate the midline, and a star marks the knife incision. P, pyramidal tract. (Bar = 500 gm.) Downloaded by guest on September 29, 2021 Neurobiology: Palkovits et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3915

of the cerebellar cortex, and, finally, in nerve terminals of The technical assistance of Maijorie Warden and Christina G. climbing fibers synapsing with dendritic spines of the Pur- Palkovits (National Institute of Mental Health) is greatly appreciat- kinje cells. ed. We also thank Larry Ostby for photographic assistance. This work is partly supported by Grant 1 PO NS 220701 (C.L.) from the Accumulation of CRF in neurons of the inferior olive after Andrew Mellon Foundation in Reproductive Neurobiology. transection of the olivocerebellar tract contralateral to the lesion and retrograde accumulation in fibers proximal to the 1. Brodal, A. (1940) Z. Ges. Neurol. Psychiat. 169, 1-153. knife cuts confirm unequivocally that CRF is transported into 2. Eccles, J. C., Ito, M. & Szentigothai, J. (1967) The Cerebel- the cerebellum via the olivocerebellar tract. On the basis of lum as a Neuronal Machine (Springer, Heidelberg). the present in situ hybridization histochemistry it can be 3. Palay, S. L. & Chan-Palay, V. (1974) Cerebellar Cortex (Spring- concluded that the CRF immunoreactivity shown in the er, New York). olivocerebellar system is translated from CRF mRNA in the 4. Brodal, A. & Kawamura, K. (1980) Adv. Anat. Embryol. Cell inferior olive. The drop in CRF mRNA levels in cells, the Biol. 64, 1-140. axons of which were cut, is similar to the effect of peripheral 5. Voogd, J. & Bigare, F. (1980) in The Inferior Olivary Nucleus: Anatomy and Physiology, eds. Courville, J., de Montigny, C. axotomy on neurofilament mRNA (26) and may be the direct & Lamarre, Y. (Raven, New York), pp. 207-234. result of injury to the cells themselves with, perhaps, an 6. Szent~gothai, J. & Rajkovits, K. (1959) Z. Anat. Entwicklungs- increase in RNase activity. Another possibility is that the gesch. 121, 130-141. lesion interrupted a positive feedback circuit. 7. Grant, G. (1970) Brain Res. 22, 236-242. Strong accumulation ofCRF immunostaining in a bundle of 8. Desclin, J. C. (1974) Brain Res. 77, 365-384. transected fibers and in many contralateral inferior olive cells 9. Groenewegen, H. J. & Voogd, J. (1977) J. Comp. Neurol. 174, suggests that CRF is generally present in the olivocerebellar 417-488. tract rather than located in only a part of it. The im- 10. Wiklund, L., Toggenburger, G. & Cudnod, M. (1982) Science munostained olivocerebellar system in lesioned animals 216, 78-80. could be followed along the surface of the medulla ventral to 11. Campbell, N. C. & Armstrong, D. M. (1983a) Brain Res. 275, the inferior olive and within the lateral reticular nucleus, as 215-233. well-exactly as these two branches of olivocerebellar tract 12. Campbell, N. C. & Armstrong, D. M. (1983) Brain Res. 275, in intact (11, 12, 15). It is 235-249. have been described animals 13. Eisenman, L. M. (1981) J. Comp. Neurol. 199, 65-76. generally accepted that the exclusive sources of climbing 14. Furber, S. E. & Watson, C. R. R. (1983) Brain Behav. Evol. fibers are the inferior olivary neurons (6-8). This fact and the 22, 132-152. present observation that almost all inferior olive cells con- 15. Wiklund, L., Toggenburger, G. & Cudnod, M. (1984) Neuro- tralateral to the lesion were immunostained for CRF indicate science 13, 441-468. that not only a certain portion but presumably all of the 16. Schipper, J., Steinbusch, H. W. M., Vermes, I. & Tilders, climbing fibers contain CRF. F. J. H. (1983) Brain Res. 267, 145-150. The climbing fibers have been recognized as important 17. Cummings, S., Elde, R. & Sharp, B. (1985) Soc. Neurosci. neuronal afferents having excitatory effects upon the Pur- Abst. 11, 683. kinje cells (2). Little is known about the neurotransmitters in 18. Palkovits, M., Brownstein, M. J. & Vale, W. (1985) Fed. Proc. climbing fibers. Because a specific uptake of D-[3H]aspartate Fed. Am. Soc. Exp. Biol. 44, 215-219. by the olivocerebellar tract has been observed (15) and a 19. Palkovits, M., Brownstein, M. J. & Vale, W. (1983) Neuroen- decrease of aspartate has been reported in the cerebellum docrinology 37, 302-305. after 6-acetylpyridine-induced destruction of the inferior 20. Merchenthaler, I., Vigh, S., Petrusz, P. & Schally, A. V. olive (10), aspartate has been implicated as one of the (1982) Am. J. Anat. 165, 385-396. neurotransmitters in the olivocerebellar system. Although 21. Ldrdnth, Cs. & Fehdr, E. (1983) Neuroscience 10, 947-958. the presence of certain neuropeptides [somatostatin, meten- 22. Young, W. S., III, Mezey, L. & Siegel, R. E. (1986) Neurosci. kephalin, substance P, phenylalanylmethionylargininylphen- Lett. 70, 198-203. ylalanylamide (FMRF)amide] in cells scattered over the 23. Jingami, H., Mizuno, N., Takahashi, H., Shibahara, S., & FEBS Lett. 192, inferior olive has been reported on the basis of immunohis- Furutani, Y., Imura, H. Numa, S. (1985) 63-66. tochemistry (27), none of these neuropeptides has been 24. Young, W. S., III, & Kuhar, M. J. (1979) Brain Res. 179, demonstrated in the olivocerebellar tract or confirmed as a 255-270. neurotransmitter in this system. CRF, which is a prominent 25. Young, W. S., III, Walker, L. C., Powers, L. E., DeSouza, member of the hypothalamic-hypophysial system, is widely E. B. & Price, D. L. (1986) Mol. Brain Res. 387, 189-192. distributed in the central nervous system (27) and may have 26. Hoffman, P. N., Cleveland, D. W., Griffin, J. W., Landes, a neurotransmitter role in addition to its well-known effect on P. W., Cowan, N. J. & Price, D. L. (1987) Proc. Natl. Acad. the anterior pituitary. Indeed, the demonstration of CRF in Sci. USA 84, 3472-3476. the olivocerebellar system (in cells, axons, and nerve termi- 27. Palkovits, M. (1987) in Peptide Hormones: Effects and Mech- nals) is morphological evidence indicating that CRF might, in anisms ofAction, eds. Negro-Vilar, A. & Conn, P. M. (CRC, fact, serve as a neurotransmitter in the central nervous Boca Raton, FL), in press. system. 28. Hsu, S. M. & Ree, H. J. (1980) Am. J. Clin. Pathol. 74, 32-40. Downloaded by guest on September 29, 2021