Coesxistence of corticotropin releasing factor and enkephalin in cerebellar afferent systems

SYNAPSE 5:167-174 (1990)

Coexistence of Corticotrox>in Releasing Factor and Enkephalin I’n Cerebella;

Merent Systems SHARON CUMMINGS AND JAMES S. KING

Department of Anatomy and Neuroscience Program, The Ohio State University, Columbus, Ohio 4321 0

KEY WORDS Cerebellum, Inferior olive, Immunohistochemistry, Climbing fibers, Mossy fibers

ABSTRACT In this study, we tested the hypothesis that corticotropin-releasing factor (CRF) and enkephalin (ENK) coexist within restricted brainstem-cerebellar circuits. Antisera for CRF and ENK were applied simultaneously or to serial sections of adult opossum brain and were processed for fluorescence or peroxidase, antiperoxidase immunohistochemistry , respectively. CRF and ENK were colocalized in 1) climbing fibers within the flocculus and in bilateral foci in the cerebellar vermis; 2) fibers with morphological characteristics of simple mossy fiber terminals or climbing fiber glomeruli that were located within the granule cell layer of the vermis underlying the foci of CRFmNK-IR climbing fibers; 3) mossy fiber terminals within the flocculus; 4) neuronal perikarya in subnucleus A and C of the medial accessory olive and in the dorsal cap of Kooy, nuclei known to project as climbing fibers; and 5 ) cell bodies in nucleus prepositus, the subtrigeminal reticular nucleus, and the reticular formation, likely origins of CRF/ENK colocalized mossy fibers. The demonstration that single climbing and mossy fibers contain two peptides extends previous studies that have described chemical heterogeneity within cerebellar afferent pathways. Furthermore, these results support suggestions that this heterogeneity may provide a substrate for differential regulation of signal transduction by chemically coded cerebellar afferents.

INTRODUCTION Eighty years ago, Ramon y Cajal(1909) described the

histological organization of the cerebellum, a structural circuitry now known as an important substrate for the coordination of movements and postural adjustments. However, investigators have only begun to identify the chemical mediators of the afferent signals in these circuits integral to motor control. Studies have implicated aspartate (Clements et al., 1986, Wiklund et al., 1982) and glutamate (Beitz et al., 1986; Clements et al., 1986) as possible transmitters in climbing and mossy fibers. Recently the distributions of the peptides corticotropin-releasing factor (CRF) and enkephalin (ENK) have been delineated within the major cerebellar afferent systems in the adult opossum (Cummings et al., 1989, King et al., 1986). Each of these peptides is present within climbing fibers, cerebellar afferents derived from the inferior olivary complex (Desclin, 19741, and mossy fibers, afferents derived from multiple nuclei of the neuraxis. The cortical distribution patterns of CRF- and ENK-immunoreactive (IR) climbing and mossy fiber populations are unique. CRF is ubiquitous within the opossum cerebellar cortex; CRF-IR climbing and mossy fibers may be found in all lobules, although the density of fibers and the level of peptide within terminal arbors varies. The distribution patterns of ENK-IR climbing and mossy fiber populations are more restricted than those containing CRF. ENK-coded affer- 0 1990 WILEY-LISS, INC.

ents are confined largely to the flocculus and focal regions of the vermis.

The distributions of CRF-IR and ENK-IR axons over- lap in restricted regions of the cerebellar cortex, and neuronal perikarya immunoreactive for CRF or ENK are found in several brainstem nuclei known to project to the cerebellum (Cummings et al., 1989; Gould, 1980; King et al., 1987). These peptides have been colocalized within the hypothalamic-pituitary axis, in cell bodies within the hypothalamic paraventricular nucleus (Hokfelt et al., 19831, and in secretory granules within the median eminence (Hisano et al., 1987). To our knowledge, there have been no reports of colocalization of CRF and ENK other than in the hypothalamus. Therefore, the current investigation was undertaken to address the possibility of coexistence of CRF and ENK within cerebellar afferent projection fibers and within brainstem cell bodies that give rise to climbing fibers or mossy fibers.

MATERIALS AND METHODS Colocalization of CRF and ENK in brainstem

Adult or juvenile opossums (n = 7) obtained from the wild or bred at the Ohio State University were used in our experiments. Animals were anesthetized with so-

Received May 10,1989; accepted in revised form August 28,1989

168 S. CUMMINGS AND J.S. KING

Figure 1.

169 CRF AND ENK COEXISTENCE

dium pentobarbital (40 mglkg; ip). While under anesthesia, some animals (n = 3) were secured in a stereo- taxic frame and injections of colchicine (10 pg/pl saline; 0.2-0.4 plhite) were placed stereotaxically into the brainstem 48 hr prior to sacrifice. After anesthesia, all animals were perfused transcardially with 1 liter of normal saline followed by 2 liters of picric acid/ phosphate (0.1 M)-buffered 2% paraformaldehyde fixative (pH 7.3) (Stefanini et al., 1967) at room tempera- ture. The brains were removed, immersed in the same fixative for 90 min, and stored overnight in 15% sucrose in 0.1 M Sorenson’s phosphate buffer, pH 7.3.

The brainstems of colchicine-treated animals were sectioned and processed for CRF and ENK immunohistochemistry by one of two methods. In the first, the brainstem was sectioned at 30 p,m on a sliding micro- tome, and sections were incubated free-floating in a solution containing the primary antiserum against rat/ human CRF (1/200) generated in rabbits (a gift from Dr. Burt Sharp, MinnesotaMedicalFoundation) andamono- clonal enkephalin antibody (MAS083, 1/200, Sera-Lab) generated in mice. Subsequently these sections were incubated in a solution containing secondary antibodies generated against either goat or mouse IgG and conju- gated to the fluorochromes fluorescein isothiocyanate (FITC) or lissamine rhodamine isothiocyanate (LRITC). Immunoreactive neuronal structures were visualized with a Zeiss standard fluorescence microscope fitted with epiillumination, using appropriate filter combinations (FITC: a 450490 nm filter was used to produce blue excitation, and the emission observed was limited to the green by a 520nm long-pass barrier filter; LRITC: a 546nm filter was used to produce green excitation, and the emission observed was limited to the red by a 590 nm long-pass barrier filter). Thus tissue sections incubated with FITC-labeled goat antibody to rabbit IgG (1/100; Antibodies Inc.) in combination with LRITC-labeled goat antibody to mouse IgG (1/100; Tago) demonstrated green-fluorescing CRF-IR and red- fluorescing ENK-IR neurons (Figs. 1 and 2).

The cerebelli of normal adult or juvenile opossums (n = 4) were sectioned in transverse or sagittal planes at 15 pm in a cryostat and incubated for 48 hr with a combination of primar antibodies as described above (rabbit antirathuman ZRF, 1/200; MAS083 mouse anti- ENK monoclonal, 1/100). Subsequently, these sections

Fig. 1. Immunofluorescence photomicrographs of opossum cerebellum (A,B,E-H) and subnucleus A of the medial accessory olivary nucleus (C,D) after incubation with the combination of an antiserum to rat CRF and a monoclonal antibody to ENK. Solid arrows indicate structures in which CRF (A,C,E,G) and ENK (B,D,F,H) are coexistent. Open arrows indicate the location of cells immunoreactive for CRF (C) but not for ENK (D). A,B: Climbing fibers (large arrows) and mossy fiber terminals (small arrows) at the base of lobule VII demonstrate immunoreactivity for both CRF and ENK when viewed with illumination appropriate for fluorescein (A)- or rhodamine (B)-labeled secondary antibodies. ML, molecular layer; PL, Purkinje cell layer; GL, granule cell layer. C,D Numerous cell bodies immunoreactive for CRF (C, solid arrows) are present within subnucleus A of the medial accessory olive. A subpopulation of these neurons also are immunoreactive for ENK (D, solid arrows). However, the majority of perikarya within the inferior olivary complex are immunoreactive for CRF (C, open arrows) but not ENK (D, open arrows). E-H: CRF (E,G) and ENK (F,H) coexist in mossy fiber terminals within the granule cell layer at the base of lobule VII. The bar in A = 25 pm and also applies to B. The bar in C = 25 pm and also applies to D. The bar in E = 25 pm and also applies to F-H.

also were processed for fluorescence immunohistochemistry with secondary antibodies of appropriate species, in order to visualize CRF- and/or ENK-IR fibers within a single section (Figs. 1 and 2).

In a second method, the brainstem was sectioned serially a t 5 pm in a cryostat and alternate sections were incubated with primary antiserum to met-ENK (156C, 1/500) (a gift from Dr. Robert Elde, University of Minnesota) or to rathuman CRF (1/500) and processed by the peroxidase-antiperoxidase (PAP) method of Sternberger (1979), with some modifications (Cum- mings et al., 1983, 1989). Immunolabeled cell bodies were drawn on transparent paper with the aid of a drawing tube attached to a microscope. Superimposi- tion of drawings of adjacent sections revealed cell bodies immunostained for both peptides. Further, comparisons of photogra hs of adjacent sections confirmed locations of cells in wRich CRF and ENK were colocalized (Fig. 3).

Antisera specificity The enkephalin antiserum 156C from Dr. Elde and

the rat CRF antiserum have been described previously (Cummings et al., 1988; Holets and Elde, 1982) and analyzed in the opossum in this laboratory (Cummings et al., 1989; King et al., 1986). Antibody MAS083 used for the fluorescence study cross reacts with leu- and met-enkephalin. Therefore, this antibody was used as a marker for ENK-like IR to confirm results of the PAP study in the brainstem and as a marker for ENK-like IR in cerebellar fibers. For brevity, the immunostaining of antiserum 156C and of monoclonal antibody MAS083 will be referred to as ENK-IR.

Since CRF and ENK antibodies, as well as secondary antibodies, were used in combination, additional con- trols for antiserum specificity were performed to confirm absence of affinity of primary antisera for the inappropriate antigen, absence of affinity of the secondary antisera for the inappropriate primary antiserum, absence of affinity of the primary antisera for each other, absence of affinity of the secondary antisera for each other, and absence of affinity of the secondary antibodies for the CRF and ENK antigens (see also Wessendorf and Elde, 1985). Effectiveness of the cut-off filters also was confirmed. Two areas of the medulla, the dorsal motor nucleus of the vagus nerve and nucleus raphe obscurus, also were examined for each of the above parameters as well as for complete separation of CRF- and ENK-IRs. In the opossum, neuronal cell bodies within the vagal nucleus contain high levels of CRF mRNA (Cummings et al., 1989) and immunostain intensely for CRF (Figs. 2 and 3), although few CRF-IR fibers are evident. A dense plexus of ENK-IR fibers consistently surrounds the CRF-IR perikarya within the vagal nucleus (Figs. 2 and 3), but ENK-IR cell bodies never have been observed there. Thus, for example, under blue illumination, intense green CRF-immunofluorescence is present in cell bodies of this nucleus; under green illumination a dense plexus of red ENK- immunofluorescent fibers surround nonfluorescing CRF-IR cell bodies (Figs. 2 and 3).

Within the raphe nuclei, particularly raphe obscurus and raphe magnus, the disposition of CRF-IR and ENK- IR cell bodies was remarkable in that the peptide-coded somata were intercalated like pieces of a puzzle, but never overlapped. The complete separation of peptide


Figure 2.

CRF AND ENK COEXISTENCE 171

IRs within the contiguous cell bodies of these nuclei served as additional confirmation of the specificity of the antibodies used in this study.

RESULTS Coexistence of CRF and ENK in the cerebellum Results of the present study have demonstrated that

CRF and ENK coexist within climbing fibers and terminals with the appearance of simple mossy fibers in restricted regions of the cerebellar vermis and within climbing and mossy fibers within the flocculus. In confirmation of previous studies (Cummings et al., 1989; Kinget al., 1986), CRF-IR climbing and mossy fibers are present in greater numbers than ENK-IR fibers within the opossum cerebellum. However, in transverse sections, dense aggregates of climbing fibers intensely immunoreactive for either CRF or ENK are prominent in a single pair of similarly located sagittal foci within lobules TI, IV, V, VII, VIII, and X of the vermis. CRF and ENK were coexistent in the majority of the climbing fibers within these restricted foci. However, in each focus, individual fibers also were present that contained only ENK or only CRF. In addition, in some instances, ENK-IR did not appear to distribute throughout a complete climbing fiber arbor delineated by CRF but was apparent only within the wider diameter primary and secondary branches. The most extensive population of coexistent climbing fibers in the vermis was located within the transition area between lobules VII and VIII. In serial transverse tissue sections, this population shifted laterally from a vermal position to the base of the paramedian lobule. Striking examples of climbing fibers immunoreactive for both CRF and ENK also were prominent within the lateral aspects of vermal lobule X and the flocculus, particularly at the ventral base of the flocculus.

Numerous mossy fiber terminals immunoreactive for CRF were present within the granule cell layer of all vermal lobules. Fewer mossy fiber terminals were immunoreactive for ENK alone. Fibers containing both CRF and ENK also were discernible in the granule cell layer of lobules 11, IV, V, VII, and X, underlying the parasagittal foci of immunoreactive climbing fibers. Enlargements or varicosities occurred at intervals along these fibers. These expansions were similar in appearance to simple mossy fiber terminals as de-

Fig. 2. Photomicrographs of three (A,B; C,D; E,F) 30 pm transverse sections through the opossum brainstem that were incubated simultaneously with CRF and ENK antisera. Panels demonstrate CRF (left column) and ENK (right column) immunofluorescent neuronal cell bodies and fibers (solid straight arrows). Open arrows indicate cell bodies immunoreactive for CRF but not ENK. A,B: Several neuronal cell bodies within nucleus prepositus (NP) are immunoreactive for CRF (A). Within the same section, one cell body also demonstrates ENK immunoreactivity (B). Curved arrows demonstrate CRF-IR somata within the dorsal motor nucleus of the vagus nerve (X) surrounded by a dense plexus of ENK-IR fibers and terminals and demonstrates separation of CRF and ENK immunostaining. C,D A single section demonstrating a cell within the medullary reticular formation in which CRF and ENK coexist (solid arrows). A second cell is immunoreactive for CRF but not ENK (open arrows). E , F CRF and ENK coexist in varicose fibers within a bundle ventral to the lateral reticular nucleus, near the ventral surface of the medulla. Fibers in this region likely are projections destined for the inferior cerebellar peduncle. Arrowheads in lower right corners indicate ventral. The bar in A = 50 pm and also applies to B, E and F. The bar in C = 50 pm and also applies to D.

scribed in Golgi preparations by Palay and Chan-Palay (1974) and/or to climbing fiber glomeruli (Chan-Palay and Palay, 1971). In these regions, highly complex mossy fiber rosettes never were observed to cocontain CRF and ENK, even though such terminals immunoreactive for CRF alone often were visible within the same microscopic field of view.

Within the flocculus, mossy fiber terminals of both simple and complex morphology exhibited immunoreactivity for CRF alone or for ENK alone or demonstrated colocalization of CRF and ENK. However, there were fewer mossy fibers demonstrating coexistence of the peptides than rosettes immunoreactive for only CRF or ENK.

In the adult, CRF and ENK also coexist within beaded fibers within the inferior cerebellar peduncle and den- tate, interpositus, and fastigial nuclei. Within the deep nuclei, the number of fibers that contained only one of the peptides greatly exceeded those in which there was coexistence.

Coexistence of CRF and ENK in precerebellar nuclei

Examination of alternate sections of brainstem tissue from opossums pretreated with colchicine and processed for CRF or ENK peroxidase immunohistochemistry as well as tissue sections incubated simultaneously with primary antisera to both peptides and processed for fluorescence immunohistochemistry revealed that CRF and ENK are coexistent within cell bodies of the inferior olivary complex. The majority of inferior olivary perikarya are immunoreactive for CRF (Cummings et al., 19891, whereas ENK-IR cell bodies appear to be restricted to subnuclei A and C of the medial accessory olive (MAO) and the dorsal cap of Kooy (King et al., 1987). The largest number of ENK-IR somata occurs in subnucleus A of MAO. The greatest population of coexistent neuronal perikarya also occurred within subnucleus A (Fig. 1); within this region each cell body immunoreactive for ENK also contained CRF. The small number of cell bodies immunoreactive for ENK within subnucleus C and the dorsal cap also were immunoreactive for CRF. No perikarya immunoreactive for ENK alone were observed in the inferior olivary complex.

Of the precerebellar nuclei known to give rise to mossy fibers (Gould 1980; Walker et al., 1988), CRF-IR and EN#-IR cell populations are found simultaneously in the lateral and subtrigeminal reticular nuclei; gigan- tocellular reticular nucleus; perihypoglossal complex; medial vestibular nucleus; locus ceruleus; and nucleus raphe pallidus, obscurus, and magnus. Each of these nuclear regions contains somata immunoreactive for CRF alone and for ENK alone. In the present study, a small number of erikarya in which CRF and ENK were

lar nucleus and nucleus prepositus and scattered within the reticular formation (Fig. 2).

The intensity of CRF and ENK immunostaining in cell bodies in the brainstem varied within and among nuclei. For example, immunoreactive staining for ENK was consistently less intense within inferior olivary somata than within the raphe nuclei and the majority of ENK-IR reticular cells. The intensity of CRF-immunostaining varied among raphe and reticular cell bodies,

coexistent were f p ound within the subtrigeminal reticu-


Figure 3.

CRF AND ENK COEXISTENCE 173

and, as has been reported previously, varied among olivary perikarya (Cummings et al., 1989). In addition, immunostaining for each peptide consistently was less in those cells that demonstrated coexistence than in perikarya that contained either peptide alone.

DISCUSSION Neuronal systems coded by multiple transmitters are

being identified within the central and peripheral nervous systems with increasing frequency, particularly with the availability of monoclonal antibodies and antibodies generated in more than one animal species. We now report the existence of cerebellar afferents in which the neuropeptides CRF and ENK are colocalized. The climbing fiber projection from the medial accessory olive to rostra1 and caudal vermis is well documented, as is the projection from the dorsal cap of Kooy to the flocculus (see Brodal, 1980; Linauts and Martin, 1978; Voogd, 1982; Voogd and Bigare, 1980). Thus the established topography of olivocerebellar projections can be corre- lated with the findings of this study: The CRF/ENK climbing fiber foci within the vermis likely derive from subnucleus A of MAO; those within the flocculus likely are derived from the dorsal cap and MAO.

In this study, the majority of climbing fibers immunoreactive for ENK also were immunoreactive for CRF. A few scattered ENK-IR fibers did not appear to demonstrate colocalization. However, with fluorescence immunohistochemistry, inferior olivary somata immunoreactive for ENK alone never were observed. Whether isolated olivary cells exist that are immunoreactive for ENK alone, or whether the level of CRF immunoreactivity within those fibers that appeared to contain only ENK was too low to be visualized by present immunohistochemical techniques could not be determined. In the present study, the levels of CRF and ENK within those neurons that contained both peptides often appeared less than in those perikarya that contained either peptide alone. The ratios of CRF to ENK within coexistent neurons also appeared to vary. As reported, climbing fibers were detected in which ENK-IR could be observed only within the larger diameter branches. We suggest in these instances that ENK-IR product was not absent from terminal branches but likely was present in concentrations below the level of detection. Studies utilizing immunohistochemistry and in situ hybridization histochemistry have demonstrated that quantita- tive differences may exist in levels of a peptide or peptide mRNA among cells within a nucleus (see Cum- mings et al., 1989; Swanson et al., 1987). For example, within the normal opossum brainstem, hybridization for CRF mRNA is consistently less within subnucleus A

Fig. 3. Photomicrographs of peroxidase-labeled adjacent 5 pm transverse sections through the opossum medulla to demonstrate CRF-IR (left column) or ENK-IR (right column) neuronal structures. A,B: Numbered arrows indicate cell bodies within subnucleus C of the medial accessory olive in which CRF and ENK coexist. Arrowheads in upper left corners indicate medial. C,D: Higher magnifications of sections in panels A and B. E,F Photomicrographs of the dorsal motor nucleus ofthe vagus nerve within tissue sections near to those shown in A-D, to demonstrate separation of CRF and ENK immunostaining and specificity of antisera. Numbered arrows indicate cell bodies immunoreactive for CRF but not ENK. A dense plexus of fibers within this nucleus in immunoreactive for ENK(so1id short arrow) but not for CRF (open short arrow). The bar in A = 50 pm and also applies to B. The bar in C = 50 pm and also applies to D-F.

than in other areas of the MAO. Such variation in levels of peptide content between neurons may be a reflection of differences in levels or rates of mRNA transcription or translation, differences in degree to which cellular pro- tein synthesis is dedicated to production of a single VS. multiple peptides, differences in rates at which proteins are transported from perikarya into axonal processes, differences in volumes of cells captured within any single tissue section, or combinations of these factors.

Mossy fiber projections from the nucleus prepositus have been demonstrated to the cerebellar flocculus in several species (rabbits, Alley et al., 1975; cat, Gould, 1980; Kotchabhakdi et al., 1978). Likewise, there is a projection to the flocculus from the subtrigeminal reticular nucleus (rabbit, Alley et al., 1978; cat, Sat0 et al., 1983; rat, Blanks et al., 1983; monkey, Langer et al., 1985). Our data suggest that some mossy fiber projections from these nuclei contain both CRF and ENK.

Based on morphological features, the axonal profiles within the granule cell layer subjacent to the vermal bands containing CRF/ENK coexistent climbing fibers could be mossy fiber terminals or climbing fiber glomeruli. In earlier studies (Cummings et al., 1989; King et al., 19871, similar profiles analyzed in 60 pm sections processed for either ENK or CRF peroxidase immunoreactivity were identified as mossy fiber terminals. How- ever, comparison of our present data with the established topography of mossy fiber projections implicated no brainstem region containing CRF/ENK colocalized cells that had a cortical distribution restricted to single narrow sagittal bands. Therefore, we suggest that many of the fiber profiles visualized within the granular layer that cocontain CRF and ENK may be climbing fibers in passage through that layer, with enlarged varicosities that correspond to climbing fiber glomerular collaterals described originally from Golgi preparations in the rat (Chan-Palay and Palay, 1971; Palay and Chan-Palay, 1974).

Colocalization of putative neurotransmittersheuro- modulators within cell bodies and their axon terminals is not sufficient evidence for cotransmission. Thus the role of single-transmitter vs. multiple-transmitter coded circuits within the cerebellar cortex remains un- determined. Climbing fibers and mossy fibers are described classically as mediating excitatory effects di- rectly or indirectly on the sole efferent projecting cerebellar cortical neuron, the Purkinje cell. However, studies in this laboratory utilizing microiontophoretic extracellular application of CRF or ENK to single isolated units within the cerebellar vermis suggest that ENK may suppress Purkinje cell firing rates (G.A. Bishop, personal communication) and that CRF en- hances the effects of aspartate and glutamate on firing rates (Bishop, 1989). In other systems, CRF has been suggested to mediate excitatory physiological effects (Eberly et al., 1983; Valentino et al., 1983; Siggins et al., 1985) and ENK to mediate inhibitory, excitatory, or modulatory effects (see Nicoll et al., 1977; Schulman, 1981). Thus, despite the rigorous homogeneity evident in the structural geometry of the cerebellar neuronal networks, the results of this study indicate that these circuits are not chemically uniform; the chemical signa- tures of climbing and mossy fibers are heterogeneous. We suggest that such variation in chemical coding may provide the essential substrate for differential regula-


tion or modulation of the well defined cerebellar circuitry.

ACKNOWLEDGMENTS We thank Dr. Georgia A. Bishop for helpful comments

and discussion. We thank Dr. Robert Elde of the Univer- sity of Minnesota for enkephalin antiserum 156C, and Dr. Burt Sharp of the Minnesota Medical Foundation for the CRF antiserum. This study was supported by NIH grant NS 08798 (J.S.K.).

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Coesxistence of corticotropin releasing factor and enkephalin in cerebellar afferent systems

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