- Email: [email protected]
The site of origin of calcitonin gene-related peptide-like immunoreactive afferents to the inferior olivary complex of the mouse
Neuroscience Research 34 (1999) 177 – 186 www.elsevier.com/locate/neures
The site of origin of calcitonin gene-related peptide-like immunoreactive afferents to the inferior olivary complex of the mouse Amanda C. Peltier, Georgia A. Bishop * Department of Cell Biology, Neurobiology and Anatomy, Di6ision of Neuroscience and the College of Medicine, The Ohio State Uni6ersity, 333 W 10th A6enue, Columbus, OH 43210, USA Received 8 March 1999; accepted 23 April 1999
Abstract The intent of the present study is to define the brainstem nuclei which give rise to CGRP-immunolabeled afferents to the inferior olivary complex of the mouse. A technique which combines retrograde transport of fluorescent microspheres with immunohistochemistry was used to address this question. In the present study, intensely labeled CGRP neurons were localized within several cranial nerve nuclei including the hypoglossal, facial, oculomotor, motor nucleus of the trigeminal nerve and nucleus ambiguus, as well as in the parabrachial nucleus, locus coeruleus and medullary and pontine reticular formation. In addition, lightly labeled CGRP neurons were identified within the deep cerebellar nuclei, the inferior olivary complex, lateral reticular nucleus, medial and lateral vestibular nuclei, nucleus Darkschewitsch, interstitial nucleus of Cajal, the central gray area adjacent to the third ventricle, and the zona incerta. The origin of the projection to the inferior olivary complex primarily arises from the deep cerebellar nuclei, the locus coeruleus, and the central gray matter of the mesodiencephalic area. In addition, a small CGRP input is derived from the superior and lateral vestibular nuclei as well as the zona incerta. In conclusion, we have identified several extrinsic sources of CGRP to the inferior olivary complex and have localized it within afferents that have been shown to have either excitatory (mesodiencephalic nuclei) or inhibitory (cerebellar nuclei) effects on olivary circuits. The presence of CGRP in these functionally diverse brainstem and cerebellar afferents suggests that the peptide may act as a co-transmitter to modulate the activity of olivary neurons. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fluorescent microspheres; Locus coeruleus; Cerebellar nuclei; Interstitial nucleus of Cajal; Nucleus of Darkschewitsch
1. Introduction Abbre6iations: CC, central canal; CG, central gray; CGRP, calcitonin gene related peptide; CP, cerebral peduncle; DAO, dorsal accessory olivary nucleus; DC, dorsal cap of Kooy; DK, nucleus of Darkschewitsch; fr, fasciculus retroflexus; III, third ventricle; INC, interstitial nucleus of Cajal; IOC, inferior olivary complex; IP, interpositus nucleus; IPN, interpeduncular nucleus; LC, locus coeruleus; LN, lateral cerebellar nucleus; LVN, lateral vestibular nucleus; MAO, medial accessory olivary nucleus; MG, medial geniculate nucleus; ML, medial lemniscus; MN, medial cerebellar nucleus; MVN, medial vestibular nucleus; PC, posterior commissure; PCR, parvicellular reticular nucleus; PO, principal olivary nucleus; RN, red nucleus; RP, raphe pallidus; SC, superior colliculus; SCP, superior cerebellar peduncle; SN, substantia nigra; SO, superior olivary complex; SpV, spinal trigeminal nucleus; TB, trapezoid body; VII, facial nucleus; VIIn, facial nerve; VTA, ventral tegmental area; XII, hypoglossal nucleus; ZI, zona incerta. * Corresponding author. Tel.: +1-614-292-8363; fax: +1-614-2927659. E-mail address: [email protected] (G.A. Bishop)
Calcitonin gene related peptide (CGRP), the alternatively spliced form of the calcitonin gene expressed in nervous tissue (Amara et al., 1982; Rosenfeld et al., 1983), has been shown to have a widespread distribution in the peripheral (Caratsch and Eusebi, 1990) nervous system where it has been shown to have numerous physiological effects including regulation of both sympathetic output (Zhao and Schwartz, 1998) and pain transmission (Franco-Cereceda et al., 1987; Inoue et al., 1993; Menard et al., 1996). The peptide also has been localized in the central nervous system (Kawai et al., 1985; Skofitsch and Jacobowitz, 1985a,b; Ishida-Yamamoto and Tohyama, 1989; Fabri and Conti, 1990; Van Rossum et al., 1997) where it has been implicated as playing a role in the regulation of body
0168-0102/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 4 5 - 0
178
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
temperature, analgesia, and food intake (Krahn et al., 1984; Saxen et al., 1994; Menard et al., 1996), altering the excitation of forebrain neurons (Twery and Moss, 1985), and modulating afferent synaptic transmission (Kangrga et al., 1990) CGRP also has been immunohistochemically localized within mossy fibers in the adult cerebellum of several species (Kawai et al., 1985; Ishida-Yamamoto and Tohyama, 1989; Bishop, 1992; Yamano and Tohyama, 1993), as well as within climbing fibers during specific stages of cerebellar development (Morara et al., 1989, 1992; Provini et al., 1992). Physiologically, CGRP has been shown to suppress spontaneous and amino acid-induced activity of cerebellar cortical neurons (Bishop, 1995). In addition to this direct effect on Purkinje cell activity, CGRP also affects cerebellar circuitry by modulating the responsiveness of neurons in the inferior olive (Gregg and Bishop, 1995), the sole source of climbing fibers to the cerebellum. In a recent study (Gregg and Bishop, 1997), CGRP was shown to be present in varicose profiles that had an extensive distribution throughout the inferior olivary complex of the mouse. An electron microscopic analysis revealed that most of these labeled varicose profiles were not synaptic terminals, but rather were dendrites or dendritic spines of olivary neurons (Gregg et al., 1997) indicating that a large portion of the CGRP observed in the olivary complex is derived from intrinsic sources. However, this ultrastructural analysis also identified some axonal profiles that contained the peptide suggesting that in addition to the intrinsic source of the peptide, there also was an extrinsic source of CGRP in the olivary complex. The inferior olive receives input from numerous spinal, medullary, pontine, mesencephalic, cerebellar, and diencephalic nuclei (Swenson et al., 1989; Nelson and Mugnaini, 1989; Fredette and Mugnaini, 1991; De Zeeuw and Ruigrok, 1994), many of which contain CGRP-positive neurons. At present, however, it is not known which of these afferent systems to the inferior olive is the source of CGRP varicosities. Thus, in the present study, a technique that combines retrograde labeling and immunohistochemistry was used to identify CGRP neurons that project to the inferior olivary complex.
2. Materials and methods
2.1. Animals All experiments were carried out in adult C57BL/6J mice. An animal use protocol for carrying out all procedures has been approved by an Ohio State University ILACUC. All experiments were carried out in deeply anesthetized animals to eliminate all pain.
2.2. Double-label experiments Fluorescently tagged microspheres, which are retrogradely transported by neurons (Katz et al., 1984), were used to label cells that project to the mouse inferior olivary complex. Adult mice (n= 6) were anesthetized with intraperitoneal injections of Avertin (0.2 ml/10 g). The mice were secured in a modified stereotaxic frame, the bone and dura over the brainstem were removed and a 5-ml Hamilton syringe filled with fluorescently tagged microspheres (LumaFluor, New York, NY) was inserted into the brainstem. A pipette tip was fixed to the syringe needle to prevent extensive damage to the tissue, to minimize leakage, and to limit the spread of the injection. Placement of the syringe was done visually; the depth was determined from histological preparations. An injection of 1–2 ml was made into the inferior olive. After the injection was completed, the wound was closed and the animal allowed to survive for 24–30 h. Alternatively, in two experiments, anesthetized animals were placed in a prone position. An incision was made in the skin over the throat, the infrahyoid muscles were separated, and the trachea and esophagus gently retracted. This procedure exposed the bone overlying the ventral brainstem; a small hole was made in the caudal aspect of the bone. The tip of the syringe, with pipette tip attached, was inserted into the brainstem at a depth of 100–200 mm from the surface and 1–2 ml of fluorescent microspheres was injected into the olivary complex. After the injection, the trachea and esophagus were released and the incision sutured close. The animals were allowed to recover for 24–30 h. Colchicine was used in some animals to insure that all neuronal somata that contained the peptide were detected. In these experiments, subcutaneous injections of colchicine (0.1 ml; 1 mg/ml) were given to four mice every hour for 8 h to block anterograde transport of peptides from the soma to the axon terminal (Sar et al., 1978). In one animal, colchicine was injected directly into the brainstem (0.1 mg/100 ml). The mice were allowed to survive overnight to obtain maximum effect of the colchicine. In some animals the colchicine procedure was eliminated. Analysis of the tissue revealed the same populations of CGRP-positive cell bodies as in the colchicine-treated animals indicating a detectable level of the peptide in most neuronal somata. Colchicine and non-colchicine treated animals were then deeply anesthetized and perfused transcardially with saline followed sequentially by 2% paraformaldehyde in 0.1 M sodium acetate buffer pH 6.5 and 2% paraformaldehyde/0.1% glutaraldehyde in 0.1 M sodium borate buffer pH 8.5 or with 4% paraformaldehyde in phosphate buffer. Comparable results were obtained regardless of the fixative used. The brains were removed and immersed in 20% sucrose in 0.1 M
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
Sorensen’s phosphate buffer (pH 7.2). The brainstem was cut in 60-mm serial transverse sections using a freezing microtome and processed for CGRP immunohistochemistry using the PAP technique. Briefly, tissue was placed in a primary antibody against CGRP (Amersham Life Science Inc., Arlington Heights, IL) diluted 1:2500 in phosphate-buffered saline/0.3% triton X-100 (PBT) for 24 – 30 h at 4°C with constant agitation. It was then placed sequentially in a secondary antibody, diluted 1:500 in PBT, and the peroxidase anti-peroxidase, diluted 1:500 in PBT, (ICN Pharmaceuticals, Aurora, OH) for 1 h each. The tissue was then processed using DAB with glucose oxidase enhancement (Shu et al., 1988), mounted on slides and coverslipped. Serial sections were analyzed and the distribution of cell bodies immunocytochemically labeled for CGRP and retrogradely labeled with fluorescent microspheres was plotted.
2.3. Data analysis Three types of labeled neurons were observed and include: (1) cells only immunolabeled for CGRP; (2) cells only retrogradely labeled, indicating the neuron projected to the IOC, but did not contain CGRP; and (3) neurons that were both immunolabeled by the CGRP antibody and also retrogradely labeled with the fluorescent microspheres indicating they were CGRP cells that projected to the injected area of the IOC. The distribution of each type of neuron was plotted on representative sections through the brainstem. Photomicrographs were taken by using a Zeiss fluorescent microscope. Transmitted and epi-illumination were used at the same time and balanced so that both the DAB reaction product and the fluorescent microspheres could be visualized and photographed simultaneously.
3. Results
3.1. Distribution of CGRP in the mouse inferior oli6ary complex In non-colchicine treated animals, CGRP-immunoreactive varicosities have an extensive distribution throughout all nuclei of the olivary complex (Fig. 1A– F). Although present in all nuclei, there are variations in the size and intensity of staining of the immunoreactive puncta in different subnuclei. At caudal levels of the inferior olivary complex (IOC), the largest profiles are present in the dorsal cap of Kooy and the lateral aspect of the medial accessory olivary nucleus. Rostrally, large profiles are present primarily in the ventral lateral outgrowth (Fig. 1B) and focal regions within the dorsal accessory olivary nucleus (Fig. 1C,D). The re-
179
maining profiles are smaller in size and more uniformly distributed in the neuropil of the inferior olive (Fig. 1B–F).
3.2. Distribution of CGRP neurons in the mouse Examination of the mouse brainstem revealed two types of CGRP-labeled neurons that can be distinguished on the basis of the intensity of the CGRP-immunoreactivity. One population is intensely stained whereas the other, while clearly above background, is less intensely immunoreactive. Intensely labeled CGRPpositive neurons are present, primarily in motor nuclei such as the hypoglossal nucleus (Fig. 2A), the nucleus ambiguus, the facial nucleus (Fig. 2B), the motor nucleus of the trigeminal nerve and the oculomotor nucleus. Additionally, intensely-labeled CGRP positive neurons also are present in the reticular formation of the medulla and pons, the locus coeruleus (Fig. 2D), the parabrachial nucleus, the basilar pons, the ventral tegmental area, and the zona incerta. Neurons that are less intensely CGRP-positive are located throughout the inferior olivary complex (Fig. 2C), the lateral reticular nucleus, the deep cerebellar nuclei (Fig. 2E), the medullary and pontine reticular formation, the medial and lateral vestibular nuclei, the spinal trigeminal nucleus, the dorsal tegmental area, the red nucleus, and the periaqueductal gray around the third ventricle adjacent to the fasiculus retroflexus in the caudal diencephalon (Fig. 2F).
3.3. Sources of CGRP to the inferior oli6ary complex Two injections made from a dorsal and one from a ventral approach were essentially confined to the inferior olive. The results from one ventral injection are representative and are illustrated in Fig. 3A–E and Fig. 4A–H. Numerous double-labeled neurons are located in the ventral aspect of the contralateral nucleus interpositus and lateral nucleus in the cerebellum (Fig. 3B; Fig. 4C,D). Labeled cells are small and interspersed with neurons that only are immunopositive for CGRP. A few double-labeled neurons are also found in the superior and lateral vestibular nuclei (Fig. 3B). A second major source of CGRP afferents to the IOC is derived from neurons located within the locus coeruleus (Fig. 3C; Fig. 4E,F). Neurons were labeled bilaterally, although the vast majority were located ipsilateral to the injection. In the midbrain and caudal diencephalon, the ipsilateral central gray matter immediately adjacent to the third ventricle and dorsal to the fasiculus retroflexus as well as the adjacent zona incerta also contain double-labeled neurons (Fig. 3E; Fig. 4G,H). Retrogradely labeled neurons are located in the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, and the red
180
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
nucleus (Fig. 3C); however, none of these cells are CGRP-positive.
4. Discussion Data from several studies now indicate that CGRP is present in the inferior olivary complex of the mouse (Gregg and Bishop, 1997) where it exerts a suppressive effect on olivary neurons (Gregg and Bishop, 1995).
Based on a fine structural analysis of CGRP-immunolabeled profiles (Gregg et al., 1997), it appears that there are both intrinsic and extrinsic sources of CGRP to the olivary complex. Results from the present study have identified several extrinsic sources of CGRP afferents to the mouse inferior olivary complex and include the lateral and interposed nuclei of the cerebellum, the locus coeruleus, and the central gray matter of the diencephalon. A comparatively small projection from the zona incerta as well as
Fig. 1. Photomicrographs that illustrate the distribution of CGRP-immunoreactive varicosities in the inferior olivary complex; (A) a low power photomicrograph of a transverse section taken through the middle of the olivary complex. The letters in (A) indicate the areas from which B–F were taken; (B) is through the ventral lateral outgrowth and (C) is through the dorsal accessory olivary nucleus. Both large, intensely stained varicosities (arrowheads) and smaller puncta (small arrows) are present in these nuclei; (D) is through the lateral dorsal accessory olivary nucleus, (E) is through the principal olivary nucleus, and (F) is through the medial accessory olivary nucleus and the ventral lamella of the principal olive. In these areas, only small immunoreactive puncta (small arrows) are present. Calibration bar in A = 200 mm; B– F = 40 mm.
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
181
Fig. 2. Photomicrographs that illustrate CGRP positive neurons in the brainstem and cerebellum. Intensely stained neurons are located within the hypoglossal nucleus (A), the facial nucleus (B) and the locus coeruleus (D). Less intensely labeled neurons are present within the inferior olivary complex (C), the cerebellar nuclei (E) and the central gray matter around the third ventricle (F). Calibration bars in A – D= 100 mm; E= 200 mm; F =50 mm.
the lateral and superior vestibular nuclei also contributes to the CGRP input to the olivary complex.
4.1. Injection site In the case of one dorsal injection there was spread to the pyramidal tract rostral to the IOC. However, because studies have shown that fibers of passage do not appear to take up the fluorescent microspheres (Katz et al., 1984), this spread to a fiber tract should not produce false-positive labeling. Further, if fibers in the pyramidal tract did take up the marker, they should be confined to the cerebral cortex where no CGRP-positive neurons were detected. The approach from the ventral side spread just rostral to the IOC. When results were compared between
all cases used for analysis, it was found that double-labeled cells were found in the same restricted areas of the rostral brainstem and only in nuclei that have previously been shown to be sources of olivary afferents. In most injections, some microspheres were observed in the area of the nucleus raphe pallidus, located immediately medial to the olivary complex. A recent study indicated there is a widespread distribution in the source of afferents to this nucleus (Hermann et al., 1997). Two of these diverse areas, namely the periaqueductal gray matter in the mesodiencephalon and the subcoerulear nucleus, are sources of afferents to this nucleus; these nuclei contained double-labeled cells. It is possible that these cells project to the nucleus raphe pallidus instead of or in addition to the inferior olivary
182
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
complex. However, if the injection in the nucleus raphe pallidus was effective, we would expect to see a more widespread distribution of retrogradelylabeled cells. In particular, there should be a large number throughout all levels of the periaqueductal grey, including the portion in the caudal mesencephalon and the pons, as well as throughout the pontine and mesencephalic reticular formation (Hermann et al., 1997). These areas were devoid of retrogradely-labeled cells, as were other areas known to be a primary source of afferents to the raphe pallidus such as the hypothalamus, preoptic areas and the Ko¨lliker – Fuse nucleus. Thus, the absence of retrograde-labeling in other areas shown to provide a large input to the nucleus raphe pallidus suggests that the spread of beads to the raphe pallidus was, at best, only minimally effective in labeling afferents to this area and that the labeling actually
represents projections to the inferior olivary complex.
4.2. Cerebellar nuclei Within the cerebellar nuclei, there are two populations of neurons delineated on the basis of their size. Large nuclear neurons, which are non-GABAergic (Ito, 1984; De Zeeuw and Ruigrok, 1994), project rostrally to the vestibular nuclei, reticular nuclei, pontine nuclei, the red nucleus, the thalamus, and the locus coeruleus (Sastry et al., 1997). Small GABAergic nuclear neurons (De Zeeuw et al., 1989), located primarily in the ventral aspect of the nucleus interpositus and lateral nucleus, project to the inferior olivary complex (Nelson and Mugnaini, 1989; Fredette and Mugnaini, 1991). These previous findings, combined with those of the present study would suggest that CGRP and GABA are co-lo-
Fig. 3. Camera lucida drawings of selected sections through the brainstem that illustrate the extent of an injection of fluorescent microspheres into the inferior olivary complex (A) and the distribution of retrogradely and double-labeled neurons (B – E). The injection illustrated was located within the inferior olivary complex and the adjacent pyramidal tract. There was slight spread into the pyramidal tract just rostral to the olivary complex. Following this injection, retrogradely labeled cells (B – E, open circles) were located in the cerebellar nuclei (B), the locus coeruleus (C), the nucleus of Darkschwetsch, the interstitial nucleus of Cajal, and the red nucleus (D). Double-labeled neurons, that is those that were CGRP-positive and that also contained retrogradely transported microspheres (B – C, closed stars) were located in the lateral and interposed cerebellar nuclei (B), the locus coeruleus (C), the superior and lateral vestibular nuclei (B), the central gray area adjacent to the third ventricle (F), and the zona incerta (F).
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
183
Fig. 3. (Continued)
calized in nuclear cell axons and axonal terminals; however, confirmation with double-label studies has yet to be carried out. Olivary neurons are functionally coupled by gap junctions (Sotelo et al., 1974) into ensembles of electrotonically coupled cells that fire in synchrony (Llinas and Yarom, 1980; Lang et al., 1996). Stimulation of the nucleo-olivary pathway has been shown to modulate the effective electrotonic coupling between olivary neurons (Fredette and Mugnaini, 1991; Lang et al., 1996). Although the effects have been attributed solely to GABA, it is likely that CGRP, when co-released from the same terminals, also plays an integral role in altering electrotonic coupling. Based on data which show that CGRP depresses olivary activity (Gregg and Bishop, 1997), we would postulate that the peptide facilitates a GABAergic effect on olivary cells leading to functional uncoupling of olivary neurons. This may be critical in regulating the synchronous output of discrete populations of olivary neurons.
Sinnamon, 1977; Moore and Bloom, 1979). There is little research to date on the function of noradrenergic fibers in the inferior olivary complex. In one study, stimulation of the brainstem close to the locus coeruleus resulted in inhibition of olivary neurons (Hesslow, 1986). Likely, this effect was due to the catecholaminergic output derived from the locus coeruleus. However, it is also possible that CGRP released from the same terminals may have played a role in suppressing olivary activity (Gregg and Bishop, 1995). It may be theorized, based on the role of norepinephrine elsewhere in the CNS (AstonJones et al., 1991, 1994; van Neerven et al., 1991; Van Bockstaele and Aston-Jones, 1995; Bremner et al., 1996), that afferents from the locus coeruleus to the inferior olivary complex and cerebellum may be involved in motor learning or in modulating the responsiveness of olivary neurons to novel sensory stimuli. Future studies will be carried out determine whether CGRP is co-localized with norepinephrine and how they interact to modulate activity of olivary neurons.
4.3. Locus coeruleus
4.4. Mesodiencephalon
The majority of norepinephrine positive neurons in the CNS are located in the locus coeruleus (Amaral and
The most rostral source of CGRP to the inferior olive is derived from neurons that are located within the
184
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
the central gray immediately adjacent to the third ventricle of the caudal diencephalon. These cells occupy the area between the nucleus of Darksche-witsch and the interstitial nucleus of Cajal, immediately dorsal to the fasciculus retroflexus. Cells in this area have been described previously as sources of afferents to the inferior olivary complex (Cintas et al., 1980; Saint-Cyr and Courville, 1981; Onodera, 1984; Rutherford et al., 1984). Little is known as to the physiological effect of this population of neurons in the inferior olive, thus it
is not possible to place them in functional context at this time. Based on an anterograde study (Cintas et al., 1980), neurons in this area project primarily to the dorsal and ventral lamella of the principal olivary nucleus, including the ventrolateral outgrowth. There is also minor projection to the caudal lateral medial accessory olivary nucleus, and the dorsal cap of Kooy. This correlates well with the distribution of large profiles tentatively identified as being derived from afferent axons (Gregg et al., 1997).
Fig. 4. Photomicrographs of a representative injection site (A,B) of fluorescent microspheres into the IOC made from a dorsal approach. The injection is located within the olivary complex and the adjacent pyramidal tract. (C – H) examples of double-labeled cells (arrows) in the nucleus interpositus (C,D), the locus coeruleus (E,F), the central gray area around the third ventricle (G), and the zona incerta (H). Calibration bars in A,B =500 mm; C– H =50 mm.
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
4.5. Functional considerations Neuropeptides such as CGRP are important signaling molecules in the nervous system as they have been shown to modulate the responsiveness of different neurons to synaptic input or to alter spontaneous patterns of activity (Kaczmarek and Levitan, 1987). CGRP has been shown to exert its effect by a variety of different mechanisms in different systems. For example, it has been shown to: (1) reduce acetylcholine release and to decrease receptor sensitivity at the neuromuscular junction (Caratsch and Eusebi, 1990); (2) increase adenylate cyclase activity, thereby increasing cyclic AMP levels (Kobayashi et al., 1987); (3) affect the release of other neurotransmitters such as glutamate and aspartate in the spinal cord (Kangrga et al., 1990); (4) facilitate Ca2 + influx in synaptosomal preparations of the spinal dorsal horn (Oku et al., 1988); and (5) directly affect membrane excitability (Twery and Moss, 1985). In the present study, we have identified several extrinsic sources of CGRP to the inferior olive and have localized it to afferents that, by themselves, have either excitatory or inhibitory effects on olivary neurons. The presence of CGRP in these same afferents together with the intrinsic source of CGRP in olivary dendrites and dendritic spines (Gregg et al., 1997) implies a more complex influence of this peptide over olivary circuits. The precise mechanisms by which CGRP alters olivary firing rate are unknown. For example, olivary neurons have been shown to have an oscillatory firing pattern that is highly dependent on Ca2 + currents (Llinas and Yarom, 1980). Release of CGRP from either intrinsic or extrinsic sources could alter these ion currents, as described in the spinal cord (Oku et al., 1988) which would change pre- and/or postsynaptic membrane excitability and ultimately change the oscillatory firing pattern characteristic of olivary neurons. Thus, CGRP, derived from diverse brainstem and cerebellar nuclei, acting as a co-transmitter, may play a complex role in modulating olivary output.
Acknowledgements This work was supported by NSF grant IBN9630867 to G. Bishop. A. Peltier was supported by funds from the Roessler Foundation from the College of Medicine, The Ohio State University. The authors gratefully acknowledge the technical assistance of Barbara DienerPhelan and the photographic assistance of Yi Fei Chen.
References Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M., 1982. Alternative RNA processing in calcitonin gene expression
185
generates mRNAs encoding different polypeptide products. Nature 289, 240 – 244. Amaral, D.G., Sinnamon, H.M., 1977. The locus coeruleus: neurobiology of a central noradrenergic nucleus. Prog. Neurobiol. 9, 147 – 196. Aston-Jones, G., Chiang, C., Alexinsky, T., 1991. Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance. Prog. Brain Res. 88, 501–520. Aston-Jones, G., Rajkowski, J., Kubiak, P., Alexinsky, T., 1994. Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task. J. Neurosci. 14, 4467 –4480. Bishop, G.A., 1992. Calcitonin gene related peptide in cerebellar afferents: Distribution and origin. J. Comp. Neurol. 322, 201– 212. Bishop, G.A., 1995. Calcitonin gene related peptide modulates neuronal activity in the mammalian cerebellar cortex. Neuropeptides 28, 85 – 97. Bremner, J.D., Krystal, J.H., Southwick, S.M., Charney, D.S., 1996. Noradrenergic mechanisms in stress and anxiety: I. Preclinical studies. Synapse 23, 28 – 38. Caratsch, C.G., Eusebi, F., 1990. Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog. Neurosci. Lett. 111, 344 – 350. Cintas, H.M., Rutherford, J.G., Gwyn, D.G., 1980. Some midbrain and diencephalic projections to the inferior olive in the rat. In: Courville, J., deMontigny, C., Lamarre, Y. (Eds.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, pp. 73 – 124. De Zeeuw, C., Ruigrok, T.J.H., 1994. Olivary projecting neurons in the nucleus of Darkschewitsch in the cat receive excitatory monosynaptic input from the cerebellar nuclei. Brain Res. 653, 345 – 350. De Zeeuw, C., Holstege, J.C., Ruigrok, T.J.H., Voogd, J., 1989. Ultrastructural study of the GABAergic, cerebellar, and mesodiencephalic innervation of the cat medial accessory olive: anterograde tracing combined with immunocytochemistry. J. Comp. Neurol. 284, 12 – 35. Fabri, M., Conti, F., 1990. Calcitonin gene-related peptide-positive neurons and fibers in the cat dorsal column nuclei. Neuroscience 35, 167 – 174. Franco-Cereceda, A., Henke, H., Lundberg, J., Petermann, J.B., Ho¨kfelt, T., Fischer, J.A., 1987. Calcitonin gene related peptide (CGRP) in capsaicin-sensitive substance P-Immunoreactive sensory neurons in animals and man: distribution and release by capsaicin. Peptide 8, 399 – 410. Fredette, B.J., Mugnaini, E., 1991. The GABAergic cerebello-olivary projection in the rat. Anat. Embryol. (Berl) 184, 225 – 243. Gregg, K.V., Bishop, G.A., 1995. Physiological effect of calcitonin gene related peptide in the mouse inferior olive. Neurosci. Abstr. 21, 1192. Gregg, K.V., Bishop, G.A., 1997. Peptide localization in the mouse inferior olive. J. Chem. Neuroanat. 12, 211 – 220. Gregg, K.V., Bishop, G.A., King, J.S., 1997. An electron microscopic analysis of the distribution of CGRP in the mouse inferior olivary complex. Neurosci. Abstr. 23, 120. Hermann, D.M., Luppi, P.-H., Peyron, C., Hinckel, P., Jouvet, M., 1997. Afferent projections to the rat nuclei raaphe magnus, raphe pallidus and reticularis gigantocellularis pars a demonstrated by iontophoretic application of choleratoxin (subunit b). J. Chem. Neuroanat. 13, 1 – 21. Hesslow, G., 1986. Inhibition of inferior olivary transmission by mesencephalic stimulation in the cat. Neurosci. Lett. 63, 76–80. Inoue, H., Nagata, N., Koshihara, Y., 1993. Profile of capsaicin-induced mouse ear oedema as neurogenic inflammatory model: comparison with arachidonic acid-induced ear oedema. Br. J. Pharmacol. 110, 1614 – 1620.
186
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
Ishida-Yamamoto, A., Tohyama, M., 1989. Calcitonin gene-related peptide in the nervous tissue. Prog. Neurobiol. 33, 335–386. Ito, M., 1984. The Cerebellum and Neural Control. Raven Press, New York. Kaczmarek, L.K., Levitan, I.B., 1987. Neuromodulation. Oxford University Press, New York. Kangrga, I., Larew, J.S.A., Randic, M., 1990. The effects of substance P and calcitonin gene-related peptide on the efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro. Neurosci. Lett. 108, 155–160. Katz, L.C., Burkhalter, A., Dreyer, W.J., 1984. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310, 498–500. Kawai, Y., Takami, K., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., MacIntyre, I., Tohyama, M., 1985. Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis. Neuroscience 15, 747–763. Kobayashi, H., Hashimoto, K., Uchida, S., Sakuma, J., Takami, K., Tohyama, M., Izumi, F., Yoshida, H., 1987. Calcitonin gene related peptide stimulates adenylate cyclase activity in rat striated muscle. Experientia 43, 314–316. Krahn, D.D., Gosnell, B.A., Levine, A.S., Morley, J.E., 1984. Effects of calcitonin gene-related peptide on food intake. Peptides 5, 861 – 864. Lang, E.J., Sugihara, I., Llinas, R., 1996. GABAergic modulation of complex spike activity by the cerebellar nucleoolivary pathway in rat. J. Neurophysiol. 76, 255–275. Llinas, R., Yarom, Y., 1980. Electrophysiological properties of mammalian inferior olivary cells in vitro. In: Courville, J. (Ed.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, pp. 379 – 388. Menard, D.P., vanRossum, D., Kar, S., St.Pierre, S., Sutak, M., Jhamandas, K., Quirion, R., 1996. A calcitonin gene-related peptide receptor antagonist prevents the development of tolerance to spinal morphine analgesia. J. Neurosci. 16, 2342–2351. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2, 113–168. Morara, S., Provini, L., Rosina, A., 1989. CGRP expression in the rat olivocerebellar system during postnatal development. Brain Res. 504, 315 – 319. Morara, S., Rosina, A., Provini, L., 1992. CGRP as a marker of the climbing fibers during the development of the cerebellum in the rat. Ann. New York Acad. Sci. 657, 461–463. Nelson, B.J., Mugnaini, E., 1989. Origins of GABAergic inputs to the inferior olive. Exp. Brain Res. 17, 86–107. Oku, R., Nanayama, T., Satoh, M., 1988. Calcitonin gene-related peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn. Brain Res. 475, 356–360. Onodera, S., 1984. Olivary projections from the mesodiencephalic structures in the cat studied by means of axonal transport of horseradish peroxidase and tritiated amino acids. J. Comp. Neurol. 227, 37 – 49. Provini, L., Morara, S., Rosina, A., Forloni, G., 1992. Expression of CGRP binding sites in the developing rat cerebellum. Ann. New York Acad. Sci. 657, 423–425.
.
Rosenfeld, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., Evans, R.M., 1983. Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature 304, 129–135. Rutherford, J.G., Anderson, W.A., Gwyn, D.G., 1984. A reevaluation of midbrain and diencephalic projections to the inferior olive in rat with particular reference to the rubro-olivary pathway. J. Comp. Neurol. 229, 285 – 300. Saint-Cyr, J., Courville, J., 1981. Sources of descending afferents to the inferior olive from the upper brain stem in the cat as revealed by the retrograde transport of horseradish peroxidase. J. Comp. Neurol. 198, 567 – 581. Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.J., Cuatrecasas, P., 1978. Immunohistochemical localization of enkephalin in rat brain and spinal cord. J. Comp. Neurol. 182, 17 – 37. Sastry, B.R., Morishita, W., Yip, S., Shew, T., 1997. Gabaergic transmission in deep cerebellar nuclei. Prog. Neurobiol. 53, 259– 271. Saxen, M.A., Smith, F.L., Dunlow, L.D., Dombrowski, D.S., Welch, S.P., 1994. The hypothermic and antinociceptive effects of intrathecal injection of CGRP (8-37) in mice. Life Sci. 55, 1665– 1674. Shu, S., Ju, G., Fan, L., 1988. The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85, 169 – 171. Skofitsch, G., Jacobowitz, D.M., 1985a. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6, 721 – 745. Skofitsch, G., Jacobowitz, D.M., 1985b. Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system. Peptides 6, 1069 – 1073. Sotelo, C., Llinas, R., Baker, R., 1974. Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling. J. Neurophysiol. 37, 541 – 559. Swenson, R.S., Sievert, C.F., Terreberry, R.R., Neafsey, E.J., Castro, A.J., 1989. Organization of cerebral cortico-olivary projections in the rat. Neurosci. Res. 7, 43 – 54. Twery, M.J., Moss, R.L., 1985. Calcitonin and calcitonin gene-related peptide alter the excitability of neurons in rat forebrain. Peptides 6, 373 – 378. Van Bockstaele, E.J., Aston-Jones, G., 1995. Integration in the ventral medulla and coordination of sympathetic, pain and arousal functions. Clin. Exp. Hypertens. 17, 153 – 165. van Neerven, J., Pompeiano, O., Collewijn, H., 1991. Effects of GABAergic and noradrenergic injections into the cerebellar flocculus on vestibulo-ocular reflexes in the rabbit. Prog. Brain Res. 88, 485 – 497. Van Rossum, D., Hanisch, U.K., Quirion, R., 1997. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci. Biobehav. Rev. 21, 649 – 678. Yamano, M., Tohyama, M., 1993. The innervation of calcitonin gene-related peptide to the Purkinje cells and granule cells in the developing mouse cerebellum. Brain Res. Dev. Brain Res. 72, 107 – 117. Zhao, B.Y., Schwartz, J.P., 1998. Involvement of cytokines in normal CNS development and neurological diseases: Recent progress and perspectives. J. Neurosci. Res. 52, 7 – 16.
The site of origin of calcitonin gene-related peptide-like immunoreactive afferents to the inferior olivary complex of the mouse Amanda C. Peltier, Georgia A. Bishop * Department of Cell Biology, Neurobiology and Anatomy, Di6ision of Neuroscience and the College of Medicine, The Ohio State Uni6ersity, 333 W 10th A6enue, Columbus, OH 43210, USA Received 8 March 1999; accepted 23 April 1999
Abstract The intent of the present study is to define the brainstem nuclei which give rise to CGRP-immunolabeled afferents to the inferior olivary complex of the mouse. A technique which combines retrograde transport of fluorescent microspheres with immunohistochemistry was used to address this question. In the present study, intensely labeled CGRP neurons were localized within several cranial nerve nuclei including the hypoglossal, facial, oculomotor, motor nucleus of the trigeminal nerve and nucleus ambiguus, as well as in the parabrachial nucleus, locus coeruleus and medullary and pontine reticular formation. In addition, lightly labeled CGRP neurons were identified within the deep cerebellar nuclei, the inferior olivary complex, lateral reticular nucleus, medial and lateral vestibular nuclei, nucleus Darkschewitsch, interstitial nucleus of Cajal, the central gray area adjacent to the third ventricle, and the zona incerta. The origin of the projection to the inferior olivary complex primarily arises from the deep cerebellar nuclei, the locus coeruleus, and the central gray matter of the mesodiencephalic area. In addition, a small CGRP input is derived from the superior and lateral vestibular nuclei as well as the zona incerta. In conclusion, we have identified several extrinsic sources of CGRP to the inferior olivary complex and have localized it within afferents that have been shown to have either excitatory (mesodiencephalic nuclei) or inhibitory (cerebellar nuclei) effects on olivary circuits. The presence of CGRP in these functionally diverse brainstem and cerebellar afferents suggests that the peptide may act as a co-transmitter to modulate the activity of olivary neurons. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fluorescent microspheres; Locus coeruleus; Cerebellar nuclei; Interstitial nucleus of Cajal; Nucleus of Darkschewitsch
1. Introduction Abbre6iations: CC, central canal; CG, central gray; CGRP, calcitonin gene related peptide; CP, cerebral peduncle; DAO, dorsal accessory olivary nucleus; DC, dorsal cap of Kooy; DK, nucleus of Darkschewitsch; fr, fasciculus retroflexus; III, third ventricle; INC, interstitial nucleus of Cajal; IOC, inferior olivary complex; IP, interpositus nucleus; IPN, interpeduncular nucleus; LC, locus coeruleus; LN, lateral cerebellar nucleus; LVN, lateral vestibular nucleus; MAO, medial accessory olivary nucleus; MG, medial geniculate nucleus; ML, medial lemniscus; MN, medial cerebellar nucleus; MVN, medial vestibular nucleus; PC, posterior commissure; PCR, parvicellular reticular nucleus; PO, principal olivary nucleus; RN, red nucleus; RP, raphe pallidus; SC, superior colliculus; SCP, superior cerebellar peduncle; SN, substantia nigra; SO, superior olivary complex; SpV, spinal trigeminal nucleus; TB, trapezoid body; VII, facial nucleus; VIIn, facial nerve; VTA, ventral tegmental area; XII, hypoglossal nucleus; ZI, zona incerta. * Corresponding author. Tel.: +1-614-292-8363; fax: +1-614-2927659. E-mail address: [email protected] (G.A. Bishop)
Calcitonin gene related peptide (CGRP), the alternatively spliced form of the calcitonin gene expressed in nervous tissue (Amara et al., 1982; Rosenfeld et al., 1983), has been shown to have a widespread distribution in the peripheral (Caratsch and Eusebi, 1990) nervous system where it has been shown to have numerous physiological effects including regulation of both sympathetic output (Zhao and Schwartz, 1998) and pain transmission (Franco-Cereceda et al., 1987; Inoue et al., 1993; Menard et al., 1996). The peptide also has been localized in the central nervous system (Kawai et al., 1985; Skofitsch and Jacobowitz, 1985a,b; Ishida-Yamamoto and Tohyama, 1989; Fabri and Conti, 1990; Van Rossum et al., 1997) where it has been implicated as playing a role in the regulation of body
0168-0102/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 4 5 - 0
178
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
temperature, analgesia, and food intake (Krahn et al., 1984; Saxen et al., 1994; Menard et al., 1996), altering the excitation of forebrain neurons (Twery and Moss, 1985), and modulating afferent synaptic transmission (Kangrga et al., 1990) CGRP also has been immunohistochemically localized within mossy fibers in the adult cerebellum of several species (Kawai et al., 1985; Ishida-Yamamoto and Tohyama, 1989; Bishop, 1992; Yamano and Tohyama, 1993), as well as within climbing fibers during specific stages of cerebellar development (Morara et al., 1989, 1992; Provini et al., 1992). Physiologically, CGRP has been shown to suppress spontaneous and amino acid-induced activity of cerebellar cortical neurons (Bishop, 1995). In addition to this direct effect on Purkinje cell activity, CGRP also affects cerebellar circuitry by modulating the responsiveness of neurons in the inferior olive (Gregg and Bishop, 1995), the sole source of climbing fibers to the cerebellum. In a recent study (Gregg and Bishop, 1997), CGRP was shown to be present in varicose profiles that had an extensive distribution throughout the inferior olivary complex of the mouse. An electron microscopic analysis revealed that most of these labeled varicose profiles were not synaptic terminals, but rather were dendrites or dendritic spines of olivary neurons (Gregg et al., 1997) indicating that a large portion of the CGRP observed in the olivary complex is derived from intrinsic sources. However, this ultrastructural analysis also identified some axonal profiles that contained the peptide suggesting that in addition to the intrinsic source of the peptide, there also was an extrinsic source of CGRP in the olivary complex. The inferior olive receives input from numerous spinal, medullary, pontine, mesencephalic, cerebellar, and diencephalic nuclei (Swenson et al., 1989; Nelson and Mugnaini, 1989; Fredette and Mugnaini, 1991; De Zeeuw and Ruigrok, 1994), many of which contain CGRP-positive neurons. At present, however, it is not known which of these afferent systems to the inferior olive is the source of CGRP varicosities. Thus, in the present study, a technique that combines retrograde labeling and immunohistochemistry was used to identify CGRP neurons that project to the inferior olivary complex.
2. Materials and methods
2.1. Animals All experiments were carried out in adult C57BL/6J mice. An animal use protocol for carrying out all procedures has been approved by an Ohio State University ILACUC. All experiments were carried out in deeply anesthetized animals to eliminate all pain.
2.2. Double-label experiments Fluorescently tagged microspheres, which are retrogradely transported by neurons (Katz et al., 1984), were used to label cells that project to the mouse inferior olivary complex. Adult mice (n= 6) were anesthetized with intraperitoneal injections of Avertin (0.2 ml/10 g). The mice were secured in a modified stereotaxic frame, the bone and dura over the brainstem were removed and a 5-ml Hamilton syringe filled with fluorescently tagged microspheres (LumaFluor, New York, NY) was inserted into the brainstem. A pipette tip was fixed to the syringe needle to prevent extensive damage to the tissue, to minimize leakage, and to limit the spread of the injection. Placement of the syringe was done visually; the depth was determined from histological preparations. An injection of 1–2 ml was made into the inferior olive. After the injection was completed, the wound was closed and the animal allowed to survive for 24–30 h. Alternatively, in two experiments, anesthetized animals were placed in a prone position. An incision was made in the skin over the throat, the infrahyoid muscles were separated, and the trachea and esophagus gently retracted. This procedure exposed the bone overlying the ventral brainstem; a small hole was made in the caudal aspect of the bone. The tip of the syringe, with pipette tip attached, was inserted into the brainstem at a depth of 100–200 mm from the surface and 1–2 ml of fluorescent microspheres was injected into the olivary complex. After the injection, the trachea and esophagus were released and the incision sutured close. The animals were allowed to recover for 24–30 h. Colchicine was used in some animals to insure that all neuronal somata that contained the peptide were detected. In these experiments, subcutaneous injections of colchicine (0.1 ml; 1 mg/ml) were given to four mice every hour for 8 h to block anterograde transport of peptides from the soma to the axon terminal (Sar et al., 1978). In one animal, colchicine was injected directly into the brainstem (0.1 mg/100 ml). The mice were allowed to survive overnight to obtain maximum effect of the colchicine. In some animals the colchicine procedure was eliminated. Analysis of the tissue revealed the same populations of CGRP-positive cell bodies as in the colchicine-treated animals indicating a detectable level of the peptide in most neuronal somata. Colchicine and non-colchicine treated animals were then deeply anesthetized and perfused transcardially with saline followed sequentially by 2% paraformaldehyde in 0.1 M sodium acetate buffer pH 6.5 and 2% paraformaldehyde/0.1% glutaraldehyde in 0.1 M sodium borate buffer pH 8.5 or with 4% paraformaldehyde in phosphate buffer. Comparable results were obtained regardless of the fixative used. The brains were removed and immersed in 20% sucrose in 0.1 M
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
Sorensen’s phosphate buffer (pH 7.2). The brainstem was cut in 60-mm serial transverse sections using a freezing microtome and processed for CGRP immunohistochemistry using the PAP technique. Briefly, tissue was placed in a primary antibody against CGRP (Amersham Life Science Inc., Arlington Heights, IL) diluted 1:2500 in phosphate-buffered saline/0.3% triton X-100 (PBT) for 24 – 30 h at 4°C with constant agitation. It was then placed sequentially in a secondary antibody, diluted 1:500 in PBT, and the peroxidase anti-peroxidase, diluted 1:500 in PBT, (ICN Pharmaceuticals, Aurora, OH) for 1 h each. The tissue was then processed using DAB with glucose oxidase enhancement (Shu et al., 1988), mounted on slides and coverslipped. Serial sections were analyzed and the distribution of cell bodies immunocytochemically labeled for CGRP and retrogradely labeled with fluorescent microspheres was plotted.
2.3. Data analysis Three types of labeled neurons were observed and include: (1) cells only immunolabeled for CGRP; (2) cells only retrogradely labeled, indicating the neuron projected to the IOC, but did not contain CGRP; and (3) neurons that were both immunolabeled by the CGRP antibody and also retrogradely labeled with the fluorescent microspheres indicating they were CGRP cells that projected to the injected area of the IOC. The distribution of each type of neuron was plotted on representative sections through the brainstem. Photomicrographs were taken by using a Zeiss fluorescent microscope. Transmitted and epi-illumination were used at the same time and balanced so that both the DAB reaction product and the fluorescent microspheres could be visualized and photographed simultaneously.
3. Results
3.1. Distribution of CGRP in the mouse inferior oli6ary complex In non-colchicine treated animals, CGRP-immunoreactive varicosities have an extensive distribution throughout all nuclei of the olivary complex (Fig. 1A– F). Although present in all nuclei, there are variations in the size and intensity of staining of the immunoreactive puncta in different subnuclei. At caudal levels of the inferior olivary complex (IOC), the largest profiles are present in the dorsal cap of Kooy and the lateral aspect of the medial accessory olivary nucleus. Rostrally, large profiles are present primarily in the ventral lateral outgrowth (Fig. 1B) and focal regions within the dorsal accessory olivary nucleus (Fig. 1C,D). The re-
179
maining profiles are smaller in size and more uniformly distributed in the neuropil of the inferior olive (Fig. 1B–F).
3.2. Distribution of CGRP neurons in the mouse Examination of the mouse brainstem revealed two types of CGRP-labeled neurons that can be distinguished on the basis of the intensity of the CGRP-immunoreactivity. One population is intensely stained whereas the other, while clearly above background, is less intensely immunoreactive. Intensely labeled CGRPpositive neurons are present, primarily in motor nuclei such as the hypoglossal nucleus (Fig. 2A), the nucleus ambiguus, the facial nucleus (Fig. 2B), the motor nucleus of the trigeminal nerve and the oculomotor nucleus. Additionally, intensely-labeled CGRP positive neurons also are present in the reticular formation of the medulla and pons, the locus coeruleus (Fig. 2D), the parabrachial nucleus, the basilar pons, the ventral tegmental area, and the zona incerta. Neurons that are less intensely CGRP-positive are located throughout the inferior olivary complex (Fig. 2C), the lateral reticular nucleus, the deep cerebellar nuclei (Fig. 2E), the medullary and pontine reticular formation, the medial and lateral vestibular nuclei, the spinal trigeminal nucleus, the dorsal tegmental area, the red nucleus, and the periaqueductal gray around the third ventricle adjacent to the fasiculus retroflexus in the caudal diencephalon (Fig. 2F).
3.3. Sources of CGRP to the inferior oli6ary complex Two injections made from a dorsal and one from a ventral approach were essentially confined to the inferior olive. The results from one ventral injection are representative and are illustrated in Fig. 3A–E and Fig. 4A–H. Numerous double-labeled neurons are located in the ventral aspect of the contralateral nucleus interpositus and lateral nucleus in the cerebellum (Fig. 3B; Fig. 4C,D). Labeled cells are small and interspersed with neurons that only are immunopositive for CGRP. A few double-labeled neurons are also found in the superior and lateral vestibular nuclei (Fig. 3B). A second major source of CGRP afferents to the IOC is derived from neurons located within the locus coeruleus (Fig. 3C; Fig. 4E,F). Neurons were labeled bilaterally, although the vast majority were located ipsilateral to the injection. In the midbrain and caudal diencephalon, the ipsilateral central gray matter immediately adjacent to the third ventricle and dorsal to the fasiculus retroflexus as well as the adjacent zona incerta also contain double-labeled neurons (Fig. 3E; Fig. 4G,H). Retrogradely labeled neurons are located in the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, and the red
180
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
nucleus (Fig. 3C); however, none of these cells are CGRP-positive.
4. Discussion Data from several studies now indicate that CGRP is present in the inferior olivary complex of the mouse (Gregg and Bishop, 1997) where it exerts a suppressive effect on olivary neurons (Gregg and Bishop, 1995).
Based on a fine structural analysis of CGRP-immunolabeled profiles (Gregg et al., 1997), it appears that there are both intrinsic and extrinsic sources of CGRP to the olivary complex. Results from the present study have identified several extrinsic sources of CGRP afferents to the mouse inferior olivary complex and include the lateral and interposed nuclei of the cerebellum, the locus coeruleus, and the central gray matter of the diencephalon. A comparatively small projection from the zona incerta as well as
Fig. 1. Photomicrographs that illustrate the distribution of CGRP-immunoreactive varicosities in the inferior olivary complex; (A) a low power photomicrograph of a transverse section taken through the middle of the olivary complex. The letters in (A) indicate the areas from which B–F were taken; (B) is through the ventral lateral outgrowth and (C) is through the dorsal accessory olivary nucleus. Both large, intensely stained varicosities (arrowheads) and smaller puncta (small arrows) are present in these nuclei; (D) is through the lateral dorsal accessory olivary nucleus, (E) is through the principal olivary nucleus, and (F) is through the medial accessory olivary nucleus and the ventral lamella of the principal olive. In these areas, only small immunoreactive puncta (small arrows) are present. Calibration bar in A = 200 mm; B– F = 40 mm.
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
181
Fig. 2. Photomicrographs that illustrate CGRP positive neurons in the brainstem and cerebellum. Intensely stained neurons are located within the hypoglossal nucleus (A), the facial nucleus (B) and the locus coeruleus (D). Less intensely labeled neurons are present within the inferior olivary complex (C), the cerebellar nuclei (E) and the central gray matter around the third ventricle (F). Calibration bars in A – D= 100 mm; E= 200 mm; F =50 mm.
the lateral and superior vestibular nuclei also contributes to the CGRP input to the olivary complex.
4.1. Injection site In the case of one dorsal injection there was spread to the pyramidal tract rostral to the IOC. However, because studies have shown that fibers of passage do not appear to take up the fluorescent microspheres (Katz et al., 1984), this spread to a fiber tract should not produce false-positive labeling. Further, if fibers in the pyramidal tract did take up the marker, they should be confined to the cerebral cortex where no CGRP-positive neurons were detected. The approach from the ventral side spread just rostral to the IOC. When results were compared between
all cases used for analysis, it was found that double-labeled cells were found in the same restricted areas of the rostral brainstem and only in nuclei that have previously been shown to be sources of olivary afferents. In most injections, some microspheres were observed in the area of the nucleus raphe pallidus, located immediately medial to the olivary complex. A recent study indicated there is a widespread distribution in the source of afferents to this nucleus (Hermann et al., 1997). Two of these diverse areas, namely the periaqueductal gray matter in the mesodiencephalon and the subcoerulear nucleus, are sources of afferents to this nucleus; these nuclei contained double-labeled cells. It is possible that these cells project to the nucleus raphe pallidus instead of or in addition to the inferior olivary
182
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
complex. However, if the injection in the nucleus raphe pallidus was effective, we would expect to see a more widespread distribution of retrogradelylabeled cells. In particular, there should be a large number throughout all levels of the periaqueductal grey, including the portion in the caudal mesencephalon and the pons, as well as throughout the pontine and mesencephalic reticular formation (Hermann et al., 1997). These areas were devoid of retrogradely-labeled cells, as were other areas known to be a primary source of afferents to the raphe pallidus such as the hypothalamus, preoptic areas and the Ko¨lliker – Fuse nucleus. Thus, the absence of retrograde-labeling in other areas shown to provide a large input to the nucleus raphe pallidus suggests that the spread of beads to the raphe pallidus was, at best, only minimally effective in labeling afferents to this area and that the labeling actually
represents projections to the inferior olivary complex.
4.2. Cerebellar nuclei Within the cerebellar nuclei, there are two populations of neurons delineated on the basis of their size. Large nuclear neurons, which are non-GABAergic (Ito, 1984; De Zeeuw and Ruigrok, 1994), project rostrally to the vestibular nuclei, reticular nuclei, pontine nuclei, the red nucleus, the thalamus, and the locus coeruleus (Sastry et al., 1997). Small GABAergic nuclear neurons (De Zeeuw et al., 1989), located primarily in the ventral aspect of the nucleus interpositus and lateral nucleus, project to the inferior olivary complex (Nelson and Mugnaini, 1989; Fredette and Mugnaini, 1991). These previous findings, combined with those of the present study would suggest that CGRP and GABA are co-lo-
Fig. 3. Camera lucida drawings of selected sections through the brainstem that illustrate the extent of an injection of fluorescent microspheres into the inferior olivary complex (A) and the distribution of retrogradely and double-labeled neurons (B – E). The injection illustrated was located within the inferior olivary complex and the adjacent pyramidal tract. There was slight spread into the pyramidal tract just rostral to the olivary complex. Following this injection, retrogradely labeled cells (B – E, open circles) were located in the cerebellar nuclei (B), the locus coeruleus (C), the nucleus of Darkschwetsch, the interstitial nucleus of Cajal, and the red nucleus (D). Double-labeled neurons, that is those that were CGRP-positive and that also contained retrogradely transported microspheres (B – C, closed stars) were located in the lateral and interposed cerebellar nuclei (B), the locus coeruleus (C), the superior and lateral vestibular nuclei (B), the central gray area adjacent to the third ventricle (F), and the zona incerta (F).
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
183
Fig. 3. (Continued)
calized in nuclear cell axons and axonal terminals; however, confirmation with double-label studies has yet to be carried out. Olivary neurons are functionally coupled by gap junctions (Sotelo et al., 1974) into ensembles of electrotonically coupled cells that fire in synchrony (Llinas and Yarom, 1980; Lang et al., 1996). Stimulation of the nucleo-olivary pathway has been shown to modulate the effective electrotonic coupling between olivary neurons (Fredette and Mugnaini, 1991; Lang et al., 1996). Although the effects have been attributed solely to GABA, it is likely that CGRP, when co-released from the same terminals, also plays an integral role in altering electrotonic coupling. Based on data which show that CGRP depresses olivary activity (Gregg and Bishop, 1997), we would postulate that the peptide facilitates a GABAergic effect on olivary cells leading to functional uncoupling of olivary neurons. This may be critical in regulating the synchronous output of discrete populations of olivary neurons.
Sinnamon, 1977; Moore and Bloom, 1979). There is little research to date on the function of noradrenergic fibers in the inferior olivary complex. In one study, stimulation of the brainstem close to the locus coeruleus resulted in inhibition of olivary neurons (Hesslow, 1986). Likely, this effect was due to the catecholaminergic output derived from the locus coeruleus. However, it is also possible that CGRP released from the same terminals may have played a role in suppressing olivary activity (Gregg and Bishop, 1995). It may be theorized, based on the role of norepinephrine elsewhere in the CNS (AstonJones et al., 1991, 1994; van Neerven et al., 1991; Van Bockstaele and Aston-Jones, 1995; Bremner et al., 1996), that afferents from the locus coeruleus to the inferior olivary complex and cerebellum may be involved in motor learning or in modulating the responsiveness of olivary neurons to novel sensory stimuli. Future studies will be carried out determine whether CGRP is co-localized with norepinephrine and how they interact to modulate activity of olivary neurons.
4.3. Locus coeruleus
4.4. Mesodiencephalon
The majority of norepinephrine positive neurons in the CNS are located in the locus coeruleus (Amaral and
The most rostral source of CGRP to the inferior olive is derived from neurons that are located within the
184
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
the central gray immediately adjacent to the third ventricle of the caudal diencephalon. These cells occupy the area between the nucleus of Darksche-witsch and the interstitial nucleus of Cajal, immediately dorsal to the fasciculus retroflexus. Cells in this area have been described previously as sources of afferents to the inferior olivary complex (Cintas et al., 1980; Saint-Cyr and Courville, 1981; Onodera, 1984; Rutherford et al., 1984). Little is known as to the physiological effect of this population of neurons in the inferior olive, thus it
is not possible to place them in functional context at this time. Based on an anterograde study (Cintas et al., 1980), neurons in this area project primarily to the dorsal and ventral lamella of the principal olivary nucleus, including the ventrolateral outgrowth. There is also minor projection to the caudal lateral medial accessory olivary nucleus, and the dorsal cap of Kooy. This correlates well with the distribution of large profiles tentatively identified as being derived from afferent axons (Gregg et al., 1997).
Fig. 4. Photomicrographs of a representative injection site (A,B) of fluorescent microspheres into the IOC made from a dorsal approach. The injection is located within the olivary complex and the adjacent pyramidal tract. (C – H) examples of double-labeled cells (arrows) in the nucleus interpositus (C,D), the locus coeruleus (E,F), the central gray area around the third ventricle (G), and the zona incerta (H). Calibration bars in A,B =500 mm; C– H =50 mm.
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
4.5. Functional considerations Neuropeptides such as CGRP are important signaling molecules in the nervous system as they have been shown to modulate the responsiveness of different neurons to synaptic input or to alter spontaneous patterns of activity (Kaczmarek and Levitan, 1987). CGRP has been shown to exert its effect by a variety of different mechanisms in different systems. For example, it has been shown to: (1) reduce acetylcholine release and to decrease receptor sensitivity at the neuromuscular junction (Caratsch and Eusebi, 1990); (2) increase adenylate cyclase activity, thereby increasing cyclic AMP levels (Kobayashi et al., 1987); (3) affect the release of other neurotransmitters such as glutamate and aspartate in the spinal cord (Kangrga et al., 1990); (4) facilitate Ca2 + influx in synaptosomal preparations of the spinal dorsal horn (Oku et al., 1988); and (5) directly affect membrane excitability (Twery and Moss, 1985). In the present study, we have identified several extrinsic sources of CGRP to the inferior olive and have localized it to afferents that, by themselves, have either excitatory or inhibitory effects on olivary neurons. The presence of CGRP in these same afferents together with the intrinsic source of CGRP in olivary dendrites and dendritic spines (Gregg et al., 1997) implies a more complex influence of this peptide over olivary circuits. The precise mechanisms by which CGRP alters olivary firing rate are unknown. For example, olivary neurons have been shown to have an oscillatory firing pattern that is highly dependent on Ca2 + currents (Llinas and Yarom, 1980). Release of CGRP from either intrinsic or extrinsic sources could alter these ion currents, as described in the spinal cord (Oku et al., 1988) which would change pre- and/or postsynaptic membrane excitability and ultimately change the oscillatory firing pattern characteristic of olivary neurons. Thus, CGRP, derived from diverse brainstem and cerebellar nuclei, acting as a co-transmitter, may play a complex role in modulating olivary output.
Acknowledgements This work was supported by NSF grant IBN9630867 to G. Bishop. A. Peltier was supported by funds from the Roessler Foundation from the College of Medicine, The Ohio State University. The authors gratefully acknowledge the technical assistance of Barbara DienerPhelan and the photographic assistance of Yi Fei Chen.
References Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M., 1982. Alternative RNA processing in calcitonin gene expression
185
generates mRNAs encoding different polypeptide products. Nature 289, 240 – 244. Amaral, D.G., Sinnamon, H.M., 1977. The locus coeruleus: neurobiology of a central noradrenergic nucleus. Prog. Neurobiol. 9, 147 – 196. Aston-Jones, G., Chiang, C., Alexinsky, T., 1991. Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance. Prog. Brain Res. 88, 501–520. Aston-Jones, G., Rajkowski, J., Kubiak, P., Alexinsky, T., 1994. Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task. J. Neurosci. 14, 4467 –4480. Bishop, G.A., 1992. Calcitonin gene related peptide in cerebellar afferents: Distribution and origin. J. Comp. Neurol. 322, 201– 212. Bishop, G.A., 1995. Calcitonin gene related peptide modulates neuronal activity in the mammalian cerebellar cortex. Neuropeptides 28, 85 – 97. Bremner, J.D., Krystal, J.H., Southwick, S.M., Charney, D.S., 1996. Noradrenergic mechanisms in stress and anxiety: I. Preclinical studies. Synapse 23, 28 – 38. Caratsch, C.G., Eusebi, F., 1990. Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog. Neurosci. Lett. 111, 344 – 350. Cintas, H.M., Rutherford, J.G., Gwyn, D.G., 1980. Some midbrain and diencephalic projections to the inferior olive in the rat. In: Courville, J., deMontigny, C., Lamarre, Y. (Eds.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, pp. 73 – 124. De Zeeuw, C., Ruigrok, T.J.H., 1994. Olivary projecting neurons in the nucleus of Darkschewitsch in the cat receive excitatory monosynaptic input from the cerebellar nuclei. Brain Res. 653, 345 – 350. De Zeeuw, C., Holstege, J.C., Ruigrok, T.J.H., Voogd, J., 1989. Ultrastructural study of the GABAergic, cerebellar, and mesodiencephalic innervation of the cat medial accessory olive: anterograde tracing combined with immunocytochemistry. J. Comp. Neurol. 284, 12 – 35. Fabri, M., Conti, F., 1990. Calcitonin gene-related peptide-positive neurons and fibers in the cat dorsal column nuclei. Neuroscience 35, 167 – 174. Franco-Cereceda, A., Henke, H., Lundberg, J., Petermann, J.B., Ho¨kfelt, T., Fischer, J.A., 1987. Calcitonin gene related peptide (CGRP) in capsaicin-sensitive substance P-Immunoreactive sensory neurons in animals and man: distribution and release by capsaicin. Peptide 8, 399 – 410. Fredette, B.J., Mugnaini, E., 1991. The GABAergic cerebello-olivary projection in the rat. Anat. Embryol. (Berl) 184, 225 – 243. Gregg, K.V., Bishop, G.A., 1995. Physiological effect of calcitonin gene related peptide in the mouse inferior olive. Neurosci. Abstr. 21, 1192. Gregg, K.V., Bishop, G.A., 1997. Peptide localization in the mouse inferior olive. J. Chem. Neuroanat. 12, 211 – 220. Gregg, K.V., Bishop, G.A., King, J.S., 1997. An electron microscopic analysis of the distribution of CGRP in the mouse inferior olivary complex. Neurosci. Abstr. 23, 120. Hermann, D.M., Luppi, P.-H., Peyron, C., Hinckel, P., Jouvet, M., 1997. Afferent projections to the rat nuclei raaphe magnus, raphe pallidus and reticularis gigantocellularis pars a demonstrated by iontophoretic application of choleratoxin (subunit b). J. Chem. Neuroanat. 13, 1 – 21. Hesslow, G., 1986. Inhibition of inferior olivary transmission by mesencephalic stimulation in the cat. Neurosci. Lett. 63, 76–80. Inoue, H., Nagata, N., Koshihara, Y., 1993. Profile of capsaicin-induced mouse ear oedema as neurogenic inflammatory model: comparison with arachidonic acid-induced ear oedema. Br. J. Pharmacol. 110, 1614 – 1620.
186
A.C. Peltier, G.A. Bishop / Neuroscience Research 34 (1999) 177–186
Ishida-Yamamoto, A., Tohyama, M., 1989. Calcitonin gene-related peptide in the nervous tissue. Prog. Neurobiol. 33, 335–386. Ito, M., 1984. The Cerebellum and Neural Control. Raven Press, New York. Kaczmarek, L.K., Levitan, I.B., 1987. Neuromodulation. Oxford University Press, New York. Kangrga, I., Larew, J.S.A., Randic, M., 1990. The effects of substance P and calcitonin gene-related peptide on the efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro. Neurosci. Lett. 108, 155–160. Katz, L.C., Burkhalter, A., Dreyer, W.J., 1984. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310, 498–500. Kawai, Y., Takami, K., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., MacIntyre, I., Tohyama, M., 1985. Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis. Neuroscience 15, 747–763. Kobayashi, H., Hashimoto, K., Uchida, S., Sakuma, J., Takami, K., Tohyama, M., Izumi, F., Yoshida, H., 1987. Calcitonin gene related peptide stimulates adenylate cyclase activity in rat striated muscle. Experientia 43, 314–316. Krahn, D.D., Gosnell, B.A., Levine, A.S., Morley, J.E., 1984. Effects of calcitonin gene-related peptide on food intake. Peptides 5, 861 – 864. Lang, E.J., Sugihara, I., Llinas, R., 1996. GABAergic modulation of complex spike activity by the cerebellar nucleoolivary pathway in rat. J. Neurophysiol. 76, 255–275. Llinas, R., Yarom, Y., 1980. Electrophysiological properties of mammalian inferior olivary cells in vitro. In: Courville, J. (Ed.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, pp. 379 – 388. Menard, D.P., vanRossum, D., Kar, S., St.Pierre, S., Sutak, M., Jhamandas, K., Quirion, R., 1996. A calcitonin gene-related peptide receptor antagonist prevents the development of tolerance to spinal morphine analgesia. J. Neurosci. 16, 2342–2351. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2, 113–168. Morara, S., Provini, L., Rosina, A., 1989. CGRP expression in the rat olivocerebellar system during postnatal development. Brain Res. 504, 315 – 319. Morara, S., Rosina, A., Provini, L., 1992. CGRP as a marker of the climbing fibers during the development of the cerebellum in the rat. Ann. New York Acad. Sci. 657, 461–463. Nelson, B.J., Mugnaini, E., 1989. Origins of GABAergic inputs to the inferior olive. Exp. Brain Res. 17, 86–107. Oku, R., Nanayama, T., Satoh, M., 1988. Calcitonin gene-related peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn. Brain Res. 475, 356–360. Onodera, S., 1984. Olivary projections from the mesodiencephalic structures in the cat studied by means of axonal transport of horseradish peroxidase and tritiated amino acids. J. Comp. Neurol. 227, 37 – 49. Provini, L., Morara, S., Rosina, A., Forloni, G., 1992. Expression of CGRP binding sites in the developing rat cerebellum. Ann. New York Acad. Sci. 657, 423–425.
.
Rosenfeld, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., Evans, R.M., 1983. Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature 304, 129–135. Rutherford, J.G., Anderson, W.A., Gwyn, D.G., 1984. A reevaluation of midbrain and diencephalic projections to the inferior olive in rat with particular reference to the rubro-olivary pathway. J. Comp. Neurol. 229, 285 – 300. Saint-Cyr, J., Courville, J., 1981. Sources of descending afferents to the inferior olive from the upper brain stem in the cat as revealed by the retrograde transport of horseradish peroxidase. J. Comp. Neurol. 198, 567 – 581. Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.J., Cuatrecasas, P., 1978. Immunohistochemical localization of enkephalin in rat brain and spinal cord. J. Comp. Neurol. 182, 17 – 37. Sastry, B.R., Morishita, W., Yip, S., Shew, T., 1997. Gabaergic transmission in deep cerebellar nuclei. Prog. Neurobiol. 53, 259– 271. Saxen, M.A., Smith, F.L., Dunlow, L.D., Dombrowski, D.S., Welch, S.P., 1994. The hypothermic and antinociceptive effects of intrathecal injection of CGRP (8-37) in mice. Life Sci. 55, 1665– 1674. Shu, S., Ju, G., Fan, L., 1988. The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85, 169 – 171. Skofitsch, G., Jacobowitz, D.M., 1985a. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6, 721 – 745. Skofitsch, G., Jacobowitz, D.M., 1985b. Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system. Peptides 6, 1069 – 1073. Sotelo, C., Llinas, R., Baker, R., 1974. Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling. J. Neurophysiol. 37, 541 – 559. Swenson, R.S., Sievert, C.F., Terreberry, R.R., Neafsey, E.J., Castro, A.J., 1989. Organization of cerebral cortico-olivary projections in the rat. Neurosci. Res. 7, 43 – 54. Twery, M.J., Moss, R.L., 1985. Calcitonin and calcitonin gene-related peptide alter the excitability of neurons in rat forebrain. Peptides 6, 373 – 378. Van Bockstaele, E.J., Aston-Jones, G., 1995. Integration in the ventral medulla and coordination of sympathetic, pain and arousal functions. Clin. Exp. Hypertens. 17, 153 – 165. van Neerven, J., Pompeiano, O., Collewijn, H., 1991. Effects of GABAergic and noradrenergic injections into the cerebellar flocculus on vestibulo-ocular reflexes in the rabbit. Prog. Brain Res. 88, 485 – 497. Van Rossum, D., Hanisch, U.K., Quirion, R., 1997. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci. Biobehav. Rev. 21, 649 – 678. Yamano, M., Tohyama, M., 1993. The innervation of calcitonin gene-related peptide to the Purkinje cells and granule cells in the developing mouse cerebellum. Brain Res. Dev. Brain Res. 72, 107 – 117. Zhao, B.Y., Schwartz, J.P., 1998. Involvement of cytokines in normal CNS development and neurological diseases: Recent progress and perspectives. J. Neurosci. Res. 52, 7 – 16.