The Distribution of Catecholamines within the Inferior Olivary Complex of the Cat and Rhesus Monkey JOHN R. SLADEK, JR. AND JAMES P. BOWMAN Departments of A n a t o m y , University of Rochester School of Medicine and Dentistry, Rochester, N e w York 14642 and Colorado State University, Fort Collins, Colorado 80521
ABSTRACT Catecholamine histofluorescence was examined in the inferior olivary complex of the cat and rhesus monkey. Species-specific patterns of catecholamine-containing varicosities were observed. In the rat, the highest concentration of catecholamine varicosities was seen within the dorsal lamella of the principal nucleus. In contrast, this same portion of the inferior olivary complex appeared void of catecholamine varicosities i n the cat and rhesus monkey. In the cat, the highest concentration of varicosities occurred within the medial one-half of the dorsal accessory nucleus while few, if any, varicosities were seen in this portion of the complex in the rat and monkey. The lateral lamella of the principal nucleus contained the highest concentration seen in the rhesus monkey, a finding which contrasts to the minimal number of varicosities seen in this area in the rat and cat. Catecholamine-containing cell bodies, reported to exist in the rat, were not observed in cat and monkey. These data extend the previous observation of species-specific distribution in rodents to include members of the more phylogenetically advanced orders; Carnivora and Primata. Catecholamines were found primarily within those portions of the olivary complex reported to be involved in harmaline-induced tremor activity in the cat.
The distribution of catecholamines (CA) monoamine oxidase inhibitor, acts directly within the mammalian inferior olivary (10) on I 0 cells or on presynaptic pontobulbar complex has been the subject of several pre- systems impinging upon them (Llinas and vious investigations from this laboratory. Volkind, '73). Comparative histofluoresHistofluorescence data have indicated a cence data are necessary for providing the species-specific distribution of CA-contain- neuroanatomical basis for such an investiing varicosities within members of the or- gation. der Rodentia (Sladek and Hoffman, '72, MATERIALS A N D METHODS '73; Hoffman and Sladek, '73). The objectives of the present study were to determine Eighteen rats, 30 cats, and ten rhesus the pattern of distribution of CA-containing monkeys (Macaca mulatta),ranging in age varicosities within the subnuclei of the cat from infants to adults and including both and monkey I 0 complex and, further, to sexes, were examined. Details of the histoascertain whether species dissimilarities chemical procedure (Falck et al., '62) are also characterize this distribution in mor- described elsewhere (Sladek, '75). Criteria phologically homologous olivary cell groups. used to evaluate varicosity density follow The impetus for this investigation was pro- those described previously (Hoffman and vided in part by recent data implicating se- Sladek, '73). To summarize the latter, claslected regions of the I 0 in the genesis of sification of the relative number of varicosharmaline-induced tremor in both cat and ities observed within a square (100 uM/ monkey (Lamarre and Mercier, '71; De- side) of brain tissue was as follows: scatMontigny and Lamarre, '73; Llinas and tered; very low (1 +); low (2 +); medium Volkind, '73). While it has been established ( 3 + ) ; high ( 4 f ) ; and very high (5+). that I 0 cells in harmaline pretreated ani- Since species comparisons were to be made mals sustain rhythmic oscillatory firing in CA localization, the capacity of the histopatterns at the tremor frequency, it has chemical procedure to produce monoamine not been resolved whether harmaline, a fluorescence of comparable quality in each J. COMP. NEUR.,163: 203-214.
20 3
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species was critical. The substantia nigra, locus coeruleus, and nucleus raphe pallidus served as control areas and were observed to possess similar patterns of monoamine cell body and varicosity fluorescence in rat, cat and monkey. Thus, the procedure was judged adequate for the purpose of this study. The subdivisions of the inferior olivary complex described and compared in the present investigation are: principal nucleus-dorsal lamella (PD); principal nucleus-ventral lamella (PV); principal nucleuslateral lamella (PL); dorsal accessory nucleus (D); medial accessory nucleus (M); and subdivisions of the medial accessory nucleus. Criteria for distinguishing subnuclei of the M followed those established in previous investigations (Kooy, '17; Brodal, '40; Walberg, '56; Taber, '61; Bowman and Sladek, '73).
cat: 6-week-old; monkey: 12-week-old)possessed adult-like patterns of CA varicosities. No apparent differences in either density or intensity were noted between infants and adults of a given species. It is presumed that the infants were examined subsequent to, or at about the age at which CA distribution is morphologically like that of adults. This phenomenon occurs in the rat, for example, at 3-4 weeks of postnatal development (Loizou, '72). Distribution of 10 varicosities
Rat. The distribution of CA-containing varicosities within the various subdivisions of the I 0 were found to be in agreement with the previous observations of Fuxe ('65a), Sladek ('71a), Loizou ('72) and Hoffman and Sladek ('73). For subsequent comparative purposes, this distribution is summarized in table 1. RESULTS Cat. Only scattered varicosities were obOntogenetic comparisons. The infants served to be distributed throughout the enin all species examined (rat: 3-week-old; tire rostrocaudal extent of the principal nuAbbreviations PD, principal nucleus-dorsal lamella dm, principal nucleus-dorsal lamella, dorsomedial portion vl, principal nucleus-dorsal lamella, ventrolateral portion PV, principal nucleus-ventral lamella PL, (rat, cat), principal nucleus-ventrolateral bend (monkey), principal nucleus-lateral lamella D, dorsal accessory nucleus dm, dorsal accessory nucleus, dorsomedial portion
vl, dorsal accessory nucleus, ventrolateral portion M (cat), Medial accessory nucleus Mv, Medial accessory nucleus, ventral part Md, Medial accessory nucleus, dorsal part B, Medial accessory nucleus, nucleus B dc, Medial accessory nucleus, dorsal cap of Kooy vlo, Medial accessory nucleus, ventrolateral outgrowth dmcc, Medial accessory nucleus, dorsomedial cell column a-g,(monkey), Medial accessory nucleus, subnuclei
TABLE 1
Summary of the distri bic tion of ca tec holam in e-conta in ing varicosities within the inferior olivary complex of the rat, cat, and rhesus monkey Rat
PD dm vl PV PL D dm vl M
dc
Cat
4+
3+ 2 f
Scattered Scattered Scattered 3+
Scattered
Scattered Scattered Scattered Scattered
Scattered Scattered Scattered 1-3
w+
Scattered Scattered
1-2
Mv Md B dc vlo dmcc
Rhesus monkey
+
0-scattered 2+
Scattered 0 0
0
+
a b C
d&e f g
0-sca ttered 0-scattered 0-sca ttered 0-scattered 0-scattered 0-scattered
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OLIVARY CATEXHOLAMINES
cleus; that is, within each of the three lamellae (figs. 1, 2A). The pattern is quite unlike that seen in the rat, where medium to high accumulations of varicosities are observed in the dorsal lamella. The pattern of varicosities within the dorsal accessory nucleus likewise appeared dissimilar to that observed in the rat. Varicosities were unevenly distributed throughout the rostrocaudal extent of the cat D, with the ventrolateral one-half possessing a 1 to 2 density of very fine varicosities and the dorsomedial one-half a 3 to 4 density (figs. 1, 3). Many of these fine varicosities, especially those in the dorsomedial region, appeared in a pericellular position. Varicosities located within the medial accessory nucleus (fig. 1) were differentially represented within its several subdivisions (as delineated by Walberg, ’56; Taber, ’61). The portion of the M designated as the ventral part (Mv) contained few, if any, varicosities at most rostrocaudal levels with the exception of an occasional linear profile of fine Varicosities. The dorsal part, or subdivision, (Md) contained a 2 density of fine varicosities throughout its rostrocaudal extent, while only scattered varicosities were seen within the nucleus B. No varicosities were observed to be distributed in the
+
+
+
+
+
RostraI dmcc
X
P r -
‘
Caudal
Fig. 1 Schematic illustration of frontal sections through the cat inferior olivary complex. Roman numerals indicate levels similar to those illustrated by Walberg (’56). The rostrocaudal distribution of C A varicosities is indicated by dots throughout the various subdivisions of the I 0 complex. Abbreviations are defined in table 1.
dorsal cap of Kooy (dc), the ventrolateral outgrowth of Kooy (vlo), or the dorsomedial cell column (dmcc). The above described varicosities all were distributed within the boundaries of the several subdivisions of the I 0 complex. However, in addition randomly distributed linear profiles, or “strings,” of larger varicosities were observed adjacent to and within the subdivisions of the complex. Rhesus monkey. Although scattered varicosities of very fine calibre were distributed throughout the rostrocaudal extent of both the dorsal and ventral lamellae of the principal nucleus, the lateral lamella contained the only pattern of varicosities in the entire I 0 complex of the monkey which was of high enough concentration to be assigned definite density ratings. Here, a 1 + to 3 density of very fine varicosities, often appearing in the form of linear profiles, was observed (figs. 2B,C, 4). In parasagittal section, the extreme lateral extent of the PL was observed to possess a n uneven distribution of varicosities, with a 1 + density in the caudal one-half and a 2 to 3 + density in the rostra1 one-half (fig. 4A). This pattern blended with a 2 to 3 density of the PL on sections just medial to the above (fig. 4B). Varicosities of very fine size were occasionally seen in the dorsal accessory olive. None of the seven subdivisions of the medial accessory nucleus (as delineated by Bowman and Sladek, ’73) possessed anything more than a n occasional very fine calibre varicosity. Olivary CA-containing cell bodies. CAcontaining cell bodies of the A 3 group (Dahlstrom and Fuxe, ’64; Fuxe, ’65b) were not seen within the I 0 of rat, cat or monkey. The existence of the A 3 group within the I 0 of rat, as reported by Dahlstrom and Fuxe (’64) and Fuxe (‘65b), is discussed below and elsewhere (Hoffman .and Sladek, ’73; Garver and Sladek, ’75). In the cat, a small number of CAsontaining cell bodies was observed immediately lateral to the ventral lamella of the principal nucleus. When discernable, such cell bodies were present at most brain stem levels which contained the I 0 complex.
+
+
DISCUSSION
The ability of the presently-employed technique to produce optimal histofluores-
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cence in different mammalian species has been discussed previously (Hoffman and Sladek, ’73; Garver and Sladek, ’75). However, an additional technical factor merits present consideration. Due to the thickness of the monkey calvaria, a greater length of time is required for brain removal following death (10-15 minutes) than in the cat (3-5 minutes) or rat (1-2 minutes). In an attempt to determine whether brain removal time represents a causative factor for the observed species-specific patterns of CA varicosities, additional cats and rats were investigated in which brain-removal-time was purposely slowed to match that of rhesus monkey. CA distribution in such animals conformed to the patterns reported above. While it is believed that extended brain-removal-time could be a factor of concern, it did not appear to affect the quality of histofluorescence within the above timespans. The general lack of dense patterns of CA varicosities in the I 0 of the rhesus monkey does not appear to be attributable to a similar lack of such varicosities in other areas of the brain of this species. For example, an extensive examination of the rhesus hypothalamus has revealed the presence of exceptionally dense and intense patterns of CA varicosities (Sladek et al., ’75). If the rhesus exhibited a general lack of histochemically-demonstrable varicosities then i t could be argued that the apparent species-specific patterns are of a lesser importance. On the contrary, initial microspectrofluorophotometric quantitative studies of CA occurrence within the hypothalamus of the rat, cat and rhesus monkey by this laboratory indicate the most intense patterns of CA varicosities occur in the primate hypothalamus. The significance of this is as yet undetermined. It is the belief of certain investigators that monoamine distribution as seen in the rat brain is representative of other mammalian species. Battista et al. (’72) reported that the principal architecture of monoamine distribution in monkey (i.e,, Macaca irus and Ceropeithecus sabaeus) is similar to that seen in the rat, rabbit and cat. 01sen et al. (’73) examined fetal human brain and indicated that the basic cytoarchitecture of monoamine distribution is similar to the rat brain. Additionally, DiCarlo et al.
(‘73) reported a lack of “significant interspecies differences between the rat and the squirrel monkey.” It is true that certain aspects of monoamine morphology as seen in the rat reflect those of other mammalian species. The existence of dopaminergic nigro-neostriatal neurons, indoleaminergic raphe neurons, noradrenergic locus coeruleus neurons, and dopaminergic arcuate neurons, all are comparable interspecies monoamine patterns. However, evidence also indicates species-specific patterns. Sladek (’71b) initially brought attention to this phenomenon in the brain stem reticular formation of the kitten, and extended the observation to include age-dependent differences in cat and macaque (Sladek, ’73; Sladek et al., ’74). Hoffman and Sladek (‘73) reported marked differences in CA distribution in the I 0 of rat, gerbil and rabbit. Felten et al. (’74) examined squirrel monkey, wherein they found three additional CA cell groups not previously reported in the rat. These later data are at variance with those of DiCarlo et al. (’73), who also examined squirrel monkey and failed to report such additional groups. A recent review by Morgane and Stern (‘74) further defines species variations in monoamine distribution. The present study lends additional support to the concept of speciesspecific patterns of CA distribution by extending definitive investigations in a particular nuclear complex to embrace members of more phylogenetically advanced orders than Rodentia. Recent investigations on the neuronal loci and pathways mediating the fast (8l2/sec) tremor induced in cats by the administration of harmaline are of signifiFig. 2 Comparative distribution of CA varicosities i n the principal nucleus in cat and rhesus monkey. A. Cat. CA varicosities are virtually absent in this portion of the I 0 complex with the exception of a n occasional varicosity (b) a s indicated. Dull lipofuscin-containing perikarya (4) are seen withi n the ventral (PV), lateral (PL), a n d dorsal lamell a e (PD). X 140. B. Rhesus m o n k e y . In contrast to the cat, the monkey principal nucleus contains a 2 to 3 + density of varicosities within the lateral lamella (PL). CA varicosities seen i n this region of the complex are characterized by linear profiles of Intense lipofuscin autofine-sized varicosities 0 ) . fluorescence is seen eccentrically placed within the cytoplasm of olivary perikarya (+). x 120. C. Rheszcs m o n k e y . Profiles of fine-sized varicosities (b) are seen i n this high power magnification of the PL. x 300.
OLIVARY CATECIIOLAMINES
Figure 2
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J O H N R. SLADEK. JR. A N D J A M E S P. B O W M A N
OLIVARY CATECHOLAMINES
209
Fig. 4 Rhesiis monkey. The schematic, taken from Bowman and Sladek ('73), illustrates a frontal section (level G ) through the principal nucleus of the olivary complex and serves to approximate the level of sections of the PL illustrated in A and B. The ventral, lateral a n d dorsal lamellae a r e stippled, unmarked, a n d cross-lined, respectively. A. Parasagittal section through the PL of the rhesus monkey. The rostral portion of the complex i s indicated ( * ) , The extreme lateral pole of the PL exhibits a 3 dendensity caudally. X 100. sity of fine-sized linear profiles of CA varicosities (b) rostrally and a 1 to 2 B. Parasagittal section slightly medial to that illustrated in A. A 1 to 2 density of CA varicosities (b) i s noted throughout all portions of the PL at this level. The rostral portion of the complex is indicated (*). Abundant lipofuscin autofluorescence is seen in a juxtanuclear position within olivary perikarya throughout the entire PL. X 80.
+
+
+
+
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J O H N R . SLADEK, JR. AND J A M E S P. B O W M A N
cance from a number of standpoints, not the least of which is that they have clearly implicated monoamines as important determinants of inferior olivary function (Lamarre and Mercier, '71; DeMontigny and Lamarre, '73; Llinis and Volkind, '73). In the context of the present study, these investigations appear to provide a basis for preliminary formulations concerning possible functional correlates associated with the "normal" pattern of olivary CA distribution by bringing to bear on a morphologic substrate both single unit and behavioral data. It thus is of interest to note that harmaline has an action apparently restricted largely to the cells of medial accessory nucleus, particularly its caudal portion, although dorsal accessory cells also have been reported to display harmaline-induced rhythmic oscillatory discharges (Llinas and Volkind, '73; DeMontigny and Lamarre, '73). The present study has shown that the accessory nuclei contain the most prominent distributions of CA-containing varicosities in the entire olivary complex of the cat, with the dorsomedial one-half of the dorsal accessory nucleus displaying a 3 to 4 + density and the dorsal part of the medial accessory having a 2 density rating. The latter nucleus, according to Taber ('61) and Walberg ('56), is represented caudally in the cat, decreases in size rostrally, and is absent oral to a midolivary level. Recognizing the hazards involved in microelectrode tract reconstruction and localization of responsive neurons and despite the fact that the olivary cells showing harmaline rhythms were not defined relative to the specific subdivisions of the medial accessory nucleus, there nevertheless appears to be correspondence between the distribution of CA varicosities in the 10 and the major sites of action of harmaline in the complex. Final determination of this correlation, however, must await further experiments in which CA distribution in the cat olive is examined under conditions of harmaline intoxic a tion. It is generally assumed that CA-containing varicosities arise from fluorescence demonstrable cells. If the pattern of varicosities observed in the present study later proves to be implicated in the harmaline induced tremor phenomenon, then the olivary varicosity fluorescence must derive
+
from cells located at pontobulbar levels of the neuraxis since cerebellectomy, combined transection a t high cervical and irtercollicular levels, and transection caudal to the red nucleus all are ineffectual in abolishing olivary harmaline cell rhythms (DeMontigny and Lamarre, '73; Llinas and Volkind, '73). DeMontigny and Lamarre ('73) conclude that such harmaline rhythms' do not derive from other medullary nuclei, leaving, by exclusion, either a driving of I 0 cells by nuclei of the pontine reticular formation or a n action of harmaline on terminals impinging on the olivary cells themselves. The marked difference in CA varicosity distribution in the I 0 complex of cat and monkey (again assuming the presence of CA reflects to some degree olivary regions likely to be implicated in harmaline tremor), suggests, among others, the following possibilities: that monoamines play a generally less dominant, although analogous, role in the function of the primate and cat olive in which case the monkey's I 0 complex may contain CA in concentrations too low to be demonstrable with formaldehydeinduced histofluorescence;' or that the fast tremor observed in monkeys under harmaline does not have an I 0 genesis. The latter suggestion is at variance with DeMontigny and Lamarre ('73) who preliminarily indicated that the 7-12/sec. harmalineinduced tremor observed in the intact monkey appears to have a similar neural substrate as that disclosed for the cat. The significance of the general lack of CA varicosity fluorescence in the I 0 complex of the monkeys we examined, especially in the accessory nuclei, thus remains to be explained. It is of interest to speculate on the location of perikarya of origin of olivary histofluorescence. Most investigators have assumed that fluorescence-demonstrable cell bodies give rise to fluorescence-demonstrable varicosities. The latter are believed to represent either terminal arborizations of I This interpretation might he favored by the newlyintroduced glyoxglic acid histofluorescence technique, which reportedlycan demonstratevaricosities offinecalibre not routinely seen with formaldehyde-induced histofluorescence (Lindvall et al., '74). However, this interpretation does not appear compatible with observations in this laboratory that the presently-employed technique i s capable of routinely demonstrating fine calibre varicosities of areas such as the cerebellar cortex, colhculi. thalamus, geniculates. and I 0 (Hoffman and Sladek, '73; Sladek. '75).
OLIVARY CATECHOLAMINES
axons or accumulations of monoamines along the preterminal portion of axons. This interpretation would require that olivary varicosities represent some portion of axons which arise from fluorescence demonstrable perikarya. A difficulty in reconciling this phenomenon with olivary histofluorescence, is that at present no known olivary afferents arise from neural loci which contain CA-perikarya. For example, sources of olivary afferents such a s parvicellular portion of the red nucleus and the spinal cord do not contain CA cell bodies. Thus, if I 0 varicosities represent axon termina:s which arise from CA-containing cells, then these cells either must be located outside of areas known to project to the olive or contain catecholamines in concentrations too low to be visualized with routine histochemistry. At present, only one site of origin of olivary afferents, the periaqueductal gray of the macaque, has been demonstrated to possess catecholaminergic perikarya. However, these perikarya as yet have only been observed in Macaca speciosa (Garver and Sladek, '75), squirrel monkey (Felten et al., '74) and Macaca mulatta (Sladek, Bowman and Felten - unpublished observations). In each species, a sparse population of CA-containing perikarya were found among the majority of non-fluorescing cells of this region. While this small number of cells could be a source of varicosities seen within the macaque 10, they appear as a less likely source in phylogenetically lower mammals wherein monoaminergic cells have not been seen in this area and wherein certain portions of the I 0 are richly supplied with varicosities. Another potential source of olivary histofluorescence might be from catecholaminergic cells reported to exist within the olivary complex (Dahlstrom and Fuxe, '64). Such cells were said to possess weak fluorescence in the rac. It has been proposed that certain catecholaminergic cells display variable fluoresc 3nce intensities depending on their state of activity, a s in the case of the dopaminergic tuberoinfundibular neurons of the neuroendocrine hypothalamus (Fuxe et al., '67). Thus, if A 3 cells, which have evaded subsequent histofluorescence demonstration (Hoffman and Sladek, '73; Garver aTid Sladek, '75), are in a state of extreme inactivity, this might account for their apparent lack of fluorescence.
21 1
Two recent investigations appear to lend support, either direct or indirect, to the hypothesis that a source of olivary afferents may arise from the locus coeruleus. Lindvall et al. ('74) demonstrated with the glyoxylic acid-vibratome technique that the rat thalamus contained a system of delicate, fine-sized varicosities, which they interpret as forming networks of terminal axonal ramifications. These varicosities are similar in appearance to those observed in this and other laboratories within the cerebellar and cerebral cortices, thalamus, colliculi, geniculates and the inferior olivary complex. It is of interest in this regard, that bilateral lesions of the locus coeruleus resulted in the absence of these fine-sized varicosities in the thalamus of the rat. Hence it is conceivable that this type of axon, classified by Lindvall et al. a s the "locus type of axon," may also project to the inferior olivary complex. Lending support to this concept is the recent study of Kobayashi et al. ('74) who performed a detailed biochemical mapping of projections of the rat locus coeruleus. Lesions were placed in the locus coeruleus and the sensitive radioisotopic enzyme assay technique was coupled with frozen section punch techniques of discrete hypothalamic nuclei (Palkovits, ' 7 3 ; Jacobowitz, '74) to observe changes in norepinephrine concentration as a result of the lesion. These investigators observed that the inferior olive, when measured bilaterally, demonstrated a significantly decreased norepinephrine concentration, suggesting a possible bilateral innervation from the locus coeruleus. Thus, based on the above, the olive may well receive afferents from the locus coeruleus. Another intriguing possibility for the interpretation of varicosity type fluorescence recently was presented by Nobin and Bjorklund ('75) who have observed apparent dendritic varicosity fluorescence within neurons of the substantia nigra-zona compacta. Such neurons possess dendritic ramifications located within the pars reticulata of the same nucleus which apparently end as dendritic varicosities, a finding in concert with both Golgi and ultrastructural studies of the same region. While these investigators indicate that this area of the rat brain is the only one a t present which displays this rather unique histofluorescence, preliminary observations in this lab-
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Falck, B., N.-A. Hillarp, G. Thieme and A. Torp 1962 Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10: 348-354. Felten, D. L.. A. M. Laties and M. B. Carpenter 1974 Monoamine-containing cell bodies in the squirrel monkey brain. Am. J. Anat., 1 3 9 : 153166. Fuxe, K. 1965a Evidence for the existence of monoamine neurons in the central nervous svstem. 111. The monoamine nerve terminal. Z. Zellforsch., 65: 573-596. 1965b Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine nerve terminals in the central nervous system. Acta Physl. Scand., Suppl., 64, p. 247. Fuxe, K., T. Hokfelt and 0. Nilsson 1967 Activity changes in the tubero-infundibular dopamine neurons of the rat during various states of the reproductive cycle. Life Sci., 6 : 2057-2061. Garver, D. L., and J . R. Sladek, Jr. 1975 Monoamine distribution in primate brain. I. Catecholamine-containing perikarya in the brain stem of Mtrctrcci speciostr. J . Comp. Neur., 159: 289-304. Hoffman, D. L., and J . R. Sladek, Jr. 1973 The distribution ofcatecholamines within the inferior olivary complex of the gerbil and rabbit. J. Comp. Neur., 1 5 1 : 101-112. ACKNOWLEDGMENTS Jacobowitz, D. 1974 Removal of discrete fresh regions o f t h e rat brain. Brain Res., 80: 111-115. This study was supported by USPHS Pro- Kobayashi, R., M. Palkovits, I. Kopin and D. Jagram Project Grant 11642. The authors cobowitz 1974 Biochemical mapping of noradrenergic nerves arising from the rat locus wish to thank Miss Yvonne Cheung for skillcoeruleus. Brain Res., 77: 269-279. ful technical assistance. Kooy, F. H . 1917 The inferior olive in vertebrates. Folia Neuro-biol., 10s:2 0 5 3 6 9 . Lamarre, Y . , a n d L. A. Mercier 1971 NeurophysLITERATURE CITED iological studies of harmaline-induced tremor in Battista, A., K. Fuxe, M. Goldstein a n d M. Ogawa the cat. Can. J. Physiol. Pharmacol., 49: 10491972 Mapping of central monoamine neurons 1058. in the monkey. Experientia, 28: 6 8 8 4 9 0 . Larochelle, L., P. Bedard, R. Boucher and L. PoirBjorklund, A,, and 0. Lindvall 1975 Dopamine ier 1970 The rubro-olivo-cerebellar loop and i n dendrites of substantia niera postural tremor in the monkey. J . Neurol. Sci., - neurons sueeestions for a role i n dendritic terminals. Brain 11 : 5344. Res., 8 3 : 531-537. Lindvall, O . , A. Bjorklund, A. Nobin and U.Stenevi Bowman, J. P., a n d J. R. Sladek, Jr. 1973 Mor1974 The adrenergic innervation of the rat thalphology of the inferior olivary complex of the amus a s revealed by the glyoxylic acid fluoresrhesus monkey ( M m c i c t i m i t l t i t t n ) . J. Comp. cence method. J . Comp. Neur., 154: 317-348. Neur., 152: 299-316. L h a s , R., a n d R. A. Volkind 1973 The olivocerBrodal, A. 1940 Experimentelle Untersuchunebellar system: functional properties as revealed by harmaline-induced tremor. Exp. Brain. Res., gen uber die olivo-cerebellare lokalization. Ztschr. 18: 69-87. ges. Nerv. u . Psychiat., 169: 1-153. Brodal, A , , F. Walberg and T. Blackstad 1950 Loizou, L. A. 1972 The postnatal ontogeny of monoamine-containing neurones i n the central Termination of spinal afferents to the inferior nervous system of the albino rat. Brain Res., olive in c a t . J. Neurophysl., 1 3 : 4 3 1 4 5 4 . 40: 3 9 5 4 1 8 . Dahlstrom, A,, and K. Fuxe 1964 Evidence for the existence of monoamine-containing neurons Morgane, P. J.. and W. C. Stern 1974 Chemical i n the CNS. I. Demonstration of monoamines in anatomy of brain circuits in relation to sleep the cell bodies of brain stem nuclei. Acta Physl. and wakefulness. I n : Advances in Sleep ReScand., Suppl., 62: p. 232. search. Vol. 1. E. Weitzman, ed. Spectrum PubDeMontigny, C., a n d Y. Lamarre 1973 Rhythmic lications Inc., New York. activity induced by harmaline in the olivo-cer- Olson, L., L. 0. Boreus and A. Seiger 1973 Histochemical demonstration and mapping of 5ebello-bulbar system of the cat. Brain Res., 53 : 81-95. hydroxytryptamine- a n d catecholamine-containDiCarlo, V . , J . E. Hubbard and P. Pate 1973 Fluing neuron systems i n the h u m a n fetal brain. Z. Anat. Entwick1:Gesch.. 139: 259-282. orescence histochemistry of monoamine-containing cell bodies i n the brain stem of the squir- Palkovits, M. 1973 Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res., rel monkey (Sttimiri sciurezts). J . Comp. Neur., 57: 307-326. 152: 347-372.
oratory (Sladek and Parnavelas, '75) indicate that other areas of the brain are characterized by such a phenomenon. Thus, if the inferior olivary complex contains dendritic ramifications of adjacent nuclei, which in themselves possess catecholamines, this may offer another possible explanation as to the source of olivary histofluorescent varicosities. It is of interest that the adjacent lateral reticular nucleus in both the cat and the rhesus monkey has been observed by this laboratory to contain such perikarya. It would be of further interest i f the lateral reticular nucleus, a source of cerebellar mossy fibers, projects dendrites into the olivary complex, a source of cerebellar climbing fibers. With the exception of the biochemical assay investigation indicated above, no attempts have been made to localize the site of origin of the species-specific patterns of olivary CA varicosities.
~
_---
OLIVARY CATECHOLAMINES Sladek, J . R., Jr. 1971a The distribution of catecholamine terminals within the mesencephalon, pons, and medulla oblongata of the immature cat brain: A fluorescence microscopic study. Dissertation Abstracts International, 32: 2489-2490. 1971b Differences in the distribution of catecholamine varicosities in cat and rat reticular formation. Sci., 174: 4101112. 1973 Age-dependent differences in catecholamine distribution within cat reticular formation. Exp. Neur., 38:520-524. (1975 submitted for publication) A new histochemical freeze-drying apparatus a n d a detailed methodological account of its use for FalckHillarp monoamine histofluorescence. Sladek, J. R., Jr., D. Garver and B. Tabakoff 1974 Histofluorescence and biochemistry of monoamines in macaque brain. Anat. Rec., 178: 465466. Sladek, J. R., Jr., and D. L. Hoffman 1972 Differences in biogenic amine distribution in mammalian brain. J. Histochem. Cytochem., 20: 848. 1973 Differences in catecholamine dis-
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tribution in the inferior olivary complex of' various mammals. Anat. Rec., 177: 444-445. Sladek, J. R., Jr., G. Hoffman, Y. Cheung and D. Felten 1975 Monoamine histofluorescence in rbesus monkey hypothalamus and pineal. Anat. Rec., 181: 482-483. Sladek, J. R., Jr., and J . Parnavelas (1975, submitted for publication) C atecholamine-con taining dendrites in primate brain. Sladek, J. R., Jr., B. Tabakoffand D. Garver 1974 Certain biochemical correlates of intense serotonin histofluorescence in the brain stem of the neonatal monkey. Brain Res., 67: 363-371. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat. J. Comp. Neur., 166: 27-70. Walberg, F. 1954 Descending connections to the inferior olive. In: Aspects of Cerebellar Anatomy. J. Jansen and A . Brodal, eds. Grundt Tanum, Oslo. 1956 Descending connections to the inferior olive. An experimental study in the cat. J. Comp. Neur., 104: 77-172.
ABSTRACT Catecholamine histofluorescence was examined in the inferior olivary complex of the cat and rhesus monkey. Species-specific patterns of catecholamine-containing varicosities were observed. In the rat, the highest concentration of catecholamine varicosities was seen within the dorsal lamella of the principal nucleus. In contrast, this same portion of the inferior olivary complex appeared void of catecholamine varicosities i n the cat and rhesus monkey. In the cat, the highest concentration of varicosities occurred within the medial one-half of the dorsal accessory nucleus while few, if any, varicosities were seen in this portion of the complex in the rat and monkey. The lateral lamella of the principal nucleus contained the highest concentration seen in the rhesus monkey, a finding which contrasts to the minimal number of varicosities seen in this area in the rat and cat. Catecholamine-containing cell bodies, reported to exist in the rat, were not observed in cat and monkey. These data extend the previous observation of species-specific distribution in rodents to include members of the more phylogenetically advanced orders; Carnivora and Primata. Catecholamines were found primarily within those portions of the olivary complex reported to be involved in harmaline-induced tremor activity in the cat.
The distribution of catecholamines (CA) monoamine oxidase inhibitor, acts directly within the mammalian inferior olivary (10) on I 0 cells or on presynaptic pontobulbar complex has been the subject of several pre- systems impinging upon them (Llinas and vious investigations from this laboratory. Volkind, '73). Comparative histofluoresHistofluorescence data have indicated a cence data are necessary for providing the species-specific distribution of CA-contain- neuroanatomical basis for such an investiing varicosities within members of the or- gation. der Rodentia (Sladek and Hoffman, '72, MATERIALS A N D METHODS '73; Hoffman and Sladek, '73). The objectives of the present study were to determine Eighteen rats, 30 cats, and ten rhesus the pattern of distribution of CA-containing monkeys (Macaca mulatta),ranging in age varicosities within the subnuclei of the cat from infants to adults and including both and monkey I 0 complex and, further, to sexes, were examined. Details of the histoascertain whether species dissimilarities chemical procedure (Falck et al., '62) are also characterize this distribution in mor- described elsewhere (Sladek, '75). Criteria phologically homologous olivary cell groups. used to evaluate varicosity density follow The impetus for this investigation was pro- those described previously (Hoffman and vided in part by recent data implicating se- Sladek, '73). To summarize the latter, claslected regions of the I 0 in the genesis of sification of the relative number of varicosharmaline-induced tremor in both cat and ities observed within a square (100 uM/ monkey (Lamarre and Mercier, '71; De- side) of brain tissue was as follows: scatMontigny and Lamarre, '73; Llinas and tered; very low (1 +); low (2 +); medium Volkind, '73). While it has been established ( 3 + ) ; high ( 4 f ) ; and very high (5+). that I 0 cells in harmaline pretreated ani- Since species comparisons were to be made mals sustain rhythmic oscillatory firing in CA localization, the capacity of the histopatterns at the tremor frequency, it has chemical procedure to produce monoamine not been resolved whether harmaline, a fluorescence of comparable quality in each J. COMP. NEUR.,163: 203-214.
20 3
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JOHN R. SLADEK, JR. AND JAMES P. BOWMAN
species was critical. The substantia nigra, locus coeruleus, and nucleus raphe pallidus served as control areas and were observed to possess similar patterns of monoamine cell body and varicosity fluorescence in rat, cat and monkey. Thus, the procedure was judged adequate for the purpose of this study. The subdivisions of the inferior olivary complex described and compared in the present investigation are: principal nucleus-dorsal lamella (PD); principal nucleus-ventral lamella (PV); principal nucleuslateral lamella (PL); dorsal accessory nucleus (D); medial accessory nucleus (M); and subdivisions of the medial accessory nucleus. Criteria for distinguishing subnuclei of the M followed those established in previous investigations (Kooy, '17; Brodal, '40; Walberg, '56; Taber, '61; Bowman and Sladek, '73).
cat: 6-week-old; monkey: 12-week-old)possessed adult-like patterns of CA varicosities. No apparent differences in either density or intensity were noted between infants and adults of a given species. It is presumed that the infants were examined subsequent to, or at about the age at which CA distribution is morphologically like that of adults. This phenomenon occurs in the rat, for example, at 3-4 weeks of postnatal development (Loizou, '72). Distribution of 10 varicosities
Rat. The distribution of CA-containing varicosities within the various subdivisions of the I 0 were found to be in agreement with the previous observations of Fuxe ('65a), Sladek ('71a), Loizou ('72) and Hoffman and Sladek ('73). For subsequent comparative purposes, this distribution is summarized in table 1. RESULTS Cat. Only scattered varicosities were obOntogenetic comparisons. The infants served to be distributed throughout the enin all species examined (rat: 3-week-old; tire rostrocaudal extent of the principal nuAbbreviations PD, principal nucleus-dorsal lamella dm, principal nucleus-dorsal lamella, dorsomedial portion vl, principal nucleus-dorsal lamella, ventrolateral portion PV, principal nucleus-ventral lamella PL, (rat, cat), principal nucleus-ventrolateral bend (monkey), principal nucleus-lateral lamella D, dorsal accessory nucleus dm, dorsal accessory nucleus, dorsomedial portion
vl, dorsal accessory nucleus, ventrolateral portion M (cat), Medial accessory nucleus Mv, Medial accessory nucleus, ventral part Md, Medial accessory nucleus, dorsal part B, Medial accessory nucleus, nucleus B dc, Medial accessory nucleus, dorsal cap of Kooy vlo, Medial accessory nucleus, ventrolateral outgrowth dmcc, Medial accessory nucleus, dorsomedial cell column a-g,(monkey), Medial accessory nucleus, subnuclei
TABLE 1
Summary of the distri bic tion of ca tec holam in e-conta in ing varicosities within the inferior olivary complex of the rat, cat, and rhesus monkey Rat
PD dm vl PV PL D dm vl M
dc
Cat
4+
3+ 2 f
Scattered Scattered Scattered 3+
Scattered
Scattered Scattered Scattered Scattered
Scattered Scattered Scattered 1-3
w+
Scattered Scattered
1-2
Mv Md B dc vlo dmcc
Rhesus monkey
+
0-scattered 2+
Scattered 0 0
0
+
a b C
d&e f g
0-sca ttered 0-scattered 0-sca ttered 0-scattered 0-scattered 0-scattered
205
OLIVARY CATEXHOLAMINES
cleus; that is, within each of the three lamellae (figs. 1, 2A). The pattern is quite unlike that seen in the rat, where medium to high accumulations of varicosities are observed in the dorsal lamella. The pattern of varicosities within the dorsal accessory nucleus likewise appeared dissimilar to that observed in the rat. Varicosities were unevenly distributed throughout the rostrocaudal extent of the cat D, with the ventrolateral one-half possessing a 1 to 2 density of very fine varicosities and the dorsomedial one-half a 3 to 4 density (figs. 1, 3). Many of these fine varicosities, especially those in the dorsomedial region, appeared in a pericellular position. Varicosities located within the medial accessory nucleus (fig. 1) were differentially represented within its several subdivisions (as delineated by Walberg, ’56; Taber, ’61). The portion of the M designated as the ventral part (Mv) contained few, if any, varicosities at most rostrocaudal levels with the exception of an occasional linear profile of fine Varicosities. The dorsal part, or subdivision, (Md) contained a 2 density of fine varicosities throughout its rostrocaudal extent, while only scattered varicosities were seen within the nucleus B. No varicosities were observed to be distributed in the
+
+
+
+
+
RostraI dmcc
X
P r -
‘
Caudal
Fig. 1 Schematic illustration of frontal sections through the cat inferior olivary complex. Roman numerals indicate levels similar to those illustrated by Walberg (’56). The rostrocaudal distribution of C A varicosities is indicated by dots throughout the various subdivisions of the I 0 complex. Abbreviations are defined in table 1.
dorsal cap of Kooy (dc), the ventrolateral outgrowth of Kooy (vlo), or the dorsomedial cell column (dmcc). The above described varicosities all were distributed within the boundaries of the several subdivisions of the I 0 complex. However, in addition randomly distributed linear profiles, or “strings,” of larger varicosities were observed adjacent to and within the subdivisions of the complex. Rhesus monkey. Although scattered varicosities of very fine calibre were distributed throughout the rostrocaudal extent of both the dorsal and ventral lamellae of the principal nucleus, the lateral lamella contained the only pattern of varicosities in the entire I 0 complex of the monkey which was of high enough concentration to be assigned definite density ratings. Here, a 1 + to 3 density of very fine varicosities, often appearing in the form of linear profiles, was observed (figs. 2B,C, 4). In parasagittal section, the extreme lateral extent of the PL was observed to possess a n uneven distribution of varicosities, with a 1 + density in the caudal one-half and a 2 to 3 + density in the rostra1 one-half (fig. 4A). This pattern blended with a 2 to 3 density of the PL on sections just medial to the above (fig. 4B). Varicosities of very fine size were occasionally seen in the dorsal accessory olive. None of the seven subdivisions of the medial accessory nucleus (as delineated by Bowman and Sladek, ’73) possessed anything more than a n occasional very fine calibre varicosity. Olivary CA-containing cell bodies. CAcontaining cell bodies of the A 3 group (Dahlstrom and Fuxe, ’64; Fuxe, ’65b) were not seen within the I 0 of rat, cat or monkey. The existence of the A 3 group within the I 0 of rat, as reported by Dahlstrom and Fuxe (’64) and Fuxe (‘65b), is discussed below and elsewhere (Hoffman .and Sladek, ’73; Garver and Sladek, ’75). In the cat, a small number of CAsontaining cell bodies was observed immediately lateral to the ventral lamella of the principal nucleus. When discernable, such cell bodies were present at most brain stem levels which contained the I 0 complex.
+
+
DISCUSSION
The ability of the presently-employed technique to produce optimal histofluores-
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JOHN R. SLADEK, JR. AND JAMES P. BOWMAN
cence in different mammalian species has been discussed previously (Hoffman and Sladek, ’73; Garver and Sladek, ’75). However, an additional technical factor merits present consideration. Due to the thickness of the monkey calvaria, a greater length of time is required for brain removal following death (10-15 minutes) than in the cat (3-5 minutes) or rat (1-2 minutes). In an attempt to determine whether brain removal time represents a causative factor for the observed species-specific patterns of CA varicosities, additional cats and rats were investigated in which brain-removal-time was purposely slowed to match that of rhesus monkey. CA distribution in such animals conformed to the patterns reported above. While it is believed that extended brain-removal-time could be a factor of concern, it did not appear to affect the quality of histofluorescence within the above timespans. The general lack of dense patterns of CA varicosities in the I 0 of the rhesus monkey does not appear to be attributable to a similar lack of such varicosities in other areas of the brain of this species. For example, an extensive examination of the rhesus hypothalamus has revealed the presence of exceptionally dense and intense patterns of CA varicosities (Sladek et al., ’75). If the rhesus exhibited a general lack of histochemically-demonstrable varicosities then i t could be argued that the apparent species-specific patterns are of a lesser importance. On the contrary, initial microspectrofluorophotometric quantitative studies of CA occurrence within the hypothalamus of the rat, cat and rhesus monkey by this laboratory indicate the most intense patterns of CA varicosities occur in the primate hypothalamus. The significance of this is as yet undetermined. It is the belief of certain investigators that monoamine distribution as seen in the rat brain is representative of other mammalian species. Battista et al. (’72) reported that the principal architecture of monoamine distribution in monkey (i.e,, Macaca irus and Ceropeithecus sabaeus) is similar to that seen in the rat, rabbit and cat. 01sen et al. (’73) examined fetal human brain and indicated that the basic cytoarchitecture of monoamine distribution is similar to the rat brain. Additionally, DiCarlo et al.
(‘73) reported a lack of “significant interspecies differences between the rat and the squirrel monkey.” It is true that certain aspects of monoamine morphology as seen in the rat reflect those of other mammalian species. The existence of dopaminergic nigro-neostriatal neurons, indoleaminergic raphe neurons, noradrenergic locus coeruleus neurons, and dopaminergic arcuate neurons, all are comparable interspecies monoamine patterns. However, evidence also indicates species-specific patterns. Sladek (’71b) initially brought attention to this phenomenon in the brain stem reticular formation of the kitten, and extended the observation to include age-dependent differences in cat and macaque (Sladek, ’73; Sladek et al., ’74). Hoffman and Sladek (‘73) reported marked differences in CA distribution in the I 0 of rat, gerbil and rabbit. Felten et al. (’74) examined squirrel monkey, wherein they found three additional CA cell groups not previously reported in the rat. These later data are at variance with those of DiCarlo et al. (’73), who also examined squirrel monkey and failed to report such additional groups. A recent review by Morgane and Stern (‘74) further defines species variations in monoamine distribution. The present study lends additional support to the concept of speciesspecific patterns of CA distribution by extending definitive investigations in a particular nuclear complex to embrace members of more phylogenetically advanced orders than Rodentia. Recent investigations on the neuronal loci and pathways mediating the fast (8l2/sec) tremor induced in cats by the administration of harmaline are of signifiFig. 2 Comparative distribution of CA varicosities i n the principal nucleus in cat and rhesus monkey. A. Cat. CA varicosities are virtually absent in this portion of the I 0 complex with the exception of a n occasional varicosity (b) a s indicated. Dull lipofuscin-containing perikarya (4) are seen withi n the ventral (PV), lateral (PL), a n d dorsal lamell a e (PD). X 140. B. Rhesus m o n k e y . In contrast to the cat, the monkey principal nucleus contains a 2 to 3 + density of varicosities within the lateral lamella (PL). CA varicosities seen i n this region of the complex are characterized by linear profiles of Intense lipofuscin autofine-sized varicosities 0 ) . fluorescence is seen eccentrically placed within the cytoplasm of olivary perikarya (+). x 120. C. Rheszcs m o n k e y . Profiles of fine-sized varicosities (b) are seen i n this high power magnification of the PL. x 300.
OLIVARY CATECIIOLAMINES
Figure 2
207
208
J O H N R. SLADEK. JR. A N D J A M E S P. B O W M A N
OLIVARY CATECHOLAMINES
209
Fig. 4 Rhesiis monkey. The schematic, taken from Bowman and Sladek ('73), illustrates a frontal section (level G ) through the principal nucleus of the olivary complex and serves to approximate the level of sections of the PL illustrated in A and B. The ventral, lateral a n d dorsal lamellae a r e stippled, unmarked, a n d cross-lined, respectively. A. Parasagittal section through the PL of the rhesus monkey. The rostral portion of the complex i s indicated ( * ) , The extreme lateral pole of the PL exhibits a 3 dendensity caudally. X 100. sity of fine-sized linear profiles of CA varicosities (b) rostrally and a 1 to 2 B. Parasagittal section slightly medial to that illustrated in A. A 1 to 2 density of CA varicosities (b) i s noted throughout all portions of the PL at this level. The rostral portion of the complex is indicated (*). Abundant lipofuscin autofluorescence is seen in a juxtanuclear position within olivary perikarya throughout the entire PL. X 80.
+
+
+
+
210
J O H N R . SLADEK, JR. AND J A M E S P. B O W M A N
cance from a number of standpoints, not the least of which is that they have clearly implicated monoamines as important determinants of inferior olivary function (Lamarre and Mercier, '71; DeMontigny and Lamarre, '73; Llinis and Volkind, '73). In the context of the present study, these investigations appear to provide a basis for preliminary formulations concerning possible functional correlates associated with the "normal" pattern of olivary CA distribution by bringing to bear on a morphologic substrate both single unit and behavioral data. It thus is of interest to note that harmaline has an action apparently restricted largely to the cells of medial accessory nucleus, particularly its caudal portion, although dorsal accessory cells also have been reported to display harmaline-induced rhythmic oscillatory discharges (Llinas and Volkind, '73; DeMontigny and Lamarre, '73). The present study has shown that the accessory nuclei contain the most prominent distributions of CA-containing varicosities in the entire olivary complex of the cat, with the dorsomedial one-half of the dorsal accessory nucleus displaying a 3 to 4 + density and the dorsal part of the medial accessory having a 2 density rating. The latter nucleus, according to Taber ('61) and Walberg ('56), is represented caudally in the cat, decreases in size rostrally, and is absent oral to a midolivary level. Recognizing the hazards involved in microelectrode tract reconstruction and localization of responsive neurons and despite the fact that the olivary cells showing harmaline rhythms were not defined relative to the specific subdivisions of the medial accessory nucleus, there nevertheless appears to be correspondence between the distribution of CA varicosities in the 10 and the major sites of action of harmaline in the complex. Final determination of this correlation, however, must await further experiments in which CA distribution in the cat olive is examined under conditions of harmaline intoxic a tion. It is generally assumed that CA-containing varicosities arise from fluorescence demonstrable cells. If the pattern of varicosities observed in the present study later proves to be implicated in the harmaline induced tremor phenomenon, then the olivary varicosity fluorescence must derive
+
from cells located at pontobulbar levels of the neuraxis since cerebellectomy, combined transection a t high cervical and irtercollicular levels, and transection caudal to the red nucleus all are ineffectual in abolishing olivary harmaline cell rhythms (DeMontigny and Lamarre, '73; Llinas and Volkind, '73). DeMontigny and Lamarre ('73) conclude that such harmaline rhythms' do not derive from other medullary nuclei, leaving, by exclusion, either a driving of I 0 cells by nuclei of the pontine reticular formation or a n action of harmaline on terminals impinging on the olivary cells themselves. The marked difference in CA varicosity distribution in the I 0 complex of cat and monkey (again assuming the presence of CA reflects to some degree olivary regions likely to be implicated in harmaline tremor), suggests, among others, the following possibilities: that monoamines play a generally less dominant, although analogous, role in the function of the primate and cat olive in which case the monkey's I 0 complex may contain CA in concentrations too low to be demonstrable with formaldehydeinduced histofluorescence;' or that the fast tremor observed in monkeys under harmaline does not have an I 0 genesis. The latter suggestion is at variance with DeMontigny and Lamarre ('73) who preliminarily indicated that the 7-12/sec. harmalineinduced tremor observed in the intact monkey appears to have a similar neural substrate as that disclosed for the cat. The significance of the general lack of CA varicosity fluorescence in the I 0 complex of the monkeys we examined, especially in the accessory nuclei, thus remains to be explained. It is of interest to speculate on the location of perikarya of origin of olivary histofluorescence. Most investigators have assumed that fluorescence-demonstrable cell bodies give rise to fluorescence-demonstrable varicosities. The latter are believed to represent either terminal arborizations of I This interpretation might he favored by the newlyintroduced glyoxglic acid histofluorescence technique, which reportedlycan demonstratevaricosities offinecalibre not routinely seen with formaldehyde-induced histofluorescence (Lindvall et al., '74). However, this interpretation does not appear compatible with observations in this laboratory that the presently-employed technique i s capable of routinely demonstrating fine calibre varicosities of areas such as the cerebellar cortex, colhculi. thalamus, geniculates. and I 0 (Hoffman and Sladek, '73; Sladek. '75).
OLIVARY CATECHOLAMINES
axons or accumulations of monoamines along the preterminal portion of axons. This interpretation would require that olivary varicosities represent some portion of axons which arise from fluorescence demonstrable perikarya. A difficulty in reconciling this phenomenon with olivary histofluorescence, is that at present no known olivary afferents arise from neural loci which contain CA-perikarya. For example, sources of olivary afferents such a s parvicellular portion of the red nucleus and the spinal cord do not contain CA cell bodies. Thus, if I 0 varicosities represent axon termina:s which arise from CA-containing cells, then these cells either must be located outside of areas known to project to the olive or contain catecholamines in concentrations too low to be visualized with routine histochemistry. At present, only one site of origin of olivary afferents, the periaqueductal gray of the macaque, has been demonstrated to possess catecholaminergic perikarya. However, these perikarya as yet have only been observed in Macaca speciosa (Garver and Sladek, '75), squirrel monkey (Felten et al., '74) and Macaca mulatta (Sladek, Bowman and Felten - unpublished observations). In each species, a sparse population of CA-containing perikarya were found among the majority of non-fluorescing cells of this region. While this small number of cells could be a source of varicosities seen within the macaque 10, they appear as a less likely source in phylogenetically lower mammals wherein monoaminergic cells have not been seen in this area and wherein certain portions of the I 0 are richly supplied with varicosities. Another potential source of olivary histofluorescence might be from catecholaminergic cells reported to exist within the olivary complex (Dahlstrom and Fuxe, '64). Such cells were said to possess weak fluorescence in the rac. It has been proposed that certain catecholaminergic cells display variable fluoresc 3nce intensities depending on their state of activity, a s in the case of the dopaminergic tuberoinfundibular neurons of the neuroendocrine hypothalamus (Fuxe et al., '67). Thus, if A 3 cells, which have evaded subsequent histofluorescence demonstration (Hoffman and Sladek, '73; Garver aTid Sladek, '75), are in a state of extreme inactivity, this might account for their apparent lack of fluorescence.
21 1
Two recent investigations appear to lend support, either direct or indirect, to the hypothesis that a source of olivary afferents may arise from the locus coeruleus. Lindvall et al. ('74) demonstrated with the glyoxylic acid-vibratome technique that the rat thalamus contained a system of delicate, fine-sized varicosities, which they interpret as forming networks of terminal axonal ramifications. These varicosities are similar in appearance to those observed in this and other laboratories within the cerebellar and cerebral cortices, thalamus, colliculi, geniculates and the inferior olivary complex. It is of interest in this regard, that bilateral lesions of the locus coeruleus resulted in the absence of these fine-sized varicosities in the thalamus of the rat. Hence it is conceivable that this type of axon, classified by Lindvall et al. a s the "locus type of axon," may also project to the inferior olivary complex. Lending support to this concept is the recent study of Kobayashi et al. ('74) who performed a detailed biochemical mapping of projections of the rat locus coeruleus. Lesions were placed in the locus coeruleus and the sensitive radioisotopic enzyme assay technique was coupled with frozen section punch techniques of discrete hypothalamic nuclei (Palkovits, ' 7 3 ; Jacobowitz, '74) to observe changes in norepinephrine concentration as a result of the lesion. These investigators observed that the inferior olive, when measured bilaterally, demonstrated a significantly decreased norepinephrine concentration, suggesting a possible bilateral innervation from the locus coeruleus. Thus, based on the above, the olive may well receive afferents from the locus coeruleus. Another intriguing possibility for the interpretation of varicosity type fluorescence recently was presented by Nobin and Bjorklund ('75) who have observed apparent dendritic varicosity fluorescence within neurons of the substantia nigra-zona compacta. Such neurons possess dendritic ramifications located within the pars reticulata of the same nucleus which apparently end as dendritic varicosities, a finding in concert with both Golgi and ultrastructural studies of the same region. While these investigators indicate that this area of the rat brain is the only one a t present which displays this rather unique histofluorescence, preliminary observations in this lab-
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JOHN R. SLADEK, JR. AND JAMES P. BOWMAN
Falck, B., N.-A. Hillarp, G. Thieme and A. Torp 1962 Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10: 348-354. Felten, D. L.. A. M. Laties and M. B. Carpenter 1974 Monoamine-containing cell bodies in the squirrel monkey brain. Am. J. Anat., 1 3 9 : 153166. Fuxe, K. 1965a Evidence for the existence of monoamine neurons in the central nervous svstem. 111. The monoamine nerve terminal. Z. Zellforsch., 65: 573-596. 1965b Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine nerve terminals in the central nervous system. Acta Physl. Scand., Suppl., 64, p. 247. Fuxe, K., T. Hokfelt and 0. Nilsson 1967 Activity changes in the tubero-infundibular dopamine neurons of the rat during various states of the reproductive cycle. Life Sci., 6 : 2057-2061. Garver, D. L., and J . R. Sladek, Jr. 1975 Monoamine distribution in primate brain. I. Catecholamine-containing perikarya in the brain stem of Mtrctrcci speciostr. J . Comp. Neur., 159: 289-304. Hoffman, D. L., and J . R. Sladek, Jr. 1973 The distribution ofcatecholamines within the inferior olivary complex of the gerbil and rabbit. J. Comp. Neur., 1 5 1 : 101-112. ACKNOWLEDGMENTS Jacobowitz, D. 1974 Removal of discrete fresh regions o f t h e rat brain. Brain Res., 80: 111-115. This study was supported by USPHS Pro- Kobayashi, R., M. Palkovits, I. Kopin and D. Jagram Project Grant 11642. The authors cobowitz 1974 Biochemical mapping of noradrenergic nerves arising from the rat locus wish to thank Miss Yvonne Cheung for skillcoeruleus. Brain Res., 77: 269-279. ful technical assistance. Kooy, F. H . 1917 The inferior olive in vertebrates. Folia Neuro-biol., 10s:2 0 5 3 6 9 . Lamarre, Y . , a n d L. A. Mercier 1971 NeurophysLITERATURE CITED iological studies of harmaline-induced tremor in Battista, A., K. Fuxe, M. Goldstein a n d M. Ogawa the cat. Can. J. Physiol. Pharmacol., 49: 10491972 Mapping of central monoamine neurons 1058. in the monkey. Experientia, 28: 6 8 8 4 9 0 . Larochelle, L., P. Bedard, R. Boucher and L. PoirBjorklund, A,, and 0. Lindvall 1975 Dopamine ier 1970 The rubro-olivo-cerebellar loop and i n dendrites of substantia niera postural tremor in the monkey. J . Neurol. Sci., - neurons sueeestions for a role i n dendritic terminals. Brain 11 : 5344. Res., 8 3 : 531-537. Lindvall, O . , A. Bjorklund, A. Nobin and U.Stenevi Bowman, J. P., a n d J. R. Sladek, Jr. 1973 Mor1974 The adrenergic innervation of the rat thalphology of the inferior olivary complex of the amus a s revealed by the glyoxylic acid fluoresrhesus monkey ( M m c i c t i m i t l t i t t n ) . J. Comp. cence method. J . Comp. Neur., 154: 317-348. Neur., 152: 299-316. L h a s , R., a n d R. A. Volkind 1973 The olivocerBrodal, A. 1940 Experimentelle Untersuchunebellar system: functional properties as revealed by harmaline-induced tremor. Exp. Brain. Res., gen uber die olivo-cerebellare lokalization. Ztschr. 18: 69-87. ges. Nerv. u . Psychiat., 169: 1-153. Brodal, A , , F. Walberg and T. Blackstad 1950 Loizou, L. A. 1972 The postnatal ontogeny of monoamine-containing neurones i n the central Termination of spinal afferents to the inferior nervous system of the albino rat. Brain Res., olive in c a t . J. Neurophysl., 1 3 : 4 3 1 4 5 4 . 40: 3 9 5 4 1 8 . Dahlstrom, A,, and K. Fuxe 1964 Evidence for the existence of monoamine-containing neurons Morgane, P. J.. and W. C. Stern 1974 Chemical i n the CNS. I. Demonstration of monoamines in anatomy of brain circuits in relation to sleep the cell bodies of brain stem nuclei. Acta Physl. and wakefulness. I n : Advances in Sleep ReScand., Suppl., 62: p. 232. search. Vol. 1. E. Weitzman, ed. Spectrum PubDeMontigny, C., a n d Y. Lamarre 1973 Rhythmic lications Inc., New York. activity induced by harmaline in the olivo-cer- Olson, L., L. 0. Boreus and A. Seiger 1973 Histochemical demonstration and mapping of 5ebello-bulbar system of the cat. Brain Res., 53 : 81-95. hydroxytryptamine- a n d catecholamine-containDiCarlo, V . , J . E. Hubbard and P. Pate 1973 Fluing neuron systems i n the h u m a n fetal brain. Z. Anat. Entwick1:Gesch.. 139: 259-282. orescence histochemistry of monoamine-containing cell bodies i n the brain stem of the squir- Palkovits, M. 1973 Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res., rel monkey (Sttimiri sciurezts). J . Comp. Neur., 57: 307-326. 152: 347-372.
oratory (Sladek and Parnavelas, '75) indicate that other areas of the brain are characterized by such a phenomenon. Thus, if the inferior olivary complex contains dendritic ramifications of adjacent nuclei, which in themselves possess catecholamines, this may offer another possible explanation as to the source of olivary histofluorescent varicosities. It is of interest that the adjacent lateral reticular nucleus in both the cat and the rhesus monkey has been observed by this laboratory to contain such perikarya. It would be of further interest i f the lateral reticular nucleus, a source of cerebellar mossy fibers, projects dendrites into the olivary complex, a source of cerebellar climbing fibers. With the exception of the biochemical assay investigation indicated above, no attempts have been made to localize the site of origin of the species-specific patterns of olivary CA varicosities.
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OLIVARY CATECHOLAMINES Sladek, J . R., Jr. 1971a The distribution of catecholamine terminals within the mesencephalon, pons, and medulla oblongata of the immature cat brain: A fluorescence microscopic study. Dissertation Abstracts International, 32: 2489-2490. 1971b Differences in the distribution of catecholamine varicosities in cat and rat reticular formation. Sci., 174: 4101112. 1973 Age-dependent differences in catecholamine distribution within cat reticular formation. Exp. Neur., 38:520-524. (1975 submitted for publication) A new histochemical freeze-drying apparatus a n d a detailed methodological account of its use for FalckHillarp monoamine histofluorescence. Sladek, J. R., Jr., D. Garver and B. Tabakoff 1974 Histofluorescence and biochemistry of monoamines in macaque brain. Anat. Rec., 178: 465466. Sladek, J. R., Jr., and D. L. Hoffman 1972 Differences in biogenic amine distribution in mammalian brain. J. Histochem. Cytochem., 20: 848. 1973 Differences in catecholamine dis-
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tribution in the inferior olivary complex of' various mammals. Anat. Rec., 177: 444-445. Sladek, J. R., Jr., G. Hoffman, Y. Cheung and D. Felten 1975 Monoamine histofluorescence in rbesus monkey hypothalamus and pineal. Anat. Rec., 181: 482-483. Sladek, J. R., Jr., and J . Parnavelas (1975, submitted for publication) C atecholamine-con taining dendrites in primate brain. Sladek, J. R., Jr., B. Tabakoffand D. Garver 1974 Certain biochemical correlates of intense serotonin histofluorescence in the brain stem of the neonatal monkey. Brain Res., 67: 363-371. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat. J. Comp. Neur., 166: 27-70. Walberg, F. 1954 Descending connections to the inferior olive. In: Aspects of Cerebellar Anatomy. J. Jansen and A . Brodal, eds. Grundt Tanum, Oslo. 1956 Descending connections to the inferior olive. An experimental study in the cat. J. Comp. Neur., 104: 77-172.
