I 77-24,614 CRUTCHER, Keith Alan, 1953- THE ORGANIZATION OF MONOAMINE NEURONS WITHIN THE BRAIN OF A GENERALIZED MARSUPIAL, Didelphis marsupial is virginiana. The Ohio State University, Ph.D., 1977 Anatomy
Xerox University MicrofilmsAnn , Arbor, Michigan 48106 THE ORGANIZATION OF MONOAMINE NEURONS WITHIN
THE BRAIN OF A GENERALIZED MARSUPIAL,
Didelphls marsupialis virginiana
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Keith Alan Crutcher, B.A.
*****
The Ohio State University
1977
Reading Committee: Approved By
Albert 0. Humbertson, Jr. George F. Martin George E. Goode
Department of Anatomy This work is dedicated to my family and to the animals from which the results were obtained.
ii ACKNOWLEDGMENTS
I would like to acknowledge the advice and encouragement of
Albert Humbertson and the many faculty members who have devoted their time to my education including George Bingham, George Martin, Jim King,
George Goode, and David Clark. The secretarial assistance provided by
Malinda Amspaugh and the photographic assistance of Gabe Palkuti are greatly appreciated.
Special thanks to Michael and Marty for being there and of course none of this would have been possible,or worth it, without
Jennifer, Tara, and the mystery child.
ili VITA
May 29, 1953 Born - Fort Lauderdale, Florida
1974 B.A., Pt. Loma College San Diego, California
1974-1976 Research Associate, Division of Neurosurgery, Department of Surgery The Ohio State University, Columbus Ohio
1976-1977 Teaching Assistant, Department of Anatomy, The Ohio State University Columbus, Ohio
PUBLICATIONS
Martin, G.F., J. Andrezik, K. Crutcher and M. Unauts. The lateral reticular nucleus of the opossum (Didelphis virginiana). II. Connec tions. Journal of Comparative Neurology (in press).
FIELDS OF STUDY
Maj or Field: Anatomy
Studies in Neuroanatomy. Dr. Albert 0. Humbertson, Jr.
Studies in Brainstem Morphology. Dr. George F. Martin
Studies in CNS Ultrastructure. Dr. James S. King
Neurosurgical Research. Dr. W. George Bingham, Jr.
iv TABLE OF CONTENTS
Page
DEDICATION...... ii
ACKNOWLEDGMENTS...... ill
VITA ...... iv
LIST OF FIGURES...... vi
LIST OF ABBREVIATIONS...... vili
INTRODUCTION ...... 1
METHODS...... 3
RESULTS...... 6
M e d u l l a ...... « . • ...... 6
Pons...... 8
Midbrain...... 11
Diencephalon...... 13
Small Intensely Fluorescent Cells ...... 13
DISCUSSION ...... 15
ILLUSTRATIONS...... 25
LIST OF REFERENCES ...... 43
v LIST OF FIGURES
Figure Page
1 Catecholamine (CA) neurons in the region of the lateral reticular nucleus (fluorescence micrograph) ...... 26
2 Indoleamine (IA) neurons within the caudal raphe nuclei (fluorescence micrograph)...... 26
3 Drawing of CA and IA neurons within the medulla and caudal p o n s ...... 28
4 CA neurons within locus coeruleus (fluorescence micrograph) ...... 30
5 IA neurons within nucleus raphe magnus (fluorescence micrograph) ...... 30
6 CA neurons within the substantia nigra (fluorescence micrograph) ...... 30
7 Drawing of CA and IA neurons within the rostral pons...... 32
8 IA neurons within the dorsal raphe nucleus (f.m.)...... 34
9 IA neurons within the superior central nucleus (f.m.)...... 34
10 CA neurons within the ventrolateral midbrain tegmentum (f.m.)...... 34
11 IA neurons within nucleus linearis (f.m.) ...... 34
12 Drawing of CA and IA neurons within the caudal midbrain and rostral pons ...... 36
vi Figures Page
13 CA neurons medial to the mesencephalic tract of the trigeminal nucleus (Nissl stain and f.m.) ...... 38
14 Small intensely fluorescent cells (f.m.)...... 38
15 CA neurons within ventral tegmental area (f.m.) . . . 38
16 CA neurons within the hypothalamus (f.m.) ...... 38
17 Drawing of CA neurons within the rostral midbrain and hypothalamus...... 40
18 Illustration of CA and IA distribution within opossum b r a i n s t e m ...... 42
vii LIST OF ABBREVIATIONS
ACf nuclei areae cuneiformis A1 nucleus alaris CA catecholamine neurons CcD 9 nucleus cochlearis dorsalis CcV nucleus cochlearis ventralis CeS nucleus centralis superior CF campi Forelli Cl colliculus inferior Coe nucleus coeruleus (Coe a = nucleus coeruleus, pars a) CS colliculus superior CuL nucleus cuneatus lateralis DLL nucleus dorsalis lemnisci lateralis Fac nucleus n. facialis GCc substantia grisea centralis: pars caudalis GCd substantia grisea centralis: pars dorsalis GCv substantia grisea centralis: pars ventralis GM nucleus corporis geniculati medialis GrP griseum pontis HDM nucleus dorsalis hypothalami medialis Hg nucleus n. hypoglossi HVM nucleus ventralis hypothalami medialis HYD area hypothalamica dorsalis HYL area hypothalamica lateralis IA indoleamine neurons IP nucleus interpeduncularis Lg lingula cerebelli LP nucleus lateralis thalami posterior Lr nucleus linearis LRC nucleus lateralis reticularis caudalis LRO nucleus lateralis reticularis oralis M nucleus mamillaris 01 nucleus olivarls inferior OcM nucleus n. oculomotor! OcMR nucleus n. oculomotor! rostralis OS nucleus olivaris superior PBr nucleus parabrachialis PHD nucleus paraventricularis hypothalami dorsalis PHg nucleus prepositus hypoglossi RaD nucleus dorsalis raphae
viii RaM nucleus magnus raphae RaO nucleus obscurus raphae RaPa nucleus pallidus raphae Rb nucleus ruber RGcv nucleus reticularis gigantocellularis pars ventralis RTg nucleus reticularis tegmenti pontis SNc substantia nigra: pars compacts SN1 substantia nigra: pars lateralis SNr substantia nigra: pars reticulata
TgV area ventralis tegmenti TrMo nucleus motorius n. trigemini TrSD nucleus sensorius n. trigemini dorsalis TrSi nucleus tractus spinalis n. trigemini: pars interpolaris TrSo nucleus tractus spinalis n. trigemini: pars oralis TrsV nucleus sensorius n. trigemini ventralis Tz nucleus corporis trapezoidei VLLd nucleus ventralis lemnisci lateralis: pars dorsolateralis VLLv nucleus ventralis lemnisci lateralis: pars ventromedialis VstI nucleus vestibularis inferior VstL nucleus vestibularis lateralis VstM nucleus vestibularis medialis ZI zona incerta
aq aqueductus Sylvii be brachium conjunctivum bp brachium pontis ccs commissura colliculi superiorls cp commissura posterior cr corpus restiformis dbc decussatio brachium conjunctivum flm fasciculus longitudinalis medialis 11 lemniscus lateralis ml lemniscus medialis osc organum subcommissurale ped pedunculus cerebri pyr tractus pyramidalis rfl fasciculus retroflexus
ix rV radix mesencephalica n. trigemini Trs tractus spinalis n. trigemini
HI oculomotor nerve VII facial nerve VIII vestibulocochlear nerve
x INTRODUCTION
The Falck-Hillarp histochemical technique for visualizing biogenic amines (Carlsson et al., f62; Falck, ’62; Falck et al., ’62) provides a powerful tool for bridging the narrowing gap between the anatomy and chemistry of the nervous system. This comparatively simple procedure has already been utilized to map specific monoamine systems in the brain of the rat (Dahlstrom and Fuxe, f64; Jacobowitz and Palkovits, '74;
Palkovits and Jacobowitz, '74), cat (Chu and Bloom, *74; Jones and Moore,
*74; Pin et al., *68; Maeda et al., '73), dog (Ishikawa et al., ’75;
Shimada et al., ’76), chicken (Ikeda and Gotoh, ’71), turtle (Parent, *73), salamander (Simms, '77), monkey (Battista et al., ’72; DiCarlo et al.,
'73; Felten et al., '74; Garver and Sladek, ’75; Hubbard and DiCarlo,
'73, ’74a, ’74b), human (de la Torre, ’72; Nobin and Bjorklund, '73;
Olson et al., ’73), and in several invertebrate species (Aramont and
Elofsson, '76; Elofsson et al., '66; Goldstone and Cooke, ’73; Osborne and Dardo, '70; Welsh, ’72; Welsh and Williams, ’70).
The North American opossum, a generalized marsupial, has main
tained a certain popularity for neuroanatomical research, not only for its significance from a comparative point of view, but also for its early availability in an external pouch, providing a unique opportunity for developmental studies (Martin et al., ’75). The distribution of
1 2
monoamine neurons in the opossum brain was studied and the results are
described herein. To our knowledge, this is the first such description ? for any representative of the marsupial radiation.
Spectrophotofluorometric analysis supports the generalization
that the green and orange-yellow fluorescence, induced by exposure to
formaldehyde vapors, represent catecholamine (CA) and indoleamine (IA)
fluorophors respectively (Corrodi and Jonsson, ' 67). Although such a designation will be followed in this paper, it should be noted that
there is an overlap in the spectral characteristics of the fluorophors depending on their concentration (Laszlo, f75). No attempt was made in this study to distinguish between the fluorescence of dopamine and norepinephrine since both of them exhibit a green fluorescence with this technique (Falck et al., ’62).
The original description of monoamine-containing neurons in the rat brain was detailed through the use of a numbering system (Dahlstrom and Fuxe, '64). Each nuclear group of fluorescent neurons was assigned a letter and number, e.g. Ag = locus coeruleus. Subsequent investiga tors have usually retained this system with some modification for different species (DiCarlo et al., *73; Garver and Sladek, ’75;
Palkovits and Jacobowitz, ’74), but other workers have abandoned it
(Swanson and Hartman, ’75). The principal advantage of employing such a system, namely, in comparing species, is overshadowed by its lack of relationship to standard anatomical terminology. As this paper is de scriptive in nature, the original numbering system will be employed only for the purpose of comparing species in the discussion. METHODS
Ten adult opossums (Didelphis marsupialis virginiana) were used for this investigation. Four animals were pretreated with one of the monoamine oxidase inhibitors, nialamide or pargyline, prior to sacri ficing with sodium pentobarbital. Six animals received no pretreatment.
The technique used in this study was essentially the same as that de scribed by other workers (Dahlstrom and Fuxe, '64; Falck, *62; Falck et al., ’62). The brain was rapidly removed and cut into blocks which were individually frozen in isopentane cooled with liquid nitrogen.
The tissue was then transferred to a Virtis lyophilizer cooled to -50°C.
The optimal freeze-drying period for this laboratory was found to be two weeks. At the end of that time the blocks were placed in an air tight glass vessel containing paraformaldehyde (relative humidity
70%). The container was placed in an oven at 80°C for one to two hours.
Following embedding in paraffin, 14pm thick sections were cut and mounted on glass slides with Entellan, a nonfluorescing mounting medium.
The sections were viewed and photographed with a Leitz fluorescence microscope equipped with an HBO 200 W/2 mercury lamp and a K530 barrier filter. Every fifth section was stained for Nissl in order to provide for precise localization of the fluorescing perikarya.
3 4
One animal was studied using the glyoxylic acid method (Batten-
berg and Bloom, *75) in order to compare the sensitivity of this tech
nique with that of the Falck-Hillarp method. No differences were
apparent when comparing the distribution of CA-containing perikarya
and for that reason the following description will be based on the
results from the Falck-Hillarp material. No comparison was possible
with IA-containing cell bodies since the glyoxylic acid method does
not demonstrate such cells.
In order to distinguish between the two types of fluorophors
developed with the formaldehyde technique both the color of the fluo
rescence and its sensitivity to ultraviolet radiation were considered.
The orange-yellow fluorescence characteristic of indoleamines decays
rapidly when exposed to ultraviolet radiation whereas the green fluo
rescence associated with catecholamines is relatively stable. A few neurons within the midbrain do not conform to these criteria, however,
and they will be discussed more fully in the section on results.
Pretreating the animal with nialamide or pargyline, both of
which Inhibit monoamine oxidase, causes a pronounced increase in the
IA fluorescence but only slightly increases the CA fluorescence.
This criterion, along with those mentioned above, served as a means
for distinguishing the CA and IA fluorophors from each other.
The location of fluorescent perikarya was determined by comparing
the sections stained for Nissl substance with the atlas of the opossum
brain prepared by Oswaldo-Cruz and Rocha-Miranda (’68). The terminology 5
used in the following description was taken from that atlas. It is important to emphasize, however, that the localization of monoaroine neurons does not conform precisely to the nuclear divisions portrayed in the atlas. RESULTS
Fluorescent neurons are present from the level of the caudal medulla to the rostral hypothalamus and will be described for each major subdivision in a caudal to rostral sequence. The reader may find it helpful to refer to the summary illustration (fig. 18) when reading the following description.
Medulla
The most caudal fluorescent neurons in the opossum brainstem are located within the ventrolateral medulla at the level of the pyramidal decussation. At such levels, and continuing rostrally through the vestibular nuclei, green fluorescent neurons appear dorsolateral (fig. 1, black-tipped arrows, fig. 3A, straight solid arrows) and, occasionally, ventrolateral (fig. 1, insert, fig. 3A curved arrow) to the lateral reticular nucleus. Such neurons are multipolar and medium-sized
(17-26ym in diameter). Many of these neurons exhibit fluorescent processes, possibly dendrites, which are oriented in various directions within the white matter (fig. 1). Although fluorescent perikarya are present in a comparable position throughout the medulla, they are most numerous in caudal sections where an average of four to six neurons per section are present. This region is also characterized by the
6 7
presence of non-fluorescing cell bodies vhich are surrounded by fluo rescent varicosities (fig. 1, solid white arrow).
Catecholamine neurons also appear ventrolateral to the dorsal vagal nucleus (nucleus alaris of Oswaldo-Cruz and Rocha-Miranda, ’68), as well as within it. Although they are fewer in number than those located more ventrally and laterally they are comparable in size and appearance (fig. 3A, open arrow). The identification of CA neurons within the dorsal vagal nucleus is difficult, however, due to the large number of fluorescent varicosities surrounding the cells in this region. These varicosities presumably represent monoaminergic terminals of fluorescent cells.
Indoleamine neurons are present within the raphe nuclei, i.e. the nucleus pallidus raphe and nucleus obscurus raphe (figs. 2, 3A,B) and continue into nucleus raphe magnus (figs. 3C, 5). In general, these cells, 17-33ym in diameter, exhibit a dorsoventral orientation and are positioned within the midline, although a few of them are located off the midline and reveal no consistent orientation (fig. 2, insert, fig. 3B, double arrow). The fluorescent neurons within the raphe usually number fifteen to twenty per section and are present throughout the entire length of the medulla and continue into the pons. The orange fluorescence of these cells is markedly increased after pretreatment with nialamide or pargyline. Even after such pretreatment, however, there are a number of neurons within the region of the raphe which do not fluoresce (fig. 2, white arrows). The presence 8
of these cells indicates either that the raphe nuclei do not constitute a homogeneous collection of serotonergic perikarya or that a certain percentage of these somata have serotonin concentrations below the de tectable limits of this technique, even after monoamine oxidase inhibition.
Pons
The CA neurons in the pons are not continuous with the ventro lateral group of the medulla and a large number of them are located ventromedial, dorsal and lateral to the motor trigeminal nucleus
(figs. 7A,B, straight closed arrows). These neurons are multipolar in appearance and range from 20-23]im in diameter. At the rostral pole of the motor trigeminal nucleus, these cells continue dorsally to merge with the ventral portion of the locus coeruleus and subcoeruleus region (see below).
Green fluorescent cells of the CA type (twelve to fourteen per section) mingle with the most rostral fibers of the exiting facial nerve which arches over the caudal end of the trigeminal motor nucleus
(fig. 7A, arrow). These neurons (23-33ym in diameter) appear to be a caudal extension of the locus coeruleus where a large number of CA neurons are located (figs. 3, 7B, 13). Almost every neuron within the locus coeruleus exhibits the green fluorescence characteristic of catecholamines although a few have a more intense yellow fluorescence
(fig. 4, black-tipped arrow) and the rostral portion of the nucleus contains more nonfluorescent perikarya than at caudal levels. The locus coeruleus is continuous, ventrolaterally, with the nucleus coeru leus, pars a where CA cell bodies are also present (fig. 4, insert, fig. 7B). The nucleus coeruleus, pars a, is probably comparable to at least part of the nucleus subcoeruleus described by some authors
(Olszewski and Baxter, ’54). Occasional fluorescent cell bodies spill over into both the dorsal principal sensory trigeminal (fig. 7B, open arrow) and the medial parabrachial nuclei (fig. 7C, open arrow).
Counts of fluorescent neurons within the locus coeruleus and subcoeru leus region reveal sixty to eighty perikarya per section.
At more rostral pontine levels (fig. 7C), and extending into the caudal mesencephalon, the CA neurons of the locus coeruleus are fewer in number (fifteen to twenty per section). At such levels, the fluo rescent neurons in the nucleus coeruleus, pars a, extend ventrally and occupy a position dorsomedial to the ventral nucleus of the lateral lemniscus (fig. 12A).
The IA neurons within the medulla continue into the pons and similar numbers occupy a position within nucleus pallidus raphe and nucleus magnus raphe (figs. 5, 3C,D, and fig. 7A). Throughout the level of the facial nucleus, and in sections caudal to it, a number of IA neurons extend laterally from the raphe proper into the ventral portion of the gigantocellular reticular nucleus (fig. 3C). These neurons exhibit a size range from 25-50ym in diameter and extend laterally to occupy a position ventral to the medial portion of the facial nucleus (fig. 3D, arrow). These more laterally situated neurons 10
are generally multipolar or exhibit a mediolateral polarity whereas the midline neurons maintain the dorsoventral orientation characteristic of the raphe neurons of the medulla. Rostrally the pontine IA neurons are fewer in number and at the level of the nucleus of the trapezoid body only a few of them appear within the nucleus magnus raphe (fig. 7A) and between the pontine tegmental reticular nuclei (fig. 7B, curved arrow).
Many of the IA cell bodies at this level exhibit eccentric nuclei
(.fig. 5, white arrows).
Just rostral to the motor trigeminal nucleus a number of small
IA neurons appear within that part of the ventro-caudal portion of the periaqueductal grey identified as the dorsal raphe nucleus (fig. 7C, solid arrow). More rostral yet, the IA neurons form a large collection within the dorsal raphe nucleus and, more ventrally, within the superior central nucleus. The cells within the dorsal raphe are 21-26um in diameter and number 80-110 cells per section (fig. 8). They are con tinuous ventrally within the superior central nucleus where they occupy its middle third (figs. 9, 12A). A few cells extend laterally at this lf?vel into the mesencephalic reticular formation where they occupy a position ventromedial to the CA neurons (fig. 12A, double arrow).
These cells range from 17-20pm in diameter.
Most of the IA cell bodies in the rostral pons are oval in shape and exhibit few fluorescent processes. The cells that do exhibit processes are mostly within the superior central nucleus and are ori ented mediolaterally (fig. 9, solid arrow). There are at least as 11
many non-fluorescent neurons within this nucleus as there are fluores cent ones (fig. 9, open arrow).
Midbrain
The greatest concentration of CA neurons, 180-200 per section, resides within the rostral midbrain of the opossum. The most caudal of these (18-23pm in diameter) are positioned within the deep tegmental area medial to the rostral fibers of the lateral lemniscus, approxi mately at the arbitrary boundary with the pons (figs. 10, 12B, arrows).
In progressively more rostral sections, comparable neurons are located lateral and ventral to the large autofluorescent neurons of the red nucleus (fig. 12C). These neurons are mostly triangular or oval in appearance, but may exhibit occasional fluorescent processes.
A second aggregate of medium-sized (11-26]im in diameter) CA neurons appears within the substantia nigra ventral to the CA tegmental neurons. These cell bodies exhibit fluorescent processes, possibly dendrites which are oriented along the long axis of the substantia nigra, i.e., from dorsolateral to ventromedial (figs. 6, 12C). Although the vast majority of them are confined to the compact portion of the substantia nigra, a few occur within its reticular and lateral divisions
(fig. 12C, arrows). The most rostral third of the pars compacts, as labelled by Oswaldo-Cruz and Rocha-Miranda does not contain fluorescent perikarya.
Continuing cranially, large numbers of CA neurons are present within nucleus linearis and the ventral tegmental area where they 12
surround the fascicles of the oculomotor nerve (fig. 17A). The fluores cent perikarya in these regions form a continuous group across the mid line dorsal and rostral to the interpeduncular nuclei (fig. 15). The
CA neurons within the ventral tegmental area extend as far rostrally as the fasciculus retroflexus where they occupy a medial position to it
(fig. 17B). Small oval neurons of the CA type are also scattered throughout the ventral portion of the rostral periaqueductal grey matter (fig. 17B, arrows) and within the rostral nucleus of the oculo motor nerve (fig. 17A).
The caudal midbrain houses the rostral continuation of pontine
IA neurons. As the dorsal raphe nucleus, described above, diminishes in size IA neurons, of the same size range, are located within the nucleus linearis where they occupy a position medial to, and surrounding, the fibers of the medial longitudinal fasciculus (figs. 11, 12B). Many of the cell bodies in this region do not lose their fluorescence as rapidly, upon exposure to ultraviolet radiation, as the other indoleamine-containing perikarya (fig. 11, arrows). The color of the fluorescence and the increase in fluorescence intensity after monoamine oxidase inhibition, however, indicate the presence of an indoleamine.
The IA neurons within the superior central nucleus and continuing into nucleus linearis mingle with the decussating fibers of the brachium conjunctiva. At this point they encroach upon the midline group of CA neurons within nucleus linearis and the ventral tegmental area de scribed above. The more lateral IA neurons extend ventrolaterally to occupy a position lateral to the interpeduncular nucleus (fig. 12B). 13
Diencephalon
A few, five to ten per section, green-fluorescent cell bodies are present within the periventricular area of the hypothalamus. These neurons are oval in appearance, 10-12ym in diameter, and exhibit little cytoplasm or fluorescence (fig. 16, bottom). They are scattered through out the rostro-caudal extent of this region and at the level of the ventromedial hypothalamic nucleus they expand into the dorsal hypothala mic paraventricular nucleus (fig. 16, top) and the ventral portion of the dorsal hypothalamic area (fig. 17C). At this level 13-20 fluores cent cells are present per section. No fluorescent perikarya were ob served within any of the thalamic nuclei and no indoleamine cells, detectable with this method, were present at any diencephalic level.
Small Intensely Fluorescent Cells
The opossum brain is remarkable for the tremendous number of small intensely fluorescent (SIF) cells present within the connective tissue surrounding the brain and its vessels. These perikarya are generally small (10-20pm in diameter) and rarely exhibit fluorescent processes (figs. 10, 14). The fluorescence is of the CA type but is usually more intense than that of neurons within the brain itself, often obscuring the presence of the nucleus. The greatest concentration of SIF cells is present within the highly vascular areas of the brain, e.g., area postrema, and around the blood vessels themselves (fig. 14, top). The cells present surrounding the blood vessels are usually 14
associated with a number of large fluorescent processes characteristic of the sympathetic innervation of these structures (fig. 14, bottom). DISCUSSION
The green fluorescence characteristic of catecholamine-containing neurons is present within four collections of nerve cells within the opossum brainstem. The IA neurons occupy a continuous midline position from the medulla through the caudal midbrain, except at rostral pontine levels where there is a gap in the distribution of these cells (fig. 18, arrow).
Within the medulla, CA neurons are located within ventrolateral
and dorsomedial areas which correspond to groups A^ and A 2 of the rat
(Dahlstrom and Fuxe, ’64) and squirrel monkey (Felten et al., *74) and
M^ and M 2 in the stump-tailed monkey (Garver and Sladek, ’75). The border between the two areas in the opossum is not distinct, a situation similar to that reported by Garver and Sladek (*75) for the stump-tailed monkey and observed in this laboratory for the rhesus monkey (unpublish ed observations).
The fluorescent perikarya within the region of the lateral reti cular nucleus (LRN) deserve particular notice. These cell bodies have been implicated as the principle source of descending catecholaminergic fibers in the spinal cord of the rat (Dahlstrom and Fuxe, ’65). Retro grade cellular changes after spinal hemisectlon and horseradish peroxi dase (HRP)-labelled cells after spinal injections of HRP are present in
15 16
the cat LRN (Torvlk and Brodal, *57; Kuypers and Maisky, '75), As in other species the opossum LRN is generally considered to be a precere- bellar nucleus (Andrezik and King, ’77; Martin et al., ?77). However, the presence of CA neurons at this level in the opossum medulla as well as recent evidence from spinal cord HRP injections (unpublished obser vations) indicates a projection from the LRN to the spinal cord.
The presence of a reticulospinal pathway originating in the LRN would provide a possible route for direct feedback modulation of the spinal cord which projects heavily to the LRN (Martin et al., ’77).
In addition, the elicitation of pressor responses from the LRN (Henry and Calaresu, ’74) and from the fastigial nucleus, which has been shown to project to the LRN (Moolenaar and Rucker, '76; Martin et al., ’77) may provide a clue to the function of LRN-spinal connections. Immuno- histochemical data demonstrates the presence of epinephrine-containing neurons in the region of the LRN which may also contribute to such a reticulospinal pathway (Hokflet et al., '74).
The dorsomedial CA neurons may also contribute to descending catecholaminergic pathways since retrograde changes in fluorescence have been observed after spinal cord transection in the rat (Dahlstrom and Fuxe, *65) and HRP-positive cells have been observed in this, and other, laboratories in the same region after spinal cord injections of
HRP (Kuypers and Maisky, *75; Crutcher, '77).
The position of IA neurons within the opossum medulla conforms to
that reported for the rat, i.e., (pallidus), B 2 (obscurus) and B^ 17
(magnus), and squirrel monkey (Hubbard and DiCarlo, f74b; Felten et al.,
*74). Such neurons have not been described in the rhesus monkey, but
unpublished evidence from our laboratory indicates a similar distribu
tion. These cell groups are major reseivoirs of serotonin (Dahlstrom
and Fuxe, *65) and have been implicated as potential sources of descend
ing pathways in the cat (Brodal et al., '60; Kuypers and Maisky, T75)f
The latter suggestion is substantiated by the presence of reaction product within them subsequent to HRP injections of the opossum spinal
cord (Crutcher, *77). Physiological experiments have also demonstrated depressor responses from stimulation of this region (Henry and Calaresu,
’74) and recent evidence implicates the region of raphe magnus and adja
cent reticular neurons as potential supraspinal control centers for pain transmission (Fields et al., ’76).
Fluorescent perikarya are not evident in the region of the opossum dorsal accessory olivary nucleus which conforms to group Ag as described
in the rat (Dahlstrom and Fuxe, *64). This group has not been identi fied in the gerbil, rabbit, rhesus monkey, stump-tailed monkey, squirrel monkey, or human fetus, although it has been reported to be present in
the chicken (Ikeda and Gotoh, '71). In light of recent immunohistochem
ical data (Swanson and Hartman, ’75) in which no comparable group was
reported, it is possible that the original observation was based on
some artifact.
The pons of the opossum contains CA-contalning perikarya within
the locus coeruleus and within an area probably comparable to the so-
called "subcoeruleus" region (the nucleus coeruleus, pars a of 18
Oswaldo-Cruz and Rocha-Miranda, ’68). The locus coeruleus neurons are homologous to group Ag in the rat (Dahlstrom and Fuxe, ’64), Mg in the stump-tailed monkey (Garver and Sladek, *75) and Cg in the squirrel monkey (DiCarlo et al., *73). The compact appearance of fluorescent perikarya in the locus coeruleus of these species differs from the diffuse collection of fluorescent neurons described in the dorsolateral tegmen tum of the cat (Chu and Bloom, *74; Jones and Moore, *74; Maeda et al.,
*73) and dog (Ishikawa et al., *75). Whether this morphological dif ference reflects a functional difference remains to be seen. The caudal extension of fluorescent neurons from the locus coeruleus may corres pond to group in the rat and M^ in the stump-tailed monkey, however, studies in the Cebus apella monkey indicate the presence of a very extensive A^ cell group (Demirjian et al., '76).
The projections of the locus coeruleus have been studied by numerous investigators (Linvall and Bjorklund, *74; Lindvall et al.,
*74; McBride and Sutin, '76; Pickel et al., *74). The locus coeruleus has been shown, by autoradiography, to project to both cerebral and cerebellar cortices as well as to numerous hypothalamic and forebrain nuclei. In addition, evidence is accumulating that this nucleus pro vides a significant input to the spinal cord in the rat, cat and monkey
(Fougerousse and Hancock, '76; Kuypers and Maisky, *75) as well as the opossum (Crutcher, *77). This area has also been shown to elicit pressor responses under appropriate stimulation conditions (Ward and
Gunn, *76). 19
The fluorescent neurons of the opossum nucleus coeruleus, pars a,
are not easily separated from those of the locus coeruleus proper nor
can they be separated into obvious rostrocaudal subdivisions. As we
employ the term in this study, the subcoeruleus group includes regions
probably comparable to and Ay of the rat (Dahlstrom and Fuxe, *64)
as well as M^, Mgc, and of the monkey (Garver and Sladek, '75). Al
though the CA neurons within the rostral ventrolateral pons and caudal mesencephalon occupy a position medial to the ventral nucleus of the
lateral lemniscus, as described for Ay in the rat (Dahlstrom and Fuxe,
'64) and Cy in the squirrel monkey (Hubbard and DiCarlo, ’73), they do not appear to be located within it, as described by Garver and Sladek
(’75) for the stump-tailed macaque. Continuity exists between CA cells of the ventrolateral medulla and those of the subcoeruleus region of the pons in the rat (Swanson and Hartman, ’75) and squirrel monkey (Hubbard and DiCarlo, ’74a), but is is not evident in the opossum (fig. 18).
Host of the neurons within the subcoeruleus region, as described for other species, contribute to the "ventral noradrenergic bundle" which
innervates numerous diencephalic and telencephalic regions (Maeda et al.,
’73).
The IA neurons in the pons, particularly those within the raphe
and those more lateral within the reticular formation, correspond to
the group in the rat. The more dorsal and rostral neurons may be homologous to group although no distinct boundary is present between
these two regions in the opossum. Fluorescent neurons have not been observed in the opossum which correspond to cell group B^. The IA 20
neurons within the caudal portion of the periaqueductal grey may cor
respond to group Bg in the rat (Dahlstrom and Fuxe, '64) and Sg in the
squirrel monkey (DiCarlo et al., *73). No adequate division could be
made in our material since these cell bodies appear the same as, and
unite rostrally with, the IA neurons within the dorsal raphe nucleus.
This latter collection corresponds to group By in the rat and Sy in the
squirrel monkey. The more ventrally situated cells within the superior
central nucleus are similar in position to Bg in rat (Sg in squirrel monkey). However, the more ventrolateral cells which in the rat and
squirrel monkey are designated Bg and Sg respectively, do not readily
distinguish themselves as a separate group in the opossum (Fig. 12A).
These cell groups reportedly give rise to projections to various dien
cephalic and limbic regions (Brodal et al., '60; Moore and Halaris, ?75).
The opossum midbrain contains the greatest collection of CA neurons. Although no distinct boundaries are present in our material,
the various CA neurons within the midbrain reticular formation, the
substantia nigra, and the ventral tegmental area correspond, for the most part, to groups Ag, Ag and A^ q in the rat and Mg, Mg and M ^ q in
the stump-tailed monkey. These nuclei have been shown, in other species,
to give rise to ascending dopaminergic pathways innervating numerous
forebrain regions (Berger et al., '76; Carpenter et al., ’76). The
Falck-Hillarp method does not allow for the differentiation between norepinephrine and dopamine since both catecholamines give rise to a green fluorescence. 21
Numerous fluorescent processes were observed In the midbraln CA cell groups. Other cell groups also revealed occasional fluorescent extensions which may be dendrites (figs. 1, 9). Similar findings have been reported previously (Dahlstrom and Fuxe, *64; Bjorklund and
Llndvall, ?75) and the suggestion has been offered that the presence of amine within a dendrite may indicate a presynaptic function or be in volved in dendritic secretion (Sladek and Parnavelas, *75). Such a sug gestion is certainly interesting and deserves further attention particu larly in light of recent evidence that the majority of aminergic ter minals identified ultrastructurally by autoradiography do not form identifiable synapses (Descarries et al., *75).
The scattered green fluorescent perikarya within the rostral peri aqueductal grey have been described in the rat (Lindvall and Bjorklund,
’74), stump-tailed monkey (group Meg of Garver and Sladek, ’75), and by
Felten et al. (group Asm3, *74) but not by DiCarlo et al. (’73) in the squirrel monkey. These cells have not been reported in either the dog or cat. The projections of these perikarya are unknown but it is likely that they are dopaminergic since studies demonstrating the presence of dopamine 3~hydroxylase (Swanson and Hartman, ’75) do not reveal their presence in the rat.
The existence of indoleamine neurons in the caudal midbrain, par ticularly within nucleus linearis, which are resistant to the decaying effects of ultraviolet radiation has been reported also in the rat
(Bjorklund et al., *71) and squirrel monkey (Hubbard and DiCarlo, f74b). 22
Whether or not these cell bodies contain a different indoleamine or
sufficient quantities of serotonin to resist decay upon ultraviolet exposure remains to be seen (Jonsson et al., ?75).
Monoamine cell bodies within the hypothalamus have not been in vestigated as extensively as in other regions. The fluorescent peri karya that we have seen in the dorsal hypothalamic paraventricular nucleus have been reported in the rat (Bjorklund and Nobin, ’73) and cat (Cheung and Sladek, *75) where they are referred to as groups A ^ and The apparently comparable neurons within the periventricular region may correspond to those described as group A14 by the same authors. The existence of fluorescent perikarya within the infundibular nucleus (group A ^ ) could not be established with certainty in our material although weakly fluorescent cell bodies were observed occa sionally in this area. Part of the difficulty in comparing hypothalamic cell groups resides in the nomenclature used for the different species.
The projections of the diencephalic CA neurons are virtually unknown.
Recent evidence from HRP injections in the spinal cord (Kuypers and
Maisky, '75; Crutcher, ’77) and H-amino acid injections in the hypothalamus (Saper et al., '76) indicate a projection from hypothalamic regions containing CA perikarya to the intermediolateral nucleus of spinal cord.
Small intensely fluorescent (SIF) cells have been studied in a number of species but usually in relation to sympathetic ganglia
(Chiba and Williams, ’75). The presence of numerous SIF cells sur rounding the blood vessels of the brain and in conjunction with vascular 23
regions, e.g., area postrema, raises the question of the function of these CA cell bodies. It has been established that they are interposed between preganglionic axons and principal ganglionic neurons (Williams,
'67). Through inhibitory postsynaptic potentials (IPSP's), SIF cells are thought to modulate superior cervical ganglionic activity in several
species (Siegrust et al., ’6 8 ; Libet, ’70; Libet and Omman, ’74).
Their role in the opossum nervous system remains to be elucidated. Un published observations from our laboratory indicate that the rhesus monkey has far fewer SIF cells in comparable regions than does the opossum.
Although differences are present, in general, the brainstem loca tion of CA and IA neurons in the opossum is comparable to that of the placental species studied to date. Species-specific differences have been observed in the terminal distribution of monoamines in certain placental mammals (Hoffman and Sladek, '73; Sladek and Bowman, ’75) making it clear that, although a general mammalian pattern exists, it is wise to use caution when extrapolating from one species to another.
In addition, the verification of proposed functions for monoamine sys
tems (Crow and Arbuthnott, *72) must await a more detailed analysis of
their numerous connections.
The similarity of the distribution of monoamine neurons in the opossum to that described for placental mammals strengthens the vali
dity of the use of this animal as a model for developmental studies
(Martin et al., '75). In particular, it would be of value to investi gate the time course of the monoaminergic innervation of the lumbosacral 24
spinal cord in the pouch young of this species. Such a study would add to our understanding of the role of these systems in the subsequent differentiation of other neuronal elements. The source of monoaminer- gic fibers in the adult opossum spinal cord is currently under investigation. 25
EXPLANATION OF FIGURES
Fig. 1 CA neurons dorsolateral to the lateral reticular nucleus (black-tipped arrows) and ventrolateral to the LRN (insert, black arrow). Non-fluorescent cell bodies surrounded by fluorescent varicosities are also present (solid white arrow). Note the presence of fluorescent processes on the CA neurons. Open arrow indicates ventromedial direction. X225.
Fig. 2 IA neurons within nucleus raphe pallidus (RaPa) at the level of Figure 3c and nucleus raphe obscurus (insert, RaO) at the level of 3b. Fluorescent perikarya (double black arrows) are interspersed with cell bodies exhib iting autofluorescent lipofuscin granules (white arrows). Most of the neurons within nucleus raphe obscurus occupy a midline position but some are paramedian in location (insert, black arrow), ml = medial lemniscus. X225.
27
Fig. 3 Drawings of cross-sections Illustrating the distribution of IA neurons black cells) and CA neurons (outlined cells) within the medulla and caudal pons. Number of cells indicates average number found at these levels. A. Level of the lateral reticular nucleus (LRC) showing the CA neurons dorsolateral (straight solid arrows) and ventrolateral (curved arrow) to this nucleus as well as CA cell bodies ventrolateral (open arrow) to the dorsal vagal nucleus (Al). B. IA neurons within nucleus raphe pallidus (RaPa) and nucleus raphe obscurus (RaO). Double arrow indicates a fluorescent cell body in a paramedian position. C. IA neurons within raphe magnus (RaM) and extending into the ventral portion of the gigantocellular re ticular nucleus (RGcv). D. Arrow indicates, an IA neuron ventral to the medial portion of the facial nucleus (Fac).
/ 28 0
V .tM 'Cjvni
\ r 1 TrSo RaM
V *tH
T rSi
RGov
T rSi Q
T rSi
RaPa #3 29
Fig. 4 CA neurons within the locus coeruleus. Occasionally, cells in this region exhibit a more intense yellow fluo rescence (black-tipped arrow) and some cells are non- fluorescent (open arrows). X450. Insert illustrates the locus coeruleus (Coe) and subcoeruleus region (Coe a) in relation to the mesencephalic tract of V (rV). X64.
Fig. 5 IA neurons within nucleus raphe magnus at the level of figure 7a Illustrating the prevalence of eccentric nuclei within many of these perikarya. Non-fluorescent cell bodies are also present (open arrow). X450.
Fig. 6 CA neurons within the pars compacts of the substantia nigra. Virtually all of the perikarya in this region are fluorescent and many exhibit fluorescent processes (white arrow, insert). X225. Insert 2450. 30 31
Fig. 7 CA (outlined neurons) and IA (black neurons) cell bodies vithin the rostral pons. A. At the level of the seventh nerve, CA neurons (arrow) are present dorsal to the caudal pole of the trigemi nal motor nucleus (TrMo). IA neurons are confined to the region nucleus raphe magnus (RaM) and adjacent reticular formation. B. Numerous CA neurons are present within the locus coeruleus (Coe) and subcoeruleus region (Coe Qt). A few extend into the dorsal trigeminal sensory nucleus (TrSD, open arrow); others being present dorsal to the superior olivary nucleus (OS, double-tailed arrow). IA neurons appear between the pontine reti cular tegmental nuclei (RTg, curved arrow). C. Occasional CA neurons appear within the medial para- brachial nucleus (open arrow) and IA neurons are present within the caudal periaqueductal grey (solid arrow). 32
P B r
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OS 33
Fig. 8 IA neurons within nucleus raphe dorsalis (RaD, insert). Host of the cells in this region are oval and do not exhibit fluorescent processes. Fluorescent varicosi ties are present in some areas (arrows). X125. Insert X64.
Fig. 9 IA neurons within the superior central nucleus (CeS, insert). Interspersed with nonflucrescent cell bodies (open arrow) some of the IA neurons reveal fluorescent processes (solid arrow). X125. Insert X64.
Fig. 10 CA neurons within the ventrolateral midbrain tegmentum
at the level of figure 1 2 b medial to fibers of the lateral lemniscus (LL). A small intensely fluorescent (SIF) cell is present along the lateral border of the midbrain (arrow). X125.
Fig. 11 IA neurons within the caudal portion of nucleus linearis surround fascicles of the medial longitudinal fasciculus (flm). Many of the cells in this region do not lose their fluorescence as rapidly when exposed to ultra violet radiation (arrows). X125. 34 35
Fig. 12 CA (outlined neurons) and IA (black neurons) cell bodies within the rostral pons and caudal midbrain. A. IA neurons extend from the dorsal raphe nucleus (RaD) through the superior central nucleus (CeS) and ventrolaterally into the reticular formation where they come in close proximity to the CA neurons (double arrows). B. The rostral continuation of IA neurons within nucleus raphe dorsalis (RaD), nucleus linearis (Lr), and lateral to the interpeduncular nucleus (IP). CA neurons occupy a position near the lateral border of the midbrain (arrows). C. A few IA neurons remain within nucleus linearis. Most of the amlnergic neurons at this level are of the CA type and extend from the pars compacts of the substantia nigra (SNc) to the lateral portion of this nucleus (SN1, arrows) and do.rsally to occupy a position beneath the red nucleus (see figure 15). 36
a q G Cv
R b
SN1 L r A
dbo Cl m
i p ped
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dbo
IP
ped
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m 37
Fig. 13 CA neurons medial to the mesencephalic tract of V (rV) (right side) with the adjacent Nissl section for com parison (left side). X450. Arrows = nonfluorescent cells.
Fig. 14 Top. Small intensely fluorescent (SIF) cells (arrows) lining a blood vessel within the caudal pons. Bottom. A SIF cell (solid arrow) and accompanying sym pathetic innervation of a blood vessel at the base of the midbrain. The intlmal lining of the vessel is autofluorescent (open arrow). X125.
Fig. 15 CA neurons within the ventral tegmental area (TgV) and nucleus linearis (Lr). X225. Insert shows the position of CA neurons (solid arrow) within the midbrain tegmentum ventral to the auto fluorescent cell bodies of the red nucleus (open arrow). X125.
Fig. 16 CA neurons (solid arrows) within the dorsal paraven tricular nucleus (top) and periventricular region (bottom). Nonfluorescent cell bodies are visible as well (open arrow), v = third ventricle. X125. 38 39
Fig. 17 CA neurons (outlined) within the rostral midbrain and hypothalamus. A. At the level of the exiting third nerve (III) numer ous CA neurons surround these fascicles and extend into the midline. B. At the level of the fasciculus retroflexus CA neurons are present within the ventral tegmental area (TgV) and within the periaqueductal grey (arrows). C. CA neurons are present within the dorsal paraventri cular nucleus (PHD) and dorsal hypothalamic area (HYD). 40
< V
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pod * SN o 41
Fig. 18 This summary illustration schematizes the distribution of CA (black areas) and IA (stippled areas) neurons within the opossum brainstem. The arrow (top illustra tion) indicates a gap in the otherwise continuous dis tribution of IA neurons along the midline. The hypo thalamic CA neurons are illustrated in figure 17C. uiuig
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