Distribution and numbers of indoleamine cell bodies in the cat brainstem determined with Falck-Hillarp fluorescence histochemistry

Brain Research

Bulletin, Vol. 9,

pp. 245-251, 1982. Printed in the U.S.A.

Distribution and Numbers of Indoleamine Cell Bodies in the Cat Brainstem Determined with Falck-Hillarp Fluorescence Histochemistry LUCIENNE *DPpartement and tDepartment

LfiGER*’

AND

LEIF

WIKLUNDt

de Mbdecine Expe’rimentale, Universite’ Claude Bernard-Lyon I 8, Avenue Rockefeller, 69373 Lyon CPdex 2 France of Histology, University of Lund, Biskopsgatan 5, 223 62 Lund, Sweden

Ll?GER, L. AND L. WIKLUND.

Distribution histochemistry.

and numbers

of indoleamine

cell bodies in the cat brainstem

determined

BRAIN RES. BULL.b9(1-6) 245-251, 1982.-Using the Falck-Hillarp method, the cat brainstem was found to contain approximately 60,000 indoleamine (IA) cells. Most of these (46,000 or 77%) are located within the raphe nuclei. Nissl-stained material demonstrated both medium- and small-sized perikarya in the raphe nuclei, and quantitation revealed that the IA cells comprise only part of the medium-sized cells. Thus, the raphe dorsalis holds about 24,000 IA cells representing some 70% of its medium&sized cells. Corresponding values were for raphe pallidus 8,000 IA cells (SS%), raphe centralis superior 7,400 (35%). raphe magnus 2,400 (l%), raphe obscurus 2,300 (33%), linearis intermedius 2,100 (23%), and raphe pontis 280 (%). A considerable number of IA cells (13,600, representing 23% of the total) were found in locations outside the raphe nuclei: in ventral brainstem as lateral extensions from the raphe, among the bundles of fasciculus longitudinalis medialis, in periventricular gray and adjacent tegmentum, mixing with the noradrenergic cells of the locus coeruleus complex, among the mesencephalic dopamine cells, and in the nucleus interpeduncularis. with Falck-Hillarp

j7uorescence

Cat Brainstem Quantitation

Falck-Hillarp fluorescence histochemistry Raphe nuclei and extra-raphe localizations

SHORTLY after the original publication by Falck and Hillarp [ 1l] of the formaldehyde-induced fluorescence method, Dahlstrijm and Fuxe [7] issued the first comprehensive description of monoamine nerve cell body distribution in mammalian central nervous system. This study of the rat brain disclosed twelve catecholaminergic (CA) and nine indoleaminergic (IA) cell groups. The CA cell groups (Al to A12) are mainly symmetrical and laterally situated in the brainstem and diencephalon, while the IA cell groups (Bl to B9) were described as predominantly medial groups located in the brainstem raphe nuclei. This principle of organization has subsequently been confirmed by many authors for other mammalian species: rabbit [5], cat [16,21], opossum [6], monkey [9], and man [20]. However, both the cat [18] and monkey [23] locus coeruleus contain a significant number of IA cells intermingled with the CA cell bodies. This observation pointed to the possibility that IA cells might exist in previously unnoticed locations and encouraged us to reinvestigate the distribution of IA cell bodies in the brainstem of the cat [29]. This review of our findings emphasizes the het-

Indoleamine nerve cell bodies

erogeneous neuronal population of the raphe nuclei and the presence of IA cell bodies in many areas outside the raphe nuclei. METHOD

These studies were performed on adult cats of both sexes. For fluorescence histochemistry, most cats were pretreated pharmacologically to increase intraneuronal serotonin content: monoamine oxidase inhibition (nialamide, 50 or 100 mg/kg, IP) and one hour later L-tryptophan (25 to 50 mg/kg, IP). One hour after the second injection, the cats were killed by decapitation under chloral hydrate or Nembutal anaesthesia. The rapidly dissected brainstems were divided into 3-4 blocks which were processed for fluorescence histochemical detection of monoamines [3,11], using a two step formaldehyde gas treatment to increase the fluorescence yield of IA [12]. Sections of the paraffin-embedded specimens were alternately mounted for fluorescence microscopy and cresyl violet staining. Microspectrofluorimetric analysis

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246

LfiGER AND WIKLUND

INDOLEAMINE

CELLS IN CAT BRAINSTEM

FIG. 2. FIGS. 1 and 2. The position of indoleamine cells (black dots) and catecholamine cells (circles) in the cat brain stem has been noted on sketches of frontal sections from Horsley-Clarke planes posterior 16 mm (Pl6) to anterior 5 mm (A5). Abbreviations: Am=nucleus ambiguus: BC=brachium conjunctivum; FLM=fasciculus longitudinalis medialis; GII=genu of facial nerve; INC=nucleus incertus; LC=nucleus locus coeruleus; LI=nucleus linearis intermedius; LM=lemniscus medialis; LR=nucleus linear-is rostralis; LRN=lateral reticular nucleus; NIP=nucleus interpeduncularis; NR=nucleus ruber; N3=oculomotor nuclei; NSm=motor trigeminal nucleus; N6=nucleus abducens; N7=nucleus facialis; OI=inferior olive; RCS=nucleus raphe centralis superior; RD=nucleus raphe dorsalis; RM=nucleus raphe magnus; RO=nucleus raphe obscurus; RP=nucleus raphe pallidus; RPo=nucleus raphe pontis; RSP=rubrospinal tract; RTP=nucleus reticularis tegmenti pontis; SC=nucleus subcoeruleus; SN=substantia nigra; SOL=lateral nucleus of superior olive; SOM=medial nucleus of superior olive; VT=ventral tegmental nucleus; VTA=ventral tegmental area of Tsai.

247

248

Ll?GER AND WIKLUND TABLE APPROXIMATE

Raphe nuclei Raphe obscurus Raphe pallidus Raphe magnus Raphe pontis Raphe centralis superior Raphe dorsalis Nucleus linearis intermedius Nucleus linearis rostralis Total

NUMBERS

1

OF INDOLEAMINE (IA) AND MEDIUM-SIZED RAPHE NUCLEI OF THE CAT*

CELLS

IN THE

Number of IA cells

IA cells vb total number of IA cells in cat brainstem

2,300 8,000 2,400 280 7,400

13’): 4% 0.4% 12%

7,100 15,000 15.000 2,900 2 I ,000

24,500 2,100

40% 3%

36,000 9.000

67% 23%

0.04%

4.000

0.6%

2.5

46,700

4%

Number of medium-sized cells

IA cells vs mediumsized cells

77%

*From Wiklund cutnl. 1291.

was performed for the IA and CA cells of the locus coeruleus complex. For conventional histology cats were perfused-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, and the brains processed for paraffin embedding, sectioning and staining with cresyl violet. Quantitation of IA cells was performed by counting fluorescent cell profiles in regularly spaced sections, correcting the counts according to Abercrombie’s formula [1] and multiplying by the sampling interval. The total number of neurons in different raphe nuclei was determined in Nisslstained perfusion-fixed material by the method of counting only cell bodies displaying a nucleolus [ 151. The IA cell bodies of the locus coeruleus complex were labelled using radioautography after injections of tritiated serotonin. The cats were pretreated with a monoamine oxidase inhibitor (Catron@, 5 mg/kg, IP) 18 and 1 hr before receiving an injectiqn of 10e2 M tritiated serotonin in the cerebral tissue at a short distance from locus coeruleus. In some animals, nonradioactive noradrenaline was added to the tracer [17]. The brains were fixed by perfusion with 1% glutaraldehyde- 1% paraformaldehyde in 0.12 M phosphate buffer, and the brainstem processed for embedding in paraffin. Regularly spaced sections were coated by dipping in Ilford K5 emulsion, exposed, developed and stained with cresyl violet. The following reports and atlases were consulted for nomenclature and delineation of anatomical structures: Taber et al. [27] for the raphe nuclei, Taber [261 and Berman [21for other brainstem nuclei. and Verhaart [28] for myelinated pathways. RESULTS

Under the short wavelength illumination of the fluorescence microscope the IA cell bodies were identified by their yellow fluorescence showing a relatively rapid photodecomposition, which contrasts with the more resistant green fluorescence of the CA cells. Yellow fluorescent cells were

observed from caudal medulla oblongata to the midlevel of mesencephalon (Figs. 1 and 2). In all locations. i.e., within and outside raphe nuclei, the IA cells were of medium size and mixed with non-fluorescent cells of medium as well as small size. In the quantitative estimates (Tables 1 and 2). the numbers of IA cells were primarily compared with the total numbers of medium-sized cells in the nuclei, since the small-sized neurons visualized with Nissl stain may represent interneurons. In medulla oblongata, the nuclei raphe pallidus (RP), raphe obscurus (RO) and raphe magnus (RM) all contain large numbers of yellow fluorescent cells. The IA cells are especially densely aggregated in the ventral RP, where only a limited number of non-fluorescent cell bodies could be distinguished in the fluorescence microscope (Fig. 3F). In contrast, the IA cells in dorsal RP (Fig. 3G), RO and especially RM (Fig. 3H), are mixed with larger numbers of nonfluorescent cells. Consistent with these observations, the quantitative estimates indicated that the 8,000 IA cells of RP consitute .55%, the 2,300 IA cells of RO 33%, and the 2,400 IA cells of RM only 15% of the total number of medium-sized cell bodies in each respective nucleus. The morphological appearance of the IA cells in the different nuclei was found to vary. The RO was marked by elongated, dorso-ventrally oriented cells, the ventral RP contained many smaller, round-oval cells (Fig. 3F), in addition to the multipolar cell type dominating dorsal RP (Fig. 3G), and the multipolar IA cells of RM (Fig. 3H) tended to be larger than those in the other locations. At all levels of medulla oblongata, IA cell bodies are found outside the raphe nuclei. Some cells are scattered among the myelinated bundles of the medial longitudinal fasciculi which border the midline. But the majority appear as lateral extensions of the RP and RM, which reach as far laterally as the rubrospinal bundle and mix with the A I group of CA cells. The rubrospinal tract is worth emphasizing since, along the length of the brainstem, it serves as a convenient landmark for the position of the lateral most IA cells. Most of the IA cells in the lateral extensions display a size

INDOLEAMINE

249

CELLS IN CAT BRAINSTEM

TABLE 2 APPROXIMATE NUMBERSOF INDOLEAMINE(IA) CELLSOUTSIDE THE RAPHENUCLEI IN CAT BRAINSTEM*

Locations Surrounding the raphe nuclei Lateral extensions of the ventral raphe: in medulla oblongata in pons

in mesencephalon Central pontine and mesencephalic tegmentum Periventricular gray and subjacent tegmentum Locus coeruleus complex Nucleus interpeduncularis

Total

Numbers of IA cells

Percentage of total number of IA cells in cat brainstem

1,400

2%

1,700

3%

1,100 60 1,100

2% 0.1% 2%

5,500

9%

1,300 1,400

2% 2%

13,600

23%

*Accumulated counts from Wiklund et al. 1291.

and morphology similar to that of cells in the corresponding raphe nuclei, but around the exit of the hypoglossal nerve a distinct population of small IA cells is located close to the ventral brainstem surface. In all, the lateral extensions in medulla oblongata contain about 1,700 IA cells (or 3% of the total number of IA cells in the brainstem). In pons yellow fluorescent cells are observed in the nuclei raphe pontis (RPo), raphe centralis superior (RCS) and raphe dorsalis (RD). RPo is the smallest IA raphe group (about 280 IA cells making up only 9% of the medium-sized cell population), while RCS (about 7,400 IA cells) and RD (about 24,500 IA cells including the part located in mesencephalon) represent, respectively, the third and largest aggregation of IA cells in cat brainstem. In RCS, the IA cells are mixed with many non-fluorescent cell bodies, and represent 35% of the medium-sized cells. In RD, quantitations indicate that the IA cells constitute 6% of the medium-sized cell population. A few green fluorescent cell bodies mixed with the IA cells of the ventral RD. While the IA cells in RCS are of a spindle or multipolar shape and among the largest IA cells (Fig. 3B), those of RD tend to be round to oval and of smaller size (Fig. 3A). The pons is very rich in IA cells outside the raphe nuclei (about 7,800 cells representing 13% of the total number pf IA cells). These IA cells roughly form two bilateral rings, dorsally extending from the raphe via the periventricular gray and subjacent tegmentum into the locus coeruleus (LC) complex, and ventrally above the superior olive and medial lemniscus (Fig. 3D) to the rubrospinal tract (Figs. 1 and 2). The IA cells which mix with the CA cells,of the LC complex were shown with radioautography to p sess uptake mechanisms for serotonin (Fig. 3E), an ? in microspectrofluorimetric analysis yielded excitation qnd emission spectri identical to those of yellow fluorescent @Is in RD and RCS [ 181.Quantitation set the number of IA cells in the LC complex at about 1,300, while 2,000 IA cells were found in pontine periventricular gray and subjacent tegmentum.

In mesencephalon, apart from the large aggregation of IA cells in RD (see above), the number of IA cells is moderate and decreases rostrally, so that the fluorescence picture is progressively dominated by the vast ventral collections of CA cells. Thus, the caudal part of nucleus linearis intermedius (LI) contains large numbers of IA cells, while the rostraI part of the nucleus is dominated by green fluorescent cells (Fig. 2). In all, the 2,100 IA cells of LI amount to 23% of the medium-sized cells in the nucleus. The most rostral of the raphe nuclei, the nucleus linearis rostralis, contains only occasional IA cells. Outside the raphe nuclei, IA cell bodies form a conspicuous group in ntieus interped~uhuis (about 1,400 IA c&s) @ii. 30 Scattered IA cells are found in the para$agittttl myelinated bun&s and mixing v&rally with the CA cells of the ventral tegmtntal area and d@rs;al substan& n&a. In all, the mesencephalon contains approximately 3,700 IA cells outside the raphe. nuclei. Most of the mesencephalic IA cells display morphological features similar to those of pons, and RCS in particular. Nucleus interpeduncularis holds, in addition, a number of small-sized IA cells (Fig. 3C). DISCUSSION With the combination of fluorescence histochemistry, radioautography and quantitation in Nissl-stained material, the present studies have enabled us to reach a detailed knowledge of the indoleamine cell distribution in the cat brainstem. The main new findings are the presence of considerable numbers of IA cells outside the raphe nuclei and the heterogeneous character of the investigated neuronal populations. As determined in the fluorescence histochemical preparations, the cat brainstem contains about 60,000 IA cells, 77% (46,700) of which are located within and 23% (13,600) outside the raphe nuclei. From RP to LI, the raphe nuclei comprise a more or less continuous series of IA cell aggregations. But the IA cells are far from being equally distributed among the different raphe nuclei. For example, the RCS contains about 7,400 IA cells (representing 16% of all IA cells in the raphe) while its rostral extension, LI, has only 2,100 IA cells (4%) and its caudal continuation, RPo, as few IA cells as 280 (0.6%). The RD alone contains 52% of the IA cells in the raphe nuclei. All raphe nuclei are composed of a heterogeneous neuronal population. Of these, the medium-sized IA as well as non-IA cell bodies are likely to be projecting neurons, while the non-IA small-sized cells may represent interneurons. The proportion of IA cells varies from one raphe nucleus to another. Thus, the IA cell bodies represent the majority (about 70%) of the medium-sized cells in RD, while they are only a small minority in RPo (9%). In the mesencephalic raphe nuclei, some of the non-IA neurons are CAcontaining, but in most raphe nuclei alI the non-IA cells are non-monoaminergic. In the rat, some evidence has been presented that the non-monoaminergic neurons of the raphe nuclei [8] comprise GABAergic [19], substance P- [13] and enkephalin-containing [25] neurons. Further, it is notable that we observed prominent morphological heterogeneity of the IA cell population in several raphe nuclei (e.g., RP and RM) where yellow fluorescent cells of different sizes and shapes are intermingled. It is conceivable that the different morphological characteristics correspond to differences in connections and functions.

LkGER

AND WIKLI JND

FIG. 3. A-D and F-H. Fluorescence micrographs showing indoleamine cell bodies in different locations. These micrographs illustrate the variations of fluorescence intensity from cell to cell in the same group, the different degrees of aggregation, the different sizes and shapes of the indoleamine cells, and the mixing of the indoleamine cell bodies with small-sized (small white arrows) and medium-sized (large white arrows) non-monoaminergic neurons. G x 140. FLM=fasciculus longitudinalis medialis; Py=pyramid. A: raphe dorsalis; B:raphe centralis superior; C:nucleus interpeduncularis; D: lateral extension from raphe centralis superior, above medial lemniscus; F: raphe pallidus, ventral part: G:raphe pallidus, dorsal part; H: raphe magnus. E: Radioautograph of locus coeruleus after injection of tritiated serotonin in the vicinity of the nucleus. Among non reactive small sized and medium-sized ceils, two labelled mediumsized cell bodies (arrows) are identified as indoleamine neurons. G x 140.

INDOLEAMINE

251

CELLS IN CAT BRAINSTEM

The presence of many IA cells outside the raphe nuclei calls for a modification of the often prevalent view of the IA systems as virtually identical to the raphe nuclei. Indeed, recent detailed investigations in the rat [lo,241 and monkey [22] brain have disclosed a distribution of IA cells which is more similar to that described by us than the original description by Dahlstriim and Fuxe [7]. Thus, these new investigations describe the occurrence of IA cells in pontine and mesencephalic periventricular gray, in LC and nucleus interpeduncularis. The interspecies differences of IA cell distribution may, therefore, be relatively limited, although the cat brainstem seems to display an especially pronounced dispersion of IA cells into lateral brainstem areas.

precise knowledge of the distribution of IA cells in the brainstem is of importance in interpreting the data of neuroanatomical tracing studies. Several studies using the horseradish peroxidase retrograde tracing technique (see [29]) have reported the presence of labelled cells in areas outside the raphe nuclei which are, in the present work, shown to contain IA cells. However, the ubiquitous mixing of IA and non-IA cells in the brainstem of the cat requires combination of retrograde tracers with monoamine histochemistry (e.g., [4,14]). Similarly, anterograde tracing techniques have to be combined with suitable complementary experiments in future delineation of the IA systems of the brain.

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