The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: Distribution of the cell bodies and some axonal projections

Brain Research, 66 (1974) 1-21

((~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Research Reports

T H E C A T E C H O L A M I N E - C O N T A I N I N G N E U R O N S IN THE CAT DORSOL A T E R A L P O N T I N E T E G M E N T U M : D I S T R I B U T I O N OF THE C E L L BODIES A N D SOME A X O N A L P R O J E C T I O N S

NAI-SHIN CHU* ANDFLOYD E. BLOOM Laboratory of Neuropharmacology, National Institute of Mental Health, St. Elizabeths Ho,spita[, Washington, D. C. 20032 ( U.S..4. )

(Accepted July 10th, 1973)

SUMMARY The distribution and the projections of the catecholamine-containing neurons in the dorsolateral pontine tegmentum of the cat was studied by the fluorescent histochemical technique of Falck and Hillarp. In contrast to the rat locus coeruleus nucleus which is well-defined and consists of densely packed norepinephrine-containing cells, the distribution of the catecholamine cells in the cat pons is more diffuse and more extensive. This extensiveness of the NE system in the pons is mainly due to the existence of catecholamine cells in the 'subcoeruleus' nucleus and in the area which surrounds the brachium conjunctivum. The catecholamine cells in the LC nucleus constitute the main source for the dorsal ascending NE pathway; the same cells also innervate the dorsal raphe nucleus and cerebellar cortex via axon collaterals. The catecholamine neurons in the medial locus coeruleus and subcoeruleus nuclei project to the pontine raphe complex, and the catecholamine cells in the lateral portion of the pontine catecholamine system project to the cerebellar cortex. The strong tendency for the catecholamine cell bodies to closely surround the brachium conjunctivum is unique in the cat.

INTRODUCTION The nucleus locus coeruleus** (LC) which lies in the dorsolateral tegmentum of the pons was so named by the Wenzels (1811) because of the dark bluish coloration * Present address: Division of Neurology, University of Colorado Medical Center, Denver, Colo. 80220, U.S.A. ** In some atlases of the cat brain stem27, the term 'nucleus dorsolateralis tegmenti' is used to designate the phylogeneticallyhomologous cell groups in the subprimate mammals in which pigmentation is usually absent5.

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the LC nucleus exhibits in man and primates 33. In the rat the LC nucleus consists of a densely packed, homogeneous cell population which exhibits green fluorescence of catecholamines when examined by the method of Falck et a l . i l . The presence of dopamine-/-]-hydroxylase in the cell bodiesT, 1"), and the capacity of these cells to synthesize norepinephrine (NE) iv suggest that these LC cells contain NE rather than dopamine. The NE cells of the LC nucleus in the rat give rise to a dorsal NE pathway to innervate extensively and preferentially the cortices of almost all the forebrain structures, including cerebral cortex, hippocampal formation and amygdala 1.1m:m. Along the ascending course, abundant axon collaterals are given off to innervate sensory relay nuclei in the thalamus and the geniculate bodies. The lateral projection of the LC neurons synapses with cerebellar Purkinje cells via the superior cerebellar peduncle ~3,'~1, whereas the descending pathway may innervate lower brain stem nuclei, i.e., the vagus nucleus 19,2°. However, the afferent inputs to this nucleus remain unknown. Despite the relative prominence of the LC nucleus in the mammals, only preliminary study of the catecholamine cell distribution has been attempted in the cat 1~,'-':~ We have investigated in detail the NE cells in the dorsolateral pontine tegmentum of the cat mainly for 2 reasons: (1) for chronic experiments in which the spontaneous activity of the cells in the LC region was studied, it became necessary to map out the distribution of the NE cells in this area in order to allow precise stereotaxic exploration by microelectrodes and to identify the recorded units specifically as N E-containing neurons ~, and (2) the cat is one of the most frequently used animals in both neurophysiological and neuroanatomical studies. Thus, detailed information regarding this monoamine-containing region is needed for further study of the cat NE synaptic projections. This report describes the diffuse nature of NE cell distribution in the dorsolateral ports and some of their fiber projections as revealed by lesioning studies.

METHODS

Thirty adult cats were used in this study. Fourteen of them were originally used in the unit recording experiments in which only one side of the brain stem was studied~L The fluorescence histochemical technique of Falck and Hillarp for monoamines 11 was used to visualize the catecholamine cell bodies and fibers. A Zeiss fluorescence microscope was used with a 100 or 200W Hg-lamp, KG1 and BGI2 filters on tile activation side and a barrier filter at 470 nm on the emission side. Under Nembutal anesthesia (45 mg/kg, i.p.), the cat brain was quickly removed in the cold room and the brain stem which included mesencephalon, pons, and the anterior medulla oblongata was sectioned either corona[ly or sagittally into blocks of about 1 2 mm thickness. Those blocks were immediately frozen in liquid nitrogen and were then freeze-dried at - 45 °C for 4-7 days. Subsequently, the blocks were reacted with vapor from paraformaldehyde (stored at 70~o humidity) at 80 C for I - I . 5 h. Finally, the blocks were embedded in v a c u o with paraffin. In some cats, a monoamine oxidase inhibitor, pargyline (50 mg/kg, i.p.), was given 1.5 2.0 h before decapitation

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to enhance fluorescence intensity in cell bodies and axon terminals 10. One out of every 8-10 sections of 10 # m thickness was mounted on the slides with Entellan for histofluorescence observation and the subsequent section was stained with Nissl's method for precise localization of the catecholamine cells on the standard stereotaxic histological map 3,2s. Unilateral transection rostral to the LC region was performed in 6 cats to deter~ mine the contribution of the N E cells in this area to the ascending NE pathways by observing the pile-up increase in fluorescence in the cell bodies 18,19- 21,24,30. By this method, the course of the ascending N E pathways through the mesencephaton and diencephalon could also be mapped out. With a knife which was slightly blunt at the tip but rather sharp on both edges, free-hand complete unilateral transection was made at different antero-posterior levels in the mesencephalon in 3 cats and in the diencephalon in 1 cat. The cats were sacrificed for fluorescence histochemistry 4-7 days postoperatively. Brain tissues both rostral and caudal to the lesion were taken for detailed examination. Cerebellum ipsilateral to the transection was included to investigate the possibility of the collateral NE fiber projection to this brain region by observing 'shunting' pile-up of catecholamine fluorescence 21. By this transection procedure, NE fiber tracts corresponding to the dorsal NE pathway in the rat 3° were located in the cat and subsequently this pathway was studied by local stereotaxic injection of 6-hydroxydopamine (6-OHDA) which causes selective degeneration of N E fibers 31. To determine the origin of the N E cells which give rise to the dorsal NE pathway, two approaches were adopted: (1) incomplete mesencephalic transection which spared the ventral part of the mesencephalic tegmentum, and (2) local injection of 6O H D A (hydrobromide base, Regis, Chicago) at various points along the dorsal NE pathway 31 whose ascending course was suggested by transection experiments. in the first approach, the knife was attached to the stereotaxic electrode carrier which was lowered to the desired depth of 3 m m below the stereotaxic horizontal zero and the unilateral transection was made by moving the knife horizontally. Two transection levels were chosen at A -- 1.0 m m and A ~ 4.5 mm. The second approach was to induce specific chemical transection of catecholamine fibers by 6-OHDA. The non-specific damage to other fiber systems or the extensive damage usually produced by the surgical method could thus be avoided. The intracerebral injection technique originally used by Ungerstedt 31 was employed in this study. 6 - O H D A was dissolved in Ringer's solution which contained 0.5 mg/ml of ascorbic acid to make a solution with final concentration of 2 #g//zl of 6-OHDA. The selectivity of 6 - O H D A in inducing specific chemical lesions is dose-dependentZl':~L Although the concentration of 6 - O H D A at 4/~g/#l is still within the safe margin for selectivity in the rat 31, we have observed in misplaced injections into the serotoninergic pathway that 6 - O H D A at 5 #g//zl clearly produced lesion of the serotonin-containing axons in the cat. Therefore, 6 - O H D A at concentration of 2/~g/#l or below was used for intracerebral injection in the cat. The stereotaxic coordinates for 6 - O H D A injection were either at A -- 4.5 mm, L -- 2.0 mm, and D = --1.0 mm, or at A - 1.0 mm. L = 1.5 ram, and D = --0.5 mm. The rate of injection was maintained at 1 /~g/10

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min and a total dose of 4-5 /~g of 6-OHDA was given. The cats were sacrificed 3-7 days after the injection for fluorescence histochemistry. The smear technique for monoamines developed by Olson and Ungerstedt '~" was used to demonstrate the projection of the dorsal NE pathway to the cerebral cortex and the hippocampus. About 1 2 cu.mm of the cortical gray matter tissue was spread on the slides which were previously cleaned with alcohol. The cortical smear on the slide was immediately dried in a hot air stream. The slides were subsequently transferred to a desiccator and dried overnight over fresh phosphorous pentoxide. Reaction with vapor from paraformaldehyde of 70% humidity was carried out at 80 ' C for 1 h on the next day. The smear was covered with Entellan for fluorescent microscopic observation. For controls, the reaction with paraformaldehyde was omitted or the specific reduction of catecholamine fluorophors by sodium borohydride 8 was used. Unilateral cerebellectomy by suction was carried out in 3 cats to localize the N E cells which innervate the cerebellum by observing the retrograde changes in the cell bodies ''1. Mechanical irritation or compression to the floor of the 4th ventricle was cautiously avoided to prevent non-specific changes in the NE cell bodies and fiber terminals. Gelfoam soaked with warm saline was packed in the empty space produced by the surgery. Four to 6 days were allowed before the cats were sacrificed. Since the main purpose of this study was to describe the distribution of the NE cell bodies in the dorsolateral ports, emphasis was primarily given to the topographical difference of the NE cells whose axons form the dorsal and ventral ascending NE pathways, and their fiber projections to the cerebellum and to the raphe complex. Detailed description of the exact course of these NE pathways was not attempted. RESULTS

Distribution of the NE-containing neurons in the dorsolateral tegmentum The NE cell distribution in the cat which will be described is based on tile nomenclature used by Taber 28,29, Berman 3, and Snider and Niemer 27 (Fig. 1). The NE cell bodies in cat dorsolateral pontine region exhibited weak to moderate intensity of green catecholamine fluorescence which could be enhanced by pretreatment with pargyline (Fig. 2). The fluorescence was usually more intense in tile perinuclear cytoplasm. but it was not so marked as that in the rat ~0. The distribution of these NE cells in the dorsolateral pons is diffuse as has been previously reported 6,1'5,')3. The NE cell bodies were mainly concentrated in the LC and LSC nuclei (Fig. 2), but they were also distributed in clusters in the nuclei parabrachialis medialis (PBM) and lateralis (PBL) and Koelliker-Fuse nucleus (KF) (Fig. 3). The fluorescent LC cells were mediuln in size and usually oriented more or less rostrocaudally. However, the medially situated NE ceils in the LC nucleus were horizontally oriented with axons often seen projecting mainly toward the nucleus centralis superior of the pontine raphe complex. The catecholamine cells in the LSC nucleus tended to be larger and usually oriented obliquely in the dorsomedial to ventrolateral direction. Again, the orientation of the medially located LSC fluorescent cells was parallel to the ventricular surface and the axons appeared to run toward the nucleus centralis superior, nucleus raphe pontis, and nucleus

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Fig. I. The pontine nuclei in the dorsolateral tegmentum and their relationship to other pontine structures. The reconstruction and the nomenclature are based on the study by Taber2s,29, Berman:~, and Snider and Niemer2v. Abbreviations: BC -= brachium conjunctivum; BP = brachium pontis; FTG -- gigantocellular tegmental field; FTP - paralemniscal tegmental field; IC ~ inferior co!liculus; KF -- Koelliker-Fuse nucleus; LC -- nucleus locus coeruleus; LL -~ lateral lemniscus; LM = medial lemniscus; SC = nucleus locus subcoeruleus; 5ME -- mesencephalic trigeminal nucleus; MLB = fasciculus longitudinalis medialis; 7N facial nerve; NR -- raphe nucleus; PbL = nucleus parabrachialis lateralis; PbM --: nucleus parabrachialis medialis; PT == pyramidal tract; SOM medial nucleus of the superior olive; 5ST -= spinal trigeminal tract; TB == trapezoid body; TD ~dorsal tegmental nucleus. Note that the PbM nucleus is omitted in left side map. For detailed descriptions see the text

raphe magnus. As c o m p a r e d to the rat, the 'subcoeruleus' N E cell g r o u p is more p r o m inent a n d well-developed in terms of the n u m b e r of the N E cells and the size of the nucleus. The N E cells in the PBL nucleus, in the medial periphery of the LC a n d LSC nuclei, and the ventral LSC were also larger. A few N E cells were always seen inside the BC, situated a m o n g the fiber bundles (Fig. 3B). One or two N E cells could often be seen even in the 5-ME nucleus, m o t o r nucleus of the trigeminal nerve, or lateral lemniscus. The tendency for the N E cells to s u r r o u n d the tip of the BC was quite r e m a r k a b l e (Fig. 3D), and is usually more p r o m i n e n t in the anterior a n d the posterior pons. The N E cells in the dorsal region of the LC nucleus also showed a tendency to s u r r o u n d the lower half of the 5-ME nucleus. The N E cells in the K F nucleus sometimes formed n u b i f o r m - l i k e c o n n e c t i o n s (Fig. 3C). Those N E cells usually orient horizontally a n d form clusters of 3-5 cells with their fibers c o n n e c t i n g a m o n g the clusters. The dorsolateral p o n t i n e area c o n t a i n i n g N E cell bodies extends rostrocaudally from P - - 1.5 m m to P - - 4.5 m m a n d mediolaterally from L = 1.5 m m to L ~-- 4:2 m m at the widest horizontal extent. However, the density of the N E cells is highest at the LC region from P = 2.0 m m to P ~ 3.0 mm. The N E cells became close to the 4th ventricle at P = 5.0 m m to P ~ 6.0 mm. Those n e u r o n s were relatively large and oriented parallel to the ventricular surface. In the sagittal view, m a n y N E cells were oriented rostrocaudally whereas the other N E cells, especially those situated ventrally

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Fig. 2, Photomicrograph of the fluorescent catecholamine cell bodies in the dorsolaleral pontine tegmenturn of the cat. Pargyline preireatment. A: sagittal view showing the distribution of the catecholamine cells 2.5 mm lateral to the midline and at P 2.5 ram. Rostral part on lhe right and tile caudal part on the left. Note that the catecholamine cells tend to orient rosirocaudally and parallel to the ventricular surface above. B : high magnification view of the catecholamine cells in the 1.(" nucleus. Note thai selective accumulation of calecholamine fluorescence arotmd the nucleus is not c o m m o n and thai the nucleus is located eccentrically in some cells. Calibration bar 30 ,-m.

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Fig. 3. P h o t o m i c r o g r a p h of the fluorescent c a t e c h o l a m i n e cell bodies in the dorsolateral puns. lpsilateral cerebellectomy in A a n d D, a n d pargyline p r e t r e a t m e n t in B a n d C. A : the c a t e c h o l a m i n e cells in nucleus parabrachialis lateralis at P ~ 2.5 ram. B: the c a t e c h o l a m i n e cells in the nucleus parabrachialis medialis at P -- 2.5 ram. Note 3 c a t e c h o l a m i n e cells inside the b r a c h i u m c o n j u n c t i v u m (BC) a n d the s t r o n g fluorescence in the neuropil. C: n u b i f o r m connections o f the catecholamine cells in the Koelliker F u s e nucleus. D: the catecholamine cells s u r r o u n d i n g the tip o f the b r a c h i u m c o n j u n c t i v u m at P ~- 2.5 ram. Note the increased intensity o f fluorescence in the s o m a t a a n d in the neuropil, x 160.

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Fig. 4. The distribution of the catecholamine cell bodies in the dorsolateral tegmentum of the pons. A : coronal view of the catecholamine cell distribution at different anterior posterior locations. The density of the crosses indicates the relative concentration of the catecholamine cell bodies. The catecholamine cells are more concentrated in the LC region from P 2.0 mm to P 3.0 mm The tendency for the catecholamine cells to surround the brachium conjunctivum is clear. At P 5.0 ram, the catecholamine cells become located close to the 4th ventricular surface. Those cells may correspond to group A4 cells of the rat. B: sagittal view of the catecholamine cell bodies at L 2.5 mm to the midline. The cross at the right upper quadrant is the stereotaxic zero. Abbreviations: CB cerebellar cortex; 5M motor trigeminal nucleus; PG pontine gray; 5SM spinal trigcminal nucleus, magnocellular division; TV . ventral tegmental nucleus; V4 4th ventricle; VI inferior vestibular nucleus; VMN medial vestibular nucletls; VS superior vestibular nucleus. For other abbreviations see the legend in Fig. 1. or medially, were oriented parallel to the ventricular surface. A s u m m a r y of the distribution of the N E cell bodies is shown in Fig. 4. It should be emphasized that the microscopic stereotaxic localization of those N E cells differed somewhat from cat to cat. By c o m p a r i s o n with histological sections, tile majority of the green fluorescent cells belong to the cell type which has p r o m i n e n t Nissl's granules in the cytoplasm. In addition to the cell bodies, the neuropil in this area also exhibited the characteristic fluorescence of the catecholamines which could be markedly enhanced by pargyline pretreatment. The neuropil fluorescence could also be differentially increased by different surgical procedures, i.e., mesencephalic transection and cerebellectomy. It is not clear whether the fluorescence in the neuropil reflects the a b u n d a n t recurrent axon collaterals, rich interconnections a m o n g the N E cells, extensive dendritic arborization24, '~s, or some c o m b i n a t i o n of these factors.

Unilateral transection at the mesencephalic-diencephalic levels The dorsal mesencephalic transection always produced intense retrograde fluorescent reaction ipsilaterally within the NE cell bodies in the LC region and, to a lesser degree, in LSC, PBM, and PBL nuclei (Fig. 5). The neuropil reaction was as intense as that seen in the perikarya. Those retrograde increases in fluorescence intensity in both perikarya and neuropil could be easily mapped out as shown in Fig. 6. The perikaryal changes went pari pa.s'.vtt with the neuropil changes except at tile level rostral to P 2.0 m m where an intense reaction was seen mainly in the NE cell bodies in tile

Fig. 5. Photomicrograph of the green fluorescent axons and cell bodies after unilateral mesencephalic transection. A : intense pile-up of fluorescence in the swollen axons of the dorsal NE pathway immediately caudal to the lesion. The NE fibers in the mediodorsal part of the pathway travel in small bundles. This tendency gradually disappears in the ventrolateral periphery. " 250. B: retrograde fluorescent reactions in the perikarya and the neuropil in the anterior LC region at P :: 2.0 ram. The LC neuropil exhibits extremely intense fluorescence in the fiber terminals and varicosities. Some NE cells arc swollen and their nucleus is obscured by the intense fluorescence. Arrows point to the ganglion cells of the mesencepbalic trigeminal nucleus which are round with a conspicuous nucleus and which always exhibit yellowish orange autofluorescence. The fluorescence of the NE fibers inside the mesencephalic trigeminal tract is also increased, especially at the tip. 160.

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Fig. 6. Distribution of catecholamine cell bodies and the fiber plexa exhibiting retrograde reactions in the dorsolateral pons 5 days after unilateral mesencephalic transection. The dots in the left side indicate the distribution of the affected NE cells and those in the right side the distribution of the NE neuropil exhibiting the retrograde changes. The density of the dots indicates the degree of the severity of the retrograde reactions. Except for the figure in the right lox~erquadrant x~hich shox~s the additional retrograde changes observed after deep mesencephalic transection, the other figures indicate the retrograde changes following dorsal mesencephalic transection. Note that the NE neurons in the LC nucleus at P 2.5 mm were most severely affected. NE cell bodies in the 'subcoeruleus" and the parabrachial nuclei were also affected as shown in the right upper figure where the distribution of affected cells and neuropil is corresponding. When the mesencephalon was completely transected, the NE cells in the "subcoeruleus" nucleus ,aere also affected. For explanation of abbreviations see legend Fig. 1.

LC nucleus. T h e fluorescent intensity o f the N E fibers inside or s u r r o u n d i n g the BC was also greatly increased at level from P :: 2.0 mm to P

3.0 mm. At the same time.

this experimental preparation revealed sparse and fine fluorescent fibers with varicosities in tile cerebellar molecular layer and a r o u n d the Purkinje cell somata (Fig. 7). This effect was most satisfactorily seen when the transection was made at the precollicular level, if the transection is made more rostrally, presumably more fluorescent catecholamine granules will be channeled to other axon collaterals destined for otker rostral brain areas, thus reducing the pile-up effect in the cerebellum. The fine fluorescent fibers and varicosities seen in the cerebellum after the dorsal brain stem lesion were not distributed evenly. They were seen in some sections but not in other sections. In many cases, the thin fluorescent fibers were seen traveling almost all the way up to the cerebellar cortical surface. When r a n d o m l y oriented N E fibers were seen. those N E fibers were usually short. The fibers exhibiting green fluorescence were seldom found in the granular cell layer. In the cerebellum o f the normal, untreated cat, the fluorescent N E fibers are usually not observed. Immediately caudal to the lesion, the swollen N E fibers with extremely intense fluorescence were seen situated ventrolaterally to the periaqueductal central gray o f

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tile m e s e n c e p h a l o n (Fig. 5A). The m e d i o d o r s a l part o f this N E p a t h w a y consisted o l m a n y small fiber bundles whe~eas in the ventrolateral part the bundle f o r m a t i o n w~, not a p p a r e n t a n d the N E fibers g r a d u a l l y dispersed into the mesencephalic reticular f o r m a t i o n (Fig. 5A). I m m e d i a t e l y rostral to the L C cell g r o u p (P .... 0.5--1.0 ram), tw(, N E fiber bundles were olien found to lie medial to the BC, one ventral and the othe~ dorsal. This situation seems similar to the o b s e r v a t i o n s o f M a e d a and Shimizu ~9 wh~, described a dorsal and an i n t e r m e d i a t e ascending N E p a t h w a y . However, rostral t(; P --- 0.5 m m these two N E p a t h w a y s in the cat became inseparable, lying lateral to the ventral tegmental nucleus o f G u d d e n and ventrolateral to the p e r i a q u e d u c t a l central gray. T h e green fluorescent fiber plexus which was n o r m a l l y seen to enter the ventrolateral p o r t i o n o f the dorsal r a p h e nucleus was m a r k e d l y increased in intensity and n u m b e r o f fluorescent fibers i n d i c a t i n g a presumptive innervation o f this structurc which contains serotonin neurons by axon collaterals from the dorsal N E pathway, Those c a t e c h o l a m i n e fibers to this nucleus were thin and the varicosities very fine, Rostral to the lesion, the fluorescence intensity in the h y p o t h a l a m u s did not s h o ~ a p p a r e n t changes. The N E cell bodies which were n o t affected by the dorsal mesencephalic transection included: (I) N E neurons which s u r r o u n d e d the lower BC, especially those in

Fig. 7. Cerebellar cortex of the cat 5 days after precollicular transection. Pile-up fluorescence of the sparse thin NE fibers with varicosities traveling through the molecular layer (M) toward the pial surface. The fluorescence of catecholamines is absent in the granular cell layer (G). Autofluorescence is seen in two Purkinje cells, on one of which (left lower) a few catecholamine fluorescent dotsare seen. x 250.

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the posterior pons, (2) the medially situated LC and LSC cells whose axons could be seen projecting horizontally toward the midline, (3) the ventrolateral group of the LSC cells, and (4) the NE cells in the PBL nucleus located rostral to P --: 2.5 ram. The axons of this cell group were seen projecting towards the inferior colliculus. When the mesencephalic transection was made more ventrally, green fluorescent fibers of larger diameter were seen scattered in the pontine reticular formation. At the same time, the NE cells and the neuropil in the LSC nucleus exhibited intense retrograde fluorescence reaction, although the neuropi[ reaction was more extensive (Fig. 6. right lower quadrant). The particular "catecholamine varicosities' seen in the kitten pons 26 seem to partly correspond to the distribution of the catecholamine-containin~, LSC region in the pons of the adult cat, although these 'catecholamine varicosities" are situated more medial and dorsal to the LSC nucleus. The retrograde changes in the LSC NE cell bodies suggest that they contribute to the ventral ascending NE pathway. The green fluorescence network around the midline raphe complex of the pons was greatly increased by this procedure. The catecholamine varicosities normally seen i,~ those raphe nuclei were usually dense and thick, especially those in the raphe magnu~, nucleus where the catecholamine terminals and varicosities were almost as dense ~s those seen in the periventricular hypothalamus (Fig. 8).

Lesioning the dorsal NE pathway by 6-OHDA Local injection of 6-OH DA into the dorsal N E pathway within the mesencephaIon as suggested by the transection study induced degeneration of the NE fibers which was manifested by swollen axons with intense fluorescence pile-up. The fluorescence exhibited by those affected NE fibers appeared to be more yellowish. The fluorescence reaction was not as intense as that produced by surgical transection, but the pattern of the fiber tracts that were affected was virtually identical to that described above. In the Nissl stained sections, the degeneration of N'E axons was accompanied by intense glial reaction at the injection site. The spread of 6-OHDA, as judged by changes in fluorescence intensity and the gila response, was about 1.0-1.2 mm from the center of the injection. The distribution of NE cell bodies affected by chemical lesioning with 6-OHDA was similar to the distribution of NE cells affected by the surgical method except that the 6-OHDA-affected area was smaller and the cellular reactions were also somewhat different. At the LC region after 6-OHDA injection, many cells exhibited yellowish

Fig. 8. Catecholamine fiber terminals and varicosities in the pontine raphe complex after mesencephalic transection. The drawing in the middle represents the posterior ports where the serotonin-containing cells are distributed as indicated by dots. Arrows point to the sites where A and B were taken. A; coronal view of raphe magnus nucleus. The picture was taken after the serotonin fluorescence had faded upon exposure to ultraviolet light. Note rich network-like catecholamine terminals and varicosities. B: sagittal view of raphe magnus nucleus. Rostral part on the right side. Note the intense catecholamine fibers coming toward the nucleus in the left Upper quadrant. Several serotonin-conraining cells are seen orienting themselves rostrocaudally. The tnedial edge of the medial lemniscus (LM) is seen situated between the catecholamine fiber plexus and the serotonin cells in the mid-lower quadrant. × 160.

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t:ig. 9. NE dorsal bundle lesion by 6-bydroxydopanline and unilateral cclebellectomy. A: swollen axons x~ith pile-up of fluoresce~ce induced by local injection of 6 - h y d r o x y d o p a m i n e at tile site of tile -,uspecied dorsal NE palh,aa~. The iniensity of lhe fluorescence reaclion is not as strong as that produced by surgical transection. In the LC region in the pons, m a n y NE cell bodies exhibited high i ntensiL,, o f c a l e c h o l a m i n e fluorescence v, hile the other NE cellsexhibited yellowish o r a n g e a u t o f l u o r e s -

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cence as s h o w n in C. B: fluorescent reactions of lhe neuropil in the dorsolateral pons 5 da5 s after ipsilateral cerebellectomy. At P 3.5 nlm level, neuropil fluorescence was increased in the area dorsomedial to the brachium conjuncfivum D: degenerating NE cells and fluorescent reactions of the N E fibers inside the bracllium conjunctivum. Arrows point to the NE ceils with pyknotic configuration and intense fluorescence. 250.

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orange autofluorescence, whereas other cells showed relative increases in the green fluorescence o f catecholamines which would fade slowly upon exposure and whose intensity could be greatly enhanced by pargyline (Fig. 9). However, the c o n t o u r and the size o f these N E cells a p p e a r e d almost normal. The NE cells in the LC nucleus showed the most intense reactions, especially those in the mid-to-lateral part of the I,C nucleus at the level from P 2.5 mm to P : 3.0 mm. By the smear technique, very fine, usaally r o u n d dots o f green fluorescence o f various intensities could be seen r a n d o m l y distributed in the smear o f the cerebral cortex (Fig. 10A). The dots with catecholamine fluorescence were absent when the step for the p a r a f o r m a l d e h y d e reaction was omitted. The specific catecholamine fluorescence could also be abolished by sodium b o r o h y d r i d e treatment. The rapidly fading yellowish orange fluorescent dots p r e s u m a b l y of serotonin varicosities could not be clearly seen in the smear p r e p a r a t i o n , even in pargyline-pretreated case. Local injection o f 6 - O H D A into the dorsal N E p a t h w a y induced a large decrease in the density of green fluorescent dots in the smears of ipsilatera[ cortex, but those fluorescent dots never d i s a p p e a r e d completely. It is possible that chemical lesioning by 6 - O H D A was not complete, or that the time between the injection and sacrifice was not long enough to allow complete a n t e r o g r a d e degeneration of the ascending N E tibet's. Since the ascending courses of the NE pathways have not yet been m a p p e d out in detail beyond

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Fig. I0. Catecholamine varicosities in the smear of the cerebral corlcx 10 days after dorsal NE pathway lesion by 6-hydroxydopamine. Pargyline pretreatment. A : the cerebral cortex contralatcral to the lesioned side. Note many small dots with catecholamine fluorescence presumably representing the calecholamine varicosities. The intensity of tile catecholamine varicosities varies. B: cerebral cortex ipsilateral to the lesioned side. Since the dots exhibiting autofluorescence were increased after 6hydroxsdopamine injection, B was deliberately chosen from a site where autofluorescent dots were virtuall3 absent in order to avoid confusion with actual catecholamine fluorescence. 500.

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Fig. I I. 6-Hydroxydopamine-induced NE dorsa! bundle lesion at A : 4.0 mm and AP : 0 mm levels. Note that the dorsal NE pathway is situated ventrolateral to the periaqueductal central gray. As it travels forward, the NE fibers become more ventrally located. Abbreviations: AQ : aqueduct: BX - decussation of the brachium conjunctivum; DR dorsal raphe nucleus; EW ..... Edinger Westphal nucleus; FTC -: central tegmental field; IP : interpeduncular nucleus; MGP : principal nucleus of the medial geniculate body; 3N oculomotor nucleus; P - pyramidal tract; PAG ~- periaqueductal central gray; PP pes pedunculi; R red nucleus; SNC -= substantia nigra, compact division; SNR : substantia nigra, reticular division; SC superior colliculus; SCC commissure of the superior colliculus. For other abbreviations see Fig, I legend.

the mesencephalon, it is not clear that the cerebral cortex is completely innervated by the dorsal N E pathway, although it contributes heavily as suggested by the smear study. A n o t h e r feature in the ipsilaterat cortex after 6 - O H D A injection was the presence of m a n y dots which exhibited yellowish orange autofluorescence. Similar findings were f o u n d in the h i p p o c a m p a l formation.

Unilateral cerebellectomy F o u r to 6 days after hemi-cerebellectomy, few degenerative changes were seen in the N E cell bodies except for those N E cells located inside the BC a n d in the PBM nucleus where the degenerating N E cells exhibited pyknotic c o n f i g u r a u o n , intense fluorescence o f yellowish green, a n d s h r u n k e n nuclei (Fig. 9D). The N E cell bodies in PBL nucleus a n d a r o u n d the tip o f the BC showed various degree of increase in fluorescence intensity (Fig. 2). The reaction was not as strong as those produced by mesencephalic transection or 6 - O H D A injection, and therefore some degree of uncertainty remains. The N E cells in the dorsolateral part of the LC nucleus also showed an increased intensity in catecholamine fluorescence after hemi-cerebellectomy. At the level of the anterior pons. the distribution of neuropil reactions was roughly similar to that caused by the mesencephalic transection but was much less prominent, suggesting either a c o m m o n source o f N E cells for b o t h the ascending N E pathway a n d the cerebellar projection, or rich interconnections between the N E cells

]8

N.-S. C H U A N D I. [ . BLOOM

giving rise respectively to those two NE projections. Tile increased axonal fluorescence in the cerebellar cortex lifter mesencephalic transection Favors tile idea of a common cellular origin to the dorsal bundle cerebral and cerebellar cortical projections. In the posterior pons, tile neuropil reaction and the retrograde changes in tile cell bodies after cerebellectomy were observed mainly in the PBM nucleus, around the tip of the BC and in the area dorsal to tile BC and 5-ME nucleus. Because tile neuropil reactions in the posterior pons after hemi-cerebellectonly had a different distribution fron~ that produced by mesencephalic transection, the NE cells which were affected by cerebellectomy in the posterior pons may project to tile cerebellum without contributing to the ascending NE pathways. By their reaction to the cerebellectomy two prominent NE fiber plexa projecting to the cerebellum could be located: one in the ventral part of the BC, especially in tile medioventral site, lind tile other in the dorsal part of the BC. The location of the medioventral plexus corresponds to the PBM nucleus where clustered NE cell bodies were always found. I)IS('USSION

It is clear that in the cat NE cell bodies exist in the phylogenetically homologous area For tile nucleus locus coeruleus of tile primates e and in the homologous region where they are concentrated in rodents ~°,19. Although cat LC NE cells do not Form a well-defined, compact, homogeneous nucleus as seen in other species, tile pontine NE cells are nevertheless more concentrated in the LC region. Tile extension of tile NE cells to tile 'subcoeruleus' nucleus and tile marginal nuclei of the BC |esults in a more diffuse NE cell distribution in tile dorsolateral pons in the cat. It should be pointed out that tile NE cell population in tile 'subcoeruleus" nucleus in the cat constitutes a substantial portion of the dorsolateral pontine N E system. This relative prominence of tile 'subcceruleus" nucleus in the cat bears more similarity to the situation in the monkey than in the rat. In tile African green monkey, many catecholamine-containing cell bodies were seen in tile 'subcoeruleus' region e, but in the rat there are only a small number of NE cells situated below the principal locus coeruleus 'e". Since the N E cells in the 'subcoeruleus" nucleus contribute heavily to tile ascending ventral NE pathway which innervates mainly hypothaiamus and tile preoptic area, the functional significance of tile prominent 'subcoeruleus' NE system in the cat and the primates remains to be determined. From the anatomical point of view, tile diffuse NE cell distribution in the cat pontine region certainly makes it rather dilIicult to consider the whole NE complex in the dorsolateral pons as one Functionally homogeneous group. The N E neurons in the cat 'subcoeruleus' and tile parabrachial nuclei seem to deserve separate grouping For their efferent projections are distinct From those originating From the LC nucleus, although some degree of overlap always exists. Furthermore, if it can be established that the afferents to those 3 pontine N E nuclei also differ, then tile subgroupings may permit a more discrete analysis of the functional involvement of the N E efferent proiections. On tile other hand, tile NE cells situated close to the tip of the BC in the anterior pons may correspond, if not exactly, to group A7 cells of the rat 1". Similarly, the NE cells

C A T E C H O L A M I N E ( ' E L L S IN ( ' A ' I PONS

!c)

lying close to the 4th ventricle in the posterior pons (P : 5.0 ram) seem to correspond to group A4 cells of the rat. Thus, clear boundaries among groups A4, A6, and A7 arc absent in the cat. It is interesting to note that in the squirrel monkey the boundaries among presumptive groups A4, A6, and A7 cells are also not clear, but the principaI LC nucleus is a well-defined, catecholamine-containing structure ~~. The NE cells in the cat LC region constitute the main source for the dorsal N~:: pathway as suggested by studies with mesencephalic transection and injection of 6O H D A . As compared to the rat, the dorsal NE pathway is situated closer to the periaqueductal central gray. Along the ascent of this NE pathway, axon collaterals are given off to innervate the dorsal raphe nucleus a~d the periaqueductal centra! gra$. Those collateral NE fiber terminals are thin and their varicosities are fine. By the smear technique, a decrease in the fluorescent varicositie~; in the cerebral cortex and the hippo-. campus after the lesion of" the dorsal NE pathway by 6-OHDA suggests a cortic~J innervation mainly by NE cells back in the LC region. Thus, for the dorsal ascendillg NE pathway, the origin of the cell bodies and the type of the terminal varicosities are similar to those in the rat :30. It appears that in the cat NE cells in the I_.C nucleus are not the major source for NE cerebellar projection, though such is the case in the rat ~. The I,C NE celis which innervate the cerebellar cortex are located mainly in dorsolateral part of the LC nucleus. The retrograde changes in catecholamine-fluorescent neurons after cerebellectomy suggest that the NE cells surrounding the BC in the parabrachial a~d Koetliker Fuse nuclei and the NE cells in the location corresponding to group A 4 ( P : 5.0 ram) contribute the major NE cerebellar innervations. It appears that in the a~nterior ports those NE cells in the LC. PBL, and PBM nuclei which send their axons to form the dorsal NE pathway also give off axen collaterals to innerwlte the cerebellum, but in the posterior pons the NE cells seem to project directly to the cerebellum without contributing to the ascending NE pathways, it is interesting to note that the NE varicosities are seen not only in the molecular layer of the cerebellar cortex but also in the Purkinje cell layer. More sensitive methods such as the autoradiographic localization of terminals labeled with [3H]NE (ref. 4) or after selective perikaryal labeling," arc needed to precisely localize those cerebellar NE synapses at the fine structural level. The strong tendency for the NE ceils to surround the brachium conjunctivun~ intimately is striking, and is certainly unique for the cat for it is absent in both rodents and primates. The close relationship of the NE cells to the BC is further indicated by the fact that a few NE cells are always seen inside the BC. Finally, abundant NE fibers can be seen situated among the fiber bundles of the BC. Two prominent NE fiber plexa which enter the BC are situated in the dorsal and the ventral parts of the B('. Similar situation has recently been found in the rat ~6.* It should be emphasized that the cat raphe complex receives rich NE projections and that different raphe nuclei appear to receive more or less separate inputs fi'om different pontine NE cell groups. For example, the dorsal NE pathway or ginating * A detailed re-examination of the rat LC -LSC complex suggests that rat NE cells may also be less homogeneous in their projections than previously thought 16.

20

N.-S. ('HU AND I. li. BLOOM

mainly from the LC nucleus synapses with the dorsal raphe nucleus, where the NE terminals and varicosities are fine. The N E cells in the medial periphery o f the LC region a p p e a r to innervate the nucleus centralis superior, and those NE cells in the medial part o f the 'subcoeruleus' nucleus seem to project to nuclei raphe pontis and magnus where the NE terminals and varicosities are usually thick. An increase in fluorescence intensity o f the catechohtmine terminals in raphe pontis and magnus nuclei after complete mesencephalic transection indicates innervations o f these raphe nuclei by ventral ascending N E pathways, it should be mentioned that the N E innerr a t i o n of the raphe magnus nucleus is unusually rich. Finally, the close association o f the N E cells of the LC nucleus with the mesencephalic trigeminal nucleus should be emphasized. A similar relationship is also observed in the squirrel m o n k e y l L and in the raO 0. In the cat, the N E cells closely surround the lower half o f the 5-ME nucleus, whereas in the squirrel m o n k e y the NE cells are situated immediately to the medial side of the 5-ME nucleus. A l t h o u g h the diffuse distribution o f the NE cells in the dorsolateral pons in the cat may offer a topological a d v a n t a g e o f m a p p i n g out the functional heterogeneity o f the NE cells, this diffuseness makes it extremely difficult to carry out experiments requiring selective stimulation or destruction o f specific pontine NE cell groups. Nevertheless, it is still possible to precisely lesion the NE pathways by the 6 - O H D A injection technique as has been described for the rat al. ACKNOWLEDGEMENTS

We t h a n k Dr. U. Ungerstedt for his d e m o n s t r a t i o n o f the smear and 6 - O H D A injection techniques, and Drs. G. R. Siggins, B. J. Hoffer, and H. Krebs for critically reading the manuscript. Assistance in histology by Mrs. E. K r a u t h a m e r is appreciated.

REFERENCES I

2 3 4 5 () 7 8

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