Brain Research Bulletin, Vol. 18, pp. 371415. Q Pergamon Journals Ltd., 1987. Printed in the U.S.A. 0361~9230/87 $3.00 + .oO

The Immunohistochemicd Localization of Choline Acetyltransferase

in the Cat Brain

STEVEN R. VINCENT AND PETER B. REINER

Division of Neurological Sciences, Department of Psychiatry The University of British Columbia, Vancouver, B.C., V6T 1 W5 Canada

Received 19 August 1986

VINCENT, S. R. AND P. B. REINER. The immunohistochemical localization of choline acetyltransferase in the cat brain. BRAIN RES BULL M(3) 371415, 1987.-The distribution of neurons displaying choline acetyltransfetase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChATpositive. Additional ChATpositive neurons were detected in the periolivsry portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic ceU groups that have been previously identified in the cat as weU as in rodent and primate brain such as motoneurons, striatal interneurons, the magnoceUular rostral cholinergic column in the basal fore- brain and the caudal cholmergic column in the midbrain and pontine tegmentum were confirmed. Together, these observa- tions sugge’st that the feline central cholinergic system may be much more extensive than previous studies have indicated.

Choline acetyltransferase Cat Acetylcholine Immunohistochemistry Central nervous system

THE localization of central cholinergic neurons was a mys- tery for many years, due to the lack of a specific method with which to identify unambiguously such cells. Although acetylcholinesterase (AChE) histochemistry has provided much valuable information, the cholinergic nature of many esterase-positive neurons has always been in doubt [26,45, 80,811. The last few years have witnessed a revolution in our understanding of central choline& anatomy, brought about primarily by the introduction of antibodies to the choline@ marker, choline acetyltransferase (ChAT).

The first major description of immunohistochemically- identified central cholinergic neurons was the mapping of the cat brain by Kimura et al. [43], who used an antiserum raised in rabbit against human ChAT. Since that time various groups have produced specific, monoclonal antibodies to ChAT [15, 19, 22, 471. These have been used to map the distribution of central cholinergic neurons in the rat [3,4, 16, 22, 23, 32, 36,48, 54, 72, 82,941 and primate [55, 60,73,74] brains. However, the cat brain has not yet been mapped with these new reagents. Therefore, in the present study a well characterized monoclonal antibody to ChAT [22] was used to examine the central cholmergic system of the cat. The data indicate that this system in the feline brain displays many features distinct from what has previously been found in other species. A preliminary report of these observations has been presented [921.

METHOD

Four adult male cats weighing three to four kg were studied. The animals were anesthetized with sodium pen- tobarbital (50 mg/kg, IP), heparinized (10,000 units, intracardiac) and perfused transcardially with 200 ml of phosphate buffered saline (0.1 M, pH 7.4) at room tempera- ture, followed by 1500 ml of ice-cold fucative. The fixatives used were 4% paraformaldehyde in 0.1 M phosphak buffer, pH 7.4 (3 cats) or the buffered picric acid pamformaldehyde solution of Zamboni and DeMartino [99] (1 cat). The brain and spinal cord were removed and post-fixed for 2 hr in the same fixative, blocked and placed in cryoprotectant (2% sucrose, 10% glycerol in 0.05 M phosphate buffer) for 48 hr at 4°C. Three of the brains were sectioned coronally and one sag&tally at 30 I.crn thickness on a freezing microtome, and collected serially in nine wells containing T&buffered saline (TBS, 0.05 M, pH 7.4) with 0.02% sodium azide.

Adjacent sets of sections were stained for Nissl with cre- syl violet and for choline acetyltransferase imrmmoreactivity using a well characterized rat monoclonal antibody to par- tine ChAT [22]. Sections were incubated in 0.3% H*O, in TBS for 1 hr at room temperature to inhibit endogenous peroxidase activity, and then rinsed in TBS. The free- floating sections were then incubated with the monoclonal antibody, diluted 1:200 in TBS containing 2% normal rabbit

371

ABBREVIATIONS

a aa ab ac AC ap aq BC BCX

& ci Cm

dc&

dbv

dh dmx

E ftm &

p ha hl hm ic IC km io ip ipi

ipo

ipp

Ic

nucleus ambiguus anterior amygdaloid nucleus basolateral nucleus of the amygdala central nucleus of the amygdala anterior commissure area postrema cerebral aqueduct brachium conjunct~vum decussation of the brachium conju~ctivum caudate nucleus CNS cerebri inferior colliculus central medial nucleus of the thalamus cuneiform nucleus horizontal limb of the nucleus of the diagonal

band of Broca vertical limb of the nucleus of the diagonal

band of Broca dorsal horn of the spinal cord dorsal motor nucleus of the vagus nerve entopeduncular nucleus central tegmental field magnoceUu1~ tegmental field nucleus gracilus globus pallidus hilus of the lateral superior otive anterior hypothalamic area lateral hypothalamic area medial habenula islands of Calleja internal capsule medial island of Calleja inferior olive interpeduncular nucleus inner division of the posterior interpeduncular

nucleus outer division of the posterior interpeduncu-

lar nucleus paramedian division of the posterior inter-

peduncular nucleus locus ceruleus

LL Ild Ilv

lot

IS0

MLF ms mso

%

; pag pb pbg PC Pg SC

si sn ST TB td TS tld tv vh III IV

4 4n

rs 6 6n

7a

% 7n 7r 12

lateral lemniscus dorsal nucleus of the lateral lemniscus ventral nucleus of the lateral lemniscus nucleus of the lateral olfactory tract lateral superior olive medial longitudinal fasieulus medial septal nucleus medial superior ofive nucleus accumbens optic tract putamen pyramidal tract periaqueductal gray parabrachial nuclei parabigeminal nucleus paracentral nucleus of the thalamus pontine gray spinal canal substantia innominata substantia nigra stria terminalis trapezoid body dorsal tegmental nucleus of Gudden solitary tract laterodorsal tegmental nucleus ventral tegmental nucleus of Gudden ventral horn of the spina! cord third ventricle fourth ventricle oculomotor nucleus trochlear nucleus trochlear nerve motor nucleus of the trigeminal nerve retrotrigeminal nucleus abducens nucleus abducens nerve facial nucleus accessory facial nucleus genu of the facial nerve facial nerve retrofacial nucleus hypoglossal nucleus

serum, 0.01% bovine serum albumin (BSA) and 0.3% Triton X-100, for 48 hr at 4°C. The sections were then processed for immunoperoxidase using the avidin-biotin complex method [34] using a rat ABC Kit (Vector Laboratories, Budingame, CA). The sections were rinsed 3x20 min in TBS, and then incubated for I hr at room temperature in biotinylated rabbit anti-rat IgG diluted I:200 in TBS containing normal rabbit sera, BSA and Triton. Next, the sections were rinsed again 3x20 min in TBS and then incubated in avidin-biot~ylated horseradish peroxidase complex, diluted 1: 100 in TBS con- taining normal rabbit sera and BSA. The sections were washed once again 3 x20 min in TBS and then reacted for peroxidase activity.

The sections from one of the coronally sectioned, aldehyde-fured cats were incubated in 50 mM Tris-Cl, pH 7.4 containing 0.025% 3,3’-diaminobenzidine, 0.01 M imidizole and 0.0075% H202 for 8 min. The sections from the other three animals were reacted using a nickel intensification technique in 50 mM Tris-Cl containing 0.02% diaminoben- zidine, 0.0015% H,O+ and 0.6% nickel ammonium sulfate.

FolIowing rinsing in TBS, the sections were mounted on ~hrorn~~~rn coated slides, dehydrated and coverslips were

applied with Permount. The sections were examined under bright and dark field illumination, and with Normarski dif- ferential interference contrast optics. Selected sections stained for ChAT were mounted in a photographic enlarger and projected directly onto 8~ IO” Ilfospeed grade 4 glossy photographic paper to illustrate the distribution of ChAT- immunoreactive cells and neuropil in the forebrain (Figs. i-8) and caudal brainstem (Figs. 17-40). The anatomical no- menclature used follows that of Berman [6,7J and Bfeier 183 in most instances, while in the brainstem, Taber [87] was also consulted.

For comparison, young adult male Wistar rats were per- fused and processed in parallel with the cats, using identical procedures throughout.

RESULTS

Cells exhibiting ChAT immunoreactivity had a wide- spread distribution throughout the central nervous system of the cat. Although cell bodies were the most obvious stained feature, numerous regions showed dense terminal fields which were best appreciated in the prints made directly from

ChAT IN THE CAT 373

the microscope slides in the photographic enlarger (Figs. 1-8, 17-40). Also, the dendritic fields of many of the cell groups were well delineated, and numerous fiber bundles, such as the cranial nerves, the fasciculus retroflexus and the dorsal tegmental pathway were well stained. No glial ele- ments were immunoreactive.

Only normal adult male cats were examined. It is possible that colchicine pretreatment might have allowed the visu- alization of additional cell groups that were not detected in the present study. However, it has been found in previous studies in rat 1211 that colchicine treatment does not improve ChAT immunostaining.

No ChAT-immunoreactive cell bodies were detected in the olfacotry bulb, hippocampus or cerebral cortex. The caudate nucleus and putamen contained a population of large, mostly multipolar ChAT-positive neurons. These were sparsely scattered throughout the entire striatum, including the nucleus accumbens (Figs. l-8). In addition, a heteroge- neous terminal field was also clearly evident throughout the striatum, with scattered, lightly-stained regions embedded in the more intensely stained neuropil. The striatal terminal field was most dense within the nucleus accumbens (Figs. 1, 2).

Between the nucleus accumbens and the septum, in the medial island of Calleja, a group of much smaller ChAT- positive cells was present (Fig. 1). These were larger (10-U iu_m diameter) than the islet granule cells among which they were scattered, but still much smaller than the ChAT- positive cells in the nucleus accumbens (25-35 pm diameter) (Fig. 9). The islet cells displayed only one or two poorly stained processes. In each of the other islands of Calleja similar clusters of small ChAT-immunoreactive cells were also found (Fig 1). These often occurred within the cup-like depression formed by the granule cells, and extended proc- esses into the islets, which exhibited dense terminal fields (Fig. 10). The size of the ChAT-immunoreactive cells asso- ciated with the islets appeared to increase in the more later- ally placed islets, with the most medial islet having the smallest ChAT-positive cells. Indeed, the cells in the most lateral islets were similar in size and morphology to those in the nucleus accumbens, caudate nucleus and putamen, and in fact appeared to be a ventral extension of the striatum, via striatal bridges across the polymorphic layer of the olfactory tubercle.

The major ChAT-immunoreactive cell group of the fore- brain was the rostra1 cholinergic column of the basal fore- brain (Figs. l-8). This extensive collection of magnocellular, intensely-immunoreactive neurons began rostrally in the medial septum. Scattered single ChAT-positive cells were found within the lateral septum, which also contained a dense terminal field. Similarly, quite a few of these large, ChAT-intense ceils could be found within and surrounding the fibers of the columns of the fornix, and caudally within and about the commissure of the fomix (Figs. 2-4). Ven-

FOLLOWING PAGES

trally, the cells of the medial septum were continuous with similar large multipolar cells in the vertical limb of the nu- cleus of the diagonal band of Broca (Fig. 2).

The cells of both the vertical and horizontal limbs of the diagonal band of Broca were large, intensely stained, and mostly multipolar (Fig. 1 lc). The ChAT-positive cells of the horizontal limb spread laterally in clumps across the polymorphic layer of the caudal olfactory tubercle and the rostra1 preoptic area to the medial edge of the lateral olfac- tory tract. Here a dorsal branch rose through the substantia innominata, penetrated the putamen, and surrounded the globus pallidus from its ventral and lateral surfaces (Figs. 3-5). A dense band of such cells coursed dorsally in the medullary lamina between the globus pallidus and putamen to reach the internal capsule. Another, sparser ascending branch arose medial to the globus pallidus beneath the anterior commissure and surrounded the medial and dorsal surfaces of the globus pallidus (Figs. 4-6).

Both these branches of the basal forebrain cholinergic column invaded the internal capsule extensively (Figs. 2-5). Many, very large, and very heavily stained cells were inter- calated among the fiber bundles dorsal to the globus pallidus, between the caudate and putamen. These cells were larger (up to 50 pm diameter) than the striatal ChAT-positive cells (25-35 pm diameter) seen at these levels (Fig. 11). It is inter- esting to note that although the globus pallidus was sur- rounded on all sides by magno~ellular ChAT-immunoreac- tive neurons, such cells only penetrated within the pallidum at its caudal pate (Figs. 6-8). A similar situation was seen in the ventral pallidum, beneath the posterior limb of the anterior commissure, which contained very few ChAT- positive cells and very little fiber staining, but was sur- rounded by the many ChAT-positive cells of the substantia innominata (Fig. 2).

Moving caudally, the ChAT-positive cells of the substan- tia innominata extended laterally above the nucleus of the lateral olfactory tract into the anterior amygdaloid area (Figs. S-8). CelIs continued to extend dorsally from here around the paliidum in the medullary lamina next to the putamen and within the external capsule. Magnoceliular ChAT-positive cells also appeared to invade the ventral third of the putamen, where they mixed with the smaller striatal ChAT cells. Both the nucleus of the lateral olfactory tract and the anterior cortical nucleus of the amygdala contained dense terminal fields (Figs. 5-8).

ChAT-positive cells extended beneath the pallidum above the central nucleus of the amygdala, and within the putamen. A few small ChAT-positive cells were found within the cen- tral nucleus of the amygdala (Figs. 8, 110. Some cells also occurred ventromedially along the surface of the globus pal- lidus beneath the rostra1 entopeduncuiar nucleus in the ansa lenticularis (Figs. 6-8). No ChAT-positive cells were found within the entopeduncular nucleus. The caudal extension of the rostra1 cholinergic coiumn continued above the dorsal tip of the optic tract to the caudal end of the entopeduncular nucleus (Fig. 8).

FIGS. l-8. Projection prints from rostra1 to caudal of ChAT-immunoreactive neurons in the cat forebrain illustrating the extent and distribu- tion of the rostra1 cholinergic column. These prints were made by mounting the immunoperoxidase-stained sections directly in a photographic enlarger and using them as “negatives” to print the images directly. The peroxidase-stained neurons and neuropil therefore appear white.

VINCEN’I AND K

FIG. 1. The rostra1 pole of the rostra1 column is found in the medial septal nucleus (ms). The striatal ChAT-positive ceils XC‘ frond in caud ate fluckus (c). putamen (p) and the nucleus accumbens (na). In addition. very small ChAT-immunoreactive cells are ~OUIK~ in ;IhsOcia with the medial island of Calleja (km) and the other small islands of Calkja (ic, arrowheads) which also shou ii dense l~rminal field.

the tion

IN THE CAT

FIG. 2. them se1 ves continuous with the cells found in the horizontal limb of the diionai band (dbh).

FIG. 3. At the rostra1 pole of the globus pafiidus (gp). the ChAT-positive neurons of the rostra1 column extend out from the ~~~~I~~)~~~I knb of the d .iagonal band (dhh) into the anterior hypothalamic area (ha). substantia innominata (si) and dorsally up into the inttrnaf capsule (IC), betw’ een the caudate nucleus (c) and the putamen (p). Note the absence of ChAT-immunoreactive neurons within the gloh~~\ pallidus.

ChAT

FIG. 4. within t stria te8 smaller

At the level of the antexior commissure (AC), many of the magnocelhdar ChiT-positive _ __“__ _ _

neurons of the mobaI cohunn a~ he internal capsule (IQ, dorsal to the globus pallidus (gp). Other cells are present in the bed nuclei of the anterior commissure. rninalis (ST), while many more are present in the horizontal limb of the diagonal band and the substantia inwminata (si). S ChAT-immunoreactive cells are present in the anterior hypothalamic area (ha).

re found , and the tattered

FIG. 5. A dense terminal field is present in the nucleus of the lateral olfactory tract (lot), which lies beneath the rohtrai ch\>ltnergic column neurons in the substantia innominata (si). The magnocellular ChAT-positive neurons can be seen surrounding the globus pallidus (gp) in the internal capsule (IO, and even invading the ventral third of the putamen (p). Medially the cells invade the tateral p~~rtr~~n $4’ ihc anterior hypothalamic area (ha), where smaller. weakly ChAT-positive cells are also found.

ChA T IN THE CAT

FIG. 6. ior

At the rostral pole of the ent~peduncular nucleus (ep), some of the &nocellular neurons invade the globus peghtus (gp) ante1 cells

amygdaloid area (aa), which also contains a rather dense terminal field. A few magnocellular neurons together with many SIX are I present in the lateral hypothalamus (hl).

d the faint

FIG. 7. Within the amygdala, dense terminal fields arc present in the central nucleus (ac) and the basolaternl nudcu~ ~.,tbi. i-ix% r-positive neurons of the rostra1 column are found mostly along the medial edge of the &bus pallidus (gp). but they extend into the gl~ihu% paliidus, and within the amygdala. Striatal C’hAT-positive neurons continue in the caudate nucleus cct and putamen ip>.

381

FIG pWl cant The

. 8. une ain sm

The caudal end of the rostd cholinergic column coiacides with the caudal end of the globus pa&&. &riatai cells are found it n (p), and some cells sre found in and around the central nucleus of the amygdala (ac) which together with the basolateral nucleus s a dense terminal field. Other terminal fields are present in the intralaminac central medial (cm) and pamcentral (pc) thalamic nu all ChAT-positive cells of the medial habenula (hm) can also be seen.

t the tab)

dei.

VINCEN I AND RElt%EK

FIG. 9. and the are sho icm am

Iis set ofmicrographs illustrates the ChAT-positive neurons present in the nucleus accumbens (na). the medial island ot’Calle :dial septal nucleus (ms). (a) is a cresyl violet section showing the granule cells of the medial island. The ChAT-immunoreactl in (b). (c.d,e) are higher power micrographs taken from (b) to illustrate the relative size\ of the Ch.Al‘-positl\c wnlons in

IS. respectively. Scale bars indicate 500 pm for (a.b) and SO pm for (c.d,e).

:ja (icm) ive cells the na,

ChATINTHECAT 383

FIG. II. These mi~rographs illustrate the relative sizes of the what-immunoreact~v~ neurons present in the caudate nucleus (a) and the putamen (b), in the nucleus of the diagonal band of Broca (c) and within the internal capsule (d). An example of the larger cells surrounding the central nucleus of the amygdala is shown (e) as are some of fhe small cells found within this nucleus (0. Scale bar indicatcs 50 pm for all figures.

ChAT IN THE CAT 385

b

FIG. 12. Many small, weakly ChAT-positive cells are scattered in the lateral hypothalamus (arrows) above some of the magnoceilular basal forebrain ChAT-positive cells which lie dorsal to the supraoptic nucleus (a). The relative sizes and staining patterns of the two hypothalamic ChAT cell populations are illustrated in (b). Some of the magnocelhilar rostra1 column cells are found embedded in the fibers of the stria terminalis (c) and positive cells are found within the bed nucleus of the stria terminalis (d). Scale bars indicate 200 +rn in (a) and 50 pm for all other figures.

3Xh

Beneath the anterior commissure in the anterior and lat- eral hypothalamic areas, two types of ChAT-positive cells were noted (Fig. 12a,b). The magnocellular cells of the lat- eral hypothalamus were continuous with those in the adja- cent horizontal limb of the diagonal band and substantia in- nominata. These were scattered in a dorsolateral direction in the lateral hy~tha~amus medial to the globus pallidus. A cluster of these magnocellu~~ ChAT-positive neurons was found just overlying the supraoptic nucleus (Figs. 6, 12a).

In addition a population of much smaller (15 pm diame- ter), less intensely stained ChAT-immunoreac~ive cells was present, which extended into the bed nuclei of the stria ter- minalis, anterior commissure and stria medullaris. This type of cell was most common medial to the internal capsule in the lateral hypothalamic area. Some similar cells also ex- tended medially into the anterior hypothalamic area. This cell group continued caudally, rising over the fornix to reach the dorsal aspect of the third ventricle. A few similar cells could also be found ventral and medial to the fornix in the anterior hypothalamic area, adjacent to the per~ventricular and arcuate nuclei. These small cells continued caudatly in the lateral hypo~h~amus to the level of the zona incerta.

No ChAT-immunorea~t~ve cell bodies were observed in the thalamus. However, a well organized pattern of terminal fields was observed. The densest terminal fields were asso- ciated with the rostra1 intralaminar nuclei, in particular the central medial and paracentral nuclei (Fig. 8). The anteroventral, reticular and dorsal lateral geniculate nuclei also contained noteworthy terminal fields.

Within the epithalamus, a moderately dense terminal field was present in the lateral habenula. In the medial habenula, a dense cluster of small ChAT-positive cells was found in the ventral half of this nucleus (Fig. 8). These were so densely clustered together that cell processes could not be distin- guished. in addition, scattered cells with clearly stained processes were observed in the medial portion of the lateral habenula (Fig. 13a,b). These extended caudally among the fibers of the habenlar commissure and descended down the fasciculus retroflexus to the level of the parafascicular nucleus.

The fibers of the fasciculus retroflexus were ChAT- positive. These ended in the interpeduncular nucleus in a dis- tinct pattern. Rostrally, the cholinergic fibers began as two compact pear-shaped bodies (Fig. 13~). These expanded caudally to fill the central interpeduncular nucleus with a unique pattern of horizontally organized fibers. This pattern extended caudally into the inner division of the posterior nucleus (Fig. 13d,e). The apical nucleus and the outer di- vision of the posterior nucleus were only poorly innervated.

The oculomotor complex began rostrally just above the supramamillary decussation. Initially only a few scattered cells were seen, but these came together caudally in a com-

FACING PAGE

pact cluster lying between the bundles of the fascicuius retro.- flexus, beneath the rostra1 pole of the aqueduct. These cells were smaller and less intensely stained than those found further caudally. These cells increased in number and moved dorsally between the medial longitudinal fasciculi at the level of the interfascicular nucleus. Scattered, larger and more in.- tensely stained cells were also found at this level, scattered among the moderately stained cells. At the level of the exit of the third nerve, the darker stained cells formed two distinct clusters lateral to the moderately immunos~~~ined cells which Iay dorsally in the midline. The moderately immunoreac~~ve cells decreased in number further caudally, while the larger. intensely labelled cells increased and formed a single large cluster lying dorsal to the medial longitudinal fasciculus f Fig. 14). Some of these cells were also found scattered within this fiber bundle ventrolateral to the main cell group. The cells of the oculomotor nucleus extended long dcndritic processes laterally into the central gray and among the fibers of the medial longitudinal fasciculus. In addition, the axons of the third nerve showed strong immunoreactivity (Fig. 14).

At its most caudal level. the cells of the ocuiomotor com- plex above and within the medial Iongitudinal fascicuius ap- peared continuous with those of the trochlear nucleus. The cells of the trochlear nucleus formed bilateral clusters above the dorsomedial surface of the fasciculus (Fig. 18). There ap- peared to be two cell populations, small intense cells capping the dorsomedial surface of the nucleus, with larger cells ven- traily and extending within the medial longitudinal fasciculus. The trochlear nucleus ended with the exit of the stained axons of the fourth nerve dorsolaterally (Fig. 22).

The superior colliculus displayed a striking pattern of ChA’I immunoreactivity (Fig. IS). A fiber network was present in lamina one of the superficial gray layer. In addition. small in- tense patches of ChAT-positive fibers were observed in the in- termediate gray layer (Fig. 17). Weakly stained ChAT-positive cell bodies were also observed in the superior collicuiu\. They were scattered sparsely in the superficial gray layers. predomi- nantly in laminae two and three. These cells were small (1.X0 pm diameter) and round, and had poorly stained processes (Fig. tSb). Often only a single process oriented dorsally was apparent. although some cells had horizontally oriented proc- esses as well.

Another group of ChAT-positive cells was detected in the parabigeminal nucleus. Essentially all of the densely packed. medium-sized (25 pm diameter) cells of this nucleus were immunoreactive (Figs. 16-l-18). The ChAT-positive neurons were mostly multipolar and fusiform in shape, and a dense network of immunoreactive processes was also present throughout the parabigeminal nucleus.

The major ChAT-positive celt group of the midbrain and pontine tegmentum comprised the caudal choIinergic col- umn. This extended from the mesencephalic reticular for- mation caudally into the pontine tegmentum (Figs. 17-25). The caudal cholinergic column was first detected at the level of the caudal oculomotor nucleus, where occasional ChAT-

FIG. 13. ChAT-immunoreactive elements in the habenulo-interpeduncular system. A dense cluster of small immunoreactive cells is present in the ventral medial habenula (a), while a few small multipolar cells are found in the lateral habenula (arrows). These two cell types are illustrated at higher magnification in (b). At the rostra1 pole of the interpeduncular nucleus, the fasciculus retroflexus fi- bers form two pear-shaped bodies (c). These fibers continue caudally and arrange in a unique pattern within the inner division of the posterior nucleus (ipi) (d), but avoid the outer (ipo) and paramedic (ipp) divisions of the posterior intc~eduncu~ar nucleus. fe) is a cresyi violet section adjacent to (d) for o~entation. Scale bars indicate 200 pm for (a), SO Nrn for (b), 100 pm for (c) and 200 pm for (d.e).

ChAT IN THE CAT 387

FIG. 14. The CbAT-positive motoneurons of the oculomotor complex, and the positive axons in the exit of the third nerve are iilustrated (a). The inset (b) shows some of the large multipolar cells extending their processes out into the medial longitudinal fasciculus. Scafe bars indicate 500 pm in (a) and 50 pm in (bf.

ChAT IN THE CAT

FIG. IS. ChAT-immummzactive elements in the superior collicuks include positive cell bodies in the superficial gray layers (arrows) and dense terminal patches in the intermediate gray layer. The cells and patches are shown at h@her magnification in (b) and (c) respectively. Scale bars indicate 200 pm in (a) and 50 pm in (b,c).

FI( S. 16. lev ,el of 1 to 1 this le for (b).

A cluster of ChAT-positive cells is present in the parabigeminal nucleus (pbg) which lies dorsolateral to the suhstantia mgra (sn the trochlear nucleus (4) and the interpeduncular nucleus (ip). The rostra1 tip of the caudal cholinergic column (arrows) also reac :vel. (b) is a higher magnification of some of the parabigeminal ChAT-positive neurons. Scale bars indicate 500 urn for (a) and

t the

sup Mm

ChAT IN THE CAT 391

FIGS. 17-40. Projection prints itlustrating the distribution of ChAT-immunoreactive neurons in the caudal midbrain, pons and medulla of the cat.

FIG. 17. The caudal cholinergic column (small arrowhead) begins the central tegmental field of the midbrain just dorsal to the caudal lateral substantia nigra (sn) and just medial to the small ChAT-immunoreactive cells of the parabigeminal nucleus (pbg). This section is at the level of the oculomotor nucleus (3). Terminal folds are present in the interpeduncular nucleus tip) and in patches in the intermediate gray layers of the superior colliculus (large arrowheads).

FIG. 18. At the level of the trochlear nucleus (4) and dorsal raphe (dr) the cells of the caudal eholinergic column extend medially into the central tegmentat field. Cells are also found in the rostra1 pole of the laterodorsal tegmental nucleus (arrowhead) in the periaqueductal gray. The small ChAT-positive cells of the parabigeminal nucleus (pbg) continue caudally to this level.

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in t

he f

loor

of

the

fou

rth

vent

ricl

e.

FIG

. 21

. T

he l

ater

odor

sal

tegm

enta

l nu

cleu

s (t

ld)

cont

ains

nu

mer

ous

ChA

T-p

ositi

ve

cells

at

this

lev

el.

Neu

rons

ex

tend

ve

ntro

late

rally

ou

t of

the

cen

tral

gr

ay

bene

ath

and

with

in

the

brac

hium

co

njun

ctiv

um.

FIG

. 22

. T

he

ChA

T-p

ositi

ve

cells

ar

e fo

und

in t

he

late

rodo

rsal

te

gmen

tal

nucl

eus

betw

een

the

dors

al

tegm

enta

l nu

cleu

s (C

iudd

en)

(td)

an

d th

e lo

cus

ceru

leus

(1

~). A

few

cel

ls e

xten

d ou

t of

the

cen

tral

gr

ay

bene

ath

the

brac

hium

’con

junc

tivum

. So

me

sim

ilar

cells

ar

e fo

und

dors

olat

eral

to

the

bra

chiu

m

conj

uact

~vum

, w

edge

d be

twee

n th

e do

rsal

nu

cleu

s of

the

la

tera

l le

mni

scus

an

d th

e ca

udal

po

le

of t

he

infe

rior

co

ilicu

lus

~a~o

whe

ad).

FIG

. 23

. T

he

leve

l of

the

ca

udal

en

d of

the

ca

udal

ch

olin

ergi

c co

lum

n,

in t

he

late

rodo

rsal

te

gmen

tal

nucl

eus

(tld

).

At

this

le

vel

a se

para

te

and

dist

inct

gr

oup

of

smal

l, w

eakl

y C

hAT

-pos

itive

ce

lls

is f

ound

in

the

pa

rabr

achi

al

nucl

eus,

es

peci

ally

in

its

do

rsol

ater

al

aspe

ct

(arr

owhe

ad).

FIG

. 24

. T

he

para

brac

hial

ce

ll gr

oup

is q

uite

ex

tens

ive

at

this

le

vel

surr

ound

ing

the

brac

hium

co

njun

ctiv

um

vent

rahy

an

d la

tera

lly

(arr

owhe

ads)

. T

hese

sm

all

cells

ar

e qu

ite

dist

inct

fr

om

the

rem

aini

ng

cells

of

th

e ca

udal

ch

olin

ergi

c co

lum

n in

the

la

tero

dors

al

tegm

enta

l nu

cleu

s.

At

this

le

vel

a fe

w

of

the

oeri

oliv

arv

cells

(a

rrow

s)

arc

foun

d la

tera

l to

th

e tr

auez

oid

body

(T

B)

at

the

rost

ra1

pole

of

th

e su

peri

or

oliv

arv

com

tdex

.

25.

TIt

e ro

stra

1 p

ole

of

the

mo

for

nu

cleu

s o

f th

e tr

igem

inal

n

erve

(51

l~?

s at

the

tau

dal

ex

trem

e o

f th

e ca

ud

at c

no

tmer

lpc

coit

imn

, w

tuch

en

ds

m t

he

late

rod

ors

al t

egm

enta

l n

ucl

eus(

tld

) in

th

e fl

oo

r o

f th

e fo

urt

h v

entr

icle

. S

om

L o

f the

smal

ler

and

fai

nte

r C

hA

T-p

osi

tive

ce

lls o

f th

e p

arab

rach

ial

nu

cleu

s (p

b)

can

als

o b

e se

en a

t th

is l

evel

, b

enea

th t

he

bra

chiu

m

con

jun

ctiv

um

(B

C)

FIG

. 26

. T

he

mai

n b

od

y o

f th

e m

oto

r n

ucl

eus

of

the

trig

emin

al

ner

ve

!i!

,:an

h

e se

en

at

this

le

vel.

Als

o

pre

sen

t ar

e so

me

of

the

per

ioliv

ary

neu

ron

s (a

rro

wh

ead

s)

surr

ou

nd

ing

th

e la

tera

l su

per

ior

oliv

ary

nu

cleu

s (I

so).

FIG

. 27

. Sm

all

cells

(ar

row

) ca

n be

see

n ex

tend

ing

betw

een

the

retr

otri

gem

inal

nu

cleu

s (r

5) n

ext

to t

he s

tain

ed

fibe

rs

of t

he d

esce

ndin

g se

vent

h ne

rve

(7n)

an

d th

e ro

stra

1 en

d of

the

abd

ucen

s nu

cleu

s (6

). M

any

peri

oliv

ary

cells

(ar

row

head

s)

are

pres

ent

at t

his

leve

l be

twee

n th

e ex

its o

f th

e si

xth

(6n)

and

sev

enth

ne

rves

.

FIG

. 28

. T

he m

ain

body

of

the

abd

ucen

s nu

cleu

s (6

) lie

s be

neat

h th

e ge

rm o

f th

e se

vent

h ne

rve

(7g)

. Sm

all

peri

oliv

ary

cells

are

pre

sent

w

ithin

th

e hi

lus

(h)

of t

he l

ater

al

supe

rior

ol

ivar

y nu

cleu

s.

Oth

er

ChA

T-p

ositi

ve

cells

ar

e pr

esen

t in

the

m

agno

cellu

lar

tegm

enta

l fi

eld

(arr

ows)

.

FIG

. 29

. A

few

sm

all

ChA

T-p

&tiv

e ce

lls

(arr

ows)

ex

tend

fr

om

the

caud

al

pofe

of

the

a&ke

ns

nucl

eus

(6)

vent

ro@

erai

fy

tow

ard

the

acce

ssor

y fa

cial

nu

cfeu

s (7

a) a

nd

the

clus

ter

of s

mat

l. ce

lls

lyin

g la

tera

l to

it

(arr

owhe

ads)

. Jo

rsal

to

the

ca

udal

en

d of

the

la

tera

l su

peri

or

&va

ry

nucl

eus

{Iso

).

FIG

. 30

. Sc

atte

red

cells

(a

rrow

s)

are

foun

d in

the

mag

noce

llula

r te

gmen

tal

kid

(ftm

) m

edia

l to

the

fac

ial

nucl

eus

(7).

Ano

ther

gr

oup

of c

ells

(,a

rrow

head

s)

is f

ound

jus

t la

tera

l to

the

ac

cess

ory

faci

al

nucl

eus

(7a)

.

31.T

~~

f8ec

rtw

lalp

obaf

tBo~

~(f

7)1s

sraa

llC

hAT

-pos

itiv

eCk

UsC

OB

fiW

G

A&

her

cIus

fer

of s

imil

ar c

elts

tla

rge

amw

~

is &

md

dozm

me&

aB

lnik

wto

~~

nucl

eusi

nthe

’

y in

the

nuc

kus

pr

epos

itus

byp

agbs

si.

mm

eew

wte

gmnt

aipE

eM:f

ftm

).

FIG

. 32

. T

he r

etro

faci

al n

ucle

us (

7r)

aris

es d

orso

late

ral

to t

he c

auda

l po

le o

f th

e fa

cial

nuc

leus

. S

mal

l C

hAT

-pos

itiv

e ce

lls

(sm

all

arro

whe

ads)

ext

end

dors

al t

o th

e re

trof

acia

l nuc

leus

to

the

rost

ral p

ole

of t

he d

orsa

l m

otor

nuc

leus

of

the

vagu

s (d

mx)

. A

clu

ster

of

sim

ilar

cel

ls (

larg

e ar

row

head

s) c

odth

ues

in

the

nucl

eus

prep

osit

us h

ypog

loss

i, a

nd t

he a

djac

ent

med

ial

long

itud

inal

fas

icul

us (

ML

F),

whi

le s

catt

ered

lar

ger

cell

s (a

rrow

s) a

re f

ound

Ven

tral

to t

his

in

the

mag

noce

llul

ar t

egm

enta

l fi

eld

abov

e th

e in

feri

or o

live

(io

).

FIG

. 33

, T

he

nuct

ews

ambi

guus

(a

) ap

pear

s as

a

caud

al

exte

nsto

n ot

th

e rC

:rot

acla

l nu

cleu

s,

an0

scat

tere

d sm

all

cells

(a

rrow

head

s)

cont

tnue

to

ex

tend

do

rsal

ly

from

it

to

the

dors

al

mot

or

nucl

eus

of

the

vagu

s (d

mx)

. 5 5;

FIG

. 34

. T

he

hypo

glos

sal

nucl

eus

(12)

mak

es

its

appe

aran

ce

med

ial

to t

he

dors

al

mot

or

nucl

eus

of

the

vagu

s (d

mx)

in

the

fl

oor

of t

he

four

th

vent

ricl

e.

A

few

sc

atte

red

cells

(a

rrow

head

s)

are

foun

d in

the

in

feri

or

vest

ibul

ar

nucl

eu\

at

this

le

vel.

FIG

. 35

. Sc

atte

red

smal

l ce

lls

(arr

owhe

ads)

co

ntin

ue

to

exte

nd

betw

een

the

dors

al

mot

or

nucl

eus

of

the

vagu

s (d

mx)

an

d th

e nu

cleu

s am

bigu

us

(a).

T

he

hypo

glos

sal

nucl

eus

(12)

is

qui

te

exte

nsiv

e at

th

is

leve

l.

FIG

. 36

. A

few

sc

atte

red

cells

(a

rrow

head

s)

lie d

orsa

l to

the

nu

cleu

s am

bigu

us

(a)

tow

ards

th

e do

rsal

m

otor

nu

cleu

s of

the

va

gus

(dm

x)

whi

ch

has

mov

ed

med

ially

to

lie

do

rsal

to

th

e hy

pogl

ossa

l nu

cleu

s (1

2)

at

the

leve

l of

th

e ar

ea

post

rem

a (a

p).

Oth

er

scat

tere

d ce

lls

(arr

ow)

lie a

long

th

e ex

it of

th

e tw

elft

h ne

rve.

immunoreactive neurons were seen in the ventrolateral mesencephalic reticular formation, just dorsal to the pars lateralis of the substantia nigra (Figs. 17, 18). At the level of the trochlear nucleus and nerve, ChAT-positive neurons were seen extending in a band across the tegmentum, within the pedunculopontine nucleus in the central tegmental field of Berman [6] (Figs. 18, 19). Still further caudal, the location of this column of ChAT-positive neurons shifted dorsome- dially; scattered ChAT-immunoreactive perikarya were seen embedded within the fiber bundles of the brachium con- junctivum which bisected the pedunculopontine nucleus at this level (Fig. 20). At the level of the dorsal and ventral tegmental nuclei of Gudden, most ChAT-positive neurons were located medial to the brachium conjunctivum within the laterodorsal tegmental nucleus and the subjacent pedun- culopontine nucleus (Fig. 21).

Further caudal, as the pedunculopontine nucleus merged with the medial parabrachial nucleus, neurons staining deeply for ChAT were seen within the laterodorsal tegmental nucleus and extended out into the medial parabrachial nu- cleus (Fig. 22). Additionally, at this level, a sheet of large spindle-shaped, horizontally oriented ChAT-positive neurons was seen capping the dorsal nucleus of the lateral lemniscus, near the lateral edge of the brachium of the in- ferior colliculus (Fig. 22). Further caudal, large ChAT- positive neurons were restricted to the laterodorsal tegmen- tal nucleus in the periventricular gray (Figs. 23, 24). At the most caudal level of this column, occasional ChAT-immuno- reactive neurons were seen in the lateral periventricular gray, apparently within the medial vestibular nucleus.

A group of small (12-15 pm diameter) spindle-shaped neurons which stained lightly for ChAT was seen within both the medial and lateral parabrachial nuclei (Figs. 21-25). In the ventral portion of the lateral parabrachial nucleus. these cells formed a distinct cluster (Fig. 24). These neurons could be distinguished clearly from the neurons of the caudal cholinergic column by both size and staining intensity (Fig. 41), even at rostra1 levels where the two cell populations intermingled (Fig. 21, 22).

Pans and Medulla

The motor nucleus of the trigeminal nerve began rostrally at the caudal pole of the laterodorsal tegmental nucleus, be- neath the small ChAT-positive cells in the parabrachial nu- clei (Fig. 25). These motoneurons formed a dense ball of large multipolar cells similar to those of the oculomotor nu- cleus. In addition, a cluster of much smaller, but strongly staining cells was found at the ventrolateral pole of the trigeminal motor nucleus beneath the exit of the fifth nerve, the axons of which were stained.

At the caudal pole of the nucleus, another group of small (25-25 pm diameter) ChAT-positive cells extended up from the dorsomedial aspect beneath the descending limb of the seventh nerve. A few such cells were also found along the dorsal surface of the seventh nerve. These continued caud- ally and formed a cluster beneath the descending limb of the seventh nerve caudal to the motor nucleus of the trigeminal. At slightly more caudal levels, these small cells seemed to form bridges between the larger motoneurons of the abdu- tens nucleus lying just beneath the genu of the seventh nerve and the retrotrigeminal nucleus embedded in the descending limb of the seventh nerve laterally and the accessory facial nucleus lying among the ascending fibers of the seventh nerve medially (Figs. 27-29).

As the caudal end of the abducens nucleus, other small

ChAT-positive cells were found scattered ventrally rn the lateral tegmental field between the descending sixth and seventh nerves, dorsal to the superior olivary complex and lateral to the accessory facial nucleus (Figs. 28, 421.

A separate group of ChAT-positive cells was found within the superior olivary complex. These first appeared rostrally as multipolar cells (20-25 pm diameter) in the preolivary nucleus beneath the lateral superior olivary nucleus (Fig. 26). Further caudal, these neurons spread out medially be- neath both the medial and lateral superior olivary nuclei (Fig. 27). Smaller (10 pm diameter). mostly bipolar cells were also found sweeping over the dorsolateral surface of the lateral superior olive and into the hilus (Figs. 28, 43). Scattered periolivary ChAT-positive cells continued to surround the principal olivary nuclei, some being present medial to the nucleus of the trapezoid body, and some just dorsal to the fibers of the trapezoid body beneath the rostra1 pole of the facial nucleus.

At the level of the facial nucleus proper, scattered medium-size cells continued to extend dorsally from the nu- cleus almost to the genu (Fig. 30). A few large, weakly- stained, multipolar cells appeared at this level in the mag- nocellular tegmental field (Figs. 30, 42~). Near the caudal pole of the facial nucleus the retrofacial nucleus made its appearance dorsally, just medial to the ascending limb of the seventh nerve (Fig. 32). Lateral to the retrofacial nucleus. the scattered medium-sized cells continued in the lateral tegmental field, while a few larger multipolar cells were scat- tered in the magnocellular tegmental field medially.

The retrofacial nucleus extended caudally and appeared to merge with the nucleus ambiguus dorsolaterally at the level of the inferior olivary complex and the exit of the twelfth nerve (Fig. 33). The nucleus ambiguus was made up of large, well stained cells similar to those in the facial and retrofacial nuclei. It appeared quite compact in coronal sec- tions, but had a considerable rostro-caudal extent. Medium-sized, weakly staining cells lay dorsal to the nucleus ambiguus, and extended up towards the dorsal motor nucleus of the vagus, which first appeared as a small dense ball of cells lying just ventromedial to the nucleus of the solitary tract (Figs. 32, 33).

Another group of ChAT-positive cells was found scat- tered in the paramedian reticular nucleus in the dorsal por- tion of the medial longitudinal fasciculus and the adjacent prepositus hypoglossal nucleus. This appeared to corre- spond to the accessory hypoglossal nucleus (Fig. 32). Some similar cells extended ventrally and were found just dorsal to the inferior olivary complex and along the exit of the twelfth nerve (Fig. 32).

The dorsal motor nucleus moved dorsomedially further caudal to enter the central gray as a dense ball ventromedial to the nucleus of the solitary tract (Figs. 32-34). A diffuse group of lighter stained cells still bridged it and the nucleus ambiguus (Fig. 33), and scattered cells of this accessory nu- cleus spread into the nucleus of the solitary tract near the tract. Some similar cells were even found dorsal to the nu- cleus of the solitary tract among the fibers of the descending root of the vestibular nerve in the inferior vestibular nucleus (Figs. 34, 44).

The hypoglossal nucleus formed a compact group just dorsolateral to the medial longitudinal fasciculus, although scattered accessory cells were still present ventrally and along the twelfth nerve (Figs. 34-38, 44).

The nucleus ambiguus became quite diffuse caudally, ending at the level of the commissural nucleus of the sohtary

FIG

. 37

. T

he h

ypo@

ossa

l nu

cleu

s (1

2),

dors

al

mot

or

nucl

eus

of t

he

vagu

s (d

mx)

an

d th

e nu

cleu

s am

bigu

us

(a)

cont

inue

ca

udal

ly

past

th

e co

mm

issu

ral

nucl

eus

of t

he

solit

ary

trac

t.

FIG

. 38

. T

he c

auda

l ex

tens

ions

of

the

hyp

oglo

ssal

nu

cleu

s (1

2) a

nd t

he d

orsa

l m

otor

nu

cleu

s of

the

vag

us (

dmx)

ex

tend

in

to t

he s

pina

l co

rd,

late

ral

to t

he

spin

al

cana

l (s

c).

FIG

. 39

. M

oton

euro

ns

are

foun

d fi

rst

at t

he m

edia

1 ed

ge o

f th

e ve

ntia

l ho

rn.

the

spin

al

cana

l.

FIG

. 40

. W

ithin

the

spi

nal

cord

, C

hAT

-pos

itive

ne

uron

s ar

e fo

und

with

in

the

vent

ral

horn

(vh

f, ju

st

late

ral

to t

he s

pin4

ca

nal

and

scat

tere

d (a

rrow

head

s)

with

in

the

dors

al

horn

(d

h).

ChAT IN THE CAT 405

FIG. 41. These micrographs illustrate the ChAT-immunoreactive neurons of the caudal cholinergic column in the pedunculopontine nucleus (a), the laterodorsal tegmental nucleus (b) and the outlying cells found dorsal to the dorsal nucleus of the lateral lemniscus Old), beneath the caudal inferior colliculus (c). Cells in the separate cell group in the parabrachial nuclei surrounding the caudal brachium conjunctivum (BC) are shown in (d) at the same magnification for comparison. Scale bar indicates 100 pm for all fgures.

FIG. 42. (a) This micrograph illustrates some of the small ChAT-positive cells associated with the motor nuclei of the caudal pans and medulla. These small cells are scattered just lateral to the abducens nucleus which lies beneath the germ of the facial nerve (7g). ‘The stained axons of the sixth nerve (6n) can also be seen. (b) This is a higher magnification of some of these smaller ChAT-positive cells next to the abducens nucleus. (c) This is an example of one of the large multipolar ChAT-immunoreactive cells found scattered in the magnocellular tegmental field at the level of the facial nucleus (see Fig. 30). Scale bars indicate 100 pm for (a), and 50 pm for fb.c).

FACING PAGE

FIG. 43. The ChAT-immunoreactive neurons (arrows) associated with the superior olivary complex beneath the accessory facial nucleus (7a) are illustrated (a). The large periohvary cells lying lateral to the exit of the sixth nerve (6n) and medial to the medial superior olive (mso) are shown in (b), while the small cells in the hilus of the lateral superior olive (Iso) are shown in (c). Scale bars indicate 250 pm in (a) and 50 pm in (bc).

ChAT IN THE CAT

FIG. 43.

408

FIG. 44. At the level of the hypoglossal nucleus (12) positive cells appear to be scattered out ventroiaterally (large arrows) fi-om the dorsal motor nucleus of the vagus (dmx), beneath the solitary tract (TS). Additional small ChAT-positive cells (small arrows) are found among the fibers of the descending root of the vestibular nerve in the inferior vestibular nucleus. These are illustrated at higher magnification in (a). Scale bars indicate 25 pm for (a) and 100 pm for (b).

FIG

. 45.

The

dis

trib

utio

n of

CbA

T-p

osit

ive

C&

J pre

sent

in th

e lu

mba

r spi

nal c

ord

is ii

luat

rate

d in

(a).

Sm

all,

wea

kly

stai

ned

cells

are

pm

saot

in th

e do

rsal

ho

rn (b

) and

larg

er ce

ils a

re fo

und

adja

cent

to th

e sp

inal

cana

l (c)

, in

addi

tion

to th

e la

rge

mul

tipo

lar c

ells

of t

he w

&al

ho

rn (d

). S

cale

bar

s ind

icat

e 25

0 pm

fo

r (a

) and

50

pm f

ar (b

,c,d

).

Q

410

tract. The dorsal motor nucleus of the vagus was quite small at this level, and lay dorsal to the hypoglossal nucleus (Fig. 37). Also, the accessory cells that were present dorsally in the nucleus of the solitary tract and the inferior vestibular nucleus had disappeared at this level.

At more caudal levels, ChAT-positive cells spread out ventrolaterally from the hypoglossal nucleus and appeared to be continuous with ventral horn cells and the cells found around the central canal and in the dorsal horn of the cervical cord (Figs. 28-40).

Spinal Cord

Sections from the lumbar and cervical cord were exam- ined, and the patterns of ChAT staining observed at these levels were similar. In the ventral horn both giant, weakly stained cells, and medium-sized, strongly stained cells were present. Both cell types also appeared to have large ChAT- positive puncta on their surface. Another group of medium- sized, strongly staining cells was present just lateral to the central canal. These, like those in the ventral horn, were often multipolar. A small population of ChAT-positive cells was also observed in the dorsal horn. These were small, weakly stained and mostly bipolar in shape. They were most com- monly observed in laminae V and VI, and were more com- mon in the cervical than in the lumbar cord (Fig. 45).

DISCUSSION

The distribution of ChAT-immunoreactive cells reported here appears to be more extensive than that which has been previously described in rodent [3, 16, 21, 32, 36, 42, 47, 48, 54, 72, 82, 94, 951 and primate (55, 60, 73, 741 brains. The present observations confirm some, but not all of the original findings of Kimura et al. [43] in their study of the feline brain using a rabbit antiserum raised against human ChAT. In ad- dition, many of the ChAT-positive cell groups which were discovered in the present study were not noted in previous reports. In the following discussion, we shall therefore com- pare our observations with those previously reported in the cat using immunohistochemistry and AChE histochemistry. Similarities and differences between what we have observed and what has been noted in other species will also be de- scribed.

The Cortex

No ChAT-immunoreactive neurons were detected in the neocortex or hippocampus of the cat. This is in agreement with the original report of Kimura et al. [43] and contrasts with the situation in the rat, where various groups have re- ported the presence of cortical ChAT-immunoreactive cells [20, 23, 32, 33, 36, 491. To date, similar cells have not been seen in primate brain [55, 73, 741. This cortical population of ChAT-positive neurons may therefore represent a peculiar feature of the rodent brain, and in the cat and primate, all cortical cholinergic innervation may be of subcortical origin.

The Striatum

The ChAT-immunoreactive neurons present in the feline caudate nucleus, putamen and nucleus accumbens appear to correspond to the large aspiny interneurons described in de- tail in various species [3, 9, 16, 26, 32, 36, 42, 43, 45, 54, 55, 67, 68, 761. The distribution of ChAT-immunoreactive neuropil observed in the present study appears similar to that recently described by Graybiel et al. [29].

As noted previously in the cat [43,941. and in agreement with what is observed in other species [?. 16. 21, 32. 36. 42. 46, 48, 53-55, 72-74, 82, 94, 951, the major ChA’I‘- immunoreactive cell group in the telencephalon is the mag- nocellular basal forebrain complex. However, the topog- raphy of the rostra1 cholinergic column appears to be differ- ent in the cat than in the monkey or rodent. In the cat. there appears to be a large dorsal extension of the rostra1 column into the internal capsule. A similar dorsal extension of the nucleus basalis has been noted in the human brain [60,71 J.

In their pharmacohistochemical study of AChE-positive cells in the cat forebrain, Parent and O’Reilly-Fromentin [66j noted the presence of large, very intensely AChE-positive cells lying dorsal to the globus pallidus within the internal capsule (see also Parent c’t rd. [65]). Bear et trl. [5] have found, using combined cresyl violet and AChE staining, that virtually all the neurons within the cat internal capsule are intensely AChE-positive. Kimura rt rrl. [43j tirst noted the presence of large ChAT-positive cells within the feline inter- nal capsule, and suggested that these could either belong to and connect with components of the neostriatum, or more caudally unite with the ChAT-positive cells of the medullary stria of the pallidum or the interstitial cells of the ansa len- ticularis. Our observations would be consistent with this lat- ter suggestion.

The ChAT-immunoreactive cells found capping the is- lands of Calleja appear to correspond to the supra-insular AChE cells noted by Parent and O’Reilly-Fromentin [66] in their pharmacohistochemical study of the cat forebrain. It is clear that these insular ChAT-positive cells are much smaller than the magnocellular cholinergic neurons of the rostra1 column.

Fallon ct al. [24] have described medium and large cells in the islands of Calleja complex of the rat that show strong AChE staining after a DFP challenge. Other groups have also noted AChE- or ChAT-positive cells associated with the islands in the rat [21,72]. In our own studies of AChE stain- ing in DFP pretreated rats, and in rat sections processed together with the present material for ChAT immunohis- tochemistry, we too observed similar cells. These appeared to be much larger than the ChAT-positive cells found in the medial cat islets, and were similar in size to the striatal cholinergic cells. In the primate brain, ChAT-positive cells have been noted around or within the islands, however, they have been included together with the striatal cholinergic in- terneurons [55]. The situation in the primate islets may there- fore be similar to that in the rat.

The small ChAT-immunoreactive cells in the cat islands of Calleja, and the larger cells observed in association with the rat and primate islets may have localized projections onto the granule cells of the islets. The neuropil of the islands stains very strongly for AChE in these species [24,73]. In addition, dense ChAT fiber staining can also be seen cover- ing the granule cells (Fig. IO; see also Mesulam ef rrl. [55]). Thus these cells may be under local cholinergic control.

In the rat, the major source of cholinergic input to the amygdaloid complex appears to arise in the basal telencepha-

ChAT IN THE CAT 411

lon [13, 59, 971. Proiections from the basal forebrain to the amygdala have also been demonstrated in the cat [62,70,88]. Kimura et al. [43] noted high concentrations of ‘cholinmp- tive’ neurons in the magnocelIular portion of the basal nu- cleus of the cat amygdala. This area also stains intensely for AChE in this species [Sal. In the present study, a dense ChAT-immunoreactive terminal field was observed in the magnocellular portion of the basolateral nucleus, and a simi- lar field was seen in the central nucleus, and rostrally within the anterior cortical nucleus and the nucleus of the lateral olfactory tract.

An important observation from the present study was the presence of intrinsic ChAT-immunoreactive neurons within the amygdaloid complex. Some of the magnocellular cholinergic neurons of the rostral column extend from the horizontal limb of the diagonal band and the substantia in- nominata laterally into the anterior amygdaloid area. In ad- dition, smaller cells, similar in size to those in the adjacent putamen, are present in the longitudinal association bundle of Johnston beneath the putamen and above the central and basolateral nuclei. ChATpositive cells are also present in the stria terminalis, medial and ventral to these cell groups. At more caudal levels some cells are found within the lateral subdivision of the central nucleus. Recently, small ChAT immunoreactive cells have also been detected in the basolat- eral nucleus of the rat [12].

Parent and O’Reilly-Fromentin [66] have noted some moderately and a few intensely AChE-positive cells in the dorsal anterior amygdaloid area, within the intercallated masses and along the dorsal aspect of the central nucleus. These appear to correspond to the ChAT-immunoreactive cells we have observed. Recently, Saper and Chelimsky [71] have described magnocellular AChE-positive neurons in the human brain surrounding the dorsal aspects of the central and basal magnocellular amygdaloid nuclei.

The Hypothalamus

There is evidence that choline&c mechanisms may regu- late hypothalamic function. Parent and Butcher [64] noted that the supraoptic and paraventricular nuclei stained for AChE following a DFP challenge, however, this reaction does not appear to be intense [26]. Instead, recent results indicate that the cholinergic input to the supraoptic nucleus arises in the basal forebrain cholinergic neurons of the rostra1 column lying just dorsal to this nucleus in the preoptic area [51,56].

A group of AChE-intense cells have been noted in various studies in the lateral hypothalamus, around the fomix and in the dorsomedial and posterior hypothalamus [21,26,48,64, 72, 731. These cells display a-melanocyte stimulating hor- mone (a-MSH)-like immunoreactivity [44] and have long projections to cortex, hippocampus and spinal cord [30,44]. In the rat [21, 48, 721 and primate [55,731, ChAT im- munoreactivity has not been detected in these AChE-intense neurons. Similarly, Kimura et al. [43] did not report any cholinergic neurons in the cat hypothalamus, although prominent ‘cholinoceptive’ cell clusters were noted in the lateral hypothalamus. This contrasts with the present obser- vation of many weakly ChAT-positive cells in the lateral hypothalamus in this species. It is not clear if these corre- spond to the AChE intense cells previously described in this species [661. Previous biochemical studies have indicated that a population of intrinsic cholinergic neurons may indeed be present in the hypothalamus 114,631.

The Thalamus

In agreement with earlier studies in the cat [43] and other species, no ChAT-immunoreactive cells were detected in the thalamus. Instead, a highly orga&ed pattern of immuno- reactive fibers was evident. The distribution of these termi- nal fields appears similar to that which has been recently noted in the rat brain [83] and in the visual thalamus of the cat 118,841. Much of this thalamic cholinergic innervation may arise from the caudal cholinergic cell column [75,83].

The Habenula-Interpeduncular System

Recent immunohistochemical studies with various monoclonal antibodies to ChAT have demonstrated the presence of immunoreactive habenular neurons in the rat [16, 32, 36, 391 and primate [55]. We have now detected a similar cell cluster in the feline medial habenula. In addition, scattered ChATpositive cells were detected within the lat- eral habenula in this species. These habenular ChAT-posi- tive neurons appear to provide the major cholinergic inner- vation to the interpeduncular nucleus, although additional cholinergic input appears to arise from some of the mag- nocellular neurons of the diagonal band [ 1,981. The staining pattern we have observed within the interpeduncular nucleus appears to be similar to that reported by Kimuh et al. [43] and agrees with the topography of the feline habenulo- interpeduncular projection [31].

The Superior Colliculus and Parabigeminal Nucleus

Dense ChATpositive fiber networks were detected in the superficial and intermediate gray layers of the superior col- liculus. These appear to correspond to the AChE staining patterns observed by Graybiel[27] in this species. The ChAT and AChE in the superficial gray layer may well arise from the parabigeminal nucleus. Graybiel[28] has shown that this cell group projects to this layer. We have found that essen- tially all of the neurons in the cat parabigeminal nucleus display ChAT immunoreactivity. This is consisOent with re- cent reports that the reptilian isthmic nucleus, the homologue of the mammalian parabigeminal nucleus, is ChAT-immunoreactive [lo,171 and that lesions of this nu- cleus decrease tectal ChAT activity [69]. Desan (personal communication cited in [69]) has also found ChAT- immunoreactive cells in the parabigeminal nuckus of the cat, while Mufson et al. [58] have retrogradely lal&ed ChAT- positive cells in this nucleus in the mouse following injec- tions of tracer into the superior colliculus.

The parabigeminal nucleus does not appear to project to the intermediate gray layers of the superior colliculus which contain AChE positive [28] and ChAT-imniunoreactive patches. AChEpositive neurons have been observed follow- ing DFP in this area in the cat [37] and we have observed some ChAT-immunoreactive cells within the superficial layers of the superior colliculus. Intrinsic ChATpositive cells have also been recently noted in the goldfish tectum [89]. The tectal ChAT-immunoreactive cells could thus con- tribute to the fiber patches seen in the cat. A more likely source for the ChAT patches may be the NADPH-dia- phorase containing cholinergic neurons of the caudal column [93]. These cells are well known to have ascending projec- tions to the tectum and thalamus 175, 81, 831 and we have observed patches showing neuropil staining for NADPH- diaphorase within the intermediate gray layers of the cat superior colliculus (in preparation).

The Oculomotor Nucleus A few small ChAT-immunoreactive neurons were found

The oculomotor complex extended rostrally above the mamillary bodies. These anterior cells were medium size and weakly stained. They appear to correspond to ChAT- positive preganglionic parasympathetic cells recently de- scribed in the cat by Brezina et al. [ 111, and included in the Edinger-Westphal nucleus by Taber [87].

in the inferior vestibular nucleus. These may be similar to the small cells extending out from the dorsal motor nucleus ol the vagus into the nucleus of the solitary tract and adjacent

regions. This contrasts with the report of Kimma r~f ol. 1431 who noted large or giant multipolar ChATpositive cells in Deiters’ nucleus. Such cells have not been observed in other species or by other groups.

The Red Nucleus

The magnocellular neurons of the feline red nucleus were found to be ChAT-immunoreactive in the original study by Kimura et al. [431. However, such cells did not display ChAT immunoreactivity in the present study. ChAT activity is quite low in the cat red nucleus [61]. Immunohistochemi- cal studies in rodent [3, 14, 32, 54, 721 and primate [ST, 73, 741 have also not noted ChAT-immunoreactive cells in the red nucleus. Thus the neurons of the red nucleus do not appear to be cholinergic.

The Caudal Column

A column of ChAT-immunoreactive neurons has been re- ported within the pedunculopontine and laterodorsal teg- mental nuclei in the rat 13, 16,21, 54,72,93], cat 141,431 and primate [55, 73, 741 and has been termed the caudal cholinergic column [72, 73, 93, 941. This column is slightly more dispersed in the cat than in other species, particularly within the mesencephalic reticular formation. In both the rat [3, 16, 21, 54, 721 and primate [SS, 72, 741, the caudal choiinergic column forms a discrete and compact cell cluster within the pars compacta of the pedunculopontine nucleus. In the cat, these cells do not coalesce to form a distinct cluster, but rather are widely dispersed throughout both the pars compacta and pars dissipatus of the pedunculopontine nucleus of Taber [87].

A band of ChAT-immunoreactive neurons was observed capping the dorsal nucleus of the lateral lemniscus. The cells here were similar in both size and staining pattern to those of the laterodorsal tegmental and pedunculopontine nuclei. Neurons identical in distribution and morphology were seen in sections stained for NADPHdiaphorase activity, which in the cat as in the rat and primate, selectively stains the caudal cholinergic column [93] (Reiner and Vincent, in preparation). The similarities between these ChATpositive neurons and those of the caudai cholinergic column in terms of size, im- munostaining intensity, and NADPH-diaphorase activity suggest that these neurons may represent a displaced com- ponent of the caudal column.

The Purabrachial Nuclei

At the caudal end of the brachium conjunctivum, small, lightly stained ChATpositive neurons were observed both medial and lateral to the brachium. These neurons were eas- ily distinguished from the ChAT-positive neurons of the caudal cholinergic column by both their size and staining intensity. Furthermore, in contrast to the strongly stained caudal cholinergic column, these parabrachial cells did not display NADPII-diaphorase activity (Reiner and Vincent, in preparation). Thus these ChAT-immunoreactive neurons may represent a group of neurons distinct from those of the caudal cholinergic column. Kimura et al. [43] described ChAT-positive and ‘cholinoceptive’ neurons surrounding the pontine portion of the brachium conjuctivum. No homolo- gous group of ChAT-immunoreactive neurons has been de- scribed in either the rodent or primate pontine tegmentum.

The Superior Oli~wy Complc.\

In their original study of the cat brain using rabbit antiserum raised against human ChAT, Kimura et (11. 1431 reported that the majority of cells in the lateral superior olive were immunoreactive, and additional cells were present in the medial nucleus, the preolivary nucleus and the nucleus of the trapezoid body. We could not confirm these observa- tions. Instead, ChAT-immunoreactive neurons were only observed within the preolivary nucleus and the periolivary portions of the superior olivary complex.

The ChAT-immunoreactive neurons found in the superior olivary complex appear similar to those recently described by Altschuler et al. [2] in the guinea pig. Similar cells have been described in the cat with AChE histochemistry, and they appear to give rise to both the medial and lateral olivocochlear efferents [96]. In the medial component in both of these species, enkephalin immunoreactivity appears to coexist with ChAT [2,25].

Motoneurons and Accessory Nuclei oj’the Pon.v (md Mrdullrr

Recent retrograde tracing studies have localized the neurons forming the superior salivatory nucleus in the cat [77]. These cells, which give rise to the parasympathetic li- bers to the submandibular and sublingual glands, are found in the dorsal portion of the rostra1 medullary reticular forma- tion 1771. These appear to correspond to some of the ChAT- immunoreactive neurons observed in the present study dor- solateral to the accessory facial nucleus at the caudal end ot the superior olivary complex, beneath the genu of the facial nerve. These multipolar neurons were smaller than those within the accessory facial nucleus and the facial nucleus itself. Some of the ChAT-positive cells in this region may also innervate the stapedius muscle in the cat [50,79].

The rostra1 pole of this cell group appears to be continu- ous with similar small ChAT-positive cells lying along the ascending and descending branches of the facial nerve, which may represent the source of innervation to the posterior belly of the digastric muscle [52,86]. These cells in turn appear to merge rostrally with a similar group of small ChAT-immunoreactive neurons found in a cluster ventral to the trigeminal motor nucleus, probably representing the motoneuron pool innervating the tensor tympani muscle [40, 79, 861. Some of the ChAT-positive cells in this region also appear to innervate the anterior belly of the digastric muscle [57]. Thus in the cat [35, 52, 57, 861, as in the rat [SS], the accessory nuclei of the trigeminal, abducens and facial nerves appear to form a continuous column of cholinergic cells.

Kimura et al. [43] reported large-to-giant ChAT- containing cells in the tegmental fields of the medullary re- ticular formation. In agreement with the present observa- tions, these cells were seen most often dorsal to the inferior olivary complex and medial to the lateral tegmental field. However, Kimura et u/. [43] also described cholinergic- cholinoceptive cells in the lateral reticular nucleus. In the

ChAT IN THE CAT

present study, no CUT-positive cells were detected within this nucleus.

Above the inferior olivary complex and the lateral reticu- lar nucleus, the retrofacial nucleus appears to form a bridge between the caudal end of the facial nucleus and the nucleus ambiguus. This agrees with recent retrograde tracing studies PO,911 and with the suggestion of Taber [87], that the retro- facial nucleus of the cat is a ventral continuation of the ros- tral nucleus ambiguus.

The small, multipolar ChAT-positive cells observed dor- sal to the nucleus ambiius beneath the dorsal motor nu- cleus of the vagus appear to correspond to the inferior salivatory nucleus. Neurons in this area have been shown to provide parasympathetic input to the parotid gland in the cat 1781.

The nucleus ambiius extends caudally from the inferior olivary complex to the pyramidal decussation. At this level, the ChAT-positive cells spread laterally forming the nucleus retroambiguus, which extends into the upper cervical cord [38]. The dorsal motor nucleus of the vagus also extends to the upper cervical cord, where it appears to be continuous with the nucleus dorsomedialis and the spinal nucieus of the accessory nerve 1381.

The Spinal Cord

The ChAT-positive neurons of the rat spinal cord have been elegantly described by Barber et al. [4). Our observa- tions in the cat are in good agreement with this report. Thus cells were seen not only in the ventral horn, but sounding the central canal and in the dorsal horn as well. In our mate- rial, the cells about the central canal could not be subdivided

413

into partition cells and central canal cluster neurons as was done in the rat [4]. Very few C&AT-positive cells were found adjacent to the ependymal layer, and thus in the cat, perhaps only partition cells are present. The OAT-positive neurons and dendritic plexus observed in the rat in lamina III 141 were not detected in the cat. Instead, only a few ChAT- immunoreactive neurons were found in the dorsal horn, pre- dominantly in laminae V-VI.

SUMMARY

This study has provided a detailed examination of ChAT- immunoreactive neurons in the feline brain. The use of a specific monoclonal antrbody in a sensitive im- munopernxidase procedure has revealed numerous ChAT- positive cell groups. Besides the well characterized cholinergic groups in the striatum, rostral and caudal col- umns and the motoneurons, other less intensely stained cell groups have been detected. These include ChAT-positive cells in the habenula, hypothalamus, parabiiminal nucleus, superior colliculus and the parabrachial nucleus. It remains an important future challenge to confirm using various exper- imental paradigms, whether all of the ~AT-~~~~~g cell groups identified immunohist~hemic~y are indeed cholinergic.

ACKNOWLEDGEMENTS

The authors thank Dr. Felix Eckenstein for generously providing the monoclonal antibody to ChAT which made this study possible. This work was supported by grants from the Medical Research Council of Canada and the British Columbia Health Care Research Foundation. S.R.V. is a Scholar and P.B.R. a Fellow ofthe M.R.C.

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