Cholinergic systems in the rat brain: IV. descending projections of the pontomesencephalic tegmentum

Brain Research Buiie#i~, Vol. 23, pp. 519-540. 0 Pergamon Press plc, 1989. Printed in the U.S.A.

0361-9230189 $3.00 + .OO

Cholinergic Systems in the Rat Brain: IV. Descending Projections of the Pontomesencephalic Tegmentum NANCY J. WOOLF’

AND LARRY L. BUTCHER

Laboratory of Chemical Neuroanatomy, Department of Psychology University of California, Los Angeles, CA 90024-1653 Received

31 August

1989

WOOLF, N. J. AND L. L. BUTCHER. Chotinergic systems in the rat brain: IV. Descending projections of the ponromesencephaiic regmenrum. BRAIN RES BULL 23(6) 519-540, 1989.-Descending projections from cholinergic neurons in the ~dunculo~ntine and laterodorsal tegmental nuclei, collectively referred to as the ~ntomesencephalote~ment~ (PMT) cholinergic complex, were studied by use of the fluorescent retrograde tracers fluorogold, true blue, or Evans Blue in combination with choline ace~l~~ferase (ChAT) immunohistochemist~ or acetylcbolinesterase (AChE) ph~acohistoche~s~. Redunculo~ntine somata positive for ChAT or staining intensely for AChE were retrogradely labeled with fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12. ChAT-positive cells in both the pedunculopontine and laterodorsal tegmental nuclei demonstrated projections to the vestibular nuclei, the spinal nucleus of the 5th cranial nerve, deep cerebellar nuclei, pontine nuclei, locus ceruleus, raphe magnus nucleus, dorsal raphe nucleus, median raphe nucleus, the medullary reticular nuclei, and the oral and caudal pontine reticular nuclei. Fluorescent tracers used in combination with AChE pharmacohistochemistIy corroborated these projections and, in addition, provided evidence for cholinergic pontomesencephalic projections to the lateral reticular nucleus and inferior olive. The majority of retrogradely labeled neurons demonstrating ChAT-like immunoreactivity were found ipsilateral to the injection site, but, in all cases, tracer-containing cholinergic cells contralateral to the infused side of the brain were detected also. More retrogradely labeled cells containing ChAT were observed in the pedunculopontine tegmental than in the laterodorsal tegmental nucleus following tracer injections at all sites with the exceptions of the locus ceruleus and dorsal raphe nucleus where the converse profile was observed. None of the pedunculopontine or laterodorsal tegmental cells immunopositive for ChAT or stained intensely for AChE contained retrogradely transported tracers following dye infusions into the cerebellar cortex or cervical spinal cord. Triple-label experiments using two tracers infused into different sites in the same animal revealed that individual ChAT-i~uno~active cells in the PMT cholinergic complex projected to more than one hindbrain site in some cases and had ascending projections as well. Certain CbAT-~sitive somata in the ~dunculo~ntine and laterodorsal tegmental nuclei were found in close association with several fiber tracts, in&ding the superior cerebellar peduncle, lateral lemniscus, dorsal tegmental tract, and medial longitudinal fasciculus. Choline acetyltransferase Pedunculopontine tegmental

Cranial nerve nuclei nucleus Laterodorsal

Reticular nuclei tegmental nucleus

THE core of the brainstem contains an elongated, essentially continuous constellation of cholinergic projection neurons ranging from caudai aspects of the substantia nigra rostrally to rostra1 domains of the locus ceruleus caudally (10,96). The component neurons, referred to collectively as the ~ntomesenc~phalotegmental (PMT) cholinergic complex (10,96), are intensely immunoreactive for choline acetyl~nsferase (ChAT) and are associated prominently with the ~dunculopontine and laterodorsal tegmental nuclei (10,96). Although many of these cholinergic cells project ascendingly to the thalamus (37, 43, 62, 76, 82, 85, 96, 99) and tectum (43, 96, 99), a significant but generally lesser number also supply ascending afferent fibers to various extrapyramidal (33,96, 99) and basal forebrain nuclei (76, 96, 99). Because of their topographic distribution and hodologies, as well as associated

Raphe nuclei Cholinergic projections

electrophysiologic properties, neurons of the PMT cholinergic complex have been considered, in part at least, components of the ascending reticular activating system (78, 86, 96). In addition to projecting ascendingly to rostra1 targets, neurons associated with the brainstem reticular fo~ation have been observed to possess descending projections as well (72), aibeit not necessarily cholinergic. ~one~eless, the existence of putative cholinergic afferents deriving from the pedunculopontine and laterodorsal tegmental nuclei and terminating in the nucleus of the tractus solitarius (68), deep cerebellar nuclei (20), and pontine reticular nucleus (56,77) is consistent with the notion of a descending PMT cholinergic system. Despite the potential physiologic significance of this latter network, however, the organization and full extent of presumed descending projections from

‘Requestsfor reprints should be addressedto Dr. Nancy J. Woolf, Department of Psychology, Angeles,

CA 90024-1653.

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WOOLF AND BUTCHER

cholinergic cells in the pedunculopontine and laterodorsal tegmental nuclei remain incompletely specified. Muscarinic and nicotinic binding sites of medium to high densities have been found in various cranial nerve nuclei, the reticular formation, the cerebellum, the inferior olive, and various pontine areas (67), suggesting that all of those regions could be possible recipients of cholinergic fibers. In an attempt to determine whether or not putative acetylcholine-containing afferents to caudally situated pontine and medullary structures derived from cholinergic somata in the pedunculopontine and laterodorsal tegmental nuclei. we infused retrogradely transported fluorescent compounds into those presumed targets and analyzed PMT somata for the colocalization of tract-tracing agents and ChAT-like immunoreactivity or acetylcholinesterase (AChE). Triple-label experiments involving demonstration of two tract-tracing labels and ChAT on the same tissue section were also performed to assess the possible existence of axon collaterals. METHOD

Eighty-six female Sprague-Dawley rats weighing 220-320 g were used. The animals were housed individually in stainless steel cages kept in rooms maintained at a constant temperature (22°C) and relative humidity (50%). A 12-hour light-dark cycle was imposed (6:00-18:OO dark; lS:OO-600 light). Food and water were provided ad lib. Surgical Procedures Rats were first anesthetized with 350 mg/kg chloral hydrate given intraperitoneally. Their heads were then shaved and mounted by use of ear plugs into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) with bregma and lambda at the same height in accordance with the procedure detailed in Paxinos and Watson (63). The fluorescent tracers Evans Blue (Matheson, Coleman and Bell, Co.. Norwood. OH), true blue (Sigma Chemical Co,. St. Louis, MO) and fluorogold (Fluorochrome, Inc.. Englewood, CO) were used. Suspensions of Evans Blue (30%) and true blue (10%) in distilled deionized water were made prior to injection. Separate l-p_1 syringes (Hamilton Co.. Reno. NV) with permanently attached stainless steel cannulae were filled with dyes. and infusions ranging from 0.05 to 0.2 PI of Evans Blue and true blue were made into various hindbrain sites at a rate of 0.01-0.05 $/min. Cannulae were left in place for at least 5 min before being slowly withdrawn in order to minimize the extent of diffusion along the cannula tract. Solutions of 2.5% fluorogold dissolved in 0.9% saline were iontophoretically applied from glass micropipettes (tip size: 15-45 pm). Alternating pulses (1 set on-l set off) of lo-20 ALAcurrent were delivered for IO-20 min. The above-mentioned tracers were infused into caudal nervous system sites at the following stereotaxic coordinates derived essentially from the atlas of Paxinos and Watson (63): a) nucleus of cranial nerve 5 (n = 3 rats); 9.3 mm posterior to bregma (P) . 2 .O mm lateral from the midline (L), and 7.0 mm ventral (V) from the surface of the brain, b) nucleus of cranial nerve 7 (n = 8 rats); I 1.3 mm P, 1.8 mm L, and 8.2 mm V. c) nucleus of cranial nerve 12 (n =3 rats); 13.8 mm P, 0.3 mm L, and 5.3 mm V, d) vestibular nuclear complex (n=6 rats): 11.3 mm P, 0.8 mm L, and 5.7 mm V, e) nucleus of spinal nerve 5 (n=4 rats): 11.3 mm P. 3.0 mm L, and 5.7 mm V, f) locus ceruleus (n=9 rats): 9.8 mm P. 1.5 mm L. and 5.8 mm V. g) raphe magnus nucleus (n = 9 rats); 10.3 mm P. 0.5 mm L. and 9.0 mm V (n = 9 rats), h) median raphe nucleus (n = 5 rats); 7.6 mm P. 0.2 mm L. and 7.8 mm V. i) dorsal raphe

nucleus (n =5 rats); 7.8 mm P, 0.2 mm L, and 5.7 mm V. j) oral pontine reticular nucleus (n = 8 rats): 8.3 mm P, 1.O mm L. and 8.0 mm V, k) caudal pontine reticular nucleus (n = 7 rats); 9.8 mm P. 1 .O mm L. and 8.3 mm V. 1) medullary reticular nucleus (n = 3 rats); 13.0 mm P, 1.5 mm L, and 5.8 mm V. m) cerebellar cortex (n=6 rats); 10.3 mm P. 0 mm L. and 3.3 mm V, n) deep cerebellar nuclei (n =4 rats); Il.3 mm P. 2.5 mm L. and 4.2 mm V. o) pontine nuclei (n=6 rats): 7.3 mm P. 1.0 mm L. and 9.2 mm V, p) inferior olive nucleus (n=3 rats): 12.3 mm P. 0.5 mm L, and 8.7 mm V, q) lateral reticular nucleus (n = 3 rats): 14.0 mm P. I .8 mm L. and 6.3 mm V. r) pontine reticulotegmental nucleus (n = 3 rats): 8.0 mm P, 0.8 mm L, and 8.0 mm V. and s) cervical spinal cord (n = 14 rats). In three sets of animals (b and j. h and i, m and o indicated in the previous paragraph). injections of tracers were made into two different targets in the same rat at the same time. In these cases, true blue was infused into one region and fluorogoId was injected into the other. In most animals, both injections were made into hindbrain sites. In 4 rats. however. tracer infusion into the raphe magnus nucleus was combined with an injection into the posterior thalamus (3.8 mm P, 2.2 mm 1,. and 5.2 mm V).

Tissue from rats which were injected with true blue or fluorogold were subsequently processed immunohistochemically for ChAT as described in previous publications from this laboratory (33.97). In brief, these animals were deeply anesthetized with 350 mg/kg chloral hydrate administered intraperitonealiy and were perfused transcardially with 50 ml cold (4°C) phosphate buffered saline (PBS) followed by 500 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.2). Brains were carefully extracted and placed into the same fixative used for perfusion and stored in that solution at 4°C for 90 min. Following postfixation, brains were blocked in the stereotaxic plane indicated in Paxinos and Watson (63). transferred into 30% sucrose in 0.1 M phosphate buffer. and kept at 4°C for 2-7 days. Brain sections were cut at 40 pm intervals on a freezing microtome and collected into PBS containing 0.3% triton-X-100. These sections were incubated in that solution for 24 hr and then transferred into a solution of a ChAT monoclonal antibody [code 111255: for characterization, see (25)] diluted 1:50 in PBS containing 0.0 I % sodium azide. Following 2-3 days’ incubation in primary antibody, tissue sections were then rinsed three times in PBS and immersed into solutions of affinity purified anti-rat IgG conjugated to fluorescein isothiocyanate (FITC, Sigma ChemicaI Co., St. Louis, MO) diluted I:50 in PBS containing 0.3% triton-X-100. Tissue sections were incubated for 2 hr at room temperature in the secondary antibodies and then rinsed 3 times in PBS. Reacted tissue was promptly mounted onto slides coated with 1.0% pig gelatin and coverslipped under a solution of glycerine and PBS (3: 1. v/v) containing 0. I M n-propyl gallate. Ten additional rats were not surgically manipulated but were processed immunohistochemically for ChAT alone in order to map the distribution of cholinergic cells in the pontomesencephalon. These animals were prepared as described previously, with the exceptions that avidin-biotin conjugated secondary antibodies were used and the sections were reacted with diamindinobenzidine (Sigma Chemical Co., St. Louis. MO), as described in detail in Gould and Butcher (32). For comparison purposes, brains from three cats were also processed for ChAT according to the avidinbiotin method. AChE Phar-maI,ohistc~chemist~?, In animals

in which Evans Blue had been injected,

tissue

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FIG. 1. Demonstration of fluorescent tracers (A, C, E), AChE (B, pharmacohistochemical regimen), and ChAT (D, F; FITC second antibody) in the pedunculopontine tegmental nucleus following infusions of Evans Blue (A), fluorogold (smaller arrowheads in C, E), or true blue (larger arrowheads in E) into the nucleus of cranial nerve 7 (A-B), the vestibular nuclear complex (smaller arrowheads, C, E), or caudal pontine reticular nucleus (larger arrowheads, E), respectively. Asterisks in C and E indicate cells that contained both fluorogold and true blue. The same tissue sections are shown in A-B, C-D, and EF. Frames A and B are from one rat and frames C-D are from another animal but at two different planes of section. Arrowheads and asterisks in all frames point to examples of neuronal somata that contained one or two fluorescent tract-tracing agents and also stained intensely for AChE (B) or were immunoreactive for ChAT (D, F). Scale bar in B is 25 p,m and applies also to A. Scale bar in D is 50 pm and applies also to frames C and E-F.

FOLLOWING PAGES FIG. 2. Distribution of somata in the rat pontomesencephalic tegmentum and adjacent method). Transverse sections at four different levels are shown at approximately 7.3 bregma’according to the stereotaxic atlas of Paxinos and Watson (63j. Abbreviations: lateral lemniscus; LDTgJaterodorsal tegmental nucleus; me5, mesencephalic trigeminal nucleus; PPTg, ~dunculo~ntine tegmental nucteus; scp, superior cerebcllar peduncle; and C are 500 pm and apply to all frames.

regions demonstrating ChAT-like immunoreactivity (avidin-biotin mm (A), 8.3 mm (B), 8.8 mm (C), and 9.3 mm (D) posterior to dtg, dorsal tegmental tract; 4, trochlear cranial nerve nucleus; 11. tract; mif, medial longitudinal fasciculus; MoS, motor trigeminal xscp, decussation of superior cerebellar peduncle. Scale bars in A

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FIG. 3. Neuronal somata and processes immunoreactive

for ChAT (avidin-biotin method) in the laterodorsal tegmental nucleus (A). in the medullary reticular formation (B), and in association with cells comprising the raphe magnus nucleus (C-D). The arrow in frame B points to a cholinergic neuron that appears to make contact with an adjacent, presumably noncholinergic cell. Arrowheads in frames B-D indicate ChAT-positive, terminal-like boutons in association with noncholinergic neurons, which, in C and D may use serotonin as a transmitter. Feline material. Scale bar in A is 50 pm and in B, 20 pm. Scaie bar in C is 15 pm and applies also to D.

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FIG. 5. Distribution of tag-blue-iabeled (BAT-positive somata in the PMT choiinergic cornpier; following infusion of that fluorescent tracer into the motor nuclei of cranial nerves 5 (solid circles). 7 (open triangles),and 12 (open circles). In this and subsequent related figures, each symbol represents one cell soma. Templates are arranged in rostrocaudal order from tap to bottom and from left to right and encompass levels 6.72-9.30 mm posterior to bregma as redrawn from Paxinos and Watson (6.1).

were processed for AChE in essential accordance with the protocol of Butcher (5,9). Briefly, such animals were intramuscularly injected with 1.8 mglkg bis-( 1-methylethyl)-phosphorofluoridate (DFP). Six hr following DFP adminis~ation, the rats were deeply anesthetized with 350 mg/kg chloral hydrate and transc~dially perfused with 120 ml 0.9% saline (4°C) followed by 120 ml 10% buffered formalin (4’C; pH = 7.2). Brains were sections

postfixed in the cold formalin solution for 2 days and then placed into cold 30% sucrose for an additional 2-7 days. Sectians were cut on a freezing microtome at 40 pm intervals in the plane indicated in Paxinos and Watson (63). cdlected into cold 0.9% sahne, mounted onto gelatinized slides, and coverslipped. Foilowing microscopic analyses (see below), coverslips were carefully removed so that tissue sections remained attached to the glass

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FIG. 6. Distribution of fluorogold-labeled ChAT-positive somata in the PMT cholinergic complex following infusion of that fluorescent tracer into the spinal trigeminal nucleus (solid circles) and the vestibular nuclei (open triangles). Templates represent rostrocaudal levels 6.72-9.30 mm posterior to bregma as redrawn from Paxinos and Watson (63).

slides. These slides with affixed brain sections were rinsed several times in 0.9% saline and then placed into solutions of 30 pM N,N’-bis-( 1-methylethyl)pyrophosphorodiamidic anhydride (K and K Laboratories, Plainview, NY) for 30 min in order to inhibit butyrylcholinesterase activity. Subsequently, slides were placed into solutions of 0.05% acetylthiocholine in 130 mM Tris-maleate buffer (pH=5.7) containing 5 mM sodium citrate, 3 mM cupric sulfate, and 0.5 mM potassium ferricyanide and incubated at room

temperature for 6 hr. Following these histochemical reactions, slides were rinsed in aqueous solutions, dehydrated in a series of increasingly concentrated alcohols, rinsed in xylenes, and coverslipped under Petmount@ (Fisher Scientific Co., Fairlawn, NJ). Data h~~y~is

The evaluation of microscopic

material was accomplished

with

BUTCHER

FIG. 7, Fluorescent tracers retrogradely transported to cholinergic somata in the PMT complex following true blue infusion into the dorsal raphe nucleus (A, compare with B) and Evans Blue injection into the raphe magnus nucleus (C. compare with D) or locus ceruleus (E, compare with F). Arrowheads point to retrogradely labeled cells in A, C, and E that colocalize the cholinergic markers ChAT (B) or AChE (D,F). The asterisk in B indicates a ChAT-positive cell that did not contain fluorescent label (compare with A). Scale in F is 50 km and applies to all frames.

Zeiss RA and Olympus Vanox microscopes containing both tungsten and fluorescent illumination capabilities. Evans Blue, which is intensely red fluorescent (Fig. IA). was viewed with ultraviolet lighting transmitted through a combination LP 520 and KP 560 excitation filter and a LP 590 barrier filter. Tungsten illumination was used to visualize AChE (Fig. 1B). Both true blue and fluorogold were viewed with an epi-illumination system containing a BG 365 exictation filter and a LP 435 barrier filter. With these lighting parameters, true blue appeared deep blue (Fig. 1E) and fluorogold was pinkish-orange (Fig. 1, C and E). Cells containing both true blue and fluorogold exhibited a steel blue to whitish fluorescence (Fig. 1, C and E), presumably resulting from the addition of the spectral features of both tracers. In some cases, both true blue and fluorogold fluorescence could be detected independently in different parts of the same cell. The FITC label was examined by use of standard Zeiss or Olympus filter packages appropriate to optimally visualize that fluorochrome. Cells cola-

beled with ChAT and true blue or fluorogold could be identified unequivocally by alternating the appropriate viewing filters, coupled with sequential photog~phy (Fig. 1, D and F; compare respectively with Fig. 1, C and E). The illumination system was alternated for the successive observation of neurons containing both AChE and Evans Blue (Fig. lB, compare with Fig. IA). Photomicrographs were made with Tri-X 400 ASA or Kodacolor 1000 ASA film. Injection sites and the localization of cells containing fluorescent tracers were mapped onto templates with the aid of a drawing tube (camera lucida) and pens containing fluorescent writing fluid, which was visualized by ambient ultraviolet light [for detailed description of methodology, see (33)J. RESULTS

Organization of‘ the Pontomesencephalotegmental Cholinergic Complex The rostrocaudal concatenation of the PMT chohnergic com-

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FIG. 8. Distribution of tracer-labeled CbAT-positive somata in the PMT eholinergic complex f&owing infusion of ffuorogold into the raphe magnus (solid circles) and median mphe nuclei (open triangles) and true blue into the dorsal raphe nucleus (open circles). Templates encompass rostrocaudal levels 6.72-9.30 mm posterior to bregma as redrawn from Paxinos and Watson (63).

plex at four different levels is shown in Fig. 2. The morphologies (Fig. 3A) and distribution of ChAT-positive cells is virtually identical to the topography of neurons staining intensely for AChE (pharmacohistochemical regimen) in the same regions of the brain [compare Figs. 2 and 3A of this report with Figs. I-? in {96).] Like ceiis of the basal forebrain cholinergic system @,37), many neuronal somata of the PMT cholinergic network and their dendritic processes @ig. 3A) are highly interdigitated with major

fiber bundles, which, in the case of the latter system, includes prominently the superior cerebellar peduncle (Fig. 2, A-C). Neurons of the PMT cholinergic complex are also found in proximity to the lateral lemniscus (Fig. 2, A-C), dorsal tegmental bundle (Fig. 2, B-C), medial long~~djn~ fasciculus (Fig. ZC), and mesencephalic ~gern~~l tract (Fig. 2D). In some areas of the brain, ChATpositive terminals deriving from cholinergic neurons could be observed in apparent associa-

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FTG, 9. Dist~bution of fluorogold-labeled ~bAT-positive somata in the PMT cholinergic complex following infusion of retrograde tracer into the locus ceruieus (solid circles). Tempiates encompass rostrocaudaI levels 6.72-9.30 mm posterior to bregma as redrawn from Raxinos and Watson 1631.

tion with nonchoiinergic cells bodies and processes in the medullary reticular formation (Fig. 3B). Particularly dense ChATpositive terminal arborizatians and boutons were found encapsulating neurons in the raphe magnus nucleus (Fig. 3, C-D). krjeetion Sites and General U~se~rat~a~s of Retrograde Labeling Patterns Typical localizations and extents of diffusion following

tracer

injection into the various brainstem sites are illustrated in Fig. 4. In the vast majority of cases, the tracer was confined substantially to the region injected. Small injections were made into the locus ceruleus in an attempt to restrict the tracer to that region; nonetheless, some tracer diffusion into the surrounding regions was unavoidable. In the case of the dorsal raphe, a small amount of tracer was observed in the adjacent periaqueductal gray. The dense cores of tracers infused into the nuclei of cranial nerves 5,

FIG. 10. Chohnergic cells in the ~dnncuiopontine (A-B, E-F) and laterodorsal (CD) tegmental nuclei following the infusion of fluorogold into the oral pontine reticular nucleus (A, compare with B) and Evans Blue into the caudal pontine reticular nucleus (C and E, compare with D and F, respectively). AtTowheads in corresponding pairs of frames in each row of photomicrographs indicate the same cells colabeled with fluorescent tracers and ChAT (B) or AChE (D,F). The asterisk in A delineates a fluorogold-labeled cell that was not immunoreactive for ChAT. Scale bar in F is 50 pm and applies to aIi frames.

7, and 12 were confined wholly to those structures, although the total extent of diffusion involved ~rnrn~i~t~ly adjacent regions of the reticular formation. It has been our experience, however, that the majority, if not all, of retrograde transport occurs from the most concentrated dye loci and not from the surrounding region of diffusion [see (96)]. Following tracer injections into the majority of hindbrain sites. several ChAT-positive or intensely stained AChE neurons pontomesencephalic tegmentum were retrogradely labeled (1-15 neurons per section). The number of retrogmdely labeled neurons did not appear to vary appreciably as a function of tracer infused; Evans Blue, true blue and fluorogold all yielded similar patterns of labeling. The amount of tracer infused, selected according to the size of the recipient structure, also did not appear to influence the number of labeled cells in a simple fashion. Medium-size infusions into the raphe magnus nucieus (Fig. 4), for example, resulted in the greatest number of retrogradely labeled cells in the PMT

cholinergic complex, whereas larger injections placed into the dorsal raphe nucleus (Fig, 4) resulted in fewer 1abeIed cells. Differences in the to~graphy of retrogradely labeled neurons additionally suggested that labeled neurons projected to the region infused rather to neural regions surrounding those structures.

ChAT-positive neurons in the pedunculopontine tegmental nucleus were labeled with retrogradely transported fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12 (Fig. 5). Although pedunculopontine neurons at virtually ail levels were labeled, however, no cholinergic cells in the laterodorsal tegmental nucleus were found to demonstrate projections to those motor nuclei (Fig. S), The vast majority of labeled cells were found ipsilateral to the side of injection. with only Z-19% of the total number of labeled cells being observed on

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FIG. I 1. Dis~ibution of tracer-labeled ChAT-~siti~re somata in the PMT cholinergic the infusion of true blue into the oral pontine reticular nucleus (solid circles) and caudal pontine reticular nucleus (open triangles) or medullary reticuiar nuclei (open encompass rostrocaudal levels 6.72-9.30 mm posterior to bregma as redrawn Watson (631.

the contralateral side. Many retrogradely labeled neurons found in the pedunculopontine tegmental nucleus following tracer infusions into the motor nuclei of cranial nerves 5, 7, and 12 also stained intensely for AChE (e.g., Fig. 1, A-B). ChAT-immunoreactive neurons were found in both the pedunculopontine and laterodorsal tegmental nuclei following infusions into the vestibular complex and spinal nucleus of nerve 5 (Figs. I.

C-D and 6). These pontomesencephalon, labeled on the side of labeled contralaterally Cholinergit

Prffjectims

complex hollowing fluorogold into the circles>. Templates from Paxinos and

neurons were distributed throughout the and, although more cells were retrogradely the infusion, a considerable proportion was (3941%). to ~~nf)a~~i~~~~ic .Nuclei

Following tracer infusions into the dorsal raphe, median raphe,

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FIG. 12. Somata of the pedunculopontine tegmental nucleus retrogradely transporting fluorogold infused into the deep cerebellar nuclei (A, compare with B) and pontine nuclei (C, compare with D) or Evans Blue injected into the lateral reticular nucleus (E, compare with F). Arrowheads point to cells in A, C, and E that colocalized ChAT (B,D) or AChE (F). The asterisks in B and D indicate ChAT-positive neurons that did not transport fluorescent label. Scale bar in F is 50 t.crn and applies to all frames.

or raphe magnus, ChAT-immunopositive cells in the ~dunculopontine and laterodorsal tegmental nuclei were labeled (Figs. 1, E-F; 7, A-B; 8). The largest projection appeared to be to the raphe magnus (Fig. 8). Projections to the median raphe and raphe magnus originated preferentially from the pedunculopontine compared to the laterodorsal tegmental nucleus (78 and 91%, respectively). In contrast, the greatest number of ChAT-positive neurons in the PMT choiinergic complex projecting to the dorsal raphe nucleus was found in the laterodorsal tegmental nucleus and represented 81% of the total number of labeled cells. Cells immunoreactive for ChAT and projecting to the raphe nuclei were predominantly ipsilateral to the side of infusion but 17-3 1% were located contralaterally. Many cells retrogradely labeled with fluorescent tracer following raphe nucleus infusions also stained intensely for AChE (e.g., Fig. 7, C-D). Infusions of tracer which were largely restricted to the locus ceruleus (see Fig. 4) resulted in the labeling of a number of

ChAT-positive neurons in the ~dunculo~ntine and laterodorsal tegmental nucleus (Fig. 9). Approximately two-thirds of these cells were ipsilateral to the injection side. Pedunculopontine and laterodorsal tegmental cells that stained intensely for AChE also were labeled following locus ceruleus infusions (Figs. 7, E-F).

Neurons immunoreactive for ChAT in the pedunculopontine and laterodorsal tegmental nuclei were labeled with fluorescent tracer following infusions into the oral pontine, caudal pontine, and medullary reticular nuclei (Figs. 10, A-B; 11; see also Fig. 3B). Compared to the laterodorsal tegmental nucleus. the majority of these neurons were located in the pedunculo~ntine tegmental nucleus (Fig. 11). Most of these tracer-labeled projection cells were located ipsilateral to the injection, with smaller proportions (18-30%) being located contralateral to the infused side of the

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FIG. 13. Distribution of retrogradely labeied ChAT-positive somata in the PMT cholinergic complex following infusion of fluorogold into the pontine nuclei (solid circles) or true blue into the deep cerebellar nuclei (open triangles). Templates encompass rostrocaudal levels 6.71-9.30 mm posterior to bregma as

redrawn frm Paxinos and Watson (63).

brain. Tracers injected into the reticular nuclei also resulted in the retrograde labeling of neurons that stained intensely for AChE in the laterodorsal (Fig. 10, C-D) and pedunculopontine tegmental nuclei (Fig. 10. E-F). Projectiom to the Ce~ebeliu?n and Associated Nuclei Infusions of tracer into the cerebellar cortex did not result in the retrograde labeling of any ChAT-positive or intensely stained

AChE somata in the pedunculopontine or laterodorsal tegmental nuclei. Cells that were retrogradely labeled were found in the pontine reticulotegmental nucleus, ventral tegmental nucleus, pons, paramedian reticular nucleus. lateral reticular nucleus, nerihvooglossal nucleus, dorsal column nuclei. inferior olive. and spinal ‘co;d in accordance with previous results (8, 24,40,49, 83). Infusions into the deep cerebellar nuclei and the pontine nuclei did result in the labeling of ChAT-positive somata in the pedunculopontine and laterodorsal tegmental nuclei (Figs. 12, A-D: 13).

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Infusions of fluorescent tracers into the pontine reticulotegmental nucleus, inferior olive, and lateral reticular nucleus (Fig. 12, E-F) resulted in the labeling of ped~c~opontine and laterodorsal tegmental nuclei cells which were intensely stained for AChE. Projections to the Spinal Cord Following tracer infusions into cervical spinal cord, no ChATpositive cells in the pedunculo~ntin~ or laterodorsal tegmental nuclei were observed. A few retrogradely labeled cell were seen in the pedunculopontine region, but these cells were not ChATpositive and did not stain intensely for AChE. As expected, retrogradely labeled cells were found in the oral and caudal pontine reticular nuclei, raphe magnus, and medullary reticular formation [e.g., see (7)]. Collateral Projections Some neurons in the PMT cholinergic complex demonstrated both ascending and descending projections (e.g., to the posterior thalamus and raphe magnus nucleus; Fig. 1, E-F). Certain ChAT-positive cells were also observed projecting to two brainstem loci, such as the vestibular nuclear configuration and the caudal pontine reticular nucleus (Fig. 1, C-D). Noncholinergic Projections to the Hindbrain In addition to the projections listed above, a number of telen~eph~ic, diencephalic, and brainstem regions demonstrated projections to the hindbrain regions which were infused with tracer. Briefly, one or more areas of cerebral cortex (frontal, parietal, temporal, insular or cingulate) contained retrogradely labeled neurons following the infusion of tracers into the pontine nuclei, inferior olive, nucleus of cranial nerve VII, raphe magnus, lateral reticular nucleus, pontine reticulotegmental nucleus, and oral and caudal pontine reticular nuclei. All of these projections have been previously documented (12, 42, 91, 94). Descending projections to the locus ceruleus, raphe magnus, and pontine reticular nuclei were demonstrated from basal forebrain nuclei including the vertical and horizontal limbs of the diagonal band, substantia innominata, central amygdala, bed nucleus of the stria terminalis, lateral h~thalamus, and the medial and lateral preoptic areas in ~o~oboration of earlier reports (13, 17. 41, 87). Although basal forebrain areas such as the diagonal band and substantia innominata contain ChAT-positive cells, no evidence was obtained in the present study that the cholinergic neurons of those regions projected to any of the hindbrain sites we examined. In the diencephalon, the zona incerta, parafascicular thalamus, and lateral habenula demonstrated descending projections to the pontine reticular nuclei. Retrograde labeling was found in the substantia nigra or ventral tegmental area, as well as in the dorsal and median raphe following tracer infusions into the reticular nuclei of the brainstem. The pontine and medullary reticular nuclei further demonstrated projections onto other reticular nuclei and onto the raphe magnus. The deep layers of the superior colliculus gave rise to projections to the inferior olive, raphe magnus, and oral and caudal pontine reticular nuclei. These pathways have been previously documented (2, 12, 26, 66, 90). DISCUSSION

Organization of Descending Chuiinergic Projections From the Ponto~esencepha~o~ The descending pathways of the PMT chohnergic complex charted in the present study are schematically represented in Fig.

535

14. Presumed cholinergic projections from the ~dunculo~ntine and laterodorsal tegmental nuclei have been reported previously to the pontine reticular formation (5677) and the deep cerebellar nuclei (20). The absence of descending cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei to the cerebellar cortex and spinal cord has also been documented in prior publications (20,3 1,68). The cerebellar cortex demonstrates binding for muscarinic cholinergic receptors (61), however, and, to the extent that cholinergic neurons innervate the cerebellar mantle, it is conceivable that the few ChAT-positive neurons in the medullary reticular formation provide that innervation (79). Although cholinergic projections to the motor nuclei of cranial nerves 5, 7, and 12 have not been previously reported, previous tract-tracing experiments indicate that the pedunculopontine region gives rise to descending projections te~inating in the trigeminal, facial, and hypoglossal motor nuclei (4457). ChATpositive terminals on hypoglossal neurons of unknown origin have also been described (16). That the dense cores of the tracers infused in the present study were contained within the borders of the relevant cranial motor nuclei, along with the fact that a unique distribution of exclusively pedunculopontine neurons provided such afferents, favors the inte~retation that those nuclei do receive a cholinergic projection from the ~dunculopontine tegmental nucleus. Cholinergic inputs to monoaminergic cell groups derived extensively from somata in the pedunculopontine and laterodorsal tegmental nuclei. Similarly, ChAT-positive projections particularly to the compact part of the substantia nigra have been demonstrated (33), and ChAT-~sitive terminals have been described in the rat dorsal raphe (34). These findings support the notion that cholinergic-monoaminergic interactions mediated by pontomesencephalic cells might be a prominent feature of the mammalian hindbrain. Of all the sites evaluated, the raphe magnus nucleus appeared to receive projections from the greatest number of PMT ChAT-positive cells. Terminals immunoreactive for the cholinergic synthetic enzyme were additionally observed to encapsulate individual somata and proximal dendrites in the region of the raphe magnus nucleus, further suggesting an important linkage between cholinergic and presumed monoaminergic neurons. The projection of ChAT-positive pedunculopontine and laterodorsal tegmental cells to the locus ceruleus is consistent with studies describing retrogradely labeled cells in regions comparable to those examined in the current study (I 3 59) but is at variance with another report (71). The fact that locus ceruleus cells are sensitive to the muscarinic agonist, carbachol (92), is compatible with the conjecture that this brain region is innervated by cholinergic cells, however. Analogous to the ascending projections of the cholinergic ~ntomesencephalon (95,96), the descending projections of the PMT cholinergic complex are also widely dist~buted, These latter projections, however, are generally sparser than their ascending counterparts [cf. (96)]. Following tracer infusions of a similar size into the thalamus, for example, many more pontomesencephalic ChAT-positive cells are retrogradely labeled (approximately 5-60 cells per brain section) than following dye injections into the hindbrain regions examined in the present study (l-15 cells per tissue section). Like ascending pathways of the PMT cholinergic complex (96), rough projection topographies were noted with respect to the descending projections as well. A proportionally larger number of somata were labeled in the laterodorsal than the pedunculopontine tegmental following tracer infusions into the locus ceruleus and dorsal and median raphe nuclei, cellular groups that are extensively connected to limbic and visceral brain regions [e.g., (13, 18, 45)]. In contrast, the pedunculopontine nucleus appeared to provide most, if not all, of cholinergic pontomesencephalic

WOOLF

AND

BUTCHER

FIG. 14. Schematic depiction of the descending projections of the PMT cholinergic

complex as demonstrated in the present study. Abbreviations: CPU, caudate-putamen nucleus; IO, inferior olive: LRt, lateral reticular nucleus; RtTg, pontine reticuIotegmental nucleus. For remaining abbreviations, see legends of Figs. 2 and 4.

afferents to such brain regions as the raphe magnus nucleus, reticulospinal nuclei, and motor nuclei of cranial nerves 5, 7, and 12, all of which are interconnected with somatosensory and motor systems (64,65).

Futmional

Cottsideratiom

The predilection of descendingly projecting cholinergic neurons of the laterodorsal tegmental nucleus for viscerally related target areas may indicate that the caudomedial component of the PMT complex comprises a node of integration for visceral info~ation. Neural pathways conveying visceral afferent signals, such as the dorsolateral tegmental bundle (IOO), are located near cholinergic neurons associated with the laterodorsal tegmental nucleus, and it is conceivable that such contiguous pathways provide vicsceral information to ChAT-positive cells in that nucleus. By analogy, pedunculopontine neurons are located near or in association with the brachium conjunctivum and lateral lemniscus, fiber bundles that convey somatosensory and auditory information. Dendrites demonstrating ChAT-like immunoreactivity have been identified in the feline lateral lemniscus, and it has been suggested that putative auditory inputs to the pedunculopontine nucleus may enable the processing of auditory signals by cholinergic pedunculopontine neurons (38). The notion that cholinergic somata of the pontomesencephalon participate in a wide range of physiologic activities that are related to their efferent projections. as well as to putative afferents from local fiber pathways, is consistent with the finding that pharmacological manipulations of different parts of the dorsal pontine tegmentum have varying and diverse effects on visceromotor or somatomotor functions (46), as would be predicted from the present anatomical results. Cells associated with the pedunculopontine and laterodorsal tegmental nuclei have also been implicated in locomotor activity (28), arousal (.53,60), sleep-wakefulness states (69). and memory (30.47). Stimulation of the mesencephalic reticular formation is known to induce locomotor behavior, and recently it has been shown that effective sites in this regard can be found within pedunculopontine regions containing ChAT-positive cells (28). A number of the descending projections mapped in the present study

may be relevant to this phenomenon and to other motor activities. Cholinergic pontomesencephalic projections were found to the nuclei of cranial nerves 5, 7. and 12, regions that have obvious motor function. These latter cholinergic pathways could modulate movements of the tongue. face, and jaw. In addition, cholinergic projections to the oral and caudal pontine reticular nuclei and the raphe magnus nucleus were demonstrated from the PMT complex, and reticulospinal neurons in similar brain loci have previously been shown to receive afferents from the mesencephalic locomotor area (29). Because reticulospinal neurons demonstrate influences over limb and axial motoneurons (65). cholinergic projections to reticulospinal nuclei are likely to pIay a critical role in locomotor behavior. A number of findings suggest that cholinergic pontomesencephalic neurons may play a pivotal role in attention and arousal, and diverse studies have focused on the neurophysiologic correlates of the ascending projections from the pontine reticular formation. Stimulation of the ascending reticular system, particularly in the regions known to contain cholinergic somata, results in cortical and hippocampal arousal (53.60). as well as in increased release of cerebral cortical acetylcholine (14,88). Although such data only implicate ascending cholinergic systems as possible generators or modulators of cortical and hippocampal arousal. a number of recent findings more directly support such a contention [e.g., see (21)]. Selective attention also could be modulated, in part. by descending cholinergic pathways from the ~ntomesencephalon onto sensory nuclei of cranial nerves, as demons~ated in the present study. It has been shown, for example, that responses to clicks in the eochlear nucleus are maximal when attention is focused on the relevant auditory stimuli (39). Descending cholinergic influences on sensory nuclei could conceivably alter thresholds for sensory stimulation, perhaps in a manner analogous to the way that acetylcholine affects sensory processing at diencephalic (80) and telencephalic sites (54.81). Involvement of the cholinergic pontomesencephalon in processes related to sleep and wakefulness has been the subject of considerable speculation. The application of cholinomimetics to neurons in the region of the pedunculopontine and laterodorsal tegmental nuclei has been shown to induce many of the signs of desynchronized sleep ( 1). and cells located in the region of pondne

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537

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ChAT-positive somata have been shown to fire during the periods of sleep desynchrony and pontogeniculate spiking (69). Although many of these effects probably can be attributable to ascending projections (19, 76, 82, 96), it is also the case that descending projections from the cholinergic somata of the pontomesencephalon might also play a role in certain aspects of sleep and wakefuiness. Muscle atonia commonly associated with desynchronized sleep has been found to depend impo~~tly upon the integrity of pontomesencephalic brain regions confining ChATpositive cells (93). and it is conceivable that descending projections from those ChAT-positive cells onto reticulospinal neurons might be involved in such atonia since reticulospinal pathways exhibit inhibitory influences on motor neurons (58,70). In the present study cholinergic projections were observed to virtually all of the reticulospinal nuclei, including the raphe magnus nucleus, lateral reticular nucleus, medullary reticular nucleus, and the oral and caudal pontine reticular nuclei. Cholinergic projections onto serotonergic neurons located in the raphe magnus nucleus, such as demonstrated in the present experiments, might also impact on the expression of muscle atonia during desynchronized sleep. Descending projections from that serotonergic structure produce marked inhibition of dorsal horn spinal neurons (55). which could be involved in the expression and modulation of muscle reflexes. Lesions in the region of cholinergic somata of the pontomesencephalon also have been associated with the production of amnesia (30) and memory impairment (47). Although damage to ascending pontomesencephalic ChAT projections are likely to contribute most saliently to such deficits (96), the descending pathways from the cholinergic somata of the pedunculopontine and laterodorsal tegmental nuclei additionally might also account for some aspects of the observed cognitive impairments. In this regard, projections to deep cerebellar nuclei might be particularly relevant, since those nuclei have been shown to be critical for the acquisition of certain conditioned responses (15). The C~olinergic Fanto~esencephafo~ curdles: ~~ecufatfve Issues

und Ca~~rigua~s Fiber

The hodologies of the PMT cholinergic complex and the relationship of its component neurons to local fiber tracts could provide clues to the fine structure of the involvement of those cells in the myriad functions indicated in the foregoing discourse. It is possible that the cholinergic neurons of the pontomesencephalic tegmentum form a concatenated nerve net with their counterparts in the basal forebrain and that together these two cholinergic complexes sample, in part, activity from a variety of fiber tracts conveying classical sensory information. How this information is processed by the cholinergic core of the brain at any moment in time would depend on indigenous patterns of electrical activity, presumably oscillatory. Rh~hmic firing patterns have been described for central cholinergic neurons (50,51), and cholinergic

cells have been implicated in diverse oscillatory activities (22, 27, 35, 36, 69, 84). Although the mechanism(s) by which these oscillatory firing patterns are initiated, maintained, and modulated has not been established, collaterals from sensory neurons, as well as loops involving other cholinergic and monoaminergic cells, might play prominent roles. Pontomescencephalic cells, possibly cholinergic, have been shown to be cholinoceptive (l), and electron microscopic studies of the basal forebrain indicate that cholinergic synapses, although sparse, do exist onto cholinergic cells (4). Like other afferents, fibers mediating cholinergiccholinergic interactions are probably preferentially associated with the distal portions of dendrites [see (75)]. In any case, sensory collateral inputs to the PMT cholinergic neurons are likely to be weak and to derive from a variety of sensory modalities (3,73). That cholinergic somata in the pontomesencephalon, as well as in the basal forebrain, may receive a number of independent inputs from classical sensorimotor systems, as well as other cholinergic cells and monoaminergic nerve networks, suggests multifactorial influences on the oscillatory activity of those cells [see (52,74)], resulting in similarly manifold modulation of the activities of the targets of the PMT complex, as might be expected in sleepwakefulness states and selective attention. M~ulation of the functions of cholinergic neurons by sensory collaterals could produce, among other sequelae, 1) immediate integration of incoming stimuli with ongoing cholinergic activity and/or 2) storage of that information within individual cholinergic neurons or within interconnected networks of cholinergic neurons for later usage. Putative reverberating circuits within the cholinergic nerve net could preserve the trace of afferent activation (see Fig. 3), or such activation could induce increased synthesis of various proteins important for the processing and storage of information in neurons. Increased electrical activity has been shown to increase ChAT activities in spinal cord cultures, for example, and accelerated uptake of trophic factors (i.e., substances that enable and maintain the differentiated state of cells) has been suggested to mediate that effect (6). An endogenous trophic agent associated with the basal forebrain cholinergic system (48,98) has been shown to increase the synthesis of ion channels (89), the syntheses of proteins associated with axonal transport and neuronal growth [for review see (1 l)], and electrical excitability and acetylcholine sensitivity (23). If similar trophiclike compounds are involved in maintaining the structurofunctional integrity of PMT cholinergic cells, then resultant changes in membrane characteristics, transmission efficacy, and/or neurite morphology could be influenced by afferent input from contiguous fiber bundles and other loci. ACKNOWLEDGEMENTS

This work was supported by USPHS grant NS 10928 to L.L.B. Dr. Felix Eckenstein is thanked for generous supplies of monoclonal autibodies. The excellent technical assistance of M. Cynthia Hemit and Justin Oh is gratefully acknowledged.

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