Brain Resenrch Bufferin, Vol. 19, pp. 705-714. D Pergamon Journals Ltd., 1987.Printed in the U.S.A. 0361~9230187$3.00 + .oo

Topographic Relations of Cholinergic and Noradrenergic in the Feline Pontomesencephalic Tegmentum: An Immunohistochemical Study

PETER B. REINER’ AND STEVEN R. VINCENT Division of Neurological Sciences, Department of Psychiatry, University of British Columbia Vancouver, British Columbia, Canada

Received 13 July 1987

REINER, P. B. AND S. R. VINCENT. ~~~pf)~ruph~~~~~at~~~ns uf~~~~linerg;~ and n~~rudrenerg~~ neurons in the feline pullt~~rs~n~~ph~~i~ t~g~~nt~~: An inzmuncthistochelnical sfudy. BRAIN RES BULL 19(6) 705-714, 1987.-The im- munohistochemic~ localization of the neurot~nsmitter synthesizing enzymes choline acetyltransfe~e, tyrosine hy- droxylase and dopamine-~-hydroxylase was examined in the feline pontomesenceph~ic tegmentum. Examination of adja- cent sections stained for either choline acetyltransferase, tyrosine hydroxylase or dopamine-@-hydroxylase immunoreac- tivity, as well as individual sections doubly stained for both choline acetyltransferase and tyrosine hydroxylase im- munoreactivity, unequivocally demonstrated that noradrenergic and cholinergic neurons were extensively intermingled in the tegmentum of the cat. This contrasts with the situation in various other species, where neurons utilizing these two are discretely localized in distinct nuclei. Furthermore, the present studies demonstrate the exist- ence of two types of choline acetyltransferase immunoreactive neurons in the feline tegmentum: the magnocellular neurons of the pedunculopontine and laterodorsal tegmental nuclei which stain histochemically for NADPH diaphorase, plus a population of small spindle-shaped neurons in the medial and lateral which do not stain positively for NADPH diaphorase. The data are discussed with respect to several influential hypotheses of sleep cycle control.

Locus ceruleus Pedunculopontine nucleus Laterodorsal tegmental nucleus Acetylcholine Noradrenaline NADPH diaphorase Sleep

THE presence of noradrenaline and acetylcholine containing contain both tyrosine hydroxylase (TH) and dopamine-p- neurons in the pontomesencephalic was hydroxylase (DBH), but not the adrenaline synthesizing originally suggested by histochemical studies which demon- enzyme phenylethanolamine-N-methyltransferase. Second, strated that selected neurons in this region exhibit catechol- acetylcholinesterase staining is not a specific marker for the amine fluorescence [lo] or acetylcholinesterase staining [60]. cholinergic neurons of the pontomesencephalic tegmentum. These data provided the structural basis for several influen- While most cholinergic neurons, as defined by choline tial hypotheses regarding the functional role of aminergic and acetyltransferase (ChAT) immunohistochemistry, in fact cholinergic neurons, particularly with respect to the neural contain acetylcholinesterase [ 12, 34, 37, 531, noradrenergic control of behavioral state [21,28]. neurons also display intense acetylcholinesterase staining, With the development of immunohistochemical tech- even using pharmachohistochemical procedures [ 1, 4, 551. niques for the localization of synthetic Although immunohistochemical probes have been exten- enzymes, it has become possible to define the dist~bution of sively used to study the anatomy of noradrenergic 19, 17,67, aminergic and cholinergic neurons in the pontomesence- 683 and cholinergic [2, 53, 56, 65, 71-731 neurons in the phalic reticular formation with ever increasing precision. brainstem of the rat, remarkably few studies have used im- Two complications of previous histochemical studies are al- munohistochemistry to examine this issue in the cat 17, 31, leviated by the use of immunohistochemistry. First, the 39, 47, 703. In most species, pontomesencephalic norad- specific catecholamine contained by neurons can be inferred renergic and cholinergic neurons are localized in distinct from the presence or absence of various synthetic enzymes. nuclei, with noradrenergic neurons concentrated within the For example, noradrenergic neurons, by definition, should nucleus locus ceruleus [14] and cholinergic neurons within

‘Requests for reprints should be addressed to Peter B. Reiner, Division of Neurological Sciences, University of British Columbia, Van- couver, B.C., V6T 1W5 Canada.

705 706 REINERANDVINCENT the laterodorsal tegmental and pedunculopontine nuclei [38]. bovine serum albumin (BSA) for 48 hours at 4°C. (2) The The feline appears to be unique with respect to the biochem- sections were washed 3x 20 min in TBS, and then incubated ical anatomy of the ~ntomesenceph~ic tegmentum. Rather in a solution of TBS cont~ning 0.5% biotinylated secondary than forming a distinct nucleus locus ceruleus, noradrenergic antiserum (goat anti-rabbit IgG for TH and DBH, rabbit neurons are topographically dispersed over several nuclei in anti-rat IgG for ChAT, both from Vector Labs), 0.3% Triton the feline brainstem, and are commonly referred to as form- X-100,2% normal serum and 0.01% BSA for 1 hour at room ing a locus ceruleus complex [8. 24, 26, 35, 42, 741. The temperature. (3) Sections were again washed 3 x 20 min, and heterogeneous distribution of noradrenergic neurons in the then incubated in TBS containing 1% avidin-biotinylated feline locus ceruleus complex suggests that these neurons horseradish peroxidase compbx (Vector Labs), 2% normal are intermingled with non-noradrenergic neurons. serum and 0.01% BSA for 1 hour at room temperature. (4) One possible transmitter candidate for the non- Sections were washed once again 3~ 20 min, and incubated noradrenergic neurons of the feline locus ceruleus complex in 50 mM Tris buffer, pH 7.4, containing 0.025% 3,3’- is acetylcholine. Kimura and Maeda 1301examined this issue diaminobenzidine (DAB), 0.01 mM imidazole and 0.0075%, using simultaneous catecholamine histofluorescence and H,O, for 8 minutes. The reaction was terminated by washing acetylcholinesterase techniques. However, their study was in TBS, and the sections were mounted on chrom-alum hampered by the fact that noradrenergic neurons, as men- coated slides, dehydrated and prepared for examination tioned previously, display intense acetylcholinesterase stain- under the light microscope. ing, thereby preventing simultaneous visualization of norad- Double immunohistochemistry for ChAT and TH in the renergic and cholinergic neurons. Thus, the present study same tissue section was accomplished by first incubating has utilized the immunohistochemical localization of TH, sections with the rat monoclonal antibody to ChAT as de- DBH and ChAT in adjacent serial sections, as well as simul- scribed above, followed by the standard DAB protocol. taneous immunohistochemical localization of TH and ChAT which resulted in a brown reaction product. The sections within the same section in order to examine directly the were then thorou~ly washed in TBS and incubated with topographic relations of noradrenergic and cholinergic rabbit antiserum to TH, and processed in a fashion similar to neurons in the feline pontomesencephalic tegmentum. Some the protocol described above until step 4 which was modified of these data have been presented in preliminary form [69]. for heavy metal intensification of the second DAB reaction. In this case the final incubation of the sections was per- formed in SOmM Tris buffer, pH 7.4, containing 0.02% DAB, METHOD 0.6% nickel ammonium sulfate, and 0.0015% HzO,. The re- The data are derived from experiments performed on four action was again terminated by washing in TBS, the sections pigmented adult male cats weighing between three and four mounted and examined under the light microscope. This kg. The animals were deeply anesthetized with sodium pen- combined protocol resulted in tissue sections with ChAT tobarbital (50 mg/kg, IP), heparinized (10,000 units, positive neurons displaying brown reaction product, while intracardiac), and perfused transcardially with 200 ml of TH positive neurons appeared blue-black. Sections stained phosphate buffered saline (0.1 M, pH 7.4) at room tempera- individually for ChAT, TH, and DBH as well as those doubly ture, followed by 1500 ml of either 4% par~o~aldehyde in labelled for TH and ChAT were used to map the dist~bution 0.1 M phosphate buffer (pH 7.4) containing I mM MgCl, of cholinergic and catecholaminergic neurons in the feline (N=3), or 2% paraformaldehyde and 15% saturated picric and . acid in 0.1 M phosphate buffer (pH 7.4, N=l) [75]. The brains were removed, postfixed for two hours in the same RESULTS fixative, blocked and placed in cryoprotectant (25% sucrose, 10% glycerol in 0.05 M phosphate buffer) for 48 hours. The TH and DBH positive neurons were found dispersed brainstem was cut either coronaily (N=3) or sagittally (NT= 1) throughout the dorsolateral within the at 30 pm thickness on a freezing microtome, and serially nuclei locus ceruleus, parabrachialis medialis and lateralis placed into a series of nine wells containing Tris buffered and Kalliker-Fuse (collectively termed the locus ceruleus saline (0.05 M, pH 7.4). Adjacent sets were stained for the complex). Although it was impossible to determine if every immunohistochemical demonstration of TH, DBH and TH positive in the locus ceruleus complex also ChAT, in that order. One (non-adjacent) set of sections was stained positively with DBH antiserum, many neurons could stained for cresyl violet. Additionally, in two cats a set of be shown to contain both antigens in adjacent sections sections (separated by a 30 pm gap from those stained for stained for each (Fig. lA,B). On the other hand, isolated ChAT) was stained for the histochemic~ demonst~tion of neurons within the (Figs. 3F, 4C), as NADPH diaphorase, which has been shown to be a reliable well as the neurons of the and sub- marker for the cholinergic neurons of the pedunculopontine stantia nigra (Fig. 4A,B) stained positively for TH only. and laterodorsal tegmental nuclei in the rat [71], according to The overall distribution of ChAT immunoreactive methods described elsewhere [57]. In one cat a set of sec- neurons in the feline brain is described elsewhere [70]. The tions was stained for the simultaneous demonstration of present report focuses upon the ChAT positive neurons of ChAT and TH (see below). the ~ntomesenceph~c reticular formation. A detailed de- Immunohistochemic~ localization of TH, DBH and scription of the ChAT positive neurons of the p~b~emin~ ChAT was accomplished by the avidin biotin complex nucleus has been published [70]. Two other readily distin- method 1231 using antibodies whose specificities have been guishable types of ChAT immunoreactive neurons were described elsewhere [ 12,471. The protocol employed was: (1) noted. Large, intensely staining ChAT positive neurons were sections were incubated free floating in diluted primary sera found in the laterodorsal tegmental and pedunculopontine (rabbit antisera to bovine adrenal TH or DBH, or rat mono- nuclei, including its caudal extension into the rostraI end of clonal antibody to C&AT) in Tris buffered saline (TBS) con- the medial p~~~h~~ nucleus. Isolated m~oce~ul~ taining 0.3% Triton X-100, 2% normal serum and 0.01% ChAT positive neurons were also found in the lateral para- ChAT AND CATECHOLS IN THE CAT BRAINSTEM

FIG. 1. Distributions of DBH (A), TH (B), ChAT (C) and NADPH diaphorase (D) positive neurons in the dorsolateral pontine tegmentum. Sections A-C are adjacent 30 pm sections, and section D is separated from C by a 30 pm gap. A large blood vessel is visible in all four sections (large arrowhead). Portions of the same neurons can be seen stained for both DBH (A) and TH (B) and two examples are marked by small arrows. A and B: TH and DBH positive neurons can be seen within the Iocus ceruleus (Ic), extending in a band across the ventral aspect of the taterodorsal tegmentai nucleus (tld) and into the medial parabrachial nucleus (pbm). Dendrites of TH positive neurons can be seen in the tld in B. C and D: ChAT and NADPH diaphorase positive neurons can be seen in the tld and pbm, but not in the Ic. In D, the large neurons of the mesencephalic trigeminal nucleus also show NADPH diaphorase staining. Calibration bar: 250 pm applies to A-D.

brachial nucleus and capping the dorsal nucleus of the lateral very similar to that of the TWDBH immunoreactive neurons lemniscus (Fig. 5A). In contrast to these magnocellul~ of the focus ceruleus complex (Fig. 3A,B,D,E). Both types ChAT positive neurons, small spine-shaped neurons which of neurons were large and multipolar; indeed, it was im- stained lightly with ChAT immunoperoxidase reaction prod- possible to distinguish the two cell types in cresyl violet uct were found in caudal portions of the medial and espe- stained sections. On the other hand, the morphology of the cially the lateral parabrachial nuclei (Fig. 5A,B). small, lightly staining, spindle-shaped ChAT positive These two types of ChAT positive neurons could be neurons seen in the medial and lateral parabrachial nuclei further distinguished on the basis of NADPH diaphorase his- was clearly distinct from the THiDBH immunoreactive tochemistry. The dist~bution and morphology of neurons neurons of the locus ceruleus complex (Fig. 30. staining positively for NADPH diaphorase were identical to The present report focuses upon topographic relations be- that of the magnocellular ChAT immunoreactive neurons of tween the ChAT positive neurons of the laterodorsal tegmen- the pontomesencephalic tegmentum (Fig. lC,D), while the tal and pedunculopontine nuclei and the THVDBH positive small spindle-shaped ChAT positive neurons of the medial neurons of the locus ceruleus complex. These were studied and lateral parabrachial nuclei did not stain positively for by examination of sets of adjacent sections (Fig. I) and sec- NADPH diaphorase. Although distinguishable on the basis tions doubly stained for ChAT and TH (Fig. 2). Within the of both mo~hology and NADPH diaphorase histochemist~, midbrain, the ChAT positive neurons of the pedunculo~n- the dist~bution of these two types of ChAT positive neurons tine nucleus were largely segregated from the TH positive within the medial and lateral parabrachial nuclei overlapped neurons of the (Fig. 4B). No ChAT positive somewhat; in the medial parabrachial nucleus the magnocel- neurons were found within the substantia nigra. lular neurons predominated while in the lateral parabrachial At the isthmus, the ChAT positive neurons of the pedun- nucleus the small spindle-shaped neurons were in the major- culopontine nucleus spread dorsomedially across the teg- ity. mentum into the laterodorsal tegmental nucleus (Fig. 4C). The mo~hology of the ChAT positive somata of the These ChAT positive neurons intermin~ed somewhat with laterodorsal tegmental and ~dunculopontine nuclei was the most rostra1 TH/DBH positive neurons of the locus 708 REINER AND VINCENT

FIG. 2. Simultaneous immunohistochemistry for ChAT and TH in the dorsolateral pontine tegmentum. This section was stained for ChAT immunoreactive neurons using the standard diaminobenzidine protocol which results in brown immunoperoxidase reaction product in the cholinergic neurons of the laterodorsal tegmental nucleus (tld) and medial parabrachial nucleus (pbm). Subsequently, the section was stained for TH immuno~active neurons using a nickel ammonium sulfate intensi~cation technique (see the Method section for details) which results in blue-black immunoperoxidase reaction product in the catecholaminergic neurons of the locus ceruleus (lc) and pbm. The dendrites of TH positive neurons in the pbm can be seen extending up into the tld. 4v-fourth ventricle. Calibration: 100 ,um.

FACING PAGE FIG. 3. Morphology of pontine ChAT, TH and DBH immunoreactive neurons. (A) Large multipolar ChAT positive neurons in the laterodor- sal tegmental nucleus. (B) DBH positive neurons in the locus ceruleus in a section adjacent to that shown in A. (C) Small, lightly stained ChAT positive neurons in the caudal medial parabrachial nucleus. (D) DBH positive neurons in the medial parabrachial nucleus in a section adjacent to that shown in C. (E) Large m~ti~iar ChAT positive neurons in the rostra1 medial ~abra~hi~ nucleus. (F) TH positive neurons in the dorsal raphe nucleus. Fourth ventricle is at the top. Calibration bar: 25 Frn for A-E; 125 pm for F. ChAT AND CATECWOLS IN THE CAT BRAINSTEM 709

“._,.” .._...... _ ____. 710 RElNER AND VINCENT

FIG. 4, ChAT AND CATECHOLS IN THE CAT BRAINSTEM 711 ceruleus complex where they extended in a thin band across into the laterodorsal tegmental nucleus. Furthermore, both the central gray. This was the only region of the central gray Golgi [67] and DBH immunohistochemical [ 171 studies in the where ChAT positive and TI-I/DBH positive neurons were rat have demonstrated extensive dendritic spread of norad- co-distributed. The ChAT positive neurons of the laterodor- renergic neurons outside of the locus ceruleus, including sal tegmental nucleus were consistently found lateral to the medially in the central gray. It is of considerable interest that TH positive neurons of the dorsal raphe nucleus (Fig. 4C). these dendrites are TH and DBH immunoreactive. Based Caudal to the isthmus, ChAT and TH/DBH positive upon similar observations, Bjorklund and Lindvall [6] hy- neurons were segregated within the central gray, with ChAT pothesized that the dendrites of substantia nigra pars com- positive neurons being found within the laterodorsal tegmen- pacta neurons, which extend ventrally for a considerable tal nucleus just medial to the THiDBH positive neurons of distance into , might release dopamine and this the locus ceruleus proper (Fig. 5A,B). However, within the has been demonstrated both in vitro [ 151and in viva [ 141. It is medial parabrachial nucleus, ChAT positive neurons were possible that the dendrites of locus ceruleus neurons release co-distributed with TH/DBH positive neurons. This phe- noradrenaline; this is supported by ultrastructural evidence nomenon was best appreciated in sections doubly stained for of presynaptic profiles on the dendrites of rodent locus both ChAT and TH (Fig. 2). Many of the small spindle- ceruleus neurons making dendro-dendritic contacts [ 16,591. shaped ChAT immunoreactive neurons were also inter- The spread of these dendrites into regions containing spersed among the THlDBH positive neurons and magnocel- cholinergic neurons raises the possibility of multiple mech- lular ChAT positive neurons of the medial and lateral para- anisms for reciprocal innervation between these cholinergic brachial nuclei (Figs. 3C, 5A,B). The dendrites of THiDRH and noradrenergic neurons. positive neurons of the locus ceruleus complex were ob- served to extend for long distances, including into the laterodorsal tegmental nucleus (Figs. !A,B, 2). ~ontomesencepha1ic tegmental neurons have long been thought to be involved in the phenomono~ogy of behavioral DISCUSSION state control. An early and influential finding was Moruzzi The major finding of the present study is the unequivocal and Magoun’s [40] demonstration that desynchronization of demonstration that noradrenergic and cholinergic neurons the cortical electroencephalogram could be evoked by elec- are extensively intermixed in the feline pontomesencephalic trical stimulation of the feline brainstem reticular formation. tegmentum. Previous studies using simultaneous acetyl- Although widespread areas of the brainstem were imph- cholinesterase histochemistry and catecholamine histo- cated, the most effective region was centered in the mid- fluorescence [30] had suggested that this might be the case in brain, essentially coincident with the pedunculopontine nu- the cat brain. The present data clearly demonstrate that this cleus. Shute and Lewis [6Oj, using acetylcholinesterase his- is so and provide details regarding the topographic relation- tochemistry in the rat, were the first to suggest that this ships of these cell groups. so-called ascending reticular activating system might origi- The present data also provide evidence for the existence nate with cholinergic neurons. The present data demonstrate of two types of chohnergic neurons in the feline dorsolateral the existence ofjust such a population of chohnergic neurons tegmentum. In addition to the magnocellular neurons of the in the feline brainstem reticular formation. Fu~hermore, pedunculopontine and Iaterodorsal tegmental nucleus, which anatomical and immunohistochemical studies have con- appear to comprise the feline homologue of the caudal firmed that at least in the rat, pontomesencephalic choliner- cholinergic column in the rat and primate [53-55, 71-731, a gic neurons have ascending projections consistent with this distinct population of small neurons displaying ChAT im- purported physiological role. In particular, brainstem munoreactivity was seen in the medial and lateral parabra- cholinergic neurons project directly to limited portions of the chial nuclei. Similar neurons have not been reported in other 152,723, to midline thalamic nuclei [56,621,as species. That these small spindle-shaped ChAT positive well as to the cholinergic basa1 forebrain [SS]. An important neurons comprise a separate population is suggested by both experimental question is to determine the extent to which their distinct morphology and the absence of NADPH thalamic 1631and basal forebrain [64] mechanisms contribute diaphorase staining of their somata. These ChAT positive to the phenomenon of EEG desynchronization produced by cells were also found to be extensively intermixed with brainstem reticular stimulation. TH/DBH positive cells in the parabrachial nuclei. Electrolytic iesion of the dorsolaterai pontine tegmentum Several lines of evidence suggest that the noradrenergic selectively abolishes the muscular atonia characteristic of neurons of the locus ceruleus may receive a cholinergic in- paradoxical sleep [20,29]. Based upon these and other data, put. Thus, these neurons contain acetylcholinesterase [l, 4, Jouvet 127,281 hypothesized that the noradrenergic neurons 32,551, possess muscarinic receptors [48], and are excited by of the caudal locus ceruleus complex were ‘“executive” ele- acetylcholine [5, 13, 18, 661. The proximity of the caudal ments in the generation of atonia during paradoxical sleep. In cholinergic column to the locus ceruleus in the rat [30] and partial support of this hypothesis, Jones rt al. [25] found that primate [S5] suggests that the presumptive cholinergic inner- large locus ceruleus complex lesions which significantly re- vation of noradrenergic neurons might arise from these duced cerebral noradrenaline content eliminated the atonia ChAT immunoreactive neurons. In the cat, this possibility is of paradoxical sleep. However, they concluded that the ef- amplified by the fact that these neurochemicaily distinct fect was not dependent upon noradrenergic neurons prr se neurons are intermingled within the dorsolateral pontine since 6-hydroxydopamine lesions did not affect the atonia of tegmentum. paradoxical sleep to any appreciable extent [33]. The present The present results also raise the possibility of a norad- finding of cholinergic neurons within the locus ceruieus renergic input to the chohnergic neurons of the laterodorsal complex suggests another cell group which might be impli- tegmental nucleus. Dendrites of TH positive neurons in the cated in the atonia of paradoxical sleep. Indeed, Hendricks medial parabrachial nucleus were observed extending up rf af. [I91 have demonstrated that much smaller electrolytic 712 REINER AND VINCENT

lesions of the region just mediai to the locus ceruleus com- there exists a subpopulation of neurons whose members ex- plex was most efficacious in producing the phenomenon. hibit stereotypical variation in their mean and absolute Jis- These lesions essentially coincide with the distribution of charge rate across behavioral states when recorded in that ChAT positive neurons in the Iaterodorsal tegmental and unanesthetized cat, falling silent during paradoxical sleep (caudal) pedunculopontine nuclei. Using retrograde tracing 121, 22, 43, 46, 49, 501. Although it has long been suspected techniques [51] it has been shown that neurochemically un- that these so-called PS-off cells are noradrenergic, the dif- identified neurons in this region project to the lower fuse distribution of noradrenergic neurons in the feline locus brainstem, including the so-called medullary inhibitory area ceruleus complex has made definitive identification difficult. of Magoun and Rhines 1361. Taken together, these observa- The results of the present study add to this c~~mplication by tions suggest that cholinergic neurons of the pontomesence- demonstrating the extensive intermingling of cholinergic phalic tegmentum might be involved in the active generation neurons with noradrenergic neurons within the locus of muscular atonia during paradoxical sleep. ceruleus complex. However. the observations that neurons Hobson et al. [22] suggested that reciprocal connections in the homogeneously noradrenergic locus ceruleus of the rat between ‘cholinergic’ neurons of the gigantocellular tegmen- also exhibit the PS-off phenemenon [3], and that PS-off cells tal field and noradrenergic neurons of the locus ceruleus in the feline locus ceruleus complex are inhibited by sys- complex might be responsible for the cyclical alteration of temic administration of clonidine [45], provide strong evi- the sleep-wake cycle. This controversial hypothesis was dence that locus ceruleus complex PS-off cells are in fact supported by the findings of Kimura c’t trl. [3 11, who reported noradrenergic. the presence of choIinergic/cholinoceptive neurons in the The physiological properties of brainstem cholinergic gigantocellular tegmental field. Although our anatomical neurons arc unknown. The present data provide the evidence in fact supports the notion of reciprocal anatomical detail required to examine this issue. The evi- cholinergic-noradrenergi~ interactions (see above), we found dence implicating brainstem cholinergic neurons in sleep re- no evidence for ChAT immunorea~tive neurons in the gigan- lated phenomena suggests that studies of the behavioral neu- tocellular tegmental field 1701 using a well characterized rophysiology of these neurons wilt significantly further our monoclonal antibody [12]. These observations, in concert understanding of the cellular and biochemicai control of be- with previous physiological data which challenged funda- havioral state. mental aspects of the hypothesis [61], provide strong evi- After submission of this manuscript, a report has ap- dence that the original reciprocal interaction hypothesis was peared by Jones and Beaudet (J Camp NPUIU~ 261: 1.5-32, incorrect in several important respects. However. it is in- 1987) who found a similar distribution of cholinergic and triguing to note that a small number of neurons with the ~ate~hoIaminergic neurons in the feline pontomesencephali~ precise state-specific firing patterns originally predicted for tegmentum using slightly different techniques. cholinergic neurons by this hypothesis, namely silent during all states but paradoxical sleep, have been recorded within the medial parabrachial nucleus of the unanesthetized cat [44,5I]. It would be premature to suggest that such neurons are cholinergic: this remains a chalIenging question for future ACKNOWLEDGEMENT study. The authors thank Dr. Felix Eckenstein for the generous gift of The most enduring and robust observation implicating ChAT antibody. Supported by the Medical Research Council of pontomesencephalic tegmental neurons in sleep cycle con- Canada and the British Columbia Health Care Research Foundation. trol is the finding that within the locus ceruleus complex, P.B.R. is a Fellow and S.R.V. a Scholar of the MRC.

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