single cholinergic mesopontine tegmental neurons...

Neuroscience Vol. 38, No. 3, pp. 643-654, 1990 Printed in Great Britain

0306-4522/90 $3.00 + 0.00 Pergamon Press plc

0 1990 IBRO

SINGLE CHOLINERGIC MESOPONTINE TEGMENTAL NEURONS PROJECT TO BOTH THE PONTINE

RETICULAR FORMATION AND THE THALAMUS IN THE RAT

K. SEMBA,*~~ P. B. I&I=* and H. C. FIBIGER*

*Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T IWS

tDepartment of Anatomy, Dalhousie University, Halifax, NS, Canada B3H 4H7

Abstract-Microinjections of the cholinergic agonist carbachol into a caudal part of the pontine reticular formation of the rat induce a rapid eye movement sleep-like state. This carbachol-sensitive region of the pontine reticular formation is innervated by choline@ neurons in the pedunculopontine and laterodorsol @mental nuclei. The same population of cholinergic neurons also project heavily to the thalamus, where there is good evidence that acetylcholine facilitates sensory transmission and blocks rhythmic thalamocortical activity. The present study was undertaken to examine the degree to which single choline@ neurons in the mesopontine tegmentum project to both the carbachol-sensitive region of the pontine reticular formation and the thalamus, by combining double fluorescent retrograde tracing and immuno- fluorescence with a monoclonal antibody to choline acetyltransferase in the rat.

The results indicated that a subpopulation (5-21% ipsilaterally) of cholinergic neurons in the mesopontine tegmentum projects to both the thalamus and the carbachol-sensitive site of the pontine reticular formation, and these neurons represented the majority (45--@3%) of choline& neurons projecting to the pontine reticular formation site. The percentage of cholinergic neurons with dual projections was higher in the pedunculopontine @mental nucleus (627%) than in the laterodorsal tegmental nucleus (4-I 1%). In addition, mixed with cholinergic neurons in the mesopontine tegmentum, there was a small population of dually projecting neurons that did not appear to be cholinergic. Mesopontine cholinergic neurons with dual projections may simultaneously modulate neuronal activity in the pontine reticular formation and the thalamus, and thereby have the potential of concurrently regulating different aspects of rapid eye movement sleep.

The presence of an ascending cholinergic pathway arising from the midbrain and pons was first suggested by Shute and Lewis? in 1967 on the basis of acetylcholinesterase histochemistry combined with lesions. These findings have recently been confirmed and extended by using immunohistochemistry with antibodies to choline acetyltransferase (CUT), a specific marker for cholinergic neurons, combined with retrograde tracing. 13~14,43*45 The ascending cholinergic projection to the thalamus has received much attention because of evidence indicating that acetylcholine has roles in the blockade of rhythmic thalamocortical activity and facilitation of sensory transmission.27~“~5s Recent studies using in vitro slice preparations have begun to unravel ionic mechanisms underlying these effects in relation to

$To whom correspondence should be addressed at: Depart- ment of Anatomy, Dalhousie University, Halifax, NS, Canada B3H 4H7.

Abbreviations: ChAT, choline acetyltransferase; FG, fluorogold; LDT, laterodorsal @mental nucleus; PGO, ponto-geniculo-occipitah PI, propidium iodide; PPT, pedunculopontine tcgmental nucleus; PRF, pontine reticular formation; REM, rapid eye movement; WGA-HRP, wheat germ agglutinin conjugated with horseradish peroxide.

the intrinsic membrane properties of thalamic neurons.27-30

Cholinergic projections arising from the mesopontine tegmentum are by no means limited to ascending pathways.45 Descending choline@ pathways also exist,4’a and there is good evidence that one such pathway is involved in the initiation of rapid eye movement (REM) sleep. Pharmacological data suggest that choline@ activation of a certain region of the pontine reticular formation (PRF) induces a REM sleep-like state accompanying muscle atonia, cortical EEG desynchronization and ponto-geniculo- occipital (PGG) spikes. ‘2*“*32*49 This region lies medial to the motor trigeminal nucleus, and includes a medial part of the nucleus subceruleus (this area is often referred to as the lateral tegmental field,*O although the nucleus subceruleus is not always re- garded as part of it,‘& as well as a lateral part of the gigantocellular tegmental field).“J4aJ”~3’~37 This region of the PRF contains moderate concentrations of muscarinic, mostly M2, receptors.4”s3 Kainate lesions in the cholinergic mesopontine tegmentum, which reduce the density of ChAT-immunoreactive fibres in both PRF and thalamus,2’ also markedly reduce the proportion of time spent in REM sleep in the cat63 (see also Refs 20, 33,42 and 62 for reviews).

643

644 K. SEMEA et al.

The crucial demonstration of cholinergic projections to the carbachol-sensitive PRF has recently been reported: this PRF region receives a cholinergic projection from the pedunculopontine (PPT) and laterodorsal (LDT) tegmental nuc1ei,39 and this descending pathway derives from IO-15% of cholinergic neurons in these nuclei in the cat.3’,48 Taken together, these findings are consistent with the notion that the activation of cholinergic input from the mesopontine tegmentum to the PRF is a key step in the initiation of REM sleep.

Perfusion

The above studies suggest that cholinergic neurons in the PPT and LDT are involved, on one hand, in REM sleep initiation through a descending projection, and on the other, through an ascending pathway, in the modulation of thalamocortical function and the blockade of rhythmic oscillatory activity during certain behavioural states. Do individual cholinergic neurons in the mesopontine tegmentum innervate both the carbachol-sensitive region of the PRF, and the thalamus? In the present study this question was investigated using a combination of double fluorescent retrograde tracing and ChAT- immunohistochemistry in the rat. Tracer injections in the PRF were aimed at the carbachol-sensitive site previously reported in rat” and cat.3’,49

Following survival times of 7-12 days, and up to 16 days in the three rats injected with FG alone, animals were deeply anaesthetized with sodium pentobarbital, and perfused transcardially, using 50 ml of 0.01 M phosphate-buffered saline (pH 7.4) at room temperature, followed by 3Ot-400ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C. Following perfusion, the brain was removed from the skull and postfixed in the same fixative for l-2 h at 4°C and then placed in a cryoprotectant solution containing 25% sucrose, 15% glycerol and 0.02% sodium azide in 0.05 M phosphate buffer for 2-4 days at 4°C. Brains were cut at 30pm on a freezing microtome into five sets of serial sections in 0.05 M Tris-buffered saline. One set was used to confirm injection sites in the thalamus and the medial pontine reticular formation, and a second set for additional immunohistochemistry as described below.

Immunqjhorescence

Animals EXPERIMENTAL PROCEDURES

Brainstem sections encompassing the PPT and LDT were immunohistochemically processed to visualize tegmental cholinergic neurons, using a rat monoclonal antibody against ChAT, the specificity of which has been previously documented in detail6 In animals with both FG and PI injections, a fluorescein isothiocyanate-conjugated anti-rat IgG raised in goat (Jackson Immunoresearch) was used as a secondary antibody; in three rats without PI injections, a secondary antibody conjugated with Texas Red was used. The two secondary antibodies produced the same results. The immunohistochemical procedure has been described in detail elsewhere.46

Thirteen male Wistar rats (Charles River) weighing 240-310 g were used.

Tracer injections

Animals were anaesthetized with sodium pentobarbital (SO mg/kg, i.p.) for microinjections of two fluorescent tracers. Fluorogold (FG, 4% in 0.9% sodium chloride, 0.05 ~1) was injected into the carbachol-sensitive sites in the PRF:“.“.49 AP = 9.4-9.6 mm from bregma, ML = 0.8-l .05, D = 9.1-9.3 from the surface of the skull. In 10 of the 13 rats, an additional two injections were made into the thalamus with propidium iodide (PI, 10% in distilled water, 0.2 ~1 each): (1) AP = 1.8, ML = 0.85-0.9, D = 5.5; and (2) AP = 3.1, ML = 0.9, D = 5.9, according to the brain atlas of Paxinos and Watson.36 All tracer injections were conducted ipsilaterally.

Following immunohistochemical processing, sections were mounted from 15% sucrose in 0.1 M phosphate buffer, air dried, coverslipped with low-fluorescence, microscopic immersion oil, and examined and photographed on a Leitz microscope. Distributions of single-, double- and triple- labelled neurons were plotted and counted using five sections matched to five standardized sections, at 1.7, I .2, 0.7, 0.2 and -0.3 mm from the interaural line, redrawn from Paxinos and Watson.Nx37 These five sections covered the entire extent of the cholinergic neurons in the mesopontine tegmentum. Cholinergic neurons in the mesopontine tegmentum are distributed in a continuum and the boundaries between the PPT and the LDT are not always well defined.45 For the purpose of data analyses, the LDT and PPT were defined as the parts of the mesopontine tegmentum populated with cholinergic neurons within and outside. respectively, of the central gray. ~~___ _-.-- -.. ~-

Abbreviations used in figures

AM APT AV CM DR DTg f Hb IC ic LC LD LG LP MD ml mlf Mo5

Z

anteromedial thalamic nucleus anterior pretectal area anteroventral thalamic nucleus central medial thalamic nucleus dorsal raphe nucleus dorsal tegmental nucleus of Gudden fornix habenula inferior colliculus internal capsule locus ceruleus laterodorsal thalamic nucleus lateral geniculate nucleus lateral posterior thalamic nucleus mediodorsal thalamic nucleus

PO postterior thalamic nucleus PC PnO Pr5 PY RMg rs Rt SC see sm so S5

VL VLL

pontine reticular formation pars caudalis pontine reticular formation pars oralis principal sensory trigeminal nucleus pyramidal tract raphe magnus rubrospinal tract reticular thalamic nucleus superior colliculus superior cerebellar peduncle stria medullaris superior olive sensory trigeminal nerve ventrolateral thalamic nucleus ventral nucleus of the lateral lemniscus

medial lemniscus medial longitudinal fasciculus motor trigeminal nucleus mammillothalamic tract parafascicular thalamic nucleus

VM ventromedial thalamic nucleus VP ventroposterior thalamic nucleus xscp decussation of the superior cerebellar peduncle ZI zona incerta 7n facial nerve

Dual projections of cholinergic tegmental neurons 645

RESULTS

The data described here are based on eight of the 13 rats, in which tracer injections were localized in and around the carbachol-sensitive site of the PRF and the thalamus, and in which ChAT immunohistochemistry was acceptable. Data analyses involved multiple counting and plotting of cells for three different fluorescent markers, singly as well as in various combinations. Although attempts were made to reduce fading of fluorescence to a minimum by frequent use of the shutter during examination as we11 as by improving coverslipping procedures (see Exper- imental Procedures), some fading was inevitable. This resulted in partial data in some rats, because only ctearly stained material was included in the analyses. One rat (No. 8) received no thalamic PI injections, and only labelling with FG and ChAT immuno- fluorescence was examined.

FG injections in the PRF in eight rats are shown in Fig. 1. The injection sites variably involved parts

of the dorsomedial tegmental area, the caudal pontine reticular nucleus and the dorsal part of the nucleus subceruleus (terminology according to Paxinos and Watson37). These injection sites corre- sponded well to the region of the PRF where microinjections of carbachol are known to induce a REM-like state.“x3’”

Minimal deposits of FG were seen in the immediate vicinity of the needle track in three of the eight rats. In rat No. 2, a faint deposit was seen in the most caudal part of the inferior colliculus, with minimum invasion of the cerebellum, and part of the locus ceruleus (Fig. 3). Similarly, in rat No. 3, deposits were found through a caudal part of the inferior colliculus and the cerebelIum. In rat No. 8, tracer diffusion was seen along the upper part of the needle track in the inferior colliculus and the dorsolateral part of the central gray. All these deposits along the needle track were fairly faint in intensity, similar to that seen in “halo” regions around the bright “core” of an injection site. Only in rat No. 2, a few ChAT-immunoreactive cells were seen close to the tracer deposit

Fig. 1. The locations of FG injections in the pontine recticular formation in eight rats. In (B)--(E) vertical hatching indicates the intensely bright “core” regions of individual injection sites in odd-nabered rats and horizontal hatching indicates those in even-numbered rats. (B) Rat Nos 1 and 2. (C) Rat Nos 3 and 4. (D) Rat Nos 5 and 6. (E) Rat Nos 7 and 8. The numbers in (A) indicate distances in mm from the

interaural line.

646 K. SEMBA er ai.

along needle track at the lateral aspect of the rats was diffusion from the deposit sufficiently close caudalmost LDT. Because it was felt impossible to or extensive to encroach ChAT-immunoreactive determine if FG labefling of these particular cells was neurons in the mesopontine tegmentum. due to damage, for example, of distal dendrites, or As iflustrated in Fig. 2, two thalamic PI injections retrograde label!ing from the injection site, these cells in each of eight rats consistently included the were excluded from the present analyses. In no other mediodorsai nucleus and the habenula, as well as

A I3 D E

Fig. 2. The locations of PI injections in the thalamus in Seven rats. In each rat, two injections were made. Only intensely labelled “core” regions of individual in&xXion sites are indicated by hatching. In (B)-(E), vertical hatching represents the injection sites in odd-numbered rats, and horizontal hatching represents those in even-numbered rats. (B) Rat Nos 1 and 2. (C) Rat Nos 3 and 4. (D) Rat Nos 5 and 6. (E) Rat No. 7. The injection sites of rat No. 8 were confhmed but the sections were subsequently lost. The numbers

in (A) indicate the distance in mm from the interaural line.

Dual pro~tions of cholinergic tegmental neurons 647

parts of the intralaminar nuclei. In addition, the injections included parts of the anterior, ventral and/or lateral cell groups of the thalamus. In most of the eight rats, injection sites also included a small part of the fimbria and/or hipp~ampus, dorsally, and part of the pretectal region, caudally.

Choline acetyltransferase-immunoreactive neurons projecting to zhe pontine reticular formation

At the level of the pons, neurons retrogradely labelled with FG from the PRF were distributed in both the rostra1 and the caudal parts of the reticular formation, the central gray, the superior colliculus, the cuneiform nucleus, the nucleus of the lateral lemniscus, and the parabrachial nuclei (Fig. 3). In the PPT/LDT ipsilateral to the injection, 1 l-22% (n = 4) of CbAT-immunoreactive neurons were retrogradely labelled with FG from the PRF (Table 1). The PPT contained about twice as great a percentage of FG-labelled, ChAT-immunoreactive neurons as that in the LDT in three rats (Nos 1, 2 and 8). In a fourth rat (No. 3), almost all double labelled neurons were found in the PPT, with only a few in the LDT. The FG injection in this rat was placed slightly more caudal compared with the other three cases.

Contralaterally, ChAT-immunoreactive neurons labelled with FG from the PRF represented 6-16% of all ChAT-immunoreactive neurons, a smaller percentage compared to the ipsilateral side (Table 1). The ratios between the PPT and LDT were similar to those found on the ipsilateral side, with higher percentages in the PPT (8-18%) than the LDT (l-12%).

Choline acetyltransferase-immunoreactive neurons projecting to the thalamus

Mesopontine tegmental neurons retrogradely labelled with PI from the thalamus accounted for 27-33% of the ChAT-immunoreactive neurons ipsilaterally, and S-15% contralaterally (n = 3, Table 1). The percentages of these double labelled neurons in the PPT (30-46%) were greater in the PPT than the LDT (19-26%) ipsilaterally; contralaterally the percentages were similar (6-14% and 13-16% for the PPT and the LDT, respectively).

Choline acetyltransferase-immunoreactive neurons projecting to both pontine reticular formation and thalamus

ChAT-immunoreactive PPT/LDT neurons that were retrogradely labelled from both PRF and thalamus represented 5-21% of the total ChAT- immunoreactive cell populations in the ipsilateral mesopontine tegmentum, whereas they represented l-14% on the contralateral side (n = 5, Table 1). Again, higher percentages of these triple labelled neurons were seen in the PFT (6-27%) than the LDT (3-11%) ipsilatecally. This was also the case contralaterally (2-24% vs O-4%). The triple labelled neurons accounted for 4588% of ChAT-immune- reactive neurons retrogradely labelled from the PRF

(n =: 3). Examples of triple labelled neurons are shown in Fig. 4.

Cholinergic neurons in the mesopontine tegmen- turn are distributed more or less in a continuum, constituting the PPT rostrally and the LDT caudaily, with the two nuclei co-existent at intermediate to caudal levels. However, the boundaries between the PPT and the LDT are not always obvious.4s To test the possibility that the aforementioned differences between the PPT and LDT in the percentages of double and triple labelled neurons are related to the Ievels along the Ion~tudinal axis, the two nuclei were combined, and percentages of triple labetled neurons were compared as a function of rostrocaudal levels. Five equally spaced sections were examined which covered the entire extent of cholinergic neurons in the tegmentum (see Experimental Procedures). The results indicated that while triple 1a~IIed neurons tended to be lower in percentage at the most caudal level in two rats (Nos 1 and 2), no obvious topography along the rostrocaudal axis was seen in the other rats (ranges for the five rostral to caudal levels in five rats; ipsilateral: O-29%, O-33%, 9-41%, 5-22%, O-20%; contralatera1: O-15%, O-20%, 2-20%, l-15%, O-8%).

Neurons retrogradely labelled from both the thalamus and PRF were rarely seen outside the PPT/LDT (Fig. 3). Occasionally, however, neurons that contained both of the retrograde markers, but lacked ChAT immuno~activity, were seen mixed with ChAT-immunoreactive neurons in the PPT] LDT. To examine the percentage of these dually projecting, but ChAT-negative PPT/LDT neurons, the PPT/LDT was first defined and demarcated in section drawings by the presence of ChAT-immunoreactive neurons. Then, neurons labelled with FG from the PRF within the demarcated PPT/LDT were examined for the presence of PI from the thalamus and ChAT immunoreactivity.

Of all PRF-projecting neurons in the PPT/LDT, 4-9% were retrogradely labelled from the ipsilateral thalamus with PI (n = 5, Table 1). Triple labelled neurons accounted for 2-6% of the total PRF- projecting cell population in the PPT/LDT. Thus, it could be derived that 50-100% of the dually projecting PPT/LDT neurons were ChAT-immunoreactive, and that O-50% were ChATnegative. ChAT- immunoreactive neurons accounted for 3-l 1% of PRF-projecting neurons in the same regions (n = 6). Generally lower percentages were obtained contralaterally for comparable cell populations.

DISCUSSION

The choline@ neurons in the PPT and LDT have previously been shown to give rise to a major ascending projection to the thalamus, and an apparently smaller, descending projection to the carbachol- sensitive PRF region (see the introduction for references). In the present study, it was shown that: (1) a

648 K. SEMBA et al.

-0.8

- PRF A ChAT l PF+F+ChAT . PRF+Thalamue 0 ChAT+Thaiamus * ~+~~+C~T

Fig. 3. An example (rat No. 2) of distributions of single, double and triple labelled neurons with retrograde labelhng with FG from the PRF and/or with PI from the thalamus, and ChAT immunoreactivity. The numbers indicate distances in mm from the interaural line. Each symbol represents one cell, except for FG-labelled and ChATpositive neurons for which one symbol represents five cells. Stippling

indicates the injection site.


Table I. Percentages of double and triple labelled neurons in the pedunculopontine tegmental nucleus/laterodorsal tegmental nucleus following fluorogold injections into the carbachol-sensitive region of the pontine reticular formation and propidium iodide injections into the thalamus, and immunohistochemistry for choline acetyltransferase in

eight rats

%CbAT+ neurons %PRF-proj~ting neurons

Projecting to Projecting to Projecting to Projecting to PRF and Projecting to thalamus and

Rat PRF thalamus thalamus thalamus ChAT+ ChAT+

Ipsilateral 1 11(13/7) 2 22 (27113) 3 17(31/l) 4 - 5 - 6 -

: &26,!14)

Contralateral

: 1~~~~) 3 7 (9/5) 4 -

27 (32/20) 29 (30/26) 33 (46/19) -

- - -

15 (14/E) 8 (6/13)

14(14/14) - - - - -

5 (6/4) 14 (19/4) 15 (25/3) 21(27/l 1) 13 (18/Q

1 fwv 5 W4) 5 (7/3)

14 (2412) 7 (12/3)

-

7 6 9 - - 6 4

-

4 5 6

- 3 2

-

11 9 7

- -

5 3

10

8 11 4

-

3 2

16

5 6 6

- - 4 2

-

2 5 3

- - 2 1

-

Tbe numbers in parentheses indicate respective numbers for the PPT and LDT for ChATpositive neurons. PRF-projecting neurons were not further divided because of relatively small percentages.

subpopulation of cholinergic tegmental neurons project to both the carbachol-~nsitive PRF site and the thalamus; (2) a majority of cholinergic tegmental neurons projecting to the PRF site also innervate the thalamus; (3) the majority of dually projecting neurons in the PPT/LDT regions are cholinergic; and (4) the number of cholinergic tegmental neurons projecting to the carbachol-sensitive PRF region appears to be smaller than that of those projecting to the thalamus.

The percentage of cholinergic PPT/LDT neurons projecting to the carbachol-sensitive PRF region ranged from 11 to 22%. The variability most likely reflects differences in the size of injections as well as the location of the injections in both the thalamus and the PRF; different degrees of fading of fluorescent markers might also have contributed to it. The present range of PRF-projecting cholinergic neurons is slightly higher than those previously reported, 10-1S??48 and S-IO%,” which were both based on wheat germ a~lutinin conjugated with horseradish peroxidase (WGA-HRP) retrograde transport combined with ChAT immunohistochemistry in the cat. The major difference, however, lies in the topography of PRF-projecting cholinergic neurons. In both previous studies using the cat, lower percentages of PRF- projecting choline@ neurons were found in the PPT compared with the LDT (10% vs 15% by Shiromani et a1.,4a and means of 5.2% vs 10.2% by Mitani et

a1.).3’ In contradistinction, in the present study using the rat, cholinergic neurons innervating the PRF appeared to be more frequent in the PPT (13-31%,

mean = 24%) than in the LDT (l-14%, mean = 9%, Table 1). The difference appears to derive primarily from the higher percentages in the PPT in the present, as compared to the previous studies; in the LDT the percentages are rather similar between the present and the previous studies. One possibility is that this is due to a species difference in the organization of mesopontine cholinergic projections. Thalamic projections from the meso~n~ne cholinergic neurons appear to be generally similar between rat and cat (see review by Semba and Fibiger).45

A second possibility, which is not mutually exclu- sive with the first, is that the difference is due to different tracers used, WGA-HRP vs FG, and related problems of uptake by ftbres of passage. This is potentially relevant because cholinergic neurons in the mesopontine tegmentum, predominantly those in the PPT (reportedly about 18% of the cholinergic cell population therein), have been shown to project caudally to innervate the medullary reticular formation in the rat,4ie*65 and one major pathway for this projection is identified as the ventromedial branch of Probst’s tract,4’a which apparently courses near the carbachol-sensitive PRF region. Although collateralization of cholinergic axons in the medulla has been suggested to be considerable,4’” that between the medullary and the pontine reticular formation remains to be investigated. Depending on the degree of this particular pattern of collateralization, the uptake by fibres of passage, if there were any, should or should not affect the number of cells which would be labelled by tracer injections into the carbachol-

650 I(. SEMBA et d.

Fig. 4. Examples of neurons (arrows) in the PPT which are retrogradely Iabelled from the PRF with FG (A), ChAT-immunoreactive (B) and retrogradely labelled from the thalamus with PI (C). An example of a neuron which is retrogradely labelled from the thalamus and immunoreactive for ChAT, but not labelled from the PRF, is indicated with arrowheads. In this particular set of micrographs, almost all PI-labelled

neurons appear to be ChAT-immunoreactive. Scale bar = JO pm.

Dual projections of choline@ tegmental neurons 651

sensitive PRF site. It remains to be clarified whether the difference in the topography of PRF-projecting neurons between the present and the previous studies is due to different species or the type of tracers used.

Twenty-seven to thirty-three per cent of all ChAT- immunoreactive neurons in the PPT/LDT were found to project to the thalamus ipsilaterally, and 8-15% contralaterally. These ranges are most likely underestimated because tracer injections did not in- volve all the thalamic nuclei. The present percentages are lower than those previously reported following large HRP injections into the thalamus (about 60%),” but comparable to the ranges of l-26% for the PPT and 3-47% for the LDT following WGA-HRP injections into selected thalamic nuclei in the rat (quoted by Mitani et a1.,31 as obtained from one of the authors from the data of Hallanger et al.13). Similar results may be seen in the ill~tra~ons of comparable data obtained from rat” and cat.“s1+s6 The variation seen in the present and previous studies most likely reflects differences in the size of tracer injections, as well as the tracer used. An additional contributing factor would be the regional differences in the density of choline& fibres within the thalamus.26

Because in the present study the tracer injections in the thalamus always involved several thalamic nuclei, a question remains as to whether the axons of these doubly projecting neurons terminate selectively within the thalamus. This would be an interesting question in relation to the role of this projection in the state-dependent regulation of thalamocortical activity (see below). The key observation relevant to this question is the previous report that cholinergic neurons in the PPT project to all thalamic nuclei, whereas those in the LDT innervate predominantly limbic parts of the thalamus.‘3*U~4s*S6 Because ChAT- positive neurons with dual projections were more frequent in the PPT than in the LDT in the present study, it is likely that doubly projecting choline@ neurons, as a cell population, innervate all thalamic nuclei. This hypothesis, however, remains to be tested directly.

The present study had added to the evidence of axonal collateralization of choline@ mesopontine tegmental neurons. In our previous study using the rat, about 10% of the neurons in the mesopontine tegmentum that were retrogradely labelled from the reticular thalamic nucleus were also labelled from the cortex, these being located mostly in the PPT; many of these dual projection neurons were also ChAT- immunoreactive.22 There is also some evidence to suggest that the collateralization of cholinergic fibres occurs within the thalamu? and between other forebrain regions.@ Axonal ~llaterali~tion of tegmentai cholinergic neurons involving the innervation of the basal ganglia has also been reported;44@ however, the cholinergic projections to the extrapyramidal system have been controversial. 4s Collateralization within medullary regions have been suggested for descend-

ing cholinergic fibres. 4ia Collateral projections to the posterior thalamus and the raphe magnus have recently been reported.6s Taken together, these findings indicate that the axons of cholinergic neurons in the m~opontine te~ent~ do collateralize to innervate different target structures, although apparently not as extensively as reported with monoaminergic axons.’

One important implication of the present findings is that some cholinergic neurons may simultaneously intluence postsynaptic neurons in the carbachol- sensitive PRF region and the thalamus. Thus, these neurons may be involved in both the initiation of REM sleep, through descending projections, and blockade of rhythmic thalamocortical activity and modulation of sensory transmission, via ascending projections. Thalamocortical oscillation is blocked in both waking (except during ~-spindles3,47) and REM sleep. One possibility, therefore, is that these dual projection cholinergic neurons are involved in the blockade of rhythmic thalamocortical activity during REM sleep but not during waking. Using chronic single unit recordings from neurons in the LDT in the cat, El Mansari et ai.’ described a sub~pulation (12%) of LDT neurons which were selectively active during REM sleep but not during waking. The same authors also reported on additional subpopulations of LDT neurons which became active during both waking and REM sleep or during waking alone. Thus, it is possible that the dual projection neurons in the present study correspond to those LDT neurons that fire only during REM sleep.

An alternative hypothesis is that mesopontine tegmental cholinergic neurons projecting to both thalamus and PRF are active during waking and REM sleep. According to this scenario, additional neuronal concomitants during REM sleep (but not during waking) permit the cholinergic input to evoIve the set of behaviours seen with pharmacological application of cholinergic agonists to the PRF. In particular, it is well documented that noradrenergic, serotonergic and histaminergic neurons are all active during waking but cease firing during REM ~leep,‘~~““* and these three transmitters have been shown to affect neurons in the carbachol-sensitive region of the PRF,9J0,s8 To the cholinergic agonist carbachol, PRF neurons respond with depolarization with a decrease in conductance,i2 which parallels the membrane potential changes seen in natural REM sleep.‘* Thus, it would be considerable interest to examine the response of PRF neurons to acetylcholine in the presence and absence of one or more of these amine transmitters.

The significance of the mesopontine cholinergic input in thalamocortical functions has begun to be understood at the cellular level using both in uim ‘s~‘7~23~24*5s and in vitru preparations.27-M These studies support the notion that with differential effects on principal relay neurons and interneurons, acetylcholine has an overall facilitatory effect on sensory transmission along with blockade of

652 K. SEMBA et al.

rhythmic oscillation. Additionally, the thalamic reticular neurons, through their widespread yet topo- graphically organized GABAergic projections to other thalamic nuclei, have an important role in regulating rhythmic thalamocortical activity.54,S5 The effects of acetylcholine on thalamic reticular neurons, however, are at present controversial.‘5.23,28 Further- more. the activity of thalamic reticular neurons, in turn, appears to be regulated by an additional cholin- ergic13.22.25.35.5l and a GABAergic?’ input from the basal forebrain, which has been suggested to play a role in cortical EEG desynchronization.3 In the basal forebrain of the cat, both waking/REM sleep-active and slow wave sleepactive neurons have been recorded.5.5y~6” Finally, in addition to the cholinergic

innervation, the thalamus receives aminergic inputs as well.27,55 Thus, again, it would be important to investigate the effects of co-activation of multiple inputs in thalamic neurons. The functional significance of the present findings of axonal collateralization of some tegmental cholinergic neurons would be best understood in view of the data regarding all these cellular events which, in concert, regulate state- dependent thalamocortical rhythmicity and sensory transmission.

Acknowledgements-We thank Felix Eckenstein for a gener- ous gift of monoclonal antibody to ChAT, and excellent technical assistance by Chui-Se Tham. This work was supported by the Medical Research Council of Canada. KS. and P.B.R. are MRC Scholars.

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(Accepted 10 May 1990)

single cholinergic mesopontine tegmental neurons...

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