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 thalamo- cortical 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 cholin- ergic projection to the thalamus has received much attention because of evidence indicating that acetyl- choline 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 mesopon- tine 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