A photographic perspective on the origins, form, course and relations of the acetylcholinesterase-containing fibres of the dorsal tegmental pathway in the rat brain

The dorsal tegmental pathway in the rat brain has been studied using acetylcholinesterase (AChe) staining alone, after lesions, and combined with the horseradish-peroxidase (HRP) tracing method. This paper characterises in photographs, diagrams and text the origins, form, extent and relations of its visible AChe-staining fibres in 3 planes. This record should provide a template for further investigations. The pathway largely takes origin from ChAT-containing pedunculopontine (PPTg) and laterodorsal (LDT) nuclei; some non-cholinergic cell groups may also contribute, notably locus coeruleus (LC). It takes the form of a horizontally disposed fan which radiates from the pontomesencephalic area to the forebrain. Its lateral portion is bunched and consists mainly of cholinergic fibres whereas the cholinergic status of its fully unfurled intermediate and partly unfurled medial contingents (which mainly accompany the central tegmental tract) is more doubtful. The changing form and relations of PPTg and LDT are adumbrated including that of the microcellular nucleus (MI) to the former and of Barrington's detrusor nucleus (B) which is unstained, to the latter. Functional overlapping between non-cholinergic and cholinergic nuclei in the peribrachial region are noted and some correlations adduced.     

The limbic circuitry underlying cocaine seeking encompasses the PPTg/LDT

The direct glutamatergic projection from the medial prefrontal cortex (mPFC) to the nucleus accumbens plays a critical role in mediating the reinstatement of cocaine seeking behavior. The mPFC also sends glutamatergic projections to the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDT), which in turn send glutamatergic and cholinergic efferents to the ventral tegmental area (VTA) where they synapse on dopaminergic cells that innervate limbic structures including the nucleus accumbens. The goal of these experiments was to examine a potential role for the PPTg/LDT in the reinstatement of cocaine seeking. All rats were trained to self-administer cocaine (0.25 mg, i.v.) on a fixed-ratio 5 schedule of reinforcement. Cocaine self-administration behavior was extinguished and a series of subsequent pharmacological experiments were performed to assess the potential role of the mPFC, PPTg/LDT and VTA in the reinstatement of cocaine seeking. Administration of the D1-like dopamine receptor agonist SKF-81297 (1.0 μg) directly into the mPFC produced a small, but statistically significant, increase in cocaine seeking behavior. Furthermore, microinjection of the ionotropic glutamate receptor antagonist CNQX (0.3 μg) into the PPTg/LDT attenuated the reinstatement of drug seeking induced by a priming injection of cocaine (10 mg/kg, i.p.). Intra-VTA administration of CNQX, the nicotinic receptor antagonist mecamylamine (10.0 μg) or the muscarinic receptor antagonist scopolamine (24.0 μg) also blocked cocaine seeking. Taken together, these results suggest that cocaine priming-induced reinstatement of drug seeking is mediated in part by a serial polysynaptic limbic subcircuit encompassing the mPFC, PPTg/LDT and VTA.     

Pontine cholinergic neurons simultaneously innervate two thalamic targets

Cholinergic neurons located in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei have been shown to principally innervate the thalamus. In order to determine whether some of these neurons might simultaneously project to two thalamic targets we made microinjections of rhodamine-conjugated microbeads into the central-lateral nucleus of the thalamus and fluorescein isothiocyanate (FITC)-conjugated microbeads into the dorso-lateral geniculate nucleus. We then determined whether both tracers were found in immunohistochemically identified cholinergic somata in the LDT and PPT nuclei. Results showed that some cholinergic and non-cholinergic neurons in the LDT and PPT nuclei projected to both thalamic sites. This finding extends our understanding of the projections of the LDT-PPT cholinergic neurons and further supports the role of these neurons in complex behaviors.     

Effects of orexin on the laterodorsal tegmental neurones

Orexin, a hypothalamic neuropeptide, has been revealed to be involved in sleep regulation. To elucidate functions of orexin in brainstem sleep regulation mechanism, we examined the effects of orexin applied from micropipettes with air pressure on neurones in and around the laterodorsal tegmental nucleus (LDT). In five of seven cholinergic neurones and six of nine non-cholinergic neurones orexin induced long-lasting excitation. These results suggest that hypothalamic orexin neurones may affect the LDT neurones directly, and thereby participate in control of sleep.     

Opioid-induced rewards,locomotion, and dopamine activation: A proposed model for control by mesopontine and rostromedial tegmental neurons

Opioids, such as morphine or heroin, increase forebrain dopamine (DA) release and locomotion, and support the acquisition of conditioned place preference (CPP) or self-administration. The most sensitive sites for these opioid effects in rodents are in the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg). Opioid inhibition of GABA neurons in these sites is hypothesized to lead to arousing and rewarding effects through disinhibition of VTA DA neurons. We review findings that the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei, which each contain cholinergic, GABAergic, and glutamatergic cells, are important for these effects. LDTg and/or PPTg cholinergic inputs to VTA mediate opioid-induced locomotion and DA activation via VTA M5 muscarinic receptors. LDTg and/or PPTg cholinergic inputs to RMTg also modulate opioid-induced locomotion. Lesions or inhibition of LDTg or PPTg neurons reduce morphine-induced increases in forebrain DA release, acquisition of morphine CPP or self-administration. We propose a circuit model that links VTA and RMTg GABA with LDTg and PPTg neurons critical for DA-dependent opioid effects in drug-naïve rodents.     

Excitotoxic lesions of the pedunculopontine tegmental nucleus of the rat. II. Examination of eating and drinking, rotation, and reaching and grasping following unilateral ibotenate or quinolinate lesions

The pedunculopontine tegmental nucleus (PPTg) contains a population of cholinergic neurons thought to be part of the ascending reticular activating system, and non-cholinergic neurons. In the previous study it was shown that various excitotoxins made effective lesions of cholinergic neurons in the PPTg but that quinolinate made smaller lesions in the non-cholinergic population, making it more selective than any other excitotoxin. The purpose of the present experiment was, first, to make lesions of cholinergic neurons throughout the length of the PPTg by infusing toxin at two different sites within it; and second, to examine simple motor activities in rats bearing either quinolinate or ibotenate lesions of the PPTg, and contrast these with the deficits seen after 6-hydroxydopamine (6-OHDA) induced lesions of mesostriatal dopamine (DA)-containing neurons. Post-mortem examination was carried out using choline acetyltransferase (ChAT) and tyrosine hydroxylase (TOH) immunohistochemistry, and routine Nissl staining. Both quinolinate and ibotenate destroyed approximately 75% of ChAT-positive neurons in the PPTg, but damage to non-cholinergic neurons (assessed by Nissl staining) was twice as great following ibotenate as quinolinate. 6-OHDA induced almost complete lesions of mesostriatal DA neurons, assessed by TOH immunohistochemistry. DA depleted rats showed deficits in drinking and spilled more food in the first 2 weeks after surgery, and were unable to reach or grasp food pellets in the staircase test. They also showed strong ipsilateral turning in response to amphetamine and contralateral turning to apomorphine. Quinolinate lesioned rats had no eating or drinking impairment in the home cage but showed a reaching (though not grasping) disability in the staircase test. They had a mild ipsilateral bias following amphetamine. Ibotenate lesioned rats, despite having larger lesions than the quinolinate, showed no deficits in eating or drinking in the home cage, or reaching or grasping disabilities in the staircase test. They did have a mild contralateral bias in response to amphetamine. This dissociation of the effects of quinolinate and ibotenate lesions of the PPTg is consistent with the suggestion that the PPTg has two functionally distinct components, and is attributed to the differential lesion of non-cholinergic neurons by the two excitotoxins.     

Neuroplasticity in cholinergic neurons of the laterodorsal tegmental nucleus contributes to the development of cocaine addiction

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus that sends cholinergic, glutamatergic, and gamma‐aminobutyric acid (GABA)‐ergic projections to the ventral tegmental area (VTA), a key brain region associated with reward information processing and reinforcement learning, and thus, with addiction induced by drugs of abuse, including cocaine. Recent studies have revealed that the LDT, in addition to the VTA, plays important roles in the development and expression of cocaine‐induced addiction and stress‐induced enhancement of addictive behaviors. Additionally, neuroplasticity induced in LDT cholinergic neurons by repeated cocaine administration critically contributes to these behaviors. Elucidation of the underlying mechanisms of cocaine‐induced neuroplasticity in the LDT that influences reward circuit activity may lead to the development of therapeutic strategies to treat cocaine addiction and stress‐induced reinstatement of cocaine use. This review summarizes recent progress in the study of the LDT, specifically neuroplasticity in LDT cholinergic neurons induced by cocaine and its functional roles in the development and modulation of addictive behaviors associated with cocaine.     

Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat

This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.     

Locomotor projections from the pedunculopontine nucleus to the medioventral medulla

Retrograde tracers were injected into the rat medioventral medulla (MED) and the injection site was identified as a locomotion- inducing area by electrical stimulation in the decerebrate preparation. Histological reconstructions showed that about 10% of cholinergic pedunculopontine (PPN) and laterodorsal tegmental (LDT) neurons project to the MED. Also, large numbers of non-cholinergic cells in and around the PPN and LDT were found to project to the MED.     

Nociceptive excited and inhibited neurons within the pedunculopontine tegmental nucleus and cuneiform nucleus

The pedunculopontine tegmental nucleus (PPTg) is defined by its collection of cholinergic neurons surrounding the lateral portion of the superior cerebellar peduncle at the midbrain pontine junction. Antinociceptive functions have been attributed to the PPTg since electrical stimulation as well as injection of cholinergic agonists in this area produces analgesia. Nociceptive neurons have also been reported in the vicinity of the PPTg and cuneiform nucleus (CN). However, specific histochemical localization of nociceptive modulatory neurons has not been determined. Thus, the goal of this study was to classify neurons according to their response to a noxious stimulus and map their location based on staining of the cholinergic neurons in the PPTg. Extracellular microelectrode recordings were conducted in 19 male Sprague-Dawley rats under light halothane anesthesia. For each neuron identified, a series of noxious tail pinches were administered. The electrode tracts were marked with ionophoresis of pontamine blue. The location of 112 recorded neurons was determined on sections stained with NADPH diaphorase to identify the cholinergic boundaries of the PPTg. Neurons were classified into one of three cell types based on their consistent response to a noxious tail pinch (excited, inhibited, and non-responsive). Tail pinch excited neurons (n=16), inhibited neurons (n=10) and non-responsive neurons (n=23) were mapped within the cholinergic boundaries of the PPTg. Excited (n=9), inhibited (n=10) and non-responsive neurons (n=10) were also found more dorsally within the cuneiform nucleus. Thus, this study localizes nociception-responsive neurons to the region of the largely cholinergic PPTg, as well as the noncholinergic cuneiform nucleus.     

Nocifensive reflex-related on- and off-cells in the pedunculopontine tegmental nucleus, cuneiform nucleus, and lateral dorsal tegmental nucleus

Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the rostral ventromedial medulla (RVM) have been implicated in nociceptive modulation. The goal of this study was to identify neurons with nocifensive reflex-related activity in the mesopontine tegmentum including the PPTg. This study used the same behavioral neurophysiological classification system to identify neurons as has been extensively described in the RVM. Extracellular microelectrode recording was conducted in lightly anesthetized rats. Changes in firing associated with the noxious heat-evoked tail flick reflex were used to classify neurons as "on-cells" (displayed a burst in neuronal activity associated with the reflex), "off-cells" (displayed a pause in activity), and neutral cells (showed no response). Of 188 neurons studied in 23 rats, 77 were classified as on-cells, 14 as off-cells, the remainder as neutral cells. Recordings during periods without noxious stimulation found that some of the on- and off-cells displayed spontaneous transitions between active and silent periods termed cell cycling. The distribution of on- and off-cells in the mesopontine tegmentum overlapped and included the cholinergic PPTg and lateral dorsal tegmental nucleus identified by NADPH diaphorase staining, as well as the cuneiform nucleus and periaqueductal gray. The mesopontine tegmentum thus contains nocifensive reflex-related neurons with neurophysiological characteristics similar to those reported in the RVM. Neurons showing reflex-related activity were frequently encountered in the cholinergic PPTg and LDTg. Further studies will be required to determine whether these neurons modulate nociception through a link to the RVM.     

Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness.

To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal tegmental nucleus (LDT) of undrugged rats, whose head was fixed painlessly, was recorded along with cortical EEG and neck EMG. Activity of some dorsal raphe (DR) neurons was also recorded for comparison. Most of the animals had been sleep-deprived for 24 h. Observation was made only on neurons generating broad spikes, presumed from previous studies to be cholinergic or monoaminergic. The position of recorded neurons was marked by Pontamine sky blue ejected from the glass pipette microelectrode, and was identified on sections processed for NADPH diaphorase histochemistry which specifically stained cholinergic neurons. According to their firing rates during wakefulness (AW), slow-wave sleep (SWS) and paradoxical sleep (PS), 46 broad-spike neurons in the LDT were classified into 4 groups: (1) neurons most active during AW and silent during PS (some of these neurons might be serotonergic rather than cholinergic, as all the 9 neurons in the DR); (2) neurons most active during PS and silent during AW; (3) neurons equally more active during AW and PS than SWS; and (4) others mainly characterized by transiently facilitated activity at awakening and/or onset of PS. Neurons of groups 2 and 3 were the major constituents of the LDT. In most neurons change in firing preceded EEG change, except at awakening from PS. These results suggest that: (1) the LDT is composed of cholinergic neurons with heterogenous characteristics in relation to sleep/wakefulness; and (2) some tegmental cholinergic neurons play a privotal role in induction and maintenance of PS.     

Firing of ‘possibly’ cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness

To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal tegmental nucleus (LDT) of undrugged rats, whose head was fixed painlessly, was recorded along with cortical EEG and neck EMG. Activity of some dorsal raphe (DR) neurons was also recorded for comparison. Most of the animals had been sleep-deprived for 24 h. Observation was made only on neurons generating broad spikes, presumed from previous studies to be cholinergic or monoaminergic. The position of recorded neurons was marked by Pontamine sky blue ejected from the glass pipette microelectrode, and was identified on sections processed for NADPH diaphorase histochemistry which specifically stained cholinergic neurons. According to their firing rates during wakefulness (AW), slow-wave sleep (SWS) and paradoxical sleep (PS), 46 broad-spike neurons in the LDT were classified into 4 groups: (1) neurons most active during AW and silent during PS (some of these neurons might be serotonergic rather than cholinergic, as all the 9 neurons in the DR); (2) neurons most active during PS and silent during AW; (3) neurons equally more active during AW and PS than SWS; and (4) others mainly characterized by transiently facilitated activity at awakening and/or onset of PS. Neurons of groups 2 and 3 were the major constituents of the LDT. In most neurons change in firing preceded EEG change, except at awakening from PS. These results suggest that: (1) the LDT is composed of cholinergic neurons with heterogenous characteristics in relation to sleep/wakefulness; and (2) some tegmental cholinergic neurons play a pivotal role in induction and maintenance of PS.     

Sound‐specific plasticity in the primary auditory cortex as induced by the cholinergic pedunculopontine tegmental nucleus

Brain cholinergic modulation is essential for learning‐induced plasticity of the auditory cortex. The pedunculopontine tegmental nucleus (PPTg) is an important cholinergic nucleus in the brainstem, and appears to be involved in learning and subcortical plasticity. This study confirms the involvement of the PPTg in the plasticity of the auditory cortex in mice. We show here that electrical stimulation of the PPTg paired with a tone induced drastic changes in the frequency tunings of auditory cortical neurons. Importantly, the changes in frequency tuning were highly specific to the frequency of the paired tone; the best frequency of auditory cortical neurons shifted towards the frequency of the paired tone. We further demonstrated that such frequency‐specific plasticity was largely eliminated by either thalamic or cortical application of the muscarinic acetylcholine receptor antagonist atropine. Our finding suggests that the PPTg significantly contributes to auditory cortical plasticity via the auditory thalamus and cholinergic basal forebrain.     

Cholinergic neurons of the feline pontomesencephalon. I. Essential role in ‘Wave A’ generation

Wave A in the cat appears to be analogous to P1 in the human. Both are positive middle-latency auditory-evoked potentials, present at slow click rates during wakefulness and REM sleep but absent during slow-wave sleep. Wave A has been recorded in the parabrachial and medial tegmental areas of the midbrain and in thalamic target projections of the reticular activating system. Two nuclei in this system, the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei, contain cholinergic cells; the cholinergic antagonist scopolamine eliminates Wave A. To test whether PPT and LDT were important in Wave A generation, we attempted to lesion these nuclei bilaterally in 11 cats. Wave A was markedly diminished or absent in all but 2 cats, in which the lesions did not include PPT. Loss of choline acetyltransferase-positive cells in PPT, but not LDT, was correlated with effects on Wave A, i.e. greatest cell loss occurred in cats in which Wave A disappeared, and least cell loss in cats with no change in Wave A. We conclude that the PPT nucleus, and particularly its cholinergic cell component, is essential for Wave A generation and suggests that a similar substrate may be significant for generation of the human P1.     

Intrinsic membrane plasticity via increased persistent sodium conductance of cholinergic neurons in the rat laterodorsal tegmental nucleus contributes to cocaine‐induced addictive behavior

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus implicated in reward processing and is one of the main sources of cholinergic afferents to the ventral tegmental area (VTA). Neuroplasticity in this structure may affect the excitability of VTA dopamine neurons and mesocorticolimbic circuitry. Here, we provide evidence that cocaine‐induced intrinsic membrane plasticity in LDT cholinergic neurons is involved in addictive behaviors. After repeated experimenter‐delivered cocaine exposure, ex vivo whole‐cell recordings obtained from LDT cholinergic neurons revealed an induction of intrinsic membrane plasticity in regular‐ but not burst‐type neurons, resulting in increased firing activity. Pharmacological examinations showed that increased riluzole‐sensitive persistent sodium currents, but not changes in Ca²⁺‐activated BK, SK or voltage‐dependent A‐type potassium conductance, mediated this plasticity. In addition, bilateral microinjection of riluzole into the LDT immediately before the test session in a cocaine‐induced conditioned place preference (CPP) paradigm inhibited the expression of cocaine‐induced CPP. These findings suggest that intrinsic membrane plasticity in LDT cholinergic neurons is causally involved in the development of cocaine‐induced addictive behaviors.     

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine‐seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath‐application of NA induced similar levels of hyperpolarization in both saline‐ and cocaine‐treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine‐ but not saline‐treated neurons through α2 adrenoceptors. This NA‐induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired‐pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.     

Cocaine exposure enhances excitatory synaptic drive to cholinergic neurons in the laterodorsal tegmental nucleus

Accumulating evidence indicates that the laterodorsal tegmental nucleus (LDT) is associated with reward processing and addiction. The cholinergic projection from the LDT to the ventral tegmental area is essential for a large dopamine release in the nucleus accumbens, which is critically involved in the reinforcing effects of addictive drugs, including cocaine. In contrast to the large number of studies on plasticity induced after cocaine exposure in the mesocorticolimbic dopaminergic system, it remains unknown whether LDT cholinergic neurons exhibit plastic changes following cocaine administration. To address this issue, we performed ex vivo whole‐cell recordings in LDT cholinergic neurons obtained from rats following cocaine administration. Neurons obtained from 1 day after 5‐day cocaine‐treated rats showed significantly smaller paired‐pulse ratios of evoked EPSCs and higher miniature EPSC frequencies than those from saline‐treated rats, indicating an induction of presynaptic plasticity of increased glutamate release. This plasticity seemed to recover after a 5‐day withdrawal from repeated cocaine exposure, and required NMDA receptor stimulation and nitric oxide production. Additionally, pharmacological suppression of activity of the medial prefrontal cortex inhibited the presynaptic plasticity in the LDT. On the other hand, AMPA/NMDA ratios were not different between saline‐ and cocaine‐treated groups, revealing an absence of postsynaptic plasticity. These findings provide the first direct evidence of cocaine‐induced synaptic plasticity in LDT cholinergic neurons and suggest that the presynaptic plasticity enhances the activity of LDT cholinergic neurons, contributing to the expression of cocaine‐induced addictive behaviors through the dysregulation of the mesocorticolimbic system.     

Pedunculopontine nucleus in the squirrel monkey: Distribution of cholinergic and monoaminergic neurons in the mesopontine tegmentum with evidence for the presence of glutamate in cholinergic neurons

The topographical relationships between cholinergic neurons, identified by their immuno-reactivity for choline acetyltransferase (ChAT) or their staining for β-nicotinamide ademine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, Nonadrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta(PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal toe the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occurnin these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are clolinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that (1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and (2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and Glutamate in primates. © 1994 Wiley-Liss, Inc.