Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high‐affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild‐type (WT) mice. This was region‐specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region‐specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up‐regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.     

Ascending projections from the pedunculopontine tegmental nucleus and the adjacent mesopontine tegmentum in the rat

Ascending projections from the pedunculopontine tegmental nucleus (PPT) and the surrounding mesopontine tegmentum to the forebrain in the rat are here examined by using both retrograde and anterograde tracing techniques combined with choline acetyltransferase (ChAT) immunohistochemistry. The anterogradely transported lectin Phaseolus vulgaris-leukoagglutinin (PHA-L) was iontophoretically injected into the PPT in 12 rats. Anterogradely labelled fibers and varicosities were observed in the thalamic nuclei, confirming the findings of our previous retrograde studies (Hallanger et al: J. Comp. Neurol. 262:105-124, '87). In addition, PHA-L-labelled fibers and varicosities suggestive of terminal fields were observed in the anterior, tuberal, and posterior lateral hypothalamic regions, the ventral pallidum in the region of the nucleus basalis of Meynert, the dorsal and intermediate lateral septal nuclei, and in the central and medial nuclei of the amygdala. To determine whether these were cholinergic projections, the retrograde tracer WGA-HRP was injected into terminal fields in the hypothalamus, septum, ventral pallidum, and amygdala. Numerous ChAT-immunoreactive neurons in the PPT and laterodorsal tegmental nucleus (LDT) were retrogradely labelled from the lateral hypothalamus. These cholinergic neurons constituted over 20% of those retrogradely labelled in the dorsolateral mesopontine tegmentum; the balance consisted of noncholinergic neurons of the central tegmental field, retrorubral field, and cuneiform nucleus. Following placement of WGA-HRP into dorsal and intermediate lateral septal regions, the vast majority (greater than 90%) of retrogradely labelled neurons were cholinergic neurons of the PPT and LDT, with few noncholinergic retrogradely labelled neurons in the adjacent tegmentum. In contrast, fewer cholinergic neurons were retrogradely labelled following placement of tracer into the nucleus basalis of Meynert or into the central, medial, and basolateral nuclei of the amygdala, while numerous noncholinergic neurons of the central tegmental field rostral to the PPT and of the retrorubral field adjacent to the PPT were retrogradely labelled in these cases. These anterograde and retrograde studies demonstrate that cholinergic PPT and LDT neurons provide a substantial proportion of mesopontine tegmental afferents to the hypothalamus and lateral septum, while projections to the nucleus basalis and the amygdala are minimal.     

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.     

Postnatal development of cholinergic neurons in the mesopontine tegmentum revealed by histochemistry

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) play a role in the regulation of several kinds of behavior. Some of them, such as locomotion, motor inhibition or sleep, show dramatic changes at a certain period of postnatal development. To understand the neural substrate for the development of these physiological functions, we studied the development of cholinergic neurons in the LDT and PPT of postnatal and adult rats using histochemical staining of NADPH-diaphorase (NADPH-d) and immunohistochemical staining of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). At postnatal day 1 (P1), ChAT- and VAChT-stained cells localized more dorsally than those of NADPH-d-stained cells, and at P7 their distributions became similar to those of NADPH-d-stained cells. The number of NADPH-d-stained cells increased rapidly after birth, reaching the adult level by P7. In contrast, the number of ChAT- and VAChT-stained cells and the intensity of their staining decreased from P1 to P3 and then increased through P21. The volume of the LDT increased during the second postnatal week. These findings indicate that cholinergic neurons in the LDT develop their cholinergic properties during the second postnatal week and mature functionally thereafter. We discuss these results in light of the several physiological functions regulated by the cholinergic neurons in the mesopontine tegmentum.     

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.     

Age-related changes in cholinergic neurons in the laterodorsal and the pedunculo-pontine tegmental nuclei of cats: a combined light and electron microscopic study

In aged cats, light microscopic studies revealed significant decrease in the soma size of choline acetyltransferase (ChAT)-positive neurons in the laterodorsal and pedunculo-pontine tegmental nuclei (LDT and PPT), compared with adult control animals. In addition, a significant reduction of the total dendritic length and total dendritic segment number of ChAT-positive neurons was detected in both the LDT and PPT of aged cats. However, in contrast to the changes of soma and dendrites, no significant changes in the number of ChAT-positive neurons in aged were found comparing to that in the control cats in both the LDT and PPT; nor were there differences in the staining intensity of the somata of neurons in the adult and aged cats. Electron microscopic analysis highlighted degenerative changes in cholinergic neurons in the LDT and PPT of aged cats which included somata with intracytoplasmic vacuoles, darkened mitochondria, depletion of dendritic microtubules and severe demyelination of axons. These data indicate that profound atrophic changes occur in cholinergic systems of the LDT and PPT as a consequence of the aging process. These alterations likely reflect the cellular bases for the age-related changes in REM sleep that occur in old animals.     

Immunohistochemical study of choline acetyltransferase-immunoreactive processes and cells innervating the pontomedullary reticular formation in the rat

The present study was undertaken to examine the cholinergic innervation of the brainstem reticular formation in an effort to understand the potential role of cholinergic neurons in processes of sensory-motor modulation and state control. The cholinergic cells and processes within the pontomedullary reticular formation were studied in the rat by application of peroxidase-antiperoxidase immunohistochemistry with silver intensification for choline-acetyltransferase (ChAT). ChAT-immunoreactive cells were located in the pontomesencephalic tegmentum within the laterodorsal and pedunculopontine tegmental (LDT and PPT) nuclei, where they numbered approximately 3,000 on each side and were scattered in the midline, medial, and lateral medullary reticular formation, where they numbered approximately 10,000 in total on each side. The cholinergic neurons within the reticular formation were commonly medium in size and gave rise to multiple dendrites that extended for considerable distances within the periventricular gray or the reticular formation, as is typical of other isodendritic reticular neurons. A prominent innervation of the entire pontomedullary reticular formation was evident by varicose ChAT-immunoreactive fibers that often surrounded large noncholinergic reticular neurons in a typical perisomatic pattern of termination, suggesting a potent influence of the cholinergic innervation on pontomedullary reticular neurons. The contribution of the pontomesencephalic cholinergic neurons to the innervation of the medial medullary and lateral pontine reticular formation was studied by retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (WGA-HRP) in combination with ChAT immunohistochemistry. A proportion of the cholinergic neurons within the laterodorsal tegmental nucleus (pars alpha) and the pedunculopontine tegmental nucleus were retrogradely labelled on the ipsilateral (10-15%) and contralateral (5-10%) sides from the medial medullary reticular formation, indicating a significant contribution to the cholinergic innervation of this region, which, however, also appeared to derive in part from intrinsic medullary cholinergic neurons. The major fiber system by which the medial medullary reticular formation was reached by the pontomesencephalic cholinergic neurons appeared to correspond to the lateral tegmentoreticular tract. Fibers passed from these cholinergic cells ventrally through the lateral pontine tegmentum, in the region of the subcoeruleus, where they also appeared to innervate by fibres en passage the noncholinergic neurons of the region. A significant proportion of the pontomesencephalic cholinergic neurons were retrogradely labelled from the lateral pontine tegmentum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colocalization of gamma-aminobutyric acid and acetylcholine in neurons in the laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and electron microscopic study

Cholinergic and gamma-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6-8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.     

Effects of chronic alcohol consumption and withdrawal on the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei of the rat: An unbiased stereological study

The brain cholinergic system comprises two main recognized subdivisions, the basal forebrain and the brainstem cholinergic systems. The effects of chronic alcohol consumption on the basal forebrain cholinergic nuclei have been investigated extensively, but there is only one study that has examined those effects on the brainstem cholinergic nuclei. The last one comprises the pedunculopontine tegmental (PPT) and the laterodorsal tegmental (LDT) nuclei, which are known to give origin to the main cholinergic projection to the ventral tegmental area, a key brain region of the neural circuit, the mesocorticolimbic system, that mediates several behavioral and physiological processes, including reward. In the present study, we have examined, using stereological methods, the effects of chronic alcohol consumption (6 months) and subsequent withdrawal (2 months) on the total number and size of PPT and LDT choline acetyltransferase (ChAT)-immunoreactive neurons. The total number of PPT and LDT ChAT-immunoreactive neurons was unchanged in ethanol-treated and withdrawn rats. However, ChAT-immunoreactive neurons were significantly hypertrophied in ethanol-treated rats, an alteration that did not revert 2 months after ethanol withdrawal. These results show that prolonged exposure to ethanol leads to long-lasting, and potentially irreversible, cytoarchitectonic and neurochemical alterations in the brainstem cholinergic nuclei. These alterations suggest that the alcohol-induced changes in the brainstem cholinergic nuclei might play a role in the mechanisms underlying the development of addictive behavior to alcohol.     

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.     

Neurotoxic lesions of the dorsolateral pontomesencephalic tegmentum-cholinergic cell area in the cat. I. Effects upon the cholinergic innervation of the brain

Kainic acid was injected bilaterally (4.8 micrograms in 1.2 microliter each side) into the dorsolateral pontomesencephalic tegmentum of cats in order to destroy cholinergic cells which are located within the pedunculopontine tegmental (PPT), laterodorsal tegmental (LDT), parabrachial (PB), and locus ceruleus (LC) nuclei in this species. The neurotoxic lesions resulted in the destruction of the majority (approximately 60%) of choline acetyltransferase (ChAT)-immunoreactive neurons and a minority (approximately 35%) of tyrosine hydroxylase (TH)-immunoreactive neurons, as well as in the destruction of other chemically unidentified neurons, in the region. The effects of these lesions upon the cholinergic innervation of the brain were investigated by comparison of brains with and without lesions which were processed for acetylcholinesterase (AChE) silver, copper thiocholine histochemistry and ChAT radio-immunohistochemistry. In the forebrain, a major and significant decrease in AChE staining, measured by microdensitometry, and associated with a decrease in ChAT immunoreactivity was found in certain thalamic nuclei, including the dorsal lateral geniculate, lateral posterior, pulvinar, intralaminar, mediodorsal and reticular nuclei. All of these nuclei receive a rich cholinergic innervation evident in both AChE histochemistry and ChAT immunohistochemistry. No significant difference in AChE staining or ChAT immunoreactivity was detected in other thalamic nuclei or in the subthalamus, hypothalamus or basal forebrain. In the brainstem, a significant decrease of AChE staining and ChAT immunoreactivity was found in the superior colliculus and the medullary reticular formation, where ChAT-immunoreactive fibers were moderately dense in the normal animal. These results indicate that the pontomesencephalic cholinergic neurons may influence the forebrain by major projections to the thalamus, involving both relay and non-specific thalamocortical projection systems, and thus act as an integral component of the ascending reticular system. They may influence the brainstem by projections onto deep tectal neurons and other reticular neurons, notably those in the medullary reticular formation, and thus also affect bulbar and bulbospinal systems.     

Activity of cholinergic neurons in the laterodorsal tegmental nucleus during emission of 22kHz vocalization in rats

It has been postulated that the ascending cholinergic tegmental system is responsible for the initiation of the aversive emotional state with a concomitant alarm vocalization in the rat. It is assumed that the activity of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) will cause release of acetylcholine in the target areas and will initiate the emission of 22 kHz vocalizations. The goal of the present study was to test the hypothesis that the cholinergic neurons of the LDT increase their activity during emission of 22 kHz alarm calls. Vocalizations were induced by an air puff or by intrahypothalamic-preoptic injection of carbachol. The activity of the LDT cholinergic neurons was studied by a double histochemical labelling for choline acetyltransferase, as a marker of cholinergic somata, and for c-Fos protein, as a marker of cells with heighten metabolic activity. Both air puff stimulation and intracerebral carbachol induced comparable 22 kHz alarm vocalizations. The activity of neurons in the LDT was significantly higher during prolonged emission of 22 kHz alarm calls induced by air puff or injection of carbachol than in the non-vocalizing or low-vocalizing controls. There were approximately two times more of all c-Fos-labelled cells in the LDT of vocalizing animals and 2.5 times more active cholinergic neurons during prolonged 22 kHz vocalization than in the control conditions without vocalization. However, the active cholinergic neurons constituted only a small proportion of all active LDT cells (2.3%). At the same time, there were no significant increases in the number of c-Fos-labelled cells in the neighbouring pedunculopontine nucleus (PPT). These findings lead to the conclusion that the neurons of the LDT, including cholinergic neurons, but not those of the PPT, significantly increased their activity during prolonged emission of alarm vocalizations, as evidenced by the c-Fos immunoreactivity.     

Afferent connections of the laterodorsal and the pedunculopontine tegmental nuclei in the rat: a retro- and antero-grade transport and immunohistochemical study.

Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH-diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents. Following injections of the retrograde tracer wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP) into either the LDT or the PPT, labelled neurons were seen in a number of limbic forebrain structures. The medial prefrontal cortex and lateral habenula contained more retrogradely labelled neurons from the LDT, whereas in the bed nucleus of the stria terminalis and central nucleus of the amygdala, more cells were labelled from the PPT. Moderate numbers of neurons were seen in the magnocellular regions of the basal forebrain, and many labelled neurons were observed in the lateral hypothalamus, the zona incerta, and the midbrain central gray from both the LDT and the PPT. Accessory oculomotor nuclei in the midbrain as well as eye movement-related structures in the lower brainstem contained some neurons labelled from the LDT, and fewer neurons from the PPT. A few labelled neurons were seen in somatosensory and other sensory relay nuclei in the brainstem and the spinal cord. Retrograde labelling was seen in a number of extrapyramidal structures, including the globus pallidus, entopenduncular and subthalamic nuclei, and substantia nigra following PPT injections; with LDT injections, labelling was similar in density in the substantia nigra but virtually absent in the entopeduncular and subthalamic nuclei. Data with the fluorescent retrograde tracer fluorogold combined with immunofluorescence indicated that many neurons in the zona incerta-lateral hypothalamic region that were retrogradely labelled from the LDT contained alpha-melanocyte-stimulating hormone. Numerous neurons were labelled throughout the reticular formation of the brainstem following either LDT or PPT injections. Many neurons retrogradely labelled in the LDT and PPT, the dorsal and median raphe nuclei, and the locus ceruleus contained choline acetyltransferase, serotonin, and tyrosine hydroxylase, respectively. The anterograde tracers WGA-HRP and phaseolus vulgaris leucoagglutinin were used to confirm some of the projections indicated by the retrograde labelling data; anterograde labelling was seen in the LDT and PPT following injections of one of these tracers into the medial prefrontal cortex, lateral hypothalamus, and the contralateral LDT.(ABSTRACT TRUNCATED AT 400 WORDS)     

The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish Danio rerio

The central nervous cholinergic system of the zebrafish (Danio rerio), a model animal for neurogenetics, is documented here using immunohistochemical methods for visualizing choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Neuronal cell bodies containing ChAT are present in the telencephalon (lateral nucleus of ventral telencephalic area), preoptic region (anterior/posterior parvocellular and magnocellular preoptic nuclei), diencephalon (habenula, dorsal thalamus, posterior tuberculum), mesencephalon (Edinger-Westphal (EW) nucleus, oculomotor nerve nucleus, rostral tegmental nucleus, tectal type XIV neurons), isthmic region (nucleus lateralis valvulae, secondary gustatory-viscerosensory nucleus, nucleus isthmi (NI), perilemniscal nucleus, superior reticular nucleus (SRN)) and rhombencephalon (trochlear, trigeminal, abducens, facial, glossopharyngeal-vagal motor nerve nuclei, rostral and caudal populations of octavolateralis efferent neurons). In addition, some ChAT positive neurons are present in the rhombencephalic reticular formation, the central gray, and in cells accompanying the descending trigeminal tract. Obvious ChAT positive terminal fields are present in the supracommissural nucleus of area ventralis telencephali and the medial zone of area dorsalis telencephali, parvocellular superficial pretectal nucleus, torus semicircularis, medial octavolateralis nucleus, facial, glossopharyngeal, and vagal lobes, and in the inferior lobe (around the periventricular nucleus of the lateral recess and in the diffuse nucleus). The identification of all central nervous cholinergic systems provided here in this model system is pivotal for future detailed studies of their development and maintenance, e.g., with regard to the zebrafish ventral telencephalic and isthmic superior reticular neuronal populations, likely representing the homologues of at least part of the cholinergic basal forebrain and pedunculopontine/laterodorsal tegmental ascending activating systems of mammals, respectively.     

Extracellular characteristics of putative cholinergic neurons in the rat laterodorsal tegmental nucleus

The extracellular electrophysiological properties of neurons in the laterodorsal tegmental nucleus (LDT), a major source of cholinergic afferents to the thalamus, were studied in chloral hydrate-anesthetized rats. A combination of antidromic activation from the thalamus and histological verification of recording sites was used to correlate the identity of extracellular recordings in the rat LDT with cholinergic neurons in that region. All neurons antidromically activated by stimulation of the anteroventral thalamus were histologically verified to be within clusters of cholinergic (NADPH-d-positive) cells in the LDT or in the adjacent nucleus locus coeruleus (LC). The thalamically projecting LDT neurons had a homogeneous neurophysiological profile consisting of long duration action potentials (mean = 2.5 ms), slow conduction velocities (mean = 0.78 m/s), and lengthy chronaxie values (mean = 0.725 ms). The appearance and axonal characteristics of these neurons resembled those of noradrenergic LC neurons, but the two populations exhibited substantially different spontaneous activity patterns and sensory responsiveness. These characteristics may be useful in the preliminary identification of putative cholinergic neurons in vivo, and thereby provide a foundation for exploring the neuropharmacology, afferent modulation, sensory responsiveness and behavioral correlates of the brainstem cholinergic system.     

Serotonergic dorsal raphe nucleus projections to the cholinergic and noncholinergic neurons of the pedunculopontine tegmental region: a light and electron microscopic anterograde tracing and immunohistochemical study

The serotonergic dorsal raphe nucleus is considered an important modulator of state-dependent neural activity via projections to cholinergic neurons of the pedunculopontine tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry at the light and electron microscopic levels, to determine whether PPT neurons receive serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3) approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites; and (4) at least 2-4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital waves that precede and accompany REM sleep is discussed. J. Comp. Neurol. 382:302-322, 1997. © 1997 Wiley-Liss, Inc.     

Basal forebrain cholinergic system of the anuran amphibian Rana perezi: evidence for a shared organization pattern with amniotes

The organization of the basal forebrain cholinergic system (BFCS) in the frog was studied by means of choline acetyltransferase (ChAT) immunohistochemistry. The BFCS was observed as a conspicuous cholinergic cell population extending through the diagonal band, medial septal nucleus, bed nucleus of the stria terminalis, and pallidal regions. Abundant fiber labeling was also found around the labeled cell bodies. The combination of retrograde tract tracing with dextran amines and ChAT immunohistochemistry revealed intraseptal and intra-BFCS cholinergic connections. In addition, an extratelencephalic cholinergic input from the laterodorsal tegemental nucleus was demonstrated. The possible influence of monoaminergic inputs on the BFCS neurons was examined by means of tyrosine hydroxylase and serotonin immunohistochemistry combined with ChAT immunolabeling. Our results showed that catecholaminergic fibers overlapped the BFCS, with the exception of the medial septal nucleus. Serotoninergic innervation was widespread, but less abundant in the caudal extent of the BFCS. Taken together, our results on the localization of the cholinergic neurons in the basal forebrain and their relationship with cholinergic, catecholaminergic, and serotoninergic afferents have shown numerous common features with amniotes. In particular, anurans and mammals (for which most data is available) share a strikingly comparable organization pattern of the BFCS.     

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.     

Acetylcholine from the mesopontine tegmental nuclei differentially affects methamphetamine induced locomotor activity and neurotransmitter levels in the mesolimbic pathway

Methamphetamine (MA) increases dopamine (DA) levels within the mesolimbic pathway and acetylcholine (ACh), a neurotransmitter known to increase DA cell firing and release and mediate reinforcement, within the ventral tegmental area (VTA). The laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei provide cholinergic input to the VTA; however, the contribution of LDT- and PPT-derived ACh to MA-induced DA and ACh levels and locomotor activation remains unknown. The first experiment examined the role of LDT-derived ACh in MA locomotor activation by reversibly inhibiting these neurons with bilateral intra-LDT microinjections of the M2 receptor agonist oxotremorine (OXO). Male C57BL/6 J mice were given a bilateral 0.1 μl OXO (0, 1, or 10 nM/side) microinjection immediately prior to IP saline or MA (2 mg/kg). The highest OXO concentration significantly inhibited both saline- and MA-primed locomotor activity. In a second set of experiments we characterized the individual contributions of ACh originating in the LDT or pedunculopontine tegmental nucleus (PPT) to MA-induced levels of ACh and DA by administering intra-LDT or PPT OXO and performing in vivo microdialysis in the VTA and NAc. Intra-LDT OXO dose-dependently attenuated the MA-induced increase in ACh within the VTA but had no effect on DA in NAc. Intra-PPT OXO had no effect on ACh or DA levels within the VTA or NAc, respectively. We conclude that LDT, but not PPT, ACh is important in locomotor behavior and the cholinergic, but not dopaminergic, response to systemic MA.     

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.