Localization of cholinergic neurons in the forebrain and brainstem that project to the suprachiasmatic nucleus of the hypothalamus in rat

In mammals, the suprachiasmatic nucleus is responsible for the generation of most circadian rhythms and their entrainment to environmental cues. Cholinergic agents can alter circadian rhythm phase, and fibres immunoreactive for choline acetyltransferase, the biosynthetic enzyme for acetylcholine, are present in the suprachiasmatic nucleus. Since there are no cholinergic somata in the suprachiasmatic nucleus, these fibres must represent the terminals of cholinergic neurons whose cell bodies are located elsewhere in the brain. This study was aimed at locating the cholinergic neurons that project to the suprachiasmatic nucleus by retrograde and anterograde tract-tracing and immunohistochemistry for choline acetyltransferase in the rat. After injection of fluorogold, a retrograde tracer, into the suprachiasmatic nucleus, retrogradely labelled neurons that were immunopositive for choline acetyltransferase were located throughout the rostrocaudal extent of the cholinergic basal nuclear complex, with highest densities in the substantia innominata and the nucleus basalis magnocellularis. A few cells were also located in the medial septum and in the vertical and horizontal limbs ofthe diagonal band of Broca. In the brainstem, double-labelled neurons were located in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus and the parabigeminal nucleus. Injections of the anterograde tracer biocytin in these three brainstem nuclei resulted in fibre labelling in the suprachiasmatic nucleus, consistent with the retrograde findings. No clearly double-labelled cells were located in the retina. These results suggest that the suprachiasmatic nucleus receives cholinergic afferents from both the basal forebrain and mesopontine tegmentum which may mediate cholinergic effects on circadian rhythms. © 1993 Wiley-Liss, Inc.     

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)     

Basal forebrain cholinergic and noncholinergic projections to the thalamus and brainstem in cats and monkeys

The projections of basal forebrain neurons to the thalamus and the brainstem were investigated in cats and primates by using retrograde transport techniques and choline acetyltransferase (ChAT) immunohistochemistry. In a first series of experiments, the lectin wheat germ-agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into all major sensory, motor, intralaminar, and reticular (RE) thalamic nuclei of cats and into the mediodorsal (MD) and pulvinar-lateroposterior thalamic nuclei of macaque monkeys. In cats numerous neurons of the vertical and horizontal limbs of the diagonal band nucleus and the substantia innominata (SI), including its rostromedial portion termed the ventral pallidum (VP), were retrogradely labeled after WGA-HRP injections in the rostral pole of the RE complex, the MD, and anteroventral/anteromedial (AV/AM) thalamic nuclei. Fewer retrogradely labeled cells were observed in the same areas after injections in the ventromedial (VM) thalamic nucleus, and none or very few after other thalamic injections. After RE, MD, and AV/AM injections, 7-20% of all retrogradely labeled cells in the basal forebrain were also ChAT positive, while none of the retrogradely labeled neurons following VM injections displayed ChAT immunoreactivity. The basal forebrain projection to the MD nucleus was shown to arise principally from VP in both cats and macaque monkeys. In a second series of experiments performed in cats, injections of WGA-HRP in the brainstem peribrachial (PB) area comprising the pedunculopontine nucleus led to retrograde labeling of a moderate number of neurons in the lateral part of the VP, SI, and preoptic area (POA), only a few of which displayed ChAT immunoreactivity. In addition, a large number of retrogradely labeled cells were observed in the bed nuclei of the anterior commissure and stria terminalis after PB injections. In a third series of experiments, the use of the retrograde double-labeling method with fluorescent tracers in squirrel monkeys allowed us to identify a significant number of basal forebrain neurons sending axon collaterals to both the RE thalamic nucleus and PB brainstem area, while no double-labeled neurons were disclosed after injections confined to the ventral anterior/ventral lateral (VA/VL) thalamic nuclei and PB area or following injections in the cerebral cortex and PB area. Our findings reveal the existence of cholinergic and noncholinergic basal forebrain projections to the thalamus and the brainstem in both cats and macaque monkeys. We suggest that these projections may play a crucial role in the control of thalamic functions in mammals.     

Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry, and electrophysiology in the rat

Brainstem afferents to the magnocellular basal forebrain were studied by using tract tracing, immunohistochemistry and extracellular recordings in the rat. WGA-HRP injections into the horizontal limb of the diagonal band (HDB) and the magnocellular preoptic area (MgPA) retrogradely labelled many neurons in the pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe nucleus, and ventral tegmental area. Areas with moderate numbers of retrogradely labelled neurons included the median raphe nucleus, and area lateral to the medial longitudinal fasciculus in the pons, the locus ceruleus, and the medial parabrachial nucleus. A few labelled neurons were seen in the substantia nigra pars compacta, mesencephalic and pontine reticular formation, a midline area in the pontine central gray, lateral parabrachial nucleus, raphe magnus, prepositus hypoglossal nucleus, nucleus of the solitary tract, and ventrolateral medulla. A similar but not identical distribution of labelled neurons was seen following WGA-HRP injections into the nucleus basalis magnocellularis. The possible neurotransmitter content of some of these afferents to the HDB/MgPA was examined by combining retrograde Fluoro-Gold labelling and immunofluorescence. In the mesopontine tegmentum, many retrogradely labelled neurons were immunoreactive for choline acetyltransferase. In the dorsal raphe nucleus, some retrogradely labelled neurons were positive for serotonin and some for tyrosine hydroxylase (TH); however, the majority of retrogradely labelled neurons in this region were not immunoreactive for either marker. The ventral tegmental area, substantia nigra pars compacta, and locus ceruleus contained retrogradely labelled neurons which were also immunoreactive for TH. Of the retrogradely labelled neurons occasionally observed in the nucleus of the solitary tract, prepositus hypoglossal nucleus, and ventrolateral medulla, some were immunoreactive for either TH or phenylethanolamine-N-methyltransferase. To characterize functionally some of these brainstem afferents, extracellular recordings were made from antidromically identified cortically projecting neurons, mostly located in the HDB and MgPA. In agreement with most previous studies, about half (48%) of these neurons were spontaneously active. Electrical stimulation in the vicinity of the pedunculopontine tegmental and dorsal raphe nuclei elicited either excitatory or inhibitory responses in 21% (13/62) of the cortically projecting neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferent connections of the anterior thalamus in rabbits

This study was designed to determine whether axons of cholinergic dorsal tegmental neurons terminate on cells in the anterior thalamus in rabbits as in other species, and to localize projecting tegmental cells for future studies of their contributions to anterior thalamic learning-relevant neuronal activity. The distribution of retrogradely labeled neurons was examined following injections of wheat germ agglutinin horseradish peroxidase (WGA-HRP) centered in the anterior ventral (AV) thalamic nucleus. The results confirm past findings in rabbits indicating projections to anterior thalamus from the mammillary nuclei, the posterior cingulate cortex, presubiculum and postsubiculum. Demonstrated for the first time in rabbits were projections from the lateral dorsal and the pedunculopontine tegmental nuclei, locus coeruleus, dorsal raphe nucleus, Gudden's dorsal tegmental nucleus, pretectum and reticular thalamic nucleus.     

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)     

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.     

Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution.

The cholinergic innervation of the human thalamus was studied with antibodies against the enzyme choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr). Acetylcholinesterase histochemistry was used to delineate nuclear boundaries. All thalamic nuclei displayed ChAT-positive axons and varicosities. Only the medial habenula contained ChAT-positive perikarya. Some intralaminar nuclei (central medial, central lateral, and paracentral), the reticular nucleus, midline nuclei (paraventricular and reuniens), some nuclei associated with the limbic system (anterodorsal nucleus and medially situated patches in the mediodorsal nucleus) and the lateral geniculate nucleus displayed the highest density of ChAT-positive axonal varicosities. The remaining sensory relay nuclei and the nuclei interconnected with the motor and association cortex displayed a lower level of innervation. Immunoreactivity for NGFr was observed in cholinergic neurons of the basal forebrain but not in cholinergic neurons of the upper brainstem. The contribution of basal forebrain afferents to the cholinergic innervation of the human thalamus was therefore studied with the aid of NGFr-immunoreactive axonal staining. The anterior intralaminar nuclei, the reticular nucleus, and medially situated patches in the mediodorsal nucleus displayed a substantial number of NGFr-positive varicose axons, presumably originating in the basal forebrain. Rare NGFr-positive axonal profiles were also seen in many of the other thalamic nuclei. These observations suggest that thalamic nuclei affiliated with limbic structures and with the ascending reticular activating system are likely to be under particularly intense cholinergic influence. While the vast majority of thalamic cholinergic input seems to come from the upper brainstem, the intralaminar and reticular nuclei, and especially medially situated patches within the mediodorsal nucleus also appear to receive substantial cholinergic innervation from the basal forebrain.     

Brainstem projections to midline and intralaminar thalamic nuclei of the rat

The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin beta -subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nuclei-the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental field. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem-thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions.     

Patterns of brainstem projection to the thalamic reticular nucleus

To understand better how the brainstem may influence thalamocortical activity, we have examined the projection patterns of different brainstem nuclei to the thalamic reticular nucleus. Iontophoretic injections of biotinylated dextran were made into various nuclei of the brainstem (superior colliculus, periaqueductal grey matter, parabrachial nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, substantia nigra, ventral tegmental area, and locus coeruleus) of Sprague-Dawley rats by using stereotaxic coordinates. Our results show that afferents from each brainstem nucleus make distinct zones within the reticular nucleus. For example, the superior colliculus projects largely to the dorsal parts of the reticular nucleus, whereas the pedunculopontine nucleus projects to the ventral parts of the reticular nucleus. The substantia nigra, on the other hand, projects to the ventrolateral edge of the reticular nucleus. We also examined the distribution of these brainstem afferents within the dorsal thalamus and compared these distributions with those seen in the reticular nucleus. We found three different patterns. First, a given brainstem nucleus projects to a particular dorsal thalamic nucleus as well as to the corresponding, functionally associated, reticular sector (e.g., from the substantia nigra). Second, a given brainstem nucleus projects to a particular dorsal thalamic nucleus but not to the corresponding reticular sector (e.g., from the superior colliculus). Finally, a given brainstem nucleus projects to a given reticular sector but not to the corresponding dorsal thalamic nucleus (e.g., from the midbrain reticular nucleus). In general, our results indicate that various brainstem nuclei project to particular territories of the thalamic reticular nucleus. Through these reticular projections, brainstem nuclei may influence distinct thalamocortical pathways in addition to those that are influenced by their direct projection to the dorsal thalamus. J. Comp. Neurol. 396:531–543, 1998. © 1998 Wiley-Liss, Inc.     

Projections from auditory cortex to cholinergic cells in the midbrain tegmentum of guinea pigs

Anterograde and retrograde tracing techniques were used to characterize projections from the auditory cortex to the pedunculopontine and laterodorsal tegmental nuclei (PPT and LDT, respectively) in the midbrain tegmentum in guinea pigs. For anterograde tracing, tetramethylrhodamine dextran (FluoroRuby) was injected at several sites within auditory cortex. After sufficient time for transport, the brain was processed for immunohistochemistry with anti-choline acetyltransferase to reveal presumptive cholinergic cells. Anterogradely labeled axons were observed ipsilaterally and, in smaller numbers, contralaterally, in both the pedunculopontine and laterodorsal tegmental nuclei. In all four nuclei, tracer-labeled boutons appeared to contact immunolabeled (i.e., cholinergic) cells. The contacts occurred on cell bodies and dendrites. The results were similar following injections that spread across multiple auditory cortical areas or injections that were within primary auditory cortex. In order to confirm the anterograde results, in a second series of experiments, retrograde tracers were deposited in the pedunculopontine tegmental nucleus. These injections labeled layer V pyramidal cells in the auditory cortex. The results suggest an excitatory projection from primary auditory cortex bilaterally to cholinergic cells in the midbrain tegmentum. Such a pathway could allow auditory cortex to activate brainstem cholinergic circuits, possibly including the cholinergic pathways associated with arousal and gating of acoustic stimuli.     

Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat.

The connections of the laterodorsal tegmental nucleus (LDTg) have been investigated using anterograde and retrograde lectin tracers with immunocytochemical detection. Inputs to LDTg were found from frontal cortex, diagonal band, preoptic areas, lateral hypothalamus, lateral mamillary nucleus, lateral habenula; the interpeduncular nucleus, ventral tegmental area, substantia nigra and retrorubral fields; the medial terminal nucleus, interstitial nucleus, supraoculomotor central grey, medial pretectum, nucleus of the posterior commissure, paramedian pontine reticular formation, paraabducens and paratrochlear region; the parabrachial nuclei and nucleus of the tractus solitarius. Terminal labelling from PHA-L injections of LDTg was found in infralimbic, cingulate and hippocampal cortex, lateral septum, septofimbrial and triangular nuclei, horizontal limb of diagonal band and preoptic areas; in the anterior, mediodorsal, reuniens, centrolateral, parafascicular, paraventricular and laterodorsal thalamic nuclei, rostral reticular thalamic nucleus, and zona incerta; the lateral habenula and the lateral hypothalamus. A number of brainstem structures apparently associated with visual functions were also innervated, mainly the superior colliculus, medial pretectum, medial terminal nucleus, paramedian pontine reticular formation, inferior olive, supraoculomotor, paraabducens and supragenual regions, prepositus hypoglossi and nucleus of the posterior commissure. Also innervated were substantia nigra compacta, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, dorsal and medial raphe, pedunculopontine tegmental region, parabrachial nuclei, and nucleus of the tractus solitarius. These findings suggest the LDTg to be a highly differentiated part of the ascending "reticular activating" system, concerned not only with specific cortical and thalamic regions, especially those associated with the limbic system, but also with the basal ganglia, and visual (particularly oculomotor) mechanisms. Additional links with the habenula-interpeduncular system are discussed in this context.     

Cholinergic systems in the rat brain: IV. Descending projections of the pontomesencephalic tegmentum

Descending projections from cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei, collectively referred to as the pontomesencephalotegmental (PMT) cholinergic complex, were studied by use of the fluorescent retrograde tracers fluorogold, true blue, or Evans Blue in combination with choline acetyltransferase (ChAT) immunohistochemistry of acetylcholinesterase (AChE) pharmacohistochemistry. Pedunculopontine somata positive for ChAT or staining intensely for AChE were retrogradely labeled with fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12. ChAT-positive cells in both the pedunculopontine and laterodorsal tegmental nuclei demonstrated projections to the vestibular nuclei, the spinal nucleus of the 5th cranial nerve, deep cerebellar nuclei, pontine nuclei, locus ceruleus, raphe magnus nucleus, dorsal raphe nucleus, median raphe nucleus, the medullary reticular nuclei, and the oral and caudal pontine reticular nuclei. Fluorescent tracers used in combination with AChE pharmacohistochemistry corroborated these projections and, in addition, provided evidence for cholinergic pontomesencephalic projections to the lateral reticular nucleus and inferior olive. The majority of retrogradely labeled neurons demonstrating ChAT-like immunoreactivity were found ipsilateral to the injection site, but, in all cases, tracer-containing cholinergic cells contralateral to the infused side of the brain were detected also. More retrogradely labeled cells containing ChAT were observed in the pedunculopontine tegmental than in the laterodorsal tegmental nucleus following tracer injections at all sites with the exceptions of the locus ceruleus and dorsal raphe nucleus where the converse profile was observed. None of the pedunculopontine or laterodorsal tegmental cells immunopositive for ChAT or stained intensely for AChE contained retrogradely transported tracers following dye infusions into the cerebellar cortex or cervical spinal cord. Triple-label experiments using two tracers infused into different sites in the same animal revealed that individual ChAT-immunoreactive cells in the PMT cholinergic complex projected to more than one hindbrain site in some cases and had ascending projections as well. Certain ChAT-positive somata in the pedunculopontine and laterodorsal tegmental nuclei were found in close association with several fiber tracts, including the superior cerebellar peduncle, lateral lemniscus, dorsal tegmental tract, and medial longitudinal fasciculus.     

Cholinergic neurons of the feline pontomesencephalon. II. Ascending anatomical projections

Immunoreactivity for choline acetyltransferase (ChAT) was analyzed in unoperated cats and in cats in which stereotaxic lesions were made in the pedunculopontine and laterodorsal tegmental nuclei. The fine reaction product revealed moderate to dense ChAT-immunoreactive fiber plexuses throughout the telencephalon, diencephalon, and midbrain. A pontomesencephalic origin of cholinergic innervation to virtually every nucleus of the diencephalon, as well as to various midbrain and basal telencephalic sites was indicated in the cats with lesions, in which the optical density of ChAT-immunoreactivity was significantly decreased as compared to controls. Pontomesencephalic lesions produced no changes, however, in the density of ChAT staining in the cerebral cortex, basolateral amygdala, or caudate nucleus. In addition to ChAT-positive terminal fiber arborizations which were widely distributed, cholinergic fibers-of-passage were traced in the unoperated and operated feline brains. The general course of ChAT fibers cut in cross-section was followed in successive transverse levels, and although pathways originating from the pedunculopontine nucleus demonstrated orientations in every direction, many demonstrated a rostral course. A particularly dense aggregate of ascending ChAT-positive fibers was localized in the dorsolateral sector of the pedunculopontine area which could be followed at more rostral levels into the central tegmental fields and the compact part of the substantia nigra. From the central tegmental fields, numerous ChAT-immunopositive fibers cut in cross-section continued to course rostrally in the intralaminar, reticular and lateroposterior nuclei of the thalamus, and a distinct bundle of ChAT fibers coursing dorsolaterally was observed medial to the optic tract ascending to the lateral geniculate. ChAT fibers with dorsolateral orientations were additionally observed in the zona incerta, ventral anterior thalamus, and ansa lenticularis on route to the reticular thalamus, the globus pallidus, and the substantia innominata. Pathways consisting of fibers traced from ChAT-containing cells in the laterodorsal tegmental nucleus could be traced to medial structures such as the periaqueductal gray, ventral tegmental area and dorsal raphe. Medially placed ChAT fibers were additionally followed through the ventral tegmental area, the midline thalamus, and the hypothalamus, up to the medial and lateral septal nuclei. The trajectories of the ascending cholinergic pathways from the pontomesencephalon are discussed in relation to locally generated electrophysiological responses in the cat.     

Cholinergic systems in the rat brain: II. Projections to the interpeduncular nucleus

The cholinergic innervation of the interpeduncular nucleus was investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Following propidium iodide or Evans Blue infusion into the interpeduncular nucleus, brains were processed for co-localization of transported fluorescent label and ChAT and AChE. Control infusions of tracers were made into the ventral tegmental area. In order to delimit the course of putative cholinergic afferents to the interpeduncular nucleus from extra-habenular sources, knife cuts surrounding the habenular nuclei were performed. Somata containing propidium iodide that were highly immunoreactive for ChAT were found primarily in the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, and the dorsolateral tegmental nucleus, also referred to as the laterodorsal tegmental nucleus. A few such co-labeled somata were also detected in the medial septal nucleus, substantia innominata, nucleus basalis, and pedunculopontine tegmental nucleus. A good correlation was observed between intensely-staining, AChE-containing and ChAT-positive neurons projecting to the interpeduncular nucleus from the aforementioned structures. Although the medial habenula contained numerous cells demonstrating transported label following interpeduncular infusion of fluorescent tracers, the ChAT-positivity associated with somata in that nucleus was weak compared to ChAT-like immunoreactivity in known cholinergic neurons in the basal forebrain and brainstem. Knife cuts that separated the habenular nuclei from the stria medullaris and neural regions lateral and posterior to those nuclei while leaving the fasciculus retroflexus intact resulted in a reduction of ChAT-like immunoreactivity in the medial habenular nucleus, fasciculus retroflexus, and interpeduncular nucleus. These data suggest (1) that the cholinergic innervation of the interpeduncular nucleus derives primarily from ChAT-positive cells in the basal forebrain and dorsolateral tegmental nucleus and (2) that putative cholinergic fibers having their origin in the medial habenula, if they exist, constitute a minor portion of the cholinergic input to the interpeduncular nucleus.     

Afferent projections to the parafascicular thalamic nucleus of the rat, as shown by the retrograde transport of wheat germ agglutinin

Afferent projections to the parafascicular nucleus of the rat have been mapped using the retrograde transport of unconjugated wheat germ agglutinin and immunohistochemistry using very short survival times. Retrogradely labelled neurones were found in laminae V and V1 of primary motor cortex, lamina V1 of primary somatosensory cortex, and deep laminae of gustatory cortex; in the reticular thalamic nucleus and zona incerta; and in the caudate-putamen, entopeduncular nucleus, mesencephalic reticular formation and pretectum. Additional label was found in the laterodorsal tegmental nucleus, nucleus tegmenti pedunculopontinus, dorsal and ventral parabrachial nuclei, vestibular nuclei and the lateral cervical, medial and interpositus nuclei of the cerebellum. These results are discussed in the context of the connections of parafascicular nucleus with the motor system, particularly the basal ganglia. Of particular interest are inputs from laterodorsal tegmental nucleus, nucleus reticularis of thalamus, mesencephalic reticular formation, nucleus tegmenti pedunculopontinus, primary motor cortex and deep cerebellar nuclei. These indicate that the parafascicular nucleus lies at an interface between the reticular activating system on the one hand, and the motor system on the other. This result thus enlarges on present concepts of the parafascicular nucleus. Comparison of afferent projections to a variety of non-specific thalamic nuclei, the parafascicular, paraventricular and mediodorsal thalamic nuclei, indicate a remarkable set of topographic parallels from cortical, reticular thalamic, hypothalamic and brainstem sites. These comparisons provide clues as to the organisational principles of these non-specific thalamic nuclei, particularly in the context of the reticular activating system.     

Cholinergic vs. noncholinergic efferents from the mesopontine tegmentum to the extrapyramidal motor system nuclei

Previous studies have suggested that the pedunculopontine tegmental nucleus (PPTn) is reciprocally connected with extrapyramidal motor system nuclei (EPMS) whereas other studies have implicated the PPTn in behavioral state control phenomena such as sleep-wakefulness cycles. Many of these studies define the nonprimate PPTn as an area of mesopontine tegmentum which is labeled from injections of anterograde tracers into the basal ganglia. Recently, we have defined the rat PPTn as a large-celled, cholinergic nucleus. The rat PPTn is cytologically distinct from a group of smaller, noncholinergic neurons that are medially adjacent to the PPTn. This noncholinergic group is further distinguished from the PPTn by its afferent input from the globus pallidus, entopeduncular nucleus, and substantia nigra. We refer to the latter area as the midbrain extrapyramidal area (MEA). Using combined choline acetyltransferase immunohistochemistry of the PPTn and WGA-HRP retrograde tracing from the EPMS, we investigated the efferent connections of the MEA and PPTn to the EPMS in the rat. The noncholinergic MEA, rather than the PPTn, is the major source of tegmental innervation to the globus pallidus, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra, and motor cortex. In contrast, the cholinergic PPTn is the major source of tegmental innervation to the ventrolateral thalamic nucleus. This finding is in contradistinction to thalamic projections from the surrounding reticular formation, which are identified only after WGA-HRP injections into "nonspecific" thalamic nuclei. This body of evidence suggests that the noncholinergic MEA represents an additional component of the EPMS and may correspond to the "mesencephalic locomotor region." The cholinergic PPTn may play a role in more global thalamic functions such as the "reticular activating system" rather than a primary role in motor function.     

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.     

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.     

Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections

The ascending projections of cholinergic neurons in the laterodorsal tegmental nucleus (TLD) were investigated in the rat by using Phaseolus vulgaris leucoagglutinin (PHA-L) and wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) anterograde tracing techniques. Two ascending pathways were identified after iontophoretic injections of PHA-L into the TLD. A long projection system courses through the dorsomedial tegmentum, caudal diencephalon, medial forebrain bundle, and diagonal band. Different branches of this system innervate the midbrain (superior colliculus, interstitial magnocellular nucleus of the posterior commissure, and anterior pretectal nucleus), the diencephalon (lateral habenular nucleus, parafascicular, anteroventral, anterodorsal, mediodorsal, and intralaminar thalamic nuclei), and the telencephalon (lateral septum and medial prefrontal cortex). The second system is shorter and more diffuse and innervates the median raphe, interpeduncular, and lateral mammillary nuclei. Retrograde tracing with WGA-HRP, combined with choline acetyltransferase immunohistochemistry, revealed that most of the TLD projections to the tectum, pretectum, thalamus, lateral septum, and medial prefrontal cortex are cholinergic. Afferents to the TLD were studied by anterograde and retrograde tracing techniques. Injection of tracers into the TLD retrogradely labelled neurons bilaterally in the midbrain reticular formation, the periaqueductal gray, the medial preoptic nucleus, the anterior hypothalamic nucleus, and the perifornical and lateral hypothalamic areas. Retrogradely labelled cells were also located bilaterally in the premammillary nucleus, paraventricular hypothalamic nucleus, zona incerta, and lateral habenular nucleus. In the telencephalon, the nucleus of the diagonal band and the medial prefrontal cortex contained retrogradely labelled neurons ipsilateral to the TLD injection site. The projections of the medial prefrontal cortex, the bed nucleus of the stria terminalis, and the lateral habenular nucleus to the TLD were confirmed in anterograde tracing studies. These findings indicate that the TLD gives rise to several ascending cholinergic projections that innervate diverse regions of the forebrain. Afferents to the TLD arise in hypothalamic and limbic forebrain regions, some of which appear to have reciprocal connections with the TLD. The latter include the lateral habenular nucleus and medial prefrontal cortex.