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)     

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.     

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)     

The origins of cholinergic and other subcortical afferents to the thalamus in the rat

The origins of the cholinergic and other afferents of several thalamic nuclei were investigated in the rat by using the retrograde transport of wheat germ agglutinin conjugated-horseradish peroxidase in combination with the immunohistochemical localization of choline acetyltransferase immunoreactivity. Small injections placed into the reticular, ventral, laterodorsal, lateroposterior, posterior, mediodorsal, geniculate, and intralaminar nuclei resulted in several distinct patterns of retrograde labelling. As expected, the appropriate specific sensory and motor-related subcortical structures were retrogradely labelled after injections into the principal thalamic nuclei. In addition, other basal forebrain and brainstem structures were also labelled, with their distribution dependent on the site of injection. A large percentage of these latter projections was cholinergic. In the brainstem, the cholinergic pedunculopontine tegmental nucleus was retrogradely labelled after all thalamic injections, suggesting that it provides a widespread innervation to the thalamus. Neurons of the cholinergic laterodorsal tegmental nucleus were retrogradely labelled after injections into the anterior, laterodorsal, central medial, and mediodorsal nuclei, suggesting that it provides a projection to limbic components of the thalamus. Significant basal forebrain labelling occurred only with injections into the reticular and mediodorsal nuclei. Only injections into the reticular nucleus resulted in retrograde labelling of the cholinergic neurons in the nucleus basalis of Meynert. The results provide evidence for an organized system of thalamic afferents arising from cholinergic and noncholinergic structures in the brainstem and basal forebrain. The brainstem structures, especially the cholinergic pedunculopontine tegmental nucleus, appear to project directly to principal thalamic nuclei, thereby providing a possible anatomical substrate for mediating the well-known facilitory effects of brainstem stimulation upon thalamocortical transmission.     

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)     

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.     

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.     

Serotonergic synaptic input to cholinergic neurons in the rat mesopontine tegmentum

Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.     

Origin of the neurotensinergic innervation of the rat basal forebrain studied by retrograde transport of cholera toxin

Basal forebrain cholinergic neurons project to the hippocampus and cerebral cortex where they play an important role in cortical activation, attention, and memory. These neurons have been shown to express functional neurotensin receptors and to receive a dense neurotensinergic innervation. In the present study, we investigated the origin of this innervation by combining retrograde transport of cholera toxin with immunohistochemical detection of neurotensin. After injection of cholera toxin in the anterior substantia innominata and diagonal band of Broca, retrogradely labelled cells were widely distributed throughout forebrain limbic structures. Only a small proportion of these cells, located (by decreasing order of frequency) in the lateral septum, medial preoptic area, rostral hypothalamus, nucleus accumbens, and rostral basal forebrain, were dually labelled for neurotensin. After injection of cholera toxin in the posterior substantia innominata and magnocellular preoptic nucleus, retrogradely labelled cells were detected throughout the limbic forebrain and ponto-mesencephalic tegmentum. Here again, only a small proportion of these cells, located (by decreasing order of frequency) in the nucleus accumbens, lateral septum, rostral basal forebrain, hypothalamus, bed nucleus of the stria terminalis, supramammilliary nucleus, ventral tegmental area, and raphe complex co-localized neurotensin. In view of the burst generating properties of neurotensin on basal forebrain cholinergic neurons, our results suggest that neurotensin projections may be part of the septo-hippocampo-septal loop regulating hippocampal theta activity. More caudally, neurotensin axons originating from the lateral hypothalamus and pontomesencephalic tegmentum may contribute to the contingent of ascending reticular formation fibers involved in the regulation of the sleep-wake cycle. J. Comp. Neurol. 391:30–41, 1998. © 1998 Wiley-Liss, Inc.     

Basal forebrain and mesopontine tegmental projections to the reticular thalamic nucleus: an axonal collateralization and immunohistochemical study in the rat

Using a double fluorescence retrograde labeling procedure, the present study sought to determine the degree to which basal forebrain and mesopontine tegmental neurons have axons that innervate both the reticular thalamic nucleus and the cerebral cortex. Immunofluorescence for choline acetyltransferase, somatostatin, and the calcium-binding protein parvalbumin was also performed to elucidate the neurochemical identity of basal forebrain and mesopontine tegmental inputs to the reticular thalamic nucleus. A significant portion (10-15%) of neurons in the basal forebrain and mesopontine tegmentum that were retrogradely labeled from the reticular thalamic nucleus were also found to be retrogradely labeled from the cortex. Many of these neurons stained positively for choline acetyltransferase. Of the basal forebrain neurons retrogradely labeled from the reticular thalamic nucleus, approximately 20% were found to be immunoreactive to choline acetyltransferase, whereas none was stained for somatostatin. A larger portion (up to 50%) of the basal forebrain neurons that were retrogradely labeled from the reticular thalamic nucleus were parvalbumin-immunoreactive, and some of these were also retrogradely labeled from the cortex. These results suggest that a subpopulation of cholinergic and non-cholinergic neurons in the basal forebrain and the mesopontine tegmentum may influence simultaneously the activity of neurons in the reticular thalamic nucleus and the cerebral cortex.     

Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum in the rat

The medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum were investigated by employing (1) the anterograde autoradiographic methodology and (2) the retrograde transport of HRP and/or WGA-HRP in combination with choline acetyltransferase immunohistochemistry. The anterograde experiments identified five descending pathways from the mesopontine tegmentum: (1) Probst's tract, which descends in the dorsolateral reticular formation in close relation to the nucleus of the solitary tract; (2) a ventrolateral branch of Probst's tract that extends ventrolaterally alongside the spinal trigeminal nucleus; (3) a ventromedial branch of Probst's tract that extends ventromedially through the gigantocellular field of the medulla; (4) the medial reticulospinal tract, which descends in parallel with the medial longitudinal fasciculus and turns ventrolaterally along the dorsal surface of the inferior olive to enter the ventrolateral funiculus of the spinal cord; and (5) a crossed ventromedial pathway, which descends in a ventral paramedian position through the magnocellular field of the medulla. The origins of these pathways reflected a rough lateral-to-medial topography of mesopontine tegmental cell groups. The parabrachial nucleus, situated furthest laterally, for example, projected primarily through Probst's tract and its ventrolateral branch. The pedunculopontine tegmental nucleus, midbrain extrapyramidal area, and the subceruleal region, situated more medially, projected descending axons largely through the ventromedial branch of Probst's tract. The pontine tegmental field, situated furthest medially and ventromedially, was the largest contributor to the medial reticulospinal tract. The retrograde transport experiments confirmed these general organizational features. The combination of retrograde transport with choline acetyltransferase immunohistochemistry established that the cholinergic pedunculopontine tegmental nucleus contributes a large portion to the mesopontine tegmental innervation of the medullary reticular formation. A much smaller number of cholinergic pedunculopontine neurons project as far as the spinal cord. Spinal projections from the mesopontine tegmentum originate largely from non-cholinergic neurons of the midbrain extrapyramidal area, subceruleal region, K?lliker-Fuse division of the parabrachial nucleus, and pontine tegmental field.     

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 and non-cholinergic mesopontine tegmental neurons projecting to the subthalamic nucleus in the rat

The subthalamic nucleus (STN) receives cholinergic and non-cholinergic projections from the mesopontine tegmentum. This study investigated the numbers and distributions of neurons involved in these projections in rats using Fluorogold retrograde tracing combined with immunostaining of choline acetyltransferase and a neuron-specific nuclear protein. The results suggest that a small population of cholinergic neurons mainly in the caudoventral part of the pedunculopontine tegmental nucleus (PPN), approximately 360 neurons (≈ 10% of the total) in the homolateral and 80 neurons (≈ 2%) in the contralateral PPN, projects to the STN. In contrast, the number of non-cholinergic neurons projecting to the STN was estimated to be nine times as much, with approximately 3300 in the homolateral side and 1300 in the contralateral side. A large gathering of the Fluorogold-labeled non-cholinergic neurons was found rostrodorsomedial to the caudolateral PPN. The biotinylated dextran amine (BDA) anterograde tracing method was used to substantiate the mesopontine-STN projections. Injection of BDA into the caudoventral PPN labeled numerous thin fibers with small en-passant varicosities in the STN. Injection of BDA into the non-cholinergic neuron-rich area labeled a moderate number of thicker fibers with patches of aggregates of larger boutons. The densities of labeled fibers and the number of retrogradely labeled cells in the mesopontine tegmentum suggested that the terminal field formed in the STN by each cholinergic neuron is more extensive than that formed by each non-cholinergic neuron. The findings suggest that cholinergic and non-cholinergic mesopontine afferents may carry different information to the STN.     

Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistochemistry

The distribution of cholinergic neurons in the midbrain and pons which project directly to the thalamus was investigated in the rat using a procedure which allows the simultaneous detection of retrogradely transported horseradish peroxidase (HRP) and immunohistochemical demonstration of choline acetyltransferase (ChAT) in the same neurons. HRP injections were placed in the dorsal half of the anterior third of the thalamus on one side which included the anteroventral nucleus as well as portions of the rostral intralaminar and reticular nuclei. These thalamic nuclei showed the highest density of immunohistochemically detectable cholinergic fibers. Neurons containing both HRP and ChAT, which represented cholinergic neurons projecting directly to the thalamus, were found in the midbrain and pons in the lateral tegmental reticular formation, parabrachial region and lateral dorsal tegmental nucleus. Ipsilateral to the injection site over 91% of the HRP labeled neurons in all of these regions were cholinergic, while an average of 60% of the cholinergic neurons had transported HRP. Contralateral to the injection site 5-6% of the cholinergic neurons in these regions were also retrogradely labeled. These findings demonstrate direct cholinergic projections to the thalamus from neurons in several regions in the tegmentum and suggest that tegmental projections to the thalamus are predominantly cholinergic.     

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.     

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.     

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.     

Neural associations of the substantia innominata in the rat: afferent connections

The afferent connections of the substantia innominata (SI) in the rat were determined employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), in combination with histochemical procedures to characterize the neuropil of the SI and identify cholinergic cells. Both neurochemical and connectional data establish that the SI is organized into a dorsal and a ventral division. Each of these divisions is strongly affiliated with a different region of the amygdala, and, together with its amygdalar affiliate, forms part of one of two largely distinct constellations of interconnected forebrain and brainstem cell groups. The dorsal SI receives selective innervation from the lateral part of the bed nucleus of the stria terminalis, the central and basolateral nuclei of the amygdala, the fundus of the striatum, distinctive perifornical and caudolateral zones of the lateral hypothalamus, and caudal brainstem structures including the dorsal raphe nucleus, parabrachial nucleus, and nucleus of the solitary tract. Projections preferentially directed to the ventral SI arise from the medial part of the bed nucleus of the stria terminalis, the rostral two-thirds of the medial nucleus of the amygdala, a large region of the rat amygdala that lies ventral to the central nucleus, the medial preoptic area, anterior hypothalamus, medialmost lateral hypothalamus, and the ventromedial hypothalamus. Both SI divisions appear to receive afferents from the dorsomedial and posterior hypothalamus, supramammillary region, ventral tegmental area, and the peripeduncular area of the midbrain. Projections to the SI whose selectivity was not determined originate from medial prefrontal, insular, perirhinal, and entorhinal cortex and from midline thalamic nuclei. Findings from both PHA-L and WGA-HRP experiments additionally indicate that cell groups preferentially innervating a single SI division maintain numerous projections to one another, thus forming a tightly linked assembly of structures. In the rat, cholinergic neurons that are scattered throughout the SI and in parts of the globus pallidus make up a cell population equivalent to the primate basal nucleus of Meynert (Mesulam et al.: Neuroscience 10:1185-1201, '83). PHA-L-filled axons, labelled from lectin deposits in the dorsal raphe nucleus, peripeduncular area, ventral tegmental area, or caudomedial hypothalamus were occasionally seen to approach individual cholinergic neurons int he SI, and to contact the surface of such cells with axonal varicosities (putative synaptic boutons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anatomical interconnections of the pedunculopontine tegmental nucleus and the nucleus prepositus hypoglossi in the cat

The Pedunculopontine tegmental nucleus (PPT) is a mesopontine structure containing predominantly cholinergic neurons, and physiological data indicate its neurons transfer eye-movement gated ponto-geniculo-occipital (PGO) waves to thalamus during the rapid eye movement phase of sleep. The present study, using anterograde and retrograde tracing of wheat germ agglutinin-conjugated horseradish peroxidase, found that the medullary nucleus Prepositus hypoglossi (PH), whose neurons are known to have eye-movement-related information, projects densely to PPT, and PPT has reciprocal projections to PH. The PH-PPT projection has some topographic organization, with rostral PH to rostral PPT and caudal PH to caudal PPT projections dominating. The PH-PPT projection may furnish the anatomical substrate for input of eye movement-related information into the rostral PGO wave system.     

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.