Subpopulations of cholinergic, GABAergic and glutamatergic neurons in the pedunculopontine nucleus contain calcium-binding proteins and are heterogeneously distributed

Neurons in the pedunculopontine nucleus (PPN) are highly heterogeneous in their discharge properties, their neurochemical markers, their pattern of connectivity and the behavioural processes in which they participate. Three main transmitter phenotypes have been described, cholinergic, GABAergic and glutamatergic, and yet electrophysiological evidence suggests heterogeneity within these subtypes. To gain further insight into the molecular composition of these three populations in the rat, we investigated the pattern of expression of calcium binding proteins (CBPs) across distinct regions of the PPN and in relation to the presence of other neurochemical markers. Calbindin- and calretinin-positive neurons are as abundant as cholinergic neurons, and their expression follows a rostro-caudal gradient, whereas parvalbumin is expressed by a low number of neurons. We observed a high degree of expression of CBPs by GABAergic and glutamatergic neurons, with a large majority of calbindin- and calretinin-positive neurons expressing GAD or VGluT2 mRNA. Notably, CBP-positive neurons expressing GAD mRNA were more concentrated in the rostral PPN, whereas the caudal PPN was characterized by a higher density of CBP-positive neurons expressing VGluT2 mRNA. In contrast to these two large populations, in cholinergic neurons expression of calretinin is observed only in low numbers and expression of calbindin is virtually non-existent. These findings thus identify novel subtypes of cholinergic, GABAergic and glutamatergic neurons based on their expression of CBPs, and further contribute to the notion of the PPN as a highly heterogeneous structure, an attribute that is likely to underlie its functional complexity.     

Evolution of the basal ganglia: dual-output pathways conserved throughout vertebrate phylogeny

The basal ganglia, including the striatum, globus pallidus interna and externa (GPe), subthalamic nucleus (STN), and substantia nigra pars compacta, are conserved throughout vertebrate phylogeny and have been suggested to form a common vertebrate mechanism for action selection. In mammals, this circuitry is further elaborated by the presence of a dual-output nucleus, the substantia nigra pars reticulata (SNr), and the presence of modulatory input from the cholinergic pedunculopontine nucleus (PPN). We sought to determine whether these additional components of the mammalian basal ganglia are also present in one of the phylogenetically oldest vertebrates, the lamprey. We show, by using immunohistochemistry, tract tracing, and whole-cell recordings, that homologs of the SNr and PPN are present in the lamprey. Thus the SNr receives direct projections from inwardly rectifying γ-aminobutyric acid (GABA)-ergic striatal neurons expressing substance P, but it is also influenced by indirect basal ganglia projections from the STN and potentially the GPe. Moreover, GABAergic SNr projection neurons are tonically active and project to the thalamus and brainstem motor areas. The homolog of the PPN contains both cholinergic and GABAergic neurons and is connected with all the nuclei of the basal ganglia, supporting its proposed role as part of an extended basal ganglia. A separate group of cholinergic neurons dorsal to the PPN corresponds to the descending mesencephalic locomotor region. Our results suggest that dual-output nuclei are part of the ancestral basal ganglia and that the PPN appears to have coevolved as part of a mechanism for action selection common to all vertebrates.     

Temporal-Spatial Profiling of Pedunculopontine Galanin-Cholinergic Neurons in the Lactacystin Rat Model of Parkinson’s Disease

Parkinson’s disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, while being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilizing the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, and increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin-lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration.     

Cholinergic and Nadph-δ neurons in the pedunculopontine and laterodorsal tegmental nuclei of human and nonhuman primates

The brainstem pedunculopontine (PPN) and laterodorsal tegmental (LDTg) nuclei are involved in multifarious activities, including motor control. Yet, their exact cytoarchitectural boundaries are still uncertain. We therefore initiated a comparative study of the topographical and neurochemical organization of the PPN and LDTg in cynomolgus monkeys (Macaca fascicularis) and humans. The distribution and morphological characteristics of neurons expressing choline acetyltransferase (ChAT) and/or nicotinamide adenine dinucleotide phosphate diaphorase (Nadph-δ) were documented. The number and density of the labeled neurons were obtained by stringent stereological methods, whereas their topographical distribution was reported upon corresponding magnetic resonance imaging (MRI) planes. In both human and nonhuman primates, the PPN and LDTg are populated by three neurochemically distinct types of neurons (ChAT-/Nadph-δ+, ChAT+/Nadph-δ-, and ChAT+/Nadph-δ+), which are distributed according to a complex spatial interplay. Three-dimensional reconstructions reveal that ChAT+ neurons in the PPN and LDTg form a continuum with some overlaps with pigmented neurons of the locus coeruleus, dorsally, and of the substantia nigra (SN) complex, ventrally. The ChAT+ neurons in the PPN and LDTg are —two to three times more numerous in humans than in monkeys but their density is —three to five times higher in monkeys than in humans. Neurons expressing both ChAT and Nadph-δ have a larger cell body and a longer primary dendritic arbor than singly labeled neurons. Stereological quantification reveals that 25.6% of ChAT+ neurons in the monkey PPN are devoid of Nadph-δ staining, a finding that questions the reliability of Nadph-δ as a marker for cholinergic neurons in primate brainstem.     

Mitochondrial DNA changes in pedunculopontine cholinergic neurons in Parkinson disease

In Parkinson disease (PD), mitochondrial dysfunction associates with nigral dopaminergic neuronal loss. Cholinergic neuronal loss co‐occurs, particularly within a brainstem structure, the pedunculopontine nucleus (PPN). We isolated single cholinergic neurons from postmortem PPNs of aged controls and PD patients. Mitochondrial DNA (mtDNA) copy number and mtDNA deletions were increased significantly in PD patients compared to controls. Furthermore, compared to controls the PD patients had significantly more PPN cholinergic neurons containing mtDNA deletion levels exceeding 60%, a level associated with deleterious effects on oxidative phosphorylation. The current results differ from studies reporting mtDNA depletion in nigral dopaminergic neurons of PD patients. Ann Neurol 2017;82:1016–1021     

The pedunculopontine nucleus in developmental disorders of the basal ganglia

The pedunculopontine nucleus (PPN), which is located in the upper brainstem, contains cholinergic and non‐cholinergic neurons, and has afferent and efferent connections to the basal ganglia and spinal cord. The PPN is known to be affected in adult‐onset basal ganglia diseases, and we speculated that the PPN might be similarly insulted in developmental basal ganglia disorders. We immunohistochemically examined the expression patterns of acetylcholine esterase and tyrosine hydroxylase, markers of acetylcholinergic and catecholaminergic neurons, respectively, in the PPN pars dissipata (PPNd) of controls and patients with bilirubin encephalopathy (BE) and perinatal hypoxic ischemic encephalopathy with localized basal ganglia lesion (HIEbg). Controls showed an age‐dependent change in the percentages of acetylcholinergic and catecholaminergic neurons. Three out of six BE cases and three out of six HIEbg cases showed a reduction in the percentage of acetylcholinergic neurons in the PPNd. Additionally, three BE cases demonstrated an increase in the percentage of catecholaminergic neurons. It is likely that the relative proportions of acetylcholinergic and catecholaminergic neurons in the PPN can be altered in developmental basal ganglia disorders.     

Distribution and organization of cholinergic neurons in the rat forebrain demonstrated by computer-aided data acquisition and three-dimensional reconstruction

An understanding of the organization of cholinergic neurons in the central nervous system has been an important objective for many years. By developing and applying a new electronic method for mapping tissue sections, we have generated original graphic and quantitative findings on forebrain cholinergic neurons that provide new insight into their distribution and organization. Satoh, Armstrong, and Fibiger (Brain Res. Bull. 11:693-720, 1983) have proposed that in the basal forebrain cholinergic neurons with long axons form a continuum rather than being arranged as a series of discrete nuclear groups. It has been difficult, however, by conventional methods of data analysis and display, to test this hypothesis. By using a digital microscopy system, the position of every cholinergic neuron was marked with 1-micron resolution in tissue sections taken at 90-microns or 180-microns intervals through the entire distribution of these neurons in the forebrain. The three-dimensional reconstruction of these neurons in context shows them to be distributed as a continuous cell column. The column twists and changes position as it is deformed by adjacent neuronal structures, such that its shape and continuity would not be apparent without reconstruction into a computer graphics model. Complementary analyses of the distribution of cholinergic interneurons in dopamine-rich regions of the forebrain indicated that there are regional differences between striatal and olfactory tubercle neurons. Cellular morphometry analyses show the population of cholinergic neurons in the rat to be surprisingly homogenous in size, but not in shape. Graphic and quantitative analyses indicated that there is a striking relationship between the distributions of projection and interneuronal cell groups. We conclude that the basal forebrain cholinergic neurons form a continuum. The chemoarchitecture of this cell group does not conform to the usual cytoarchitectural divisions. The present results, however, taken together with the findings based on Nissl-stained sections and connectional and biochemical data, suggest that the region of this neurochemically defined continuum should be reexamined for consideration as a single functional entity or nucleus: a cholinergic basal nuclear complex.     

Muscarinic and nicotinic responses in the developing pedunculopontine nucleus (PPN)

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system (RAS), is known to modulate waking and rapid eye movement (REM) sleep. REM sleep decreases between 10 and 30 days postnatally in the rat, with the majority occurring between 12 and 21 days. We investigated the possibility that changes in the cholinergic, muscarinic and/or nicotinic, input to PPN neurons could explain at least part of the developmental decrease in REM sleep. We recorded intracellularly from PPN neurons in 12-21 day rat brainstem slices maintained in artificial cerebrospinal fluid (aCSF) and found that application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) depolarized PPN neurons early in development, then hyperpolarized PPN neurons by day 21. Most of the effects of DMPP persisted following application of the sodium channel blocker tetrodotoxin (TTX), and in the presence of glutamatergic, serotonergic, noradrenergic and GABAergic antagonists, but were blocked by the nicotinic antagonist mecamylamine (MEC). The mixed muscarinic agonist carbachol (CAR) hyperpolarized all type II (A current) PPN cells and depolarized all type I (low threshold spike-LTS current) and type III (A+LTS current) PPN cells, but did not change effects during the period known for the developmental decrease in REM sleep. The effects of CAR persisted in the presence of TTX but were mostly blocked by the muscarinic antagonist atropine (ATR), and the remainder by MEC. We conclude that, while the nicotinic inputs to the PPN may help modulate the developmental decrease in REM sleep, the muscarinic inputs appear to modulate different types of cells differentially.     

Long-term effects of striatal lesions on c-Fos immunoreactivity in the pedunculopontine nucleus

The basal ganglia are a group of subcortical nuclei classically thought to be involved in the control of movement, and they have reciprocal connections with the cortex, thalamus and structures in the brainstem. Recent findings suggest that the basal ganglia interact with structures involved in the control of the sleep-waking cycle. The pedunculopontine tegmental nucleus (PPN) maintains a close relationship with the basal ganglia and is intimately involved in the regulation of wakefulness and REM sleep. This study evaluated changes in activity of PPN neurons following striatal kainic acid-induced lesions. Rats were injected in the anterodorsal striatum with either kainic acid or vehicle and allowed to recover for 7 or 30 days. The results showed an increase in the number of c-Fos+ cells in the PPN 30 days but not 7 days after the striatal lesion, when motor hyperactivity was no longer detected. In addition, we found a significant correlation between the ventricular brain ratio, as an indicator of lesion size, and the number of c-Fos+ cells in the PPN. Furthermore, the spatial distribution of cell types suggested that most c-Fos+ cells in the PPN were not cholinergic. These results provide new insights into the functional relationship between the basal ganglia and the PPN and suggest that the striatum, through its indirect influence on the PPN, may contribute to the regulation of wakefulness and cortical activation.     

Sub-regions of the dorsal raphé nucleus receive different inputs from the brainstem

The dorsal raphe nucleus (DRN) through its extensive efferent projections has been implicated in a great variety of physiological and behavioral functions including the regulation of the sleep-wake cycle. This nucleus is composed of five sub-regions defined according to the distribution of its serotonergic (5-HT) neurons. In addition to its heterogeneity in neuronal populations, the DRN contains a great diversity of 5-HT neuronal subtypes identified based on their electrophysiological characteristics, morphology and sub-regional distribution. This suggests that the DRN sub-regions may play different functional roles. Recent studies reported long-range inputs specific to the 5-HT neurons of the DRN; but they did not differentiate whether some inputs were specific to a DRN sub-region, or another region. To fulfill this gap, we have previously described the forebrain afferents to the different sub-regions of the DRN using cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin, as retrograde and anterograde tracers respectively. In the present work, we provide a detailed map of the brainstem projections to these different sub-regions. We show that if some brainstem structures project homogeneously to all sub-regions, most of the brainstem long-range inputs project in a topographically organized manner onto the DRN and, moreover, that a rich interconnected network is present within the DRN.     

Expression of superoxide dismutase messenger RNA in adult rat brain cholinergic neurons

Superoxide dismutase (SOD) protects cells exposed to an excess of the free radical nitric oxide, by preventing the formation of peroxynitrite. Certain central cholinergic neurons express constitutive nitric oxide synthase (nNOS), and presumably they are at risk from peroxynitrite intoxication. Immunocytochemistry for choline acetyltransferase (ChAT) was combined with in situ hybridization histochemistry (ISHH) to examine whether brain cholinergic populations differ with respect to their expression of the messenger RNA molecules (mRNAs) for the manganese-dependent (Mn-SOD) and copper/zinc-dependent superoxide dismutases (Cu/Zn-SOD). The cholinergic neurons located in the reticular formation of the upper brainstem (the laterodorsal tegmental nucleus [LDTN] and the pedunculopontine nucleus [PPN]) were found to express relatively high levels of Mn-SOD mRNA, whereas cholinergic neurons located in the basal forebrain (substantia innominata [SI], diagonal band [DB], medial septum [MS], and the nucleus basalis magnocellularis [nBM]), and the striatal cholinergic interneurons expressed low to intermediate levels of Mn-SOD mRNA. The rank order of median Mn-SOD mRNA density per cholinergic cell was LDTN > PPN > SI > striatum = nBM = DB > MS. This is similar to the rank order of nNOS mRNA densities in the cholinergic cells in these regions (R=0.9, p<0.02). The rank order of Cu/Zn-SOD mRNA levels in cholinergic populations (DB > LDTN = PPN = MS > SI = nBM = striatum) was not correlated with nNOS mRNA (R = 0.29, P>0.05). Thus, for cholinergic neurons, Mn-SOD may be important for protection from NO-related oxidative stress.     

Cholinergic and noncholinergic brainstem neurons expressing Fos after paradoxical (REM) sleep deprivation and recovery

It is well accepted that populations of neurons responsible for the onset and maintenance of paradoxical sleep (PS) are restricted to the brainstem. To localize the structures involved and to reexamine the role of mesopontine cholinergic neurons, we compared the distribution of Fos- and choline acetyltransferase-labelled neurons in the brainstem of control rats, rats selectively deprived of PS for approximately 72 h and rats allowed to recover from such deprivation. Only a few cholinergic neurons from the laterodorsal (LDTg) and pedunculopontine tegmental nuclei were Fos-labelled after PS recovery. In contrast, a large number of noncholinergic Fos-labelled cells positively correlated with the percentage of time spent in PS was observed in the LDTg, sublaterodorsal, alpha and ventral gigantocellular reticular nuclei, structures known to contain neurons specifically active during PS. In addition, a large number of Fos-labelled cells were seen after PS rebound in the lateral, ventrolateral and dorsal periaqueductal grey, dorsal and lateral paragigantocellular reticular nuclei and the nucleus raphe obscurus. Interestingly, half of the cells in the latter nucleus were immunoreactive to choline acetyltransferase. In contrast to the well-accepted hypothesis, our results strongly suggest that neurons active during PS, recorded in the mesopontine cholinergic nuclei, are in the great majority noncholinergic. Our findings further demonstrate that many brainstem structures not previously identified as containing neurons active during PS contain cholinergic or noncholinergic neurons active during PS, and these structures may therefore play a key role during this state. Altogether, our results open a new avenue of research to identify the specific role of the populations of neurons revealed, their interrelations and their neurochemical identity.     

Identification of a subpopulation of neuropeptide Y-containing locus coeruleus neurons that project to the entorhinal cortex

A fundamental question important to the understanding of the neurochemical organization of the central nervous system focuses on the relationships between the differential phenotypic expression of multiple neurotransmitter markers in individual neuronal populations and the factors that regulate their expression. The first approach in studying this phenomenon is the determination of specific relationships between neurochemically distinct neuronal subpopulations and their efferent targets. The pontine nucleus locus coeruleus (LC) provides a useful model for addressing this question since the projections of LC neurons are topographically organized and several neuropeptides are expressed along with noradrenergic markers in subsets of these neurons. In these studies, we have focused on defining the efferent targets of LC neurons that contain neuropeptide tyrosine (NPY)-like immunoreactivity. This has been accomplished by injecting the retrograde fluorescent tracer fluorogold into specific cortical and hippocampal targets in adult rats and identifying the proportion of retrogradely labeled LC neurons that are positive for NPY-like immunoreactivity. In agreement with other investigators, no preferential cortical projections of NPY-positive LC neurons were observed. However, when fluorogold injections included or were limited to the entorhinal cortex, a discrete cluster of round or ovoid neurons in the dorsomedial portion of the LC approximately 9.8 mm posterior to bregma were found to contain NPY-like immunoreactivity. This observation demonstrates that some topographic organization of NPY-containing LC neurons does exist. In fact, these data indicate that morphologic and topographic organization exists even within neurochemically distinct subsets of neuronal populations.     

Expression of calcium‐binding proteins and nNOS in the human vestibular and precerebellar brainstem

Information about the position and movement of the head in space is coded by vestibular receptors and relayed to four nuclei that comprise the vestibular nuclear complex (VNC). Many additional brainstem nuclei are involved in the processing of vestibular information, receiving signals either directly from the eighth nerve or indirectly via projections from the VNC. In cats, squirrel monkeys, and macaque monkeys, we found neurochemically defined subdivisions within the medial vestibular nucleus (MVe) and within the functionally related nucleus prepositus hypoglossi (PrH). In humans, different studies disagree about the borders, sizes, and possible subdivisions of the vestibular brainstem. In an attempt to clarify this organization, we have begun an analysis of the neurochemical characteristics of the human using brains from the Witelson Normal Brain Collection and standard techniques for antigen retrieval and immunohistochemistry. Using antibodies to calbindin, calretinin, parvalbumin, and nitric oxide synthase, we find neurochemically defined subdivisions within the MVe similar to the subdivisions described in cats and monkeys. The neurochemical organization of PrH is different. We also find unique neurochemical profiles for several structures that suggest reclassification of nuclei. These data suggest both quantitative and qualitative differences among cats, monkeys, and humans in the organization of the vestibular brainstem. These results have important implications for the analysis of changes in that organization subsequent to aging, disease, or loss of input. J. Comp. Neurol. 518:872–895, 2010. © 2009 Wiley‐Liss, Inc.     

A cholinergic projection to the rat superior colliculus demonstrated by retrograde transport of horseradish peroxidase and choline acetyltransferase immunohistochemistry

Acetylcholinesterase (AChE) has been localized by histochemistry in the superior colliculus and in the tegmentum of the caudal midbrain and rostral pons of the rat. The pattern of AChE localization in the superior colliculus was characterized by homogeneous staining in the superficial layers and patchlike staining in the intermediate gray layer. In the tegmentum, AChE was localized in the pedunculopontine nucleus (PPN), beginning rostrally at the caudal pole of the substantia nigra and extending caudally to the level of the parabrachial nuclei, and in the lateral dorsal tegmental nucleus (LDTN) of the central gray. The localization of AChE in these nuclei overlapped the distribution of neurons stained by immunohistochemistry using an antibody to choline acetyltransferase (ChAT), the synthesizing enzyme of the neurotransmitter acetylcholine. Other neighboring areas that were stained with AChE, but that did not contain ChAT-immunoreactive neurons, included the microcellular tegmental nucleus and the ventral tegmental nucleus. Neurons in the PPN and LDTN were determined to be potential sources of the cholinergic projection to the intermediate gray layer of the rat superior colliculus by double labelling with retrograde transport of horseradish peroxidase (HRP) combined with the immunohistochemical localization of ChAT. Three populations of neurons were identified. A predominantly ipsilateral ChAT-immunoreactive population was located in the pars compacta subdivision of PPN (PPNpc). Retrograde HRP-labelled neurons in the pars dissipata subdivision of the PPN (PPNpd), located ventral to the superior cerebellar peduncle (SCP) at the level of the inferior colliculus, composed a second population that was predominantly contralateral but was not ChAT immunoreactive. A third population of retrogradely labelled neurons was predominantly ipsilateral and ChAT immunoreactive and was located in the LDTN. These findings compared favorably with the full extent of the projection from this tegmental region revealed by retrograde transport of HRP from the superior colliculus when more compatible fixation and chromogen procedures were used. The results suggest that the PPN and the LDTN are two sources of the cholinergic input to the superior colliculus. Since the PPN also has extensive efferent, and afferent, connections with basal-ganglia-related structures, this cholinergic excitatory input to the superior colliculus, like the GABA-ergic inhibitory input from the substantia nigra pars reticulata, may provide the basis for an additional influence of the basal ganglia on visuomotor behavior.     

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.     

Brainstem dopaminergic, cholinergic and serotoninergic afferents to the pallidum in the squirrel monkey

The retrograde tracer cholera toxin B subunit (CTb) was used in combination with immunohistochemistry for tyrosine hydroxylase (TH), calbindin D-28k (CaBP), choline acetyltransferase (ChAT) and 5-hydroxytryptamine (5-HT) to determine the distribution and relative proportion of brainstem chemospecific neurons that project to the pallidum in the squirrel monkey (Saimiri sciureus). Large injections of CTb involving both pallidal segments produce numerous retrogradely labeled neurons in the substantia nigra (SN), the pedunculopontine tegmental nucleus (PPN) and the dorsal raphe nucleus (DR). Labeled neurons are distributed uniformly in SN with a slight numerical increase at the junction between the pars compacta (SNc) and the ventral tegmental area (VTA). Retrogradely labeled neurons abound also in PPN, principally in its pars dissipata, whereas other CTb-labeled cells are scattered throughout the rostrocaudal extent of DR. After CTb injection involving specifically the internal pallidal segment (GPi), the same pattern of cell distribution is found in SN, PPN and DR, except that the number of retrogradely labeled cells is lower than after large pallidal complex injections. Approximately 70% of all CTb-labeled neurons in SNc-VTA complex display TH immunoreactivity, whereas 20% are immunoreactive for CaBP. About 39% of all retrogradely labeled neurons in PPN are immunoreactive for ChAT, whereas approximately 38% of the labeled neurons in DR display 5-HT immunoreactivity. Following CTb injection in the external pallidal segment (GPe), the number of labeled cells is much smaller than after GPi injection. The majority of CTb-labeled cells in SNc-VTA complex are located in the lateral half of SNc and approximately 93% of these neurons display TH immunoreactivity compared to 10% that are immunoreactive for CaBP; very few CTb-labeled cells occur in PPN. Retrogradely labeled cells in DR are located more laterally than those that projects to the GPi and about 25% of them are immunoreactive for 5-HT. These results suggest that, in addition to their action at striatal and/or nigral levels, the brainstem dopaminergic, cholinergic and serotoninergic neurons influence the output of the primate basal ganglia by acting directly upon GPi neurons.     

Glutamatergic neuronal populations in the brainstem of the sea lamprey,Petromyzon marinus: An in situ hybridization and immunocytochemical study

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium‐sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract‐tracing methods or γ‐aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium‐sized cells of some reticulospinal‐projecting groups were often glutamate‐immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal‐projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non‐glutamatergic cells or neurons that co‐release both GABA and glutamate. J. Comp. Neurol. 521:522–557, 2013. © 2012 Wiley Periodicals, Inc.     

GABAergic interneurons in human subthalamic nucleus.

The subthalamic nucleus (STN) is considered a homogeneous structure composed essentially of projection neurons that exert a profound glutamate-mediated excitatory influence upon the main output structures of the basal ganglia. It is currently the most efficient target for deep brain stimulations designed to alleviate symptoms of Parkinson's disease. STN neurons were analyzed by applying stereological methods and single/double-immunostaining procedures to postmortem material obtained from normal individuals. Besides a multitude of closely packed projection neurons ( approximately 24.7 mum in diameter), the human STN (mean volume, 174.5 +/- 20.4 mm3; total neuronal density, 239.5 +/- 31.9 x 10(3)) contained smaller neurons (approximately 12.2 microm), which displayed glutamic acid decarboxylase (GAD)(65/67) immunoreactivity and shared the morphological features of interneurons described in Golgi studies of primate STN. These putative gamma-aminobutyric acid (GABA)ergic interneurons accounted for 7.5% of the total neuronal population of the STN. Although present throughout the nucleus, they were significantly more numerous in its posterior-ventral-medial sector, which belongs to the limbic/associative functional territory. Many projection neurons located dorsolaterally in the STN showed parvalbumin immunoreactivity and others lying ventromedially displayed calretinin immunostaining, but none of the GAD-positive interneurons expressed these calcium-binding proteins. Although less abundant than projection neurons, GABAergic interneurons might play a important role in the intrinsic organization of the STN. The morphological and chemical heterogeneity of the human STN reported here might have important implications in the functional organization of the basal ganglia.     

Neuropathological analysis of brainstem cholinergic and catecholaminergic nuclei in relation to rapid eye movement (REM) sleep behaviour disorder

B. N. Dugger, M. E. Murray, B. F. Boeve, J. E. Parisi, E. E. Benarroch, T. J. Ferman and D. W. Dickson (2012) Neuropathology and Applied Neurobiology 38, 142–152 Neuropathological analysis of brainstem cholinergic and catecholaminergic nuclei in relation to rapid eye movement (REM) sleep behaviour disorder Aims: Rapid eye movement sleep behaviour disorder (RBD) is characterized by loss of muscle atonia during rapid eye movement sleep and is associated with dream enactment behaviour. RBD is often associated with α‐synuclein pathology, and we examined if there is a relationship of RBD with cholinergic neuronal loss in the pedunculopontine/laterodorsal tegmental nucleus (PPN/LDT), compared to catecholaminergic neurones in a neighbouring nucleus, the locus coeruleus (LC). Methods: This retrospective study utilized human brain banked tissues of 11 Lewy body disease (LBD) cases with RBD, 10 LBD without RBD, 19 Alzheimer's disease (AD) and 10 neurologically normal controls. Tissues were stained with choline acetyl transferase immunohistochemistry to label neurones of PPN/LDT and tyrosine hydroxylase for the LC. The burden of tau and α‐synuclein pathology was measured in the same regions with immunohistochemistry. Results: Both the LC and PPN/LDT were vulnerable to α‐synuclein pathology in LBD and tau pathology in AD, but significant neuronal loss was only detected in these nuclei in LBD. Greater cholinergic depletion was found in both LBD groups, regardless of RBD status, when compared with normals and AD. There were no differences in either degree of neuronal loss or burden of α‐synuclein pathology in LBD with and without RBD. Conclusions: Whether decreases in brainstem cholinergic neurones in LBD contribute to RBD is uncertain, but our findings indicate these neurones are highly vulnerable to α‐synuclein pathology in LBD and tau pathology in AD. The mechanism of selective α‐synuclein‐mediated neuronal loss in these nuclei remains to be determined.