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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
Abstract
The topographical relationships between cholinergic neurons, identified by their immunoreactivity for choline acetyltransferase (ChAT) or their staining for beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, noradrenergic, 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 to 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 occur in 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 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.
... Employing ChAT immunostaining, Mesulam and colleagues described two main ACh cell groups in the human and nonhuman primate brainstem: group Ch5, which referred to the ACh neurons in the PPN, and group Ch6, corresponding to the LDTg (Mesulam et al., 1984(Mesulam et al., , 1989. This ChAT immunostaining approach has since been largely employed to delineate these brainstem reticular nuclei in various species, including monkeys (Lavoie & Parent, 1994;, mice (Mufson et al., 1986), rats Hallanger et al., 1987;Mesulam et al., 1983;Rye et al., 1987;Skinner et al., 1989;Sofroniew et al., 1985;Woolf & Butcher, 1986), cats (Jones & Beaudet, 1987;Kimura et al., 1981;Mizukawa et al., 1989;Vincent & Reiner, 1987) and dogs (Isaacson & Tanaka, 1986). Altogether, these studies have shown that the exact boundaries of the PPN and LDTg within the mesencephalic reticular formation are difficult to trace largely because these two ACh groups overlap partially (Alam et al., 2011;Martinez-Gonzalez et al., 2011;Mazzone et al., 2005Mazzone et al., , 2012Mena-Segovia & Bolam, 2017;Nowacki et al., 2019;Stefani et al., 2007;Wang & Morales, 2009;Zrinzo et al., 2007Zrinzo et al., , 2008Zrinzo et al., , 2011. ...
... Classically defined as the major ACh neuronal populations of the brainstem, the ACh neurons of the PPN and LDTg are now known to be intermingled with glutamatergic and GABAergic neurons (Boucetta & Jones, 2009;Lavoie & Parent, 1994;Luquin et al., 2018;Mena-Segovia & Bolam, 2017;Mena-Segovia et al., 2009;Wang & Morales, 2009), the density of which varies significantly across the rostrocaudal axis of the brainstem (Mena-Segovia et al., 2009;Wang & Morales, 2009). The ACh neurons of the PPN and LDTg also express a mitochondrial enzyme called nicotinamide adenine dinucleotide phosphate diaphorase (Nadph-δ) (Geula et al., 1993;Lavoie & Parent, 1994;Mesulam et al., 1989). ...
... Classically defined as the major ACh neuronal populations of the brainstem, the ACh neurons of the PPN and LDTg are now known to be intermingled with glutamatergic and GABAergic neurons (Boucetta & Jones, 2009;Lavoie & Parent, 1994;Luquin et al., 2018;Mena-Segovia & Bolam, 2017;Mena-Segovia et al., 2009;Wang & Morales, 2009), the density of which varies significantly across the rostrocaudal axis of the brainstem (Mena-Segovia et al., 2009;Wang & Morales, 2009). The ACh neurons of the PPN and LDTg also express a mitochondrial enzyme called nicotinamide adenine dinucleotide phosphate diaphorase (Nadph-δ) (Geula et al., 1993;Lavoie & Parent, 1994;Mesulam et al., 1989). Hence, staining for Nadph-δ has therefore often been used to delineate the PPN and LDTg largely because this histochemical procedure is simpler than ChAT immunostaining (Vincent et al., 1983(Vincent et al., , 1986. ...
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.
... Detailed immunohistochemical mapping studies of the nuclear organization of the catecholaminergic system, most using antibodies directed against tyrosine hydroxylase (TH), have been conducted in over 40 nonprimate mammal species (e.g., Calvey et al., 2013;Calvey, Alagaili et al., 2015;Dell et al., 2010;Dell, Karlsson, et al., 2016;Dell, Patzke, Spocter, Bertelsen et al., 2016;Imam et al., 2018;Maseko et al., 2013;Patzke et al., 2014;Pillay et al., 2017). Similar studies of this system across nonhuman primates are more limited, with studies having been undertaken on three species of strepsirrhine (Calvey, Patzke, Kaswera-Kyamayaka, et al., 2015), two species of platyrrhine (Felten et al., 1974;Hubbard & Di Carlo, 1974;Jacobowitz & MacLean, 1978;Lavoie & Parent, 1994), and three species of catarrhine primates (Garver & Sladek, 1975;Satoh & Fibiger, 1985;Schofield & Everitt, 1981), including humans (Bogerts, 1981;Halliday et al., 1988;Kitahama et al., 1996;Pearson et al., 1983). More recently, a systematic quantitative study of neuronal numbers in the locus coeruleus of primates, including rhesus monkeys (Macaca mulatta), lar gibbon (Hylobates lar), Bornean gibbon (Hylobates muelleri), Western lowland gorillas (Gorilla gorilla), chimpanzees (Pan troglodytes) and humans (Homo sapiens), indicated that neuronal numbers within the locus coeruleus scale with the size of the medulla oblongata (Sharma et al., 2010). ...
... No predominant dendritic orientation could be ascertained for these A8 neurons (Figures 5h and 6j), although they appear to run between the fascicles of passage in the midbrain tegmentum. In comparison to other nonhominoid primates (Calvey, Patzke, Kaswera-Kyamayaka et al., 2015;Felten et al., 1974;Garver & Sladek, 1975;Hubbard & Di Carlo, 1974;Jacobowitz & MacLean, 1978;Lavoie & Parent, 1994;Satoh & Fibiger, 1985;Schofield & Everitt, 1981), the neurons forming the A8 nucleus appear to be distributed over a greater proportion of the midbrain tegmentum. ...
... The organization of the locus coeruleus complex in the primates, as exemplified in the current study of a lar gibbon and a chimpanzee, is a feature that distinguishes the primates from most other mammalian species that have been investigated. As mentioned above, the A4 in the two apes studied is extensive in comparison to other primates, but the presence of both diffuse and compact divisions of the A6 is a specific trait that has a restricted phylogenetic occurrence, occurring only in primates (Bogerts, 1981;Calvey, Patzke, Kaswera-Kyamayaka, 2015;Felten et al., 1974;Garver & Sladek, 1975;Halliday et al., 1988;Hubbard & Di Carlo, 1974;Jacobowitz & MacLean, 1978;Kitahama et al., 1996;Lavoie & Parent, 1994;Pearson et al., 1983;Satoh & Fibiger, 1985;Schofield & Everitt, 1981;Sharma et al., 2010) and megachiropteran bats Maseko et al., 2007). This type of A6 parcellation is not observed in the nonprimate members of the classically defined Euarchontoglires (Blessing et al., 1978;Calvey, Alagaili et al., 2015;Murray et al., 1982). ...
Using tyrosine hydroxylase immunohistochemistry, we describe the nuclear parcellation of the catecholaminergic system in the brains of a lar gibbon (Hylobates lar) and a chimpanzee (Pan troglodytes). The parcellation of catecholaminergic nuclei in the brains of both apes is virtually identical to that observed in humans and shows very strong similarities to that observed in mammals more generally, particularly other primates. Specific variations of this system in the apes studied include an unusual high‐density cluster of A10dc neurons, an enlarged retrorubral nucleus (A8), and an expanded distribution of the neurons forming the dorsolateral division of the locus coeruleus (A4). The additional A10dc neurons may improve dopaminergic modulation of the extended amygdala, the enlarged A8 nucleus may be related to the increased use of communicative facial expressions in the hominoids compared to other primates, while the expansion of the A4 nucleus appears to be related to accelerated evolution of the cerebellum in the hominoids compared to other primates. In addition, we report the presence of a compact division of the locus coeruleus proper (A6c), as seen in other primates, that is not present in other mammals apart from megachiropteran bats. The presence of this nucleus in primates and megachiropteran bats may reflect homology or homoplasy, depending on the evolutionary scenario adopted. The fact that the complement of homologous catecholaminergic nuclei is mostly consistent across mammals, including primates, is advantageous for the selection of model animals for the study of specific dysfunctions of the catecholaminergic system in humans.
... Despite the vast majority of human studies focused especially on striatofugal and pallidofugal pathways, connections between the GP and brainstem nuclei are widely documented [14]. Among these, ipsilateral and contralateral projections between the pedunculopontine tegmental nucleus (PPN) and pallidal segments have been described in rats and monkeys [15][16][17][18][19][20][21]. The PPN is a mostly cholinergic nucleus located in the mesencephalic locomotor region, within the mesopontine tegmentum [22], sharing connections with the basal ganglia, thalamus, cerebral cortex and other brainstem regions [23]. ...
... We successfully reconstructed pallidotegmental pathways already described in non-human primates [16,21,65] and also in humans [32,35,66]. To the best of our knowledge, this is the first time that the crossed pallidotegmental pathway between the PPN and GPi has been reconstructed in vivo in humans by means of tractography. ...
... To the best of our knowledge, this is the first time that the crossed pallidotegmental pathway between the PPN and GPi has been reconstructed in vivo in humans by means of tractography. We found that both the ipsilateral and contralateral pallidotegmental bundles here reconstructed followed an anatomical course similar to those described in primates [16,21]. Ipsilateral tracts traversed the medial portion of mesopontine tegmentum, ascending through the cerebral peduncle and finally reaching the ipsilateral pallidal complex [21]. ...
Background and objectives: The internal (GPi) and external segments (GPe) of the globus pallidus represent key nodes in the basal ganglia system. Connections to and from pallidal segments are topographically organized, delineating limbic, associative and sensorimotor territories. The topography of pallidal afferent and efferent connections with brainstem structures has been poorly investigated. In this study we sought to characterize in-vivo connections between the globus pallidus and the pedunculopontine nucleus (PPN) via diffusion tractography. Materials and Methods: We employed structural and diffusion data of 100 subjects from the Human Connectome Project repository in order to reconstruct the connections between the PPN and the globus pallidus, employing higher order tractography techniques. We assessed streamline count of the reconstructed bundles and investigated spatial relations between pallidal voxels connected to the PPN and pallidal limbic, associative and sensorimotor functional territories. Results: We successfully reconstructed pallidotegmental tracts for the GPi and GPe in all subjects. The number of streamlines connecting the PPN with the GPi was greater than the number of those joining it with the GPe. PPN maps within pallidal segments exhibited a distinctive spatial organization, being localized in the ventromedial portion of the GPi and in the ventral-anterior portion in the GPe. Regarding their spatial relations with tractography-derived maps of pallidal functional territories, the highest value of percentage overlap was noticed between PPN maps and the associative territory. Conclusions: We successfully reconstructed the anatomical course of the pallidotegmental pathways and comprehensively characterized their topographical arrangement within both pallidal segments. PPM maps were localized in the ventromedial aspect of the GPi, while they occupied the anterior pole and the most ventral portion of the GPe. A better understanding of the spatial and topographical arrangement of the pallidotegmental pathways may have pathophysiological and therapeutic implications in movement disorders.
... In situ hybridization labelling studies and immunohistochemistry demonstrate that the PPN contains distinct neuronal populations of glutamatergic, GABAergic, apart from cholinergic cells (Wang & Morales, 2009). Coexpression of markers for glutamate and GABA with markers for ACh implies that the cholinergic PPN neurons may also release glutamate and GABA (Lavoie & Parent, 1994). ...
... Both cholinergic and glutamatergic neurons contact nigral neurons (Lavoie & Parent, 1994). It has been also shown that nigral DA neurons receive glutamatergic and possibly GABAergic inputs from the PPN (Charara et al., 1996). ...
... Noradrenergic neurons of the LC are quite segregated from PPN neurons but overlapping has been also reported (Lavoie & Parent, 1994). In the PPN, cells became fewer medially and are intertwined with neurons of the LC. ...
The pedunculopontine nucleus (PPN) is part of the mesencephalic locomotor region (MLR) and has been involved in the control of gait, posture, locomotion, sleep, and arousal. It likely participates in some motor and non-motor symptoms of Parkinson's disease and is regularly proposed as a surgical target to ameliorate gait, posture and sleep disorders in parkinsonian patients. The PPN overlaps with the monoaminergic systems including dopamine, serotonin and noradrenaline in the modulation of the above-mentioned functions. All these systems are involved in Parkinson's disease and the mechanism of the antiparkinsonian agent, mostly L-DOPA. This suggests that PPN interacts with monoaminergic neurons and vice versa, but the data are not so clear. Some evidence indicate that the PPN sends cholinergic, glutamatergic and even gabaergic to mesencephalic dopaminergic cells, the data regarding serotonergic or noradrenergic cells being less known. Similarly, the controls exerted by PPN on dopaminergic neurons, multiple and complex, is better known than the other monoaminergic systems. The data regarding the influence of monoaminergic systems on PPN neuron activity are rather scarce. While there is evidence that the PPN influences the therapeutic response of L-DOPA, the antiparkinsonian agent whom the dopaminergic effects rely on the three monoaminergic systems, it is still difficult to emphasize the reciprocal action of PPN and monoaminergic systems in this action. Additional data are required to better understand the functional organization of monoaminergic in the MLR including the PPN to get a clearer picture on their interaction.
... Ascending PPN outputs project via the ventral and dorsal tegmental bundle pathways carry major cholinergic projections (Garcia-Rill, 1991) to all thalamic nuclei (Lavoie and Parent, 1994b). Strong cholinergic innervations to the intralaminar and reticular nuclei were also revealed (Mesulam et al., 1992a). ...
... The globus pallidus interna (GPi) of the globus pallidum (GP) sends inhibitory efferent fibers to the ipsilateral PPN. Anterograde tracer studies reveal that the PPN sends substantial efferent fibers to the GPi (Lavoie and Parent, 1994b) rather than the globus pallidus externa (GPe). In humans, the GP receives cholinergic innervations from the brainstem (Mesulam et al., 1983). ...
... Furthermore, cell bodies analogous to the dopaminergic peri-and retrorubral cell clusters decrease rapidly posteriorly in the anterior PPN. Lavoie and Parent (1994b) also report that DA and cholinergic cells dominate adjoining but definite regions, with the dopaminergic population more anteriorly and laterodorsally located. Thus, the PPN along with the CuN receives dopaminergic innervation, endorsing that DA has a role in neural activity modulation of these structures. ...
The pedunculopontine nucleus (PPN) is situated in the upper pons in the dorsolateral portion of the ponto-mesencephalic tegmentum. Its main mass is positioned at the trochlear nucleus level, and is part of the mesenphalic locomotor region (MLR) in the upper brainstem. The human PPN is divided into two subnuclei, the pars compacta (PPNc) and pars dissipatus (PPNd), and constitutes both cholinergic and non-cholinergic neurons with afferent and efferent projections to the cerebral cortex, thalamus, basal ganglia (BG), cerebellum, and spinal cord. The BG controls locomotion and posture via GABAergic output of the substantia nigra pars reticulate (SNr). In PD patients, GABAergic BG output levels are abnormally increased, and gait disturbances are produced via abnormal increases in SNr-induced inhibition of the MLR. Since the PPN is vastly connected with the BG and the brainstem, dysfunction within these systems lead to advanced symptomatic progression in Parkinson's disease (PD), including sleep and cognitive issues. To date, the best treatment is to perform deep brain stimulation (DBS) on PD patients as outcomes have shown positive effects in ameliorating the debilitating symptoms of this disease by treating pathological circuitries within the parkinsonian brain. It is therefore important to address the challenges and develop this procedure to improve the quality of life of PD patients.
... One of the medullary nuclei affected relatively early in PD is the pedunculopontine nucleus (PPN) (Hirsch et al., 1987;Shinotoh et al., 1999;Tubert et al., 2019;Zweig et al., 1989). The neurons found in the PPN can be divided into cholinergic, glutamatergic, and GABAergic subsets (Clements et al., 1991;Clements and Grant, 1990;Ford et al., 1995;Lavoie and Parent, 1994). The PPN subset of cholinergic neurons (CNs) has long, highly branched axons that stretch rostrally to the di-and telencephalon, as well as caudally into the brainstem (Dautan et al., 2016;Lavoie and Parent, 1994;Mena-Segovia et al., 2008;Semba and Fibiger, 1992;Takakusaki et al., 1996). ...
... The neurons found in the PPN can be divided into cholinergic, glutamatergic, and GABAergic subsets (Clements et al., 1991;Clements and Grant, 1990;Ford et al., 1995;Lavoie and Parent, 1994). The PPN subset of cholinergic neurons (CNs) has long, highly branched axons that stretch rostrally to the di-and telencephalon, as well as caudally into the brainstem (Dautan et al., 2016;Lavoie and Parent, 1994;Mena-Segovia et al., 2008;Semba and Fibiger, 1992;Takakusaki et al., 1996). These PPN CNs are more vulnerable in PD than neighboring glutamatergic and GABAergic neurons (Hirsch et al., 1987). ...
Like a handful of other neuronal types in the brain, cholinergic neurons (CNs) in the pedunculopontine nucleus (PPN) are lost during Parkinson’s disease (PD). Why this is the case is unknown. One neuronal trait implicated in PD selective neuronal vulnerability is the engagement of feed-forward stimulation of mitochondrial oxidative phosphorylation (OXPHOS) to meet high bioenergetic demand, leading to sustained oxidant stress and ultimately degeneration. The extent to which this trait is shared by PPN CNs is unresolved. To address this question, a combination of molecular and physiological approaches were used. These studies revealed that PPN CNs are autonomous pacemakers with modest spike-associated cytosolic Ca²⁺ transients. These Ca²⁺ transients were partly attributable to the opening of high-threshold Cav1.2 Ca²⁺ channels, but not Cav1.3 channels. Cav1.2 channel signaling through endoplasmic reticulum ryanodine receptors stimulated mitochondrial OXPHOS to help maintain cytosolic adenosine triphosphate (ATP) levels necessary for pacemaking. Inhibition of Cav1.2 channels led to the recruitment of ATP-sensitive K⁺ channels and the slowing of pacemaking. A ‘side-effect’ of Cav1.2 channel-mediated stimulation of mitochondria was increased oxidant stress. Thus, PPN CNs have a distinctive physiological phenotype that shares some, but not all, of the features of other neurons that are selectively vulnerable in PD.
... This diverse functionality of the cholinergic system has provided the impetus for the anatomical analyses of this system in strepsirrhine (e.g., Calvey et al., 2015), platyrrhine (e.g., Everitt et al., 1988;Lavoie & Parent, 1994;Wu et al., 2000), and catarrhine primates (e.g., Ichikawa & Shimizu, 1998;Mesulam et al., 1984;Raghanti et al., 2011;Rico & Cavada, 1998;Satoh & Fibiger, 1985), with partial mapping of the neuronal portions of this system being made in non-human apes (e.g., Benzing et al., 1993;Raghanti et al., 2011), with focussed studies examining the cholinergic axonal terminal fields in the cerebral cortex and basal ganglia of apes and other primates (e.g., Lewis, 1991;Mesulam et al., 1992;Raghanti et al., 2008;Stephenson et al., 2017). Despite this research, no full description of the neurons forming the cholinergic system in the brain and spinal cord of non-human apes has been provided to date (e.g., Delucchi, 1965). ...
... The major conclusion from the current study is that, across mammals, variations in the organization and nuclear parcellation of the cholinergic system are quite rare. For example, the human, chimpanzee, and lar gibbon show virtually identical nuclear organization of the cholinergic system to that seen in other primates (Benzing et al., 1993;Calvey et al., 2015;Everitt et al., 1988;Ichikawa & Shimizu, 1998;Lavoie & Parent, 1994;Mesulam et al., 1984;Raghanti et al., 2011;Rico & Cavada, 1998;Satoh & Fibiger, 1985;Wu et al., 2000), apart from the minor variations in expression of the different components discussed above. This indicates that the projection patterns of the cholinergic system are also likely to be very similar, although variations in the density and distribution of cholinergic axon terminal fields have been observed in primates (e.g., Lewis, 1991;Mesulam et al., 1992;Raghanti et al., 2008;Stephenson et al., 2017). ...
Using choline acetyltransferase immunohistochemistry, we describe the nuclear parcellation of the cholinergic system in the brains of two apes, a lar gibbon (Hylobates lar) and a chimpanzee (Pan troglodytes). The cholinergic nuclei observed in both apes studied are virtually identical to that observed in humans and show very strong similarity to the cholinergic nuclei observed in other primates and mammals more generally. One specific difference between humans and the two apes studied is that, with the specific choline acetyltransferase antibody used, the cholinergic pyramidal neurons observed in human cerebral cortex were not labeled. When comparing the two apes studied and humans to other primates, the presence of a greatly expanded cholinergic medullary tegmental field, and the presence of cholinergic neurons in the intermediate and dorsal horns of the cervical spinal cord are notable variations of the distribution of cholinergic neurons in apes compared to other primates. These neurons may play an important role in the modulation of ascending and descending neural transmissions through the spinal cord and caudal medulla, potentially related to the differing modes of locomotion in apes compared to other primates. Our observations also indicate that the average soma volume of the neurons forming the laterodorsal tegmental nucleus (LDT) is larger than those of the pedunculopontine nucleus (PPT) in both the lar gibbon and chimpanzee. This variability in soma volume appears to be related to the size of the adult derivatives of the alar and basal plate across mammalian species.
... 6,7 In the monkey PPN, the existence of GABAergic neurons remains controversial, GABA immunoreactivity being observed in terminal axons, 8 but not in the cell bodies. 9 GABAergic and glycinergic neurons have been visualized and quantified only once in the human PPN. 10 Colocalization of acetylcholine with either glutamate or GABA has been suggested in animals [11][12][13][14] but recently refuted. 15 Thus, the composition of neuronal populations within the MLR together with their 3-dimensional (3D) organization remains unknown in humans. ...
... They were 2.6 times more numerous than the number of cholinergic neurons, confirming previous results. 10 The existence of glutamatergic neurons has already been reported, 9,11 but its detection by using an antibody against glutamate is problematic because glutamate is a metabolic precursor of GABA. In the present study, the presence of glutamatergic neurons was identified in both the PPN and CuN of the normal human brainstem by the expression of VGluT2 mRNA using ISH. ...
Background
Deep brain stimulation of the pedunculopontine nucleus has been performed to treat dopamine-resistant gait and balance disorders in patients with degenerative diseases. The outcomes, however, are variable, which may be the result of the lack of a well-defined anatomical target.
Objectives
The objectives of this study were to identify the main neuronal populations of the pedunculopontine and the cuneiform nuclei that compose the human mesencephalic locomotor region and to compare their 3-dimensional distribution with those found in patients with Parkinson's disease and progressive supranuclear palsy.
Methods
We used high-field MRI, immunohistochemistry, and in situ hybridization to characterize the distribution of the different cell types, and we developed software to merge all data within a common 3-dimensional space.
Results
We found that cholinergic, GABAergic, and glutamatergic neurons comprised the main cell types of the mesencephalic locomotor region, with the peak densities of cholinergic and GABAergic neurons similarly located within the rostral pedunculopontine nucleus. Cholinergic and noncholinergic neuronal losses were homogeneous in the mesencephalic locomotor region of patients, with the peak density of remaining neurons at the same location as in controls. The degree of denervation of the pedunculopontine nucleus was highest in patients with progressive supranuclear palsy, followed by Parkinson's disease patients with falls.
Conclusions
The peak density of cholinergic and GABAergic neurons was located similarly within the rostral pedunculopontine nucleus not only in controls but also in pathological cases. The neuronal loss was homogeneously distributed and highest in the pedunculopontine nucleus of patients with falls, which suggests a potential pathophysiological link.
... Finally, several studies have reported the presence of glutamate in PPN cholinergic cells in different species (Clements et al., 1991;Lavoie and Parent, 1994). In parallel, electrophysiological studies have identified substantial functional heterogeneity both within cholinergic and non-cholinergic neurons in the PPN (Steriade et al., 1990;Takakusaki et al., 1996Takakusaki et al., , 1997Mena-Segovia et al., 2008). ...
... A lower percentage of coexpression (2% in PPN and 1% in LDT) was reported previously (Wang and Morales, 2009), which may be due to the different quantitative method, the dual labeling protocol used, or to both. Earlier studies had illustrated the presence of glutamate in PPN cholinergic neurons in several species (Clements et al., 1991;Lavoie and Parent, 1994). The coexpression of the vesicular glutamate transporter reported here, however, adds to these data the potential to actually release glutamate. ...
The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.
... Le PPN innerve également l'ensemble des structures des ganglions de la base, incluant le STN [Lavoie andParent, 1994c, Muthusamy et al., 2007], le GPi [Lavoie and Parent, 1994c], la substance noire [Charara et al., 1996, Kitai et al., 1999, Rohrbacher et al., 2000, l'aire tegmentale ventrale [Alderson et al., 2006] et dans une très faible mesure le striatum [Nakano et al., 1990, Lavoie and Parent, 1994b, Dautan et al., 2014. ...
... Our data demonstrate that PPN axons innervate the whole extent of these basal ganglia nuclei, including all the three anatomo-functional territories of each nucleus. Thus numerous labelled PPN fibers innervated the SNc and the adjacent limbic VTA in both monkey and human, as reported using tract tracing (Lavoie and Parent, 1994b) and tractography (Aravamuthan et al., 2008). The fact that no PPN connection to the nigra was previously been reported in humans (Aravamuthan et al., 2008) contrary to our present results is probably due to differences in the method used as we discussed above. ...
Le vieillissement de la population a vu émerger des maladies liées à l'âge telles que les maladies neurodégénératives. La neuromodulation peut être proposée à certains patients lorsque les médicaments ne sont plus efficaces ou qu'ils entraînent des effets secondaires invalidants. L’objectif de cette thèse est de mieux caractériser les structures cérébrales pour optimiser le ciblage de la neuromodulation et, ainsi augmenter les bénéfices thérapeutiques.Le premier axe de recherche porte sur la région locomotrice mésencéphalique (MLR) qui est une cible en cours d'évaluation pour les patients parkinsoniens souffrant de troubles de la marche et de l'équilibre. Nous avons exploré la connectivité de la MLR et les résultats nous ont amené à considérer que le noyau pédonculopontin (PPN), qui est une région constituante de la MLR, est la cible à privilégier. Or, une perte des neurones cholinergiques du PPN a été montrée chez les patients parkinsoniens. Le second projet a consisté à étudier la topographie de la perte de neurones chez différents groupes pathologiques. Nos résultats montrent que le maximum de densité des neurones cholinergiques se situe à +3 mm du début supérieur du PPN et serait la cible optimale de sa neuromodulation. Enfin, nous avons construit un atlas 3D du tronc cérébral humain afin de guider l’implantation d'électrode dans la MLR.Le second axe de recherche concerne le Vim qui est la cible usuelle pour les tremblements essentiels. Nous avons appliqué différentes méthodes de ciblage et comparé les localisations. Nous avons trouvé des différences de distance entre cibles, pouvant affecter les résultats de la neuromodulation, supérieures à 1.5 mm.
... PPN glutamatergic and GABAergic neurons are observed in the rat (Clements et al. 1991;Wang and Morales 2009;Martinez-Gonzalez et al. 2011) and cat (Jia et al. 2003). In monkeys, GABAergic neurons were not found in the PPN (Lavoie and Parent 1994a), whereas both glutamatergic and GABAergic PPN neurons appear to project to midbrain dopamine neurons (Charara et al. 1996). The possible colocalization of acetylcholine with either glutamate or GABA in PPN neurons is still controversial. ...
... The possible colocalization of acetylcholine with either glutamate or GABA in PPN neurons is still controversial. About 50% of cholinergic neurons were reported to express GABA (Jia et al. 2003), and about 40% seem to express glutamate in rat and monkey (Clements et al. 1991;Lavoie and Parent 1994a). However, no cholinergic neurons were observed to coexpress GABA or glutamate using a combination of in situ hybridization and immunohistochemistry (Wang and Morales 2009). ...
Patients with Parkinson’s disease (PD) develop cardinal motor symptoms, including akinesia, rigidity, and tremor, that are alleviated by dopaminergic medication and/or subthalamic deep brain stimulation. Over the time course of the disease, gait and balance disorders worsen and become resistant to pharmacological and surgical treatments. These disorders generate debilitating motor symptoms leading to increased dependency, morbidity, and mortality. PD patients also experience sleep disturbance that raise the question of a common physiological basis. An extensive experimental and clinical body of work has highlighted the crucial role of the pedunculopontine nucleus (PPN) in the control of gait and sleep, and its potential major role in PD. Here, we summarise our investigations in the monkey PPN in the normal and parkinsonian states. We first examined the anatomy and connectivity of the PPN and the cuneiform nucleus which both belong to the mesencephalic locomotor region. Second, we conducted experiments to demonstrate the specific effects of PPN cholinergic lesions on locomotion in the normal and parkinsonian monkey. Third, we aimed to understand how PPN cholinergic lesions impair sleep in parkinsonian monkeys. Our final goal was to develop a novel model of advanced PD with gait and sleep disorders. We believe that this monkey model, even if it does not attempt to reproduce the exact human disease with all its complexities, represents a good biomedical model to characterise locomotion and sleep in the context of PD.
... In this context, the work by Janickova and coworkers in this issue of the Journal of Neurochemistry addresses two of the most pressing questions: (i) Is the beneficial effect of rivastigmine mediated by the cholinergic brainstem nucleus PPN? (ii) Is the beneficial effect of PPN stimulation really mediated by the release of acetylcholine? The second question is of particular importance since the PPN is highly heterogenous with cholinergic, GABAergic and glutamatergic neurons in varying densities and even evidence of corelease of glutamate and acetylcholine from the same neuron (Martinez-Gonzalez et al. 2012;Lavoie and Parent 1994). Janickova and coworkers engineered mice lacking the vesicular acetylcholine transporter selectively in the PPN and in the laterodorsal tegmental nucleus by crossing a line with 'floxed' vesicular acetylcholine transporter alleles (Martins-Silva et al. 2011) to a line expressing Cre recombinase in the mesencephalon (Kimmel et al. 2000). ...
... This study complements a recent inverse study that tested the effects of selectively activating cholinergic neurons in the PPN using designer receptors exclusively activated by designer drugs, showing beneficial effects in a rat model of PD (Pienaar et al. 2015). Because of the corelease of glutamate from cholinergic neurons (Lavoie and Parent 1994), the study by Janickova confirms that it is really the release of acetylcholine that underlies the beneficial effect. This is important for the attribution of the beneficial effect of the cholinergic drug rivastigmine to the PPN. ...
Gait impairment is one of the most intractable symptoms of Parkinson's disease, responding poorly to dopaminergic medication. Promising therapeutic strategies include deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) and enhancing cholinergic neurotransmission by acetylcholine esterase inhibitors. This Editorial discusses an elegant study by Janickova and coworkers in the current issue of the Journal of Neurochemistry, in which the authors engineered mice lacking cholinergic transmission selectively in the PPN and demonstrate that cholinergic neurons of the PPN are critical for gait. These findings are important for therapeutic approaches that aim at gait improvement in Parkinson's disease. Read the highlighted article 'Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait' on doi: 10.1111/jnc.13910.
... Furthermore, any methodology that eliminates cholinergic PPT/LDT neurons might not exactly pinpoint the role of acetylcholine (ACh) released by these neurons on behavior. This is because PPT/LDT cholinergic neurons are thought to co-release glutamate (Glu) or GABA (Clements et al. 1991;Lavoie and Parent 1994;Clarke et al. 1997;Jia et al. 2003;Wang and Morales 2009). In the striatum, ACh and glutamate co-released by cholinergic neurons have distinct contributions to brain functions Sakae et al. 2015;Gangarossa et al. 2016). ...
... Our data suggest that cholinergic activity originating from PPT/LDT cholinergic neurons is important for the modulation of balance, gait and locomotion. Future experiments will be necessary to determine whether glutamate and GABA that seem to be co-released from these cholinergic neurons (Clements et al. 1991;Lavoie and Parent 1994;Clarke et al. 1997;Jia et al. 2003;Wang and Morales 2009) also have a role in the modulation of these behavioral functions. Importantly, our results support the hypothesis that long-term degeneration of cholinergic neurons from PPT/LDT in PD and other parkinsonian disorders has a causal role to increase severity of motor symptoms. ...
Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long‐term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1‐Cre‐flox/flox). VAChTEn1‐Cre‐flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire‐hang and grip‐force tests. Young VAChTEn1‐Cre‐flox/flox mice (2–5 months‐old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1‐Creflox/flox mice (13–16 months‐old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT‐deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients.
Read the Editorial Highlight for this article on page 688.
... Among these neuronal groups, most authors distinguish the dorsal (DR) and central superior (CS) raphe nuclei, which contain serotonergic neurons; the locus coeruleus (LC), rich in noradrenergic neurons; and the pedunculopontine (PpT) and laterodorsal (LdT) tegmental nuclei, containing cholinergic neurons (Reinoso-Suárez 1997). Nevertheless, and along with many of other types of neurons, most of these nuclei contain a great variety of neuropeptide-containing neurons, and an appreciable amount of GABAergic and glutamatergic neurons (Sutin and Jacobowitz 1988;Clements and Grant 1990;Jones 1991;Sakai 1991;Lavoie and Parent 1994;Nitz and Siegel 1997;Maloney et al. 1999;Gervasoni et al. 2000;Brown et al. 2008). ...
... Using single-unit recording in unanesthetized cats and rats, three different dorsal mesopontine tegmentum cholinergic neuron firing patterns have been identified in the SWC (Nelson et al. 1983;El Mansari et al. 1989;Kayama et al. 1992). These patterns are expressed by: (1) a small group (5-9%) of mesopontine cholinergic neurons that show phasic activities in relation to PGO waves, with phasic field potentials recorded during REM sleep (PGO-on neurons) (Steriade et al. 1990); (2) a larger group of cholinergic neurons (more than 40%), which show higher firing rates in REM sleep than in W and NREM sleep (REM-on neurons); and (3) the largest group of mesopontine cholinergic neurons (more than 50%), which show higher firing rates in W and REM sleep than in NREM sleep (W/REM-on neurons) with most of the latter exhibiting the highest firing rates in W. In addition, it is important to emphasize that most mesopontine neurons may utilize more than one neurotransmitter; for example, in cholinergic neurons, a great variety of neurotransmitters colocalize with acetylcholine such as glutamate, atriopeptin, CRF, substance P, or nitric oxide, etc. (Lavoie and Parent 1994;Semba 1999). Glutamate appears to be the main excitatory neurotransmitter of many dorsal mesopontine tegmentum cholinergic neurons and this is also supposed to be true in the basal forebrain and oral pontine reticular formation (Rasmusson et al. 1996;Semba 1999;Fournier et al. 2004). ...
Wakefulness (W) is necessary for a thoughtful and precise knowledge of things, allowing us to recognize our essential attributes and the changes that we experience in ourselves. We spend about two-thirds of our life in W. This state is circadian and homeostatically regulated and precisely meshed with sleep into the sleep–wakefulness cycle (SWC). Sleep is also a necessary, active, periodic, and diverse condition. Although five different stages have been described for sleep in man, most experimental studies have curtailed them into two sleeping stages: Slow wave sleep (SWS), also called non-REM sleep (NREM sleep); and rapid eye movement sleep (REM sleep or paradoxical sleep). Together with W, these three phases constitute the SWC. The hypothalamic suprachiasmatic nucleus is the pacemaker for SWC circadian rhythmicity. Photic retinal stimulation by light modulates suprachiasmatic nucleus activity through the retino-hypothalamic pathway, tuning the SWC to a circadian rhythm with a nocturnal sleep time in adult humans.
... One of the medullary nuclei affected relatively early in PD is the pedunculopontine nucleus (PPN) (Hirsch et al., 1987;Zweig et al., 1989;Shinotoh et al., 1999;Tubert et al., 2019). The neurons found in the PPN can be divided into cholinergic, glutamatergic and GABAergic subsets (Clements et al., 1991;Clements & Grant, 1990;Ford et al., 1995;Lavoie & Parent, 1994). The PPN subset of cholinergic neurons (CNs) have long, highly branched axons that stretch rostrally to the di-and telencephalon, as well as caudally into the brainstem (Semba & Fibiger, 1992;Lavoie & Paren, 1994;Takakusaki et al., 1996;Mena-Segovia et al., 2008;Dautan et al., 2014;2016). ...
Like a handful of other neuronal types in the brain, cholinergic neurons (CNs) in the pedunculopontine nucleus (PPN) are lost in the course of Parkinson's disease (PD). Why this is the case is unknown. One neuronal trait implicated in PD selective neuronal vulnerability is the engagement of feed-forward stimulation of mitochondrial oxidative phosphorylation (OXPHOS) to meet high bioenergetic demand, leading to sustained oxidant stress and ultimately degeneration. The extent to which this trait is shared by PPN CNs is unresolved. To address this question, a combination of molecular and physiological approaches were used. These studies revealed that PPN CNs are autonomous pacemakers with modest spike-associated cytosolic Ca2+ transients. These Ca2+ transients were attributable in part to the opening of high-threshold Cav1.2 Ca2+ channels, but not Cav1.3 channels. Nevertheless, Cav1.2 channel signaling through endoplasmic reticulum ryanodine receptors stimulated mitochondrial OXPHOS to help maintain cytosolic adenosine triphosphate (ATP) levels necessary for pacemaking. Inhibition of Cav1.2 channels led to recruitment of ATP-sensitive K+ channels and slowing of pacemaking. Cav1.2 channel-mediated stimulation of mitochondria increased oxidant stress. Thus, PPN CNs have a distinctive physiological phenotype that shares some, but not all, of the features of other neurons that are selectively vulnerable in PD.
... The pontomesencephalic cholinergic nuclei appear to be most diffusely connected to all of the other brainstem neuromodulatory systems including dorsal raphe nucleus, LC, and SN/VTA, and dorsal striatum (Woolf and Butcher, 1989). PPN sends extensive cholinergic projections onto DA neurons of the SNc (Clarke et al., 1987;Lavoie and Parent, 1994), which, via nicotinic cholinergic receptors, controls DA release at the striatal level (Blaha et al., 1996). LDTg send cholinergic projections to SN, dorsal striatum, and VTA (Clarke et al., 1987;Oakman et al., 1995). ...
To adapt to the sustained demands of chronic stress, discrete brain circuits undergo structural and functional changes often resulting in anxiety disorders. In some individuals, anxiety disorders precede the development of motor symptoms of Parkinson's disease (PD) caused by degeneration of neurons in the substantia nigra (SN). Here, we present a circuit framework for probing a causal link between chronic stress, anxiety, and PD, which postulates a central role of abnormal neuromodulation of the SN's axon initial segment by brainstem inputs. It is grounded in findings demonstrating that the earliest PD pathologies occur in the stress-responsive, emotion regulation network of the brainstem, which provides the SN with dense aminergic and cholinergic innervation. SN's axon initial segment (AIS) has unique features that support the sustained and bidirectional propagation of activity in response to synaptic inputs. It is therefore, especially sensitive to circuit-mediated stress-induced imbalance of neuromodulation, and thus a plausible initiating site of neurodegeneration. This could explain why, although secondary to pathophysiologies in other brainstem nuclei, SN degeneration is the most extensive. Consequently, the cardinal symptom of PD, severe motor deficits, arise from degeneration of the nigrostriatal pathway rather than other brainstem nuclei. Understanding when and how circuit dysfunctions underlying anxiety can progress to neurodegeneration, raises the prospect of timed interventions for reversing, or at least impeding, the early pathophysiologies that lead to PD and possibly other neurodegenerative disorders.
... The pedunculopontine tegmental nucleus (PTg) is located in the mesopontine tegmentum. Morphological and electrophysiological studies have demonstrated that the PTg receives projections from the ipsilateral side of the substantia nigra pars reticulata and from the internal globus pallidus, which are the output nuclei in the basal ganglia (Nakamura et al., 1989;Spann and Grofova, 1991;Semba and Fibiger, 1992;Lavoie and Parent, 1994;Takakusaki et al., 1997;Saitoh et al., 2003). The PTg projects to the to the pars compacta of the substantia nigra (SNC) (Jackson and Crossman, 1983;Beninato and Spencer, 1987) and to the lateral reticular formation above the nucleus ambiguous (Rye et al., 1988;Yasui et al., 1990). ...
This study investigates whether the swallowing reflex is modulated by stimulation of the pedunculopontine tegmental nucleus (PTg). Sprague–Dawley rats under urethane anesthesia were used. The swallowing reflex was induced by electrical stimulation of the superior laryngeal nerve and was identified by the electromyographic activities from the mylohyoid muscle. The number of swallows was reduced by electrical stimulation of the PTg. The latency of the onset of the first swallow was increased during stimulation of the PTg. The duration of electromyogram bursts of the mylohyoid muscle was significantly shorter during the PTg stimulation than with no stimulation. The number of swallows was reduced, latency of onset of the first swallow increased, the duration of electromyogram bursts of the mylohyoid muscle was significantly shorter and the peak-to-peak amplitude of electromyogram bursts of the mylohyoid muscle was significantly suppressed after microinjection of glutamate into the PTg. These results suggest that the PTg is involved in the control of swallowing.
... Neurons in MLR are excitatory. The pedunculopontine nucleus (PPN) is located in the ventrolateral portion of caudal MLR and is composed of neurons comprising glutamate and acetylcholine (ACh) (Clements & Grant, 1990;Lavoie & Parent, 1994;Takakusaki et al., 1996;Mena-Segovia et al., 2008;Takakusaki et al., 2016). Previous studies have suggested that cholinergic neurons are important in maintaining the rhythm of locomotion and postural muscle tone (Bohnen & Albin, 2011;Takakusaki et al., 2011). ...
Background: Mammalian locomotor behaviour called fictive locomotion can be elicited in an isolated spinal cord in the absence of higher brain center or sensory input. This relatively simple behaviour is produced by the motoneuronal rhythmic activity which is under the control of spinal neuronal networks called central pattern generators (CPGs). Disturbance of this rhythmic motor output can occur following spinal cord injury (SCI). This elementary isolated spinal cord model gives us an opportunity to study the basic physiology of locomotion during control conditions, the pathological processes following lesion (which can be induced chemically), and eventually the application of therapeutic approaches curbing injury.
Objectives: Multiple aspects of spinal functions can be demonstrated by stimulating or/and blocking specific inputs and measuring the outputs using electrophysiological, immunohistochemical and calcium imaging tools. Using isolated neonatal rat spinal cords and organotypic spinal slices as SCI models, the basic mechanisms (such as dysmetabolic state or excitotoxicity) which can develop during the early phase of the lesion were addressed and studied. The injury was evoked chemically by applying either pathological medium (to mimic dysmetabolic/hypoxic conditions) or kainate (to produce excitotoxicity that completely abolishes fictive locomotion and network synaptic transmission) for 1 h. Fictive locomotion was examined stimulating the lumbar dorsal root and recording from the ipsilateral and ipsi-segmental ventral roots. Other network parameters were also studied such as synaptic transmission and rhythmicity. Various therapeutic drugs such as methylprednisolone sodium succinate (MPSS), propofol, nicotine and celastrol were used during or after the injury (to produce neuroprotection) and network properties were characterized during the treatment and after 24 h as well. Subsequently, the structural properties were monitored using different biomarkers (isolated spinal cord sectioned slices) and calcium imaging (here organotypic spinal slices were used).
Results and conclusions: We found that dose-dependent application of MPSS produced modest recovery of white matter damage evoked by pathological medium resulting in the emergence of sluggish chemically induced fictive locomotor patterns. However, it could not prevent damage (to gray matter) evoked by the excitotoxic agent kainate. Therefore, to provide better neuroprotection to gray matter, we tested the widely used intravenous anaesthetic propofol. This drug has shown comparatively good protection to spinal neurons and motoneurons in the gray matter. As it is an anesthetic it acted by depressing the functional network characteristics by lowering the N-methyl-D-aspartate (NMDA) and potentiating the γ aminobutyric acid (GABAA) mediated receptor responses.
The next issue we addressed was to study the neuroprotective roles of nicotinic acetylcholine receptors (nAChRs) by using the receptor agonist nicotine. Recent studies have shown that nicotine could provide good neuroprotection to the rat brainstem. To further investigate its effect on the spinal cord, we applied nicotine at the same concentration used in previous studies in the brainstem: such a concentration was toxic to spinal ventral motoneurons. Therefore the correct dose of nicotine was optimized and was found to be ten times lower. Thus, satisfactory protective effects to spinal neurons and motoneurons and the fictive locomotor patterns were observed. These neuroprotective effects were replicated with calcium imaging by using organotypic spinal slice cultures. The mechanism of protection predominantly involved α4β2 and less α7 nAChRs.
In addition, the subsequent goal of our study was to explore whether the motoneuron survival after excitotoxicity relies on cell expression of heat shock protein 70 (HSP70) or some other mechanisms. To test this hypothesis we used a bioactive drug, celastrol which induces the expression of HSP70. Prior application of the drug followed by kainate preserved network polysynaptic transmission and fictive locomotion, however, it could not reverse the depression of monosynaptic reflex responses. In vivo studies are necessary in the future to further investigate the long-term neuroprotective role of these drugs.
... These findings 96 suggest that many central cholinergic subpopulations can co-transmit other classical 97 neurotransmitters besides ACh. While some reports suggest the potential for co-release from 98 midbrain and hindbrain cholinergic projection neurons, it has not yet been systematically 99 investigated (Aitta-Aho et al., 2018;Clements and Beitz, 1991;Estakhr et al., 2017;Ford et al., 100 1995;Lavoie and Parent, 1994;Wang and Morales, 2009;Xiao et al., 2016). 101 ...
Vesicular glutamate transporters (VGLUTs) mediate the synaptic uptake of glutamate from the cytosol into synaptic vesicles and are considered unambiguous neurochemical markers of glutamate neurons. However, many neurons not classically thought of as glutamatergic also express a VGLUT and co-release glutamate. Using a genetic fate-mapping strategy we found that most cholinergic neurons in the mouse mesopontine tegmentum express VGLUT2 at some point during development, including the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus, and parabigeminal nucleus (PBG), but not the oculomotor nucleus. In contrast, very few of these cholinergic neurons displayed evidence of vesicular GABA transporter expression. Using multiplex fluorescent in situ hybridization, we determined that only PBG cholinergic neurons are also predominantly positive for VGLUT2 mRNA in the adult, with only small numbers of PPTg cholinergic neurons overlapping with VGLUT2 mRNA. Using Cre-dependent viral vectors we confirm these in situ hybridization data, and demonstrate projection patterns of cholinergic and glutamatergic populations. These results demonstrate that most mesopontine cholinergic neurons may transiently express VGLUT2, but that a large majority of PBG neurons retain VGLUT2 expression throughout adulthood, and support a growing body of literature indicating that distinct cholinergic populations have differing potential for GABA or glutamate co-release.
... Additionally, cholinergic neurons from different brain regions are known to corelease either glutamate or GABA with acetylcholine (ACh) (13)(14)(15)(16)(17)(18), and even though the physiologic relevance of ACh and GABA or ACh and glutamate corelease has just started to be appreciated (13,19), recent studies suggest that each of these neurotransmitters can differentially influence behavior (17,18,20). There is evidence indicating that in the PPT/LDT some cholinergic neurons have the potential to corelease GABA (1), whereas others have the potential to release glutamate (1,21). Furthermore, a recent optogenetic study showed that photoactivation of PPT cholinergic neurons excites the central amygdala through ACh-independent glutamate release (22), giving further support for ACh and glutamate corelease from PPT cholinergic neurons. ...
The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT‐associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y‐maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety‐like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT‐mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.—Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress. FASEB J. 33, 7018–7036 (2019). www.fasebj.org
... Within the VTA there are GABAergic, glutamatergic, and dopaminergic neurons-mainly D1 DA which are excitatory and D2 DA which are inhibitory (Nair- Roberts et al. 2008;Olson and Nestler 2007;Ranaldi and Wise 2001). The glutamatergic afferents to the VTA originate from prefrontal cortex (PFC) primarily but also from the subthalamic nucleus, the pedunculopontine-and laterodorsal-tegmental nuclei, the bed nucleus of the stria terminalis, and the superior colliculus (Morikawa and Paladini 2011;Kita and Kitai 1987;Clements et al. 1991;Lavoie and Parent 1994;Charara et al. 1996;Georges and Aston-Jones 2002;Comoli et al. 2003;Dommett et al. 2005). The GABAergic afferents come from the nucleus accumbens (NAc), ventral pallidum, and rostromedial tegmental nucleus (Morikawa and Paladini 2011). ...
Methylphenidate (MPD) is a psychostimulant used for the treatment of ADHD and works by increasing the bioavailability of dopamine (DA) in the brain. As a major source of DA, the ventral tegmental area (VTA) served as the principal target in this study as we aimed to understand its role in modulating the acute and chronic MPD effect. Forty-eight male Sprague–Dawley rats were divided into control, sham, electrical lesion, and 6-OHDA lesion groups. Given the VTA’s implication in the locomotive circuit, three locomotor indices—horizontal activity, number of stereotypy, and total distance—were used to measure the animals’ behavioral response to the drug. Baseline recording was obtained on experimental day 1 (ED 1) followed by surgery on ED 2. After recovery, the behavioral recordings were resumed on ED 8. All groups received daily intraperitoneal injections of 2.5 mg/kg MPD for six days after which the animals received no treatment for 3 days. On ED 18, 2.5 mg/kg MPD was re-administered to assess for the chronic effect of the psychostimulant. Except for one index, there was an increase in locomotive activity in all experimental groups after surgery (in comparison to baseline activity), acute MPD exposure, induction with six daily doses, and after MPD re-challenge. Furthermore, the increase was greatest in the electrical VTA lesion group and lowest in the 6-OHDA VTA lesion group. In conclusion, the results of this study suggest that the VTA may not be the primary nucleus of MPD action, and the VTA plays an inhibitory role in the locomotive circuit.
... Historically defined as a population of cholinergic neurons in the rostral brainstem, the pedunculopontine nucleus (PPN) is now known to be not only neurochemically heterogenous but functionally complex. In addition to cholinergic neurons, the PPN is populated by glutamatergic and GABAergic neurons (Clements and Grant, 1990;Clements et al., 1991;Ford et al., 1995;Lavoie and Parent, 1994b). Cholinergic neurons, which express choline acetyltransferase (ChAT) and NADPH (Clements and Grant, 1990), have medium to large fusiform, triangular, multipolar or round somas (20-40 μm in diameter) with 2 to 6 primary dendrites (Honda and Semba, 1995;Ichinohe et al., 2000;Rye et al., 1987). ...
In the last decade, scientific and clinical interest in the pedunculopontine nucleus (PPN) has grown dramatically. This growth is largely a consequence of experimental work demonstrating its connection to the control of gait and of clinical work implicating PPN pathology in levodopa-insensitive gait symptoms of Parkinson's disease (PD). In addition, the development of optogenetic and chemogenetic approaches has made experimental analysis of PPN circuitry and function more tractable. In this brief review, recent findings in the field linking PPN to the basal ganglia and PD are summarized; in addition, an attempt is made to identify key gaps in our understanding and challenges this field faces in moving forward.
... Two areas can be established according to cell density: a compact zone with high neuronal density, located in the caudal half of the nucleus towards the dorsolateral region; and another zone with lower neuronal density, called dissipated, which is scattered and partially within the superior cerebellar peduncle and central tegmental tract (Mesulam et al. 1983;García-Rill, 1991). According to the type of neurotransmission, there are two fundamental classes of neuronal populations in the PPTg: the majority is cholinergic, with large neuronal somas (Mesulam et al. 1989;Lavoie and Parent 1994a); The other population, noncholinergic, with smaller cell bodies, show glutamatergic (Lavoie and Parent 1994b;Rye et al. 1987;Takakusaki et al. Fig. 8 Level of excitatory amino acid neurotransmitters in the PnC following lesion of the SNc. a Levels of glutamate, arginine, and aspartate expressed as a percentage of the total amount of glutamate found in the PnC of control animals. ...
The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.
... The electrophysiological and neurochemical heterogeneity of neurons that form the PPTg [17,63, [67][68][69][70][71][72][73][74] raises the question of whether each neuronal population plays a specific role in REM sleep and NREM sleep. ...
... Selective loss of cholinergic neurons in the pedunculopontine nucleus (PPN) is another characteristic of PD pathology (Zweig et al. 1989;Jellinger 1991). PPN contains two populations of neurons, cholinergic neurons in pars compacta (PPNc) and glutamatergic neurons in pars dissipata (PPNd) (Nakano et al. 1990; Lavoie and Parent 1994). The cholinergic neurons project to thalamus and to globus pallidus pars interna/substantia nigra pars reticulata. ...
Parkinson’s disease (PD) is a multisystem neurodegenerative disorder affecting, besides the dopaminergic function, multiple neurotransmission systems, including the cholinergic system. Central cholinergic circuits of human brain can be tested non-invasively by coupling peripheral nerve stimulation with transcranial magnetic stimulation (TMS) of motor cortex; this test is named short latency afferent inhibition (SAI). SAI abnormalities have been reported in PD patients with gait disturbances and many non-motor symptoms, such as visual hallucinations (VHs), REM sleep behavior disorder (RBD), dysphagia, and olfactory impairment. The findings of these TMS studies strongly suggest that cholinergic degeneration is an important contributor to a number of clinical features of PD. TMS and neuropsychological raise the possibility that the presence of RBD, VHs and olfactory dysfunction indicate increased risk of cognitive impairment in patients with PD. Longitudinal studies of the patients are required to verify whether SAI abnormalities can predict a future severe cognitive decline. TMS can provide simple measures that may represent suitable biomarkers of cholinergic neurotransmission in PD. SAI studies enable an early recognition of PD patients with cholinergic system degeneration, and this might allow future targeted cholinergic treatment approaches, in addition to dopaminergic therapy, to ameliorate non-motor and motor clinical symptoms in PD patients.
... Interestingly, specific lesion of cholinergic neurons in rat was found to be associated with deficit in sustained attention in a proportional manner, suggesting that these neurons would have an active role in the neuronal network associated with the control of attentionnal process (Cyr et al. 2015). By means of their strong projections onto intralaminar thalamic nuclei and BG (Paré et al. 1988;Lavoie and Parent 1994a, b;Benarroch 2008;Martinez-Gonzalez et al. 2011;Smith et al. 2011) the MRF neurons could in fact be involved in the maintenance of the optimal level of attention to improve task performance Smith et al. 2011;Okada and Kobayashi 2015) including locomotion. This hypothesis supports optogenetic data showing that MLR is in a position to regulate both locomotion and changes in brain state (Lee et al. 2014). ...
The mesencephalic reticular formation (MRF) mainly composed by the pedunculopontine and the cuneiform nuclei is involved in the control of several fundamental brain functions such as locomotion, rapid eye movement sleep and waking state. On the one hand, the role of MRF neurons in locomotion has been investigated for decades in different animal models, including in behaving nonhuman primate (NHP) using extracellular recordings. On the other hand, MRF neurons involved in the control of waking state have been consistently shown to constitute the cholinergic component of the reticular ascending system. However, a dual control of the locomotion and waking state by the same groups of neurons in NHP has never been demonstrated in NHP. Here, using microelectrode recordings in behaving NHP, we recorded 38 neurons in the MRF that were followed during transition between wakefulness (TWS) and sleep, i.e., until the emergence of sleep episodes characterized by typical cortical slow wave activity (SWA). We found that the MRF neurons, mainly located in the pedunculopontine nucleus region, modulated their activity during TWS with a decrease in firing rate during SWA. Of interest, we could follow some MRF neurons from locomotion to SWA and found that they also modulated their firing rate during locomotion and TWS. These new findings confirm the role of MRF neurons in both functions. They suggest that the MRF is an integration center that potentially allows to fine tune waking state and locomotor signals in order to establish an efficient locomotion.
... The PPN is a very heterogeneous structure, consisting of a caudolateral pars compacta (PPNc) and an anteromedial pars dissipata. Cholinergic cells predominate in PPNc, but PPNc and anteromedial pars dissipata also contain large populations of GABAergic or glutamatergic neurons [77][78][79]. The input and output relationships of the various neuron groups in the PPN have not been precisely determined, but it is known that the nucleus gives rise to projections to the basal ganglia, thalamus, basal forebrain, reticular formation, and spinal cord [69,74,[80][81][82][83][84][85][86][87][88][89][90][91][92][93], thus being, at the same time, part of the extended basal ganglia family of nuclei [74], and a conduit of descending basal ganglia outputs. ...
Deep brain stimulation (DBS) is highly effective for both hypo- and hyperkinetic movement disorders of basal ganglia origin. The clinical use of DBS is, in part, empiric, based on the experience with prior surgical ablative therapies for these disorders, and, in part, driven by scientific discoveries made decades ago. In this review, we consider anatomical and functional concepts of the basal ganglia relevant to our understanding of DBS mechanisms, as well as our current understanding of the pathophysiology of two of the most commonly DBS-treated conditions, Parkinson's disease and dystonia. Finally, we discuss the proposed mechanism(s) of action of DBS in restoring function in patients with movement disorders. The signs and symptoms of the various disorders appear to result from signature disordered activity in the basal ganglia output, which disrupts the activity in thalamocortical and brainstem networks. The available evidence suggests that the effects of DBS are strongly dependent on targeting sensorimotor portions of specific nodes of the basal ganglia-thalamocortical motor circuit, that is, the subthalamic nucleus and the internal segment of the globus pallidus. There is little evidence to suggest that DBS in patients with movement disorders restores normal basal ganglia functions (e.g., their role in movement or reinforcement learning). Instead, it appears that high-frequency DBS replaces the abnormal basal ganglia output with a more tolerable pattern, which helps to restore the functionality of downstream networks.
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A hypothetical mechanism of the basal ganglia involvement in the occurrence of paradoxical sleep dreams and rapid eye movements is proposed. According to this mechanism, paradoxical sleep is provided by facilitation of activation of cholinergic neurons in the pedunculopontine nucleus as a result of suppression of their inhibition from the output basal ganglia nuclei, This disinhibition is promoted by activation of dopaminergic cells by pedunculopontine neurons, subsequent rise in dopamine concentration in the input basal ganglia structure, striatum, and modulation of the efficacy of cortico-striatal inputs. In the absence of signals from retina, a disinhibition of neurons in the pedunculopontine nucleus and superior colliculus allows them to excite neurons in the lateral geniculate body and other thalamic nuclei projecting to the primary and higher visual cortical areas, prefrontal cortex and back into the striatum. Dreams as visual images and "motor hallucinations" are the result of an increase in activity of definitely selected groups of thalamic and neocortical neurons. This selection is caused by modifiable action of dopamine on long-term changes in the efficacy of synaptic transmission during circulation of signals in closed interconnected loops, each of which includes one of the visual cortical areas (motor cortex), one of the thalamic nuclei, limbic and one of the visual areas (motor area) of the basal ganglia, pedunculopontine nucleus, and superior colliculus. Simultaneous modification and modulation of synapses in diverse units of neuronal loops is provided by PGO waves. Disinhibition of superioir colliculus neurons and their excitation by pedunculopontine nucleus lead to an appearance of rapid eye movements during paradoxical sleep.
Spinal cord injury (SCI) results in a disruption of information between the brain and the spinal circuit. Electrical stimulation of the mesencephalic locomotor region (MLR) can promote locomotor recovery in acute and chronic SCI rodent models. Although clinical trials are currently under way, there is still debate about the organization of this supraspinal center and which anatomic correlate of the MLR should be targeted to promote recovery. Combining kinematics, electromyographic recordings, anatomic analysis, and mouse genetics, our study reveals that glutamatergic neurons of the cuneiform nucleus contribute to locomotor recovery by enhancing motor efficacy in hindlimb muscles, and by increasing locomotor rhythm and speed on a treadmill, over ground, and during swimming in chronic SCI mice. In contrast, glutamatergic neurons of the pedunculopontine nucleus slow down locomotion. Therefore, our study identifies the cuneiform nucleus and its glutamatergic neurons as a therapeutical target to improve locomotor recovery in patients living with SCI.
Parkinson's disease (PD) is a motor disorder resulting from degeneration of dopaminergic neurons of substantia nigra pars compacta (SNpc), with classical and non-classical symptoms such as respiratory instability. An important region for breathing control, the Pedunculopontine Tegmental Nucleus (PPTg), is composed of cholinergic, glutamatergic, and GABAergic neurons. We hypothesize that degenerated PPTg neurons in a PD model contribute to the blunted respiratory activity. Adult mice (40 males and 29 females) that express the fluorescent green protein in cholinergic, glutamatergic or GABAergic cells were used (Chat-cre Ai6, Vglut2-cre Ai6 and Vgat-cre Ai6) and received bilateral intrastriatal injections of vehicle or 6-hydroxydopamine (6-OHDA). Ten days later, the animals were exposed to hypercapnia or hypoxia to activate PPTg neurons. Vglut2-cre Ai6 animals also received retrograde tracer injections (cholera toxin b) into the retrotrapezoid nucleus (RTN) or preBötzinger Complex (preBötC) and anterograde tracer injections (AAV-mCherry) into the SNpc. In 6-OHDA-injected mice, there is a 77% reduction in the number of dopaminergic neurons in SNpc without changing the number of neurons in the PPTg. Hypercapnia activated fewer Vglut2 neurons in PD, and hypoxia did not activate PPTg neurons. PPTg neurons do not input RTN or preBötC regions but receive projections from SNpc. Although our results did not show a reduction in the number of glutamatergic neurons in PPTg, we observed a reduction in the number of neurons activated by hypercapnia in the PD animal model, suggesting that PPTg may participate in the hypercapnia ventilatory response.
The basal ganglia are several synaptically interconnected subcortical structures that play important roles in regulating various aspects of psychomotor behaviors, and are central to the pathophysiology of common human movement disorders such as Parkinson’s and Huntington’s diseases (PD/HD). These structures classically include: 1) the striatum, which comprises the caudate nucleus (CD), putamen (PUT), and nucleus accumbens (Acc); 2) the globus pallidus, which includes the external (GPe; globus pallidus in nonprimates) and internal (GPi; entopeduncular nucleus [EPN] in nonprimates) segments; 3) the subthalamic nucleus (STN); and 4) the substantia nigra, which comprises the pars compacta (SNc) and pars reticulata (SNr) (Fig. 1).
The basal ganglia are a group of closely connected cell masses, forming a more or less continuum, extending from the telencephalon to the midbrain tegmentum (Sect. 11.3). A few notes on the development of the basal ganglia are presented in Sect. 11.2. This complex comprises the striatum (the nucleus caudatus and the putamen, largely separated by the internal capsule), the globus pallidus, the subthalamic nucleus and the substantia nigra. The output of the basal ganglia is aimed at the ventral anterior (VA) and ventrolateral (VL) thalamic nuclei or VA-VL complex, the centromedian thalamic nucleus, the habenula, the pedunculopontine tegmental nucleus and the superior colliculus. In most non-primate mammals, the caudate and putamen are not clearly separated by an internal capsule and are known as the caudate-putamen complex or striatum. In primates, the globus pallidus consists of external or lateral and internal or medial segments. In other mammals, the entopeduncular nucleus is the homologue of the internal segment. The caudate nucleus, the putamen and the globus pallidus form the dorsal part of the striatal complex. The nucleus accumbens and the olfactory tubercle form the ventral striatum. The rostral part of the substantia innominata forms a ventral extension of the globus pallidus and is known as the ventral pallidum.
Almost a century ago, Constantin von Economo observed that in patients with encephalitis lethargica lesions in the upper brain stem and posterior hypothalamus impaired consciousness. From lesion studies in cats and anatomical data, the idea arose that the brain stem reticular formation is the origin of the ascending reticular activating system (ARAS) that would operate through the intralaminar nuclei and activate widespread regions of the cerebral cortex. This view of the reticular formation has been extensively modified, and nowadays the reticular formation is viewed as a series of highly specific cell groups, which closely surround the individual motor and sensory nuclei of the brain stem (► Sects. 5.2 and 5.4). The diffuse system, driving arousal and consciousness, is now attributed to the neuromodulatory system, including the serotonergic raphe nuclei, the locus coeruleus and other noradrenergic or adrenergic cell groups and cholinergic cell groups, all close to the reticular formation (► Sects. 5.3 and 5.5). The English terms of the Terminologia Neuroanatomica are used throughout.
The Subthalamic Nucleus (STh) is an oval-shaped diencephalic structure located ventrally to the thalamus, playing a fundamental role in the circuitry of the basal ganglia. In addition to being involved in the pathophysiology of several neurodegenerative disorders, such as Huntington’s and Parkinson’s disease, the STh is one of the target nuclei for deep brain stimulation. However, most of the anatomical evidence available derives from non-human primate studies. In this review, we will present the topographical and morphological organization of the nucleus and its connections to structurally and functionally related regions of the basal ganglia circuitry. We will also highlight the importance of additional research in humans focused on validating STh connectivity, cytoarchitectural organization, and its functional subdivision.
The interest in the pedunculopontine tegmental nucleus (PPTg), a structure located in the brainstem at the level of the pontomesencephalic junction, has greatly increased in recent years because it is involved in the regulation of physiological functions that fail in Parkinson's disease and because it is a promising target for deep brain stimulation in movement disorders. The PPTg is highly interconnected with the main basal ganglia nuclei and relays basal ganglia activity to thalamic and brainstem nuclei and to spinal effectors. In this review, we address the functional role of the main PPTg outputs directed to the basal ganglia, thalamus, cerebellum and spinal cord. Together, the data that we discuss show that the PPTg may influence thalamocortical activity and spinal motoneuron excitability through its ascending and descending output fibers, respectively. Cerebellar nuclei may also relay signals from the PPTg to thalamic and brainstem nuclei. In addition to participating in motor functions, the PPTg participates in arousal, attention, action selection and reward mechanisms. Finally, we discuss the possibility that the PPTg may be involved in excitotoxic degeneration of the dopaminergic neurons of the substantia nigra through the glutamatergic monosynaptic input that it provides to these neurons.
The pedunculopontine nucleus (PPN) is a rostral brainstem structure that has extensive connections with basal ganglia nuclei and the thalamus. Through these the PPN contributes to neural circuits that effect cortical and hippocampal activity. The PPN also has descending connections to nuclei of the pontine and medullary reticular formations, deep cerebellar nuclei, and the spinal cord. Interest in the PPN has increased dramatically since it was first suggested to be a novel target for treating patients with Parkinson’s disease who are refractory to medication. However, application of frequency-specific electrical stimulation of the PPN has produced inconsistent results. A central reason for this is that the PPN is not a heterogeneous structure. In this article, we review current knowledge of the neurochemical identity and topographical distribution of neurons within the PPN of both humans and experimental animals, focusing on studies that used neuronally selective targeting strategies to ascertain how the neurochemical heterogeneity of the PPN relates to its diverse functions in relation to movement and cognitive processes. If the therapeutic potential of the PPN is to be realized, it is critical to understand the complex structure-function relationships that exist here.
Amaç: Diyabet, her geçen gün yaygınlığı artan önemli bir hastalıktır. Diyabet hastaları fiziksel sorunlar yanında pek çok ruhsal sorunla karşılaşmakta ve bu sorunlar hastaların tedavi süreçlerini de olumsuz etkileyebilmektedir. Hastaların yaşadığı ruhsal sorunların düzeyi ve ilişkili olduğu faktörler belirlenerek tedavi edilirse fiziksel tedavileri de daha sağlıklı yürütülebilir. Bu nedenle, bu araştırmanın amacı Tip 2 diyabet hastalarının kaygı, depresyon ve genel ruhsal sorunlarıyla (kaygı+depresyon toplam puanı) ilişkili faktörlerin (baş etme yolları, algılanan sosyal destek, öz yeterlik, duygu dışa vurumu ve kaynak kaybı) Kaynakların Korunması kuramı kapsamında incelenmesidir.
Hastalar ve Yöntem: Araştırmaya toplam 116 diyabet hastası katılmıştır. Sosyo-demografik ve Hastalıkla İlgili Bilgi Formu’nun yanı sıra 6 ayrı ölçek uygulanmıştır. Değişkenler arası ilişkilerin incelenmesi için bağımsız gruplar için t testi, korelasyon ve regresyon analizleri yapılmıştır. Bulgular: Diyabet hastalarının yaklaşık yarısının yüksek düzeyde kaygı ve depresyon yaşadıkları bulunmuştur. Çaresiz baş etme ve kaynak kaybının ruhsal sorunların tümüyle pozitif yönde ilişkili olduğu, iyimser baş etmenin ise tüm ruhsal sorunlarla negatif yönde ilişkili olduğu bulunmuştur. Algılanan duygu dışavurumunun, duygusal aşırı ilgilenme boyutundaki artış ise sadece genel ruhsal sorunlardaki azalmayla ilişkili bulunmuştur.
Sonuç: Psikolojik müdahale ve destek programlarında araştırmada belirlenen konulara odaklanılması yararlı olacaktır
Gait and posture disorders are frequent signs of Parkinson's disease. Authors reviewed clinical and pathophysiological results reported for these disorders as well as the methods of investigation and treatment approaches including rehabilitation measures.
Traditionell werden die Bewegungsstörungen (Parkinsonsyndrome, Dyskinesien, Dystonien, Tics, Tremor, Chorea) zu den „Basalganglien- erkrankungen“ gezählt, womit ein gemeinsamer Nenner zumindest für die grobe anatomische Lokalisation der Funktionsstörung oder der pathologischen Auffälligkeit für einige der Störungen vorgegeben wird. Für eine große Anzahl von Bewegungsstörungen ist der primäre Ort der Funktionsstörung aber weiterhin nicht bekannt, verschiedene Lokalisationen kommen in Betracht bzw. die Störung kann nur im Rahmen der Wechselwirkungen in einem übergeordneten neuronalen System verstanden werden. Die Zielpunkte der Hirnstimulation liegen bei Bewegungsstörungen in den Basalganglien und dem Thalamus bzw. seiner Verbindungen. Die kortikale Stimulation hat lediglich eine Rolle in der Schmerztherapie, und nur vereinzelt wurde über den Einsatz in der Tremor- und Parkinsontherapie berichtet. Es ist jedoch zu bemerken, dass die sich weiter entwickelnden Regelkreismodelle zwischen subkortikalen und kortikalen Strukturen es durchaus denkbar erscheinen lassen, dass in nicht allzu ferner Zukunft andere Zielpunkte in den subkortikalen-kortikalen Netzwerken zum Einsatz kommen werden; zusätzlich zu den heutigen Eingriffen im Ncl. subthalamicus (STN), Ncl. ventralis intermedius (Vim) und Globus pallidus internus (GPi). Ohne den Erkenntnisgewinn der letzten zwanzig Jahre im Bereich der Basalganglienorganisation und ihrer thalamischen und kortikalen Verbindungen ist die Renaissance der Stereotaxie und damit der tiefen Hirnstimulation (THS) gar nicht denkbar. Die anatomischen Verbindungen zwischen Basalganglien, Thalamus und dem frontalen Kortex sind die Grundlage für das Konzept der basalganglienthalamokortikalen Regelkreise (Abb. 1). Dieses Regelkreismodell gilt heute als eines der wichtigsten neuronalen Netzwerke für die Steuerung der Motorik, aber auch anderer Hirnfunktionen wie Emotion, Kognition und Verhalten. In diesem Kapitel wird vor allem auf die systemphysiologischen Modelle und die funktioneile Anatomie eingegangen werden.
Recently we incorporated an organotypic culture method in our basal ganglia research. In our preparation, we were successful in co-culturing more than two structures of interest1–3. This in vitro organotypic preparation combines the advantage of in vitro slice preparation for ease of intracellular sharp or patch recording under improved controlled experimental chemical environment with the in vivo preparation in which the structure of interest is not isolated from the source of their major afferents. In this report, we would like to present a triple culture preparation consisting of the tegmental pedunculopontine nucleus (PPN), the subthalamic nucleus (STN) and the substantia nigra (SN).
A new posteroventral pallidotomy has been introduced for patients with Parkinson’s disease (Laitinen, 1992). By this pallidotomy, a muscle rigidity and akinesia can be effectively abolished. A gait disturbance and balance problems could also be improved. The effects of the new pallidotomy is assumed to be the results of the blockade of the increased inhibitory effect from internal segment of the globus pallidus on the motor thalamus, as well as on the brain stem target, that is the pedunculopontine tegmental nucleus (PPN)(Ohye, 1995).
Anatomical studies have revealed dense connections between the pedunculopontine tegmental nucleus (PPN) and the basal ganglia (Figure 1). In primates, PPN receives massive afferents from the two output structures of the basal ganglia, that is, the substantia nigra pars reticulata and the internal pallidum. In turn, PPN projects profusely to the substantia nigra pars compacta and the subthalamic nucleus (Filion and Harnois, 1978; Harnois and Filion, 1980; Harnois and Filion, 1982; Parent and Hazrati, 1995). These ascending projections were found both ipsilaterally and contralaterally (Lavoie and Parent, 1994b). The existence of cerebello-tegmental projections, which are ipsilateral collaterals of the cerebello-thalamic projection, was also reported (Hazrati and Parent, 1992). Motor cortex (area 4) also sends fibers to PPN (Hartmann-Von Monakow et al., 1979; Moon Edley and Graybiel, 1983). PPN is a part of the mesencephalic locomotor region, from which locomotor movements are induced by its electrical stimulation (Garcia-Rill, 1986; Garcia-Rill, 1991).
The present review was attempted to analyze the multiple channels of basal ganglia-thalamocortical connections, and the connections of their related nuclei. The prefrontal and motor areas consist of a number of modules, which seem to provide multiple subloops of the basal ganglia-thalamocortical connections in subhuman primates. There may be a gnat degree of convergence of the limbic, associative and motor loops at the level of the striatum, substantia nigra, pallidum, and the subthalamic nucleus as well as the pedunculopontine nucleus. Nigral dopaminergic neurons receive limbic input directly as well as indirectly through the striosomes in the striatum. Dopamine contributes to behavioral learning by signaling motivation and reinforcement. The pedunculopontine nucleus might be involved in behavioral state control, learning and reinforcement processes, locomotion and autonomic functions. Each subdivision of the motor areas receives a mixed and weighted transthalamic input from both the cerebellum and basal ganglia. In particular, based on the author's data, the hand/arm motor area and adjacent premotor area receive strong superficial basal ganglia-thalamocortical projections as well as the deep cerebello-thalamocortical projections. These areas, have very dense corticocotrical connections with other cortical areas, receive polymodal afferents from the parietal and temporal cortices, and integrated information, via multiple routes, from the prefrontal cortex. The author suggests that the ventrolateral part of the caudal medial pallidal segment (GPi) and the ventromedial part of the GPi are linked directly to these areas by ways of the oral part of ventral lateral nucleus (VLo) and the Ventral part of the parvicellular part of ventral anterior nucleus (VApc), respectively. These connections are thought to be involved in the acquisition and coordination of motor sequences.
We present data from animal studies showing that the pedunculopontine tegmental nucleus-conserved through evolution, compartmentalized, and with a complex pattern of inputs and outputs-has functions that involve formation and updates of action-outcome associations, attention, and rapid decision making. This is in contrast to previous hypotheses about pedunculopontine function, which has served as a basis for clinical interest in the pedunculopontine in movement disorders. Current animal literature points to it being neither a specifically motor structure nor a master switch for sleep regulation. The pedunculopontine is connected to basal ganglia circuitry but also has primary sensory input across modalities and descending connections to pontomedullary, cerebellar, and spinal motor and autonomic control systems. Functional and anatomical studies in animals suggest strongly that, in addition to the pedunculopontine being an input and output station for the basal ganglia and key regulator of thalamic (and consequently cortical) activity, an additional major function is participation in the generation of actions on the basis of a first-pass analysis of incoming sensory data. Such a function-rapid decision making-has very high adaptive value for any vertebrate. We argue that in developing clinical strategies for treating basal ganglia disorders, it is necessary to take an account of the normal functions of the pedunculopontine. We believe that it is possible to use our hypothesis to explain why pedunculopontine deep brain stimulation used clinically has had variable outcomes in the treatment of parkinsonism motor symptoms and effects on cognitive processing. © 2016 International Parkinson and Movement Disorder Society.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease, affecting up to 1% of individuals aged 65–69 years and 3% of those over 80 years of age [1]. Among the cardinal features of parkinsonism (resting tremor, bradykinesia, rigidity, and postural instability), postural and gait disfunction leading to falls represents the largest single contributor to the number of emergency room visits and overall cost to the healthcare system relating to PD [2–4]. In addition, the fear of falling is associated with its recurrence, and frequently leads to a loss of independence and depression [5]. Postural and gait disfunction have proven particularly resistant to current dopamine and surgical therapies, which suggests a greater involvement of non-dopaminergic pathways and other brain loci distinct from the pallidal and subthalamic nuclei in their pathophysiology [6–14].
In spite of the existence of pedunculopontine tegmental nucleus (PPTg) projections to cerebellar nuclei, their nature and functional role is unknown. These fibers may play a crucial role in postural control and may be involved in the beneficial effects induced by deep brain stimulation of brainstem structures in motor disorders. We investigated the effects of PPTg microstimulation on single unit activity of dentate, fastigial and interpositus nuclei. The effects of PPTg stimulation were also studied in rats whose PPTg neurons were destroyed by ibotenic acid and subsequently subjected to iontophoretically applied cholinergic antagonists. The main response recorded in cerebellar nuclei was a short latency (1.5-2 ms) and brief (13-15 ms) orthodromic activation. The dentate nucleus was the most responsive to PPTg stimulation. The destruction of PPTg cells reduced the occurrence of PPTg-evoked activation of dentate neurons, suggesting that the effect was due to stimulation of cell bodies and not due to fibers passing through or close to the PPTg. Application of cholinergic antagonists reduced or eliminated the PPTg-evoked response recorded in the dentate nucleus. The results show that excitation is exerted by the PPTg on the cerebellar nuclei, in particular on the dentate nucleus. Taken together with the reduction of NADPH-diaphorase positive neurons in lesioned animals, the iontophoretic experiments suggest that the activation of dentate neurons is due to cholinergic fibers. These data help to explain the effects of deep brain stimulation of the PPTg on axial motor disabilities in neurodegenerative disorders.
The targets of internal pallidal efferents have attracted considerable attention given the central role proposed for the internal segment of the globus pallidus (GPi) in models of normal and pathological movement.1–3 The previous emphasis of these models on basal ganglia-thalamocortical circuitry, has left pathways between the GPi and the midbrain tegmentum largely unexplored. In the primate, the size and functional import of pallidofugal projections upon the mesopontine tegmentum are nonetheless likely to be significant. A majority of neurons in the primate GPi contribute to this pathway via collateralization from pallidothalamic fibers,4–6 and its terminl zone has been described as “extensive”7. Experimental and pathophysiological observations implicate the mesopontine tegmental region in receipt of basal ganglia output as important in modulating normal and pathological movement. Electrical stimulation and micro infusions of substance-P or NMDA8 into the mesopontine tegmentum in decerebrate subprimate preparations elicit treadmill locomotion, while GABAergic pathways play an inhibitory role8, 9 (i. e. the “mesencephalic locomotor region” (MLR).10–12 In awake behaving subprimates, cytotoxic lesions including, but not restricted to, midbrain tegmental/basal ganglia circuitry produce incomplete hindlimb extension, bradykinesia and dyscoordination.13 Depending on the locus and the electrical or pharamacological stimulus parameters applied, motor effects ranging from decreased “postural support” to increased spontaneous motor activity have also been reported.14–21 Enhanced utilization of 2-deoxyglucose in the mesopontine tegmentum in primate models of Parkinsons disease (PD)22 suggests that excessive pallidotegmental inhibition might contribute to hypokinesia, while decreased utilization in a model of hemiballismus23 suggests that disinhibition of the mesopontine tegmentum might contribute to hyperkinetic disorders.
The new neuroscience data rapidly accumulating by the end of the second millennium calls for radical revision of many long-established and widely accepted postulates. This paper reviews some data leading to new concepts of life and work of neurons. The adult brain contains stem cells which are the source of the precursors for all main types of the brain cells: neurons, astrocytes, and oligodendroglia. These cells can substitute the deteriorating elements in the adult and even old brain. The neurons occur to be highly resistant to lesion of their processes as well to anoxia, and inhibitory neurons are shown to be especially stable in some pathological conditions. Changes in the affrent inputs result in various types of rapid compensatory morphological and functional reorganizations at different levels. Thus, the previous fatalistic view of the nervous system is substituted now for an optimistic one regarding various possibilities of prolongation and restoration of normal functioning of the brain. Simultaneously, our concepts of the neurons changed drastically. An unitary neuron may operate by several neurotransmitting substances; their synaptic influences upon the dendrites may evoke the active propagation of calcium and sodium spikes, their axons may differentially release transmitter substances depending on parameters of excitation. All neuronal functions are helped and controlled by astroglia, which participates in the synthesis of transmitters and protects the neurons from the excitotoxic death. Besides the synaptic interactions between the neurons, there exist other types of communications, such as volume conduction of transmitters after their spillover from the excited synapses and non-synaptic (varicose) zones, as well as exchange of molecules and ions through the gap junctions. A complex picture of interneuronal communications with multiple synaptic, presynaptic, and parasynaptic interactions is further complicated by the intimate participation of neurotrophic substances and "mediatros of the immune system" - cytokines in these processes. The mutual regulatory influences between neurotransmitters, neurotrophic, and neuroimmune systems show that in normal conditions all they are working in concert. This increase in number of factors determining the final result of interaction between the neurons contributes new difficulties to the development of theoretical concepts or simulation of brain functions. In this context it is possible to speak about a certain crisis of theoretical neurobiology at present, because multiplicity of fine details obtained by molecular neurobiology and neurogenetics cannot be integrated in a coherent view of the brain functions. Overcoming the present gap between the analytic and synthetic approaches to understanding the brain work will be the main aim for the neurobiologists of the third millennium.
The neurons of the laterodorsal and pedunculopontine tegmental nuclei (LDT–PPT) have a dual function in the control of behavioral states: they promote either wakefulness (W) or REM sleep. During W, these neurons are also related to cognitive and motor functions. In fact, the PPT is the main output station of the basal ganglia circuit and has a major role in the akinesia of Parkinson´s disease (PD). Interest in this area has grown tremendously following recent demonstrations that the PPT is a promising target for treating PD symptoms by deep brain stimulation. Further progress in treating PD will be greatly assisted by a clear understanding of the structure and function of the LDT–PPT.
Cholinergic neurons are the principal mediators of activity originating in the LDT–PPT. These neurons project widely to the forebrain and brainstem and increase their firing rate either during W and REM sleep or during REM sleep exclusively. Microinjections of cholinergic agonists into the nucleus pontis oralis, which is a recipient of LDT–PPT cholinergic neuronal projections, prolongs the duration of the REM sleep state. Although GABAergic neurons in the LDT–PPT outnumber cholinergic neurons by two to one, the function of these neurons is mostly unknown. These neurons may have local functions such as controlling the input and the output of cholinergic neurons; however, these cells also project outside the LDT–PPT. Utilizing Fos immunoreactivity as a marker of neuronal activity, it was shown in cats and rats that GABAergic neurons of the LDT–PPT are active during REM sleep. In addition, microinjections of muscimol (GABAA agonist) within this area generate REM sleep both in cats and rats; on the contrary, GABAA antagonists induce W. These data suggest that GABAergic neurons of the LDT–PPT promote REM sleep. In the present report, we review the role of GABA within the LDT–PPT in REM sleep regulation.
This study was performed to examine the hypothesis that thalamic-projecting neurons of mesopontine cholinergic nuclei display activity patterns that are compatible with their role in inducing and maintaining activation processes in thalamocortical systems during the states of waking (W) and rapid-eye-movement (REM) sleep associated with desynchronization of the electroencephalogram (EEG). A sample of 780 neurons located in the peribrachial (PB) area of the pedunculopontine tegmental nucleus and in the laterodorsal tegmental (LDT) nucleus were recorded extracellularly in unanesthetized, chronically implanted cats. Of those neurons, 82 were antidromically invaded from medial, intralaminar, and lateral thalamic nuclei: 570 were orthodromically driven at short latencies from various thalamic sites: and 45 of the latter elements are also part of the 82 cell group, as they were activated both antidromically and synaptically from the thalamus. There were no statistically significant differences between firing rates in the PB and LDT neuronal samples. Rate analyses in 2 distinct groups of PB/LDT neurons, with fast (greater than 10 Hz) and slow (less than 2 Hz) discharge rates in W, indicated that (1) the fast-discharging cell group had higher firing rates in W and REM sleep compared to EEG-synchronized sleep (S), the differences between all states being significant (p less than 0.0005); (2) the slow-discharging cell group increased firing rates from W to S and further to REM sleep, with significant difference between W and S (p less than 0.01), as well as between W or S and REM sleep (p less than 0.0005). Interspike interval histograms of PB and LDT neurons showed that 75% of them have tonic firing patterns, with virtually no high-frequency spike bursts in any state of the wake-sleep cycle. We found 22 PB cells that discharged rhythmic spike trains with recurring periods of 0.8-1 sec. Autocorrelograms revealed that this oscillatory behavior disappeared when their firing rate increased during REM sleep. Dynamic analyses of sequential firing rates throughout the waking-sleep cycle showed that none of the full-blown states of vigilance is associated with a uniform level of spontaneous firing rate. Signs of decreased discharge frequencies of mesopontine neurons appeared toward the end of quiet W, preceding by about 10-20 sec the most precocious signs of EEG synchronization heralding the sleep onset. During transition from S to W, rates of spontaneous discharges increased 20 sec before the onset of EEG desynchronization.(ABSTRACT TRUNCATED AT 400 WORDS)
The distribution of acetylcholine neurons in the brainstem of the cat was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared to that of catecholamine neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. The largest group of ChAT-positive (+) neurons was located in the lateral pontomesencephalic tegmentum within the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus rostrally and within the parabrachial nuclei and locus coeruleus nucleus more caudally. TH+ neurons were found to be coextensive and intermingled with ChAT+ neurons in the dorsolateral pontomesencephalic tegmentum, where the number of ChAT+ cells (approximately 18,500) exceeded that of the TH+ cells (approximately 12,000). In the caudal pons, scattered ChAT+ neurons were situated in the ventrolateral tegmentum together with TH+ neurons. In the medulla, numerous ChAT+ cells were located in the lateral tegmental field, where they extended in a radial column from the dorsal motor nucleus of the vagus to the ventrolateral tegmentum around the facial and ambiguus nuclei, occupying the position of preganglionic parasympathetic neurons of the 7th, 9th, and 10th cranial nerves. TH+ cells were also present in this field. Neurons within the general visceral, special visceral, and somatic motor cranial nerve nuclei were all immunoreactive to ChAT. Scattered ChAT+ neurons were also present within the medullary gigantocellular and magnocellular tegmental fields together with a small number of TH+ neurons. Other groups of ChAT+ cells were identified within the periolivary nuclei, parabigeminal nucleus, prepositus hypoglossi nucleus, and the medial and inferior vestibular nuclei. Acetylcholine neurons thus constitute a heterogeneous population of cells in the brainstem, which in addition to including the somatic and visceral efferent systems, comprises many other discrete systems and represents an important component of the brainstem reticular formation. The proximity to and interdigitation with catecholamine neurons within these systems may be of important functional significance.
This book stemmed from an IBRO symposium that took place in Leipzig, from 12 to 14 August 1987. Local organizers were D. Biesold and V. Bigl from the Department of Neurochemistry at the Paul Flechsig Institute for Brain Research. Some of the contributors to this book were members of the International Program Committee: L. L. Butcher, M.-M. Mesulam, G. Pepeu, and M. Steriade. Leipzig was chosen as the site of a meeting devoted to brain cholinergic systems because some historical steps in this domain are related to the German city. Indeed, after the initial description of the substantia innominata (die ungennante Marksubstanz) by J. C. Reil (1809), T. Meynert, one of the founders of scientifically oriented psychiatry, designated this structure as Ganglion der Hirnschenkelschlinge in a volume published in Leipzig (1872). The name of Meynert was linked to the nucleus basalis by A. Koelliker in a Handbook, also published in Leipzig (1896).
Since edited volumes rarely allow the expression of coherence between the numerous authors, we decided to ask for chapters from only a limited number of participants and we invited other colleagues, who could not attend the symposium, to submit their contribution. Our goal was to present current data and concepts about the morphology, physiology, and pathology of brainstem and basal forebrain cholinergic systems controlling the excitability of the thalamus and cerebral cortex. It is a pleasure to extend our thanks to all colleagues, whose names and affiliations are listed, for taking time from busy lives to survey their fields of interest. We also express our appreciation to Oxford University Press for an agreeable collaboration and support in the preparation of this book.
In an effort to account for a large number of reported functions mediated by a small portion of the midbrain, a hypothesis is advanced as a basis for discussion and not as established fact and is guided by reports from a large number of laboratories working on the same region but using widely disparate preparations. Overall, the hypothesized model suggests an underlying mechanism of action for what is essentially the ascending reticular activating system. The model proposed will hopefully be tested stringently in order to arrive at a better understanding of brain stem mechanisms modulating a host of rhythmic functions.
Choline acetyltransferase immunohistochemistry has identified a large group of cholinergic neurons in the pontine tegmentum. By combined immunohistochemical and enzyme histochemical studies this particular cholinergic cell group was found to contain an enzyme, NADPH-diaphorase, that can be visualized histochemically. Thus NADPH-diaphorase histochemistry provides a simple, reliable method to selectively stain the cholinergic neurons of the brainstem reticular formation. The resolution obtained by this novel histochemical technique is similar to that found with the Golgi stain, and it should therefore be of great value in morphological studies of this cholinergic cell group.
A major group of cholinergic neurons is present in the midbrain and pontine tegmentum. These cells could be selectively stained using either monoclonal antibodies to choline acetyltransferase, the pharmacohistochemical acetylcholinesterase procedure, or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Using these three techniques, the precise distribution of this cell group was determined. By combining these techniques with immunohistochemical staining for various neuropeptides, examples of peptide-cholinergic coexistence could be demonstrated in this cell group. Approximately 30% of these cholinergic neurons displayed substance P immunoreactivity. Most of these cells also showed corticotropin-releasing factor immunoreactivity and bombesin/gastrin-releasing peptide immunoreactivity. These results therefore provide evidence for the coexistence of various neuropeptides together with NADPH-diaphorase activity in the ascending cholinergic reticular system.
No Abstract. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/49930/1/900780303_ftp.pdf
The afferent connections of the pedunculopontine tegmental nucleus (PPT) and the adjacent midbrain extrapyramidal area (MEA) were examined by retrograde tracing with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Major afferents to the PPT originate in the periaqueductal gray, central tegmental field, lateral hypothalamic area, dorsal raphe nucleus, superior colliculus, and pontine and medullary reticular fields. Other putative inputs originate in the paraventricular and preoptic hypothalamic nuclei, the zona incerta, nucleus of the solitary tract, central superior raphe nucleus, substantia innominata, posterior hypothalamic area, and thalamic parafascicular nucleus. The major afferent to the medially adjacent MEA originates in the lateral habenula, while other putative afferents include the perifornical and lateral hypothalamic area, periaqueductal gray, superior colliculus, pontine reticular formation, and dorsal raphe nucleus. MEA inputs from basal ganglia nuclei include moderate projections from the substantia nigra pars reticulata, entopeduncular nucleus, and a small projection from the globus pallidus, but not the subthalamic nucleus. Dense anterograde labeling was observed in the substantia nigra pars compacta, entopeduncular nucleus, subthalamic nucleus, globus pallidus, and caudate-putamen only following WGA-HRP injections involving the MEA.
Choline acetyltransferase immunhistochemistry was employed at light and electron microscopic levels in order to determine the distribution of cholinergic neurons in two subdivisions of the rat pedunculopontine tegmental nucleus that were previously defined on cytoarchitectonic grounds, and to compare the synaptic inputs to cholinergic and non-cholinergic somata in the subnucleus dissipatus, which receives major input from the substantia nigra. Large cholinergic neurons were found in both the pars compacta and the pars dissipata of the pedunculopontine nucleus. However, they were intermingled with non-cholinergic neurons and did not respect the cytoarchitectural boundaries of the nucleus. Ultrastructural study showed that all cholinergic neurons in the subnucleus dissipatus exhibited similar features. The majority had large somata (largest diameter ⩾20 μm) containing abundant cytoplasmic organelles and nuclei displaying a few shallow invaginations. Synaptic terminals on the cholinergic cell bodies were scarce and unlabeled boutons containing spherical synaptic vesicles and establishing asymmetric synaptic junctions were the dominant type. In contrast, the non-cholinergic neurons presented prominent differences in the size of their somata as well as in the distribution of axosomatic synapses. Two almost equally represented classes of non-cholinergic neurons which are referred to as large (largest diameter ⩾20 μm) and small (largest diameter
Single- and double-immunostaining procedures were used to study the distribution of the acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) and the calcium binding protein calbindin D-28k in the nucleus basalis of Meynert (nbM) and in the pedunculopontine nucleus (PPN) of the squirrel monkey (Saimiri sciureus). As expected from previous studies in other primates, including humans, the nbM in the squirrel monkey is enriched with large ChAT-immunoreactive neurons that form clusters in the substantia innominata. Some ChAT-positive neurons are also scattered more dorsally within the internal and external medullary laminae of the pallidal complex. A smaller number of calbindin-immunoreactive cells occur in the same locations and their mean cross-sectional somatic area (424 microns 2) is not significantly different from that of the ChAT-immunoreactive cells (450 microns 2). Furthermore, 60% of the ChAT-immunopositive cells in the nbM display calbindin immunoreactivity. Most of these double-immunoreactive neurons occur in the typical clusters of the nbM, whereas the large neurons scattered in between the clusters display ChAT immunoreactivity only. In the PPN, ChAT-positive neurons are scattered around and partly within the superior cerebellar peduncle and also form a dense cluster in the lateral portion of the mesopontine tegmentum. Calbindin-immunoreactive cells also abound around the superior cerebellar peduncle, but they are more sparsely distributed and cover a larger sector of the tegmentum than the ChAT-positive neurons. These calbindin-immunoreactive cells are significantly smaller (200 microns 2) than the ChAT-immunoreactive cells (471 microns 2) and no double-immunostained neurons are present in the PPN.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent neuroanatomical studies undertaken with various powerful neural tracing methods have radically changed our concept of the organization of the basal ganglia. This paper briefly reviews some of the findings that have led to the conclusion that the major components of the basal ganglia can no longer be considered as single undifferentiated entities. Instead, each of these structures is characterized by several distinct afferent and efferent chemospecific subsystems by which they can modulate and convey the multifarious information that flows through the basal ganglia. This paper focuses mainly on data obtained in primates, but also stresses the importance of comparison with non-primate species.
For the last decade the functional organization pf cholinergic neurons has dominated studies of the basal forebrain. Cholinergic neurons in the brain, exclusive of motor neurons and interneurons, are found in two spatially separate groups (Armstrong et al., 1983, Mesulam et al., 1984). The rostral group, located in the basal forebrain, has received substantial attention because of its corticopedal projections (Mesulam et al., 1984) and its’ degeneration in Alzheimer’s disease (Coyle et al., 1983). The caudal group is found in the laterodorsal tegmental nucleus (LDT) and pedunculopontine nucleus (PPT) within the pontine tegmentum (Vincent et al., 1983; Mesulam et al., 1984; Satoh and Fibiger, 1986), and is the source of cholinergic innervation to the basal forebrain, thalamus and brainstem (Sofroniew et al., 1985; Hallenger et al., 1987; Maley et al., 1988; Rye et al., 1988; Jones, 1990).
In an effort to account for a large number of reported functions mediated by a small portion of the midbrain, a hypothesis is advanced as a basis for discussion and not as established fact and is guided by reports from a large number of laboratories working on the same region but using widely disparate preparations. Overall, the hypothesized model suggests an underlying mechanism of action for what is essentially the ascending reticular activating system. The model proposed will hopefully be tested stringently in order to arrive at a better understanding of brain stem mechanisms modulating a host of rhythmic functions.
A specific antibody raised against 5-hydroxytryptamine (5-HT) conjugated to bovine serum albumin was used to study the serotoninergic innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus). At midbrain level, numerous fine 5-HT-immunoreactive axons were seen to arise from the immunopositive neurons of the dorsal raphe nucleus and less abundantly from those of the nucleus centralis superior. The bulk of these axons formed a rather loosely arranged bundle that arched ventrorostrally through the central portion of the midbrain tegmentum and ascended toward the ventral tegmental area. Several fascicles detached themselves from this bundle to reach the substantia nigra where they arborized into a multitude of heterogeneously distributed 5-HT terminals. The 5-HT innervation was particularly dense in the pars reticulata but much less so in the pars compacta of the substantia nigra. More rostrally other 5-HT fibers swept dorsolaterally and formed a remarkably dense network of varicose fibers within the subthalamic nucleus. A multitude of 5-HT axons continued their ascending course within the lateral hypothalamic area, and many of them swept laterally to invade the lenticular nucleus. At pallidal levels, the 5-HT axons arborized much less profusely in the external segment than in the internal segment, which contained numerous 5-HT varicose fibers and terminals arranged in a typical bandlike pattern. At striatal levels, the 5-HT terminals were particularly abundant in the ventral striatum, including the nucleus accumbens and deep layers of the olfactory tubercle. They also abounded in the ventrolateral region of the putamen and the ventromedial aspect of the caudate nucleus. Overall, the number of 5-HT fibers and terminals decreased progressively along the rostrocaudal axis of the striatum and several large and elongated zones rather devoid of 5-HT immunoreactivity were visualized, particularly in the caudate nucleus and the dorsal putamen. These zones of poor 5-HT immunoreactivity were in register with similar areas devoid of tyrosine hydroxylase immunoreactivity as seen on contiguous sections. These findings reveal that all the core structures of the basal ganglia in primates receive a significant serotoninergic input, but that the densities and patterns of innervation vary markedly from one structure to the other.
Membrane properties and postsynaptic responses to stimulation of the substantia nigra reticulata (SNr) of the neurons in rat pedunculopontine nucleus (PPN) were studied in an in vitro parasagittal slice preparation using intracellular recording techniques. Based on electrical membrane properties, PPN neurons were classified into 3 types (types I, II and II). The unique feature of the type I neuron was the low threshold calcium spike while the type II neuron had various inward and outward rectifications. The type III neuron showed no such features as those observed in type I or II neurons. Some recorded neurons were intracellularly labeled with biocytin to study their morphology, and their transmitter phenotype was investigated by immunocytochemistry for choline acetyltransferase (ChAT). The type I and III neurons were found to be non-cholinergic, but 50% of the labeled type II neurons were immunopositive for ChAT. Morphological features of type II neurons were also different from type I or III neurons. The soma of the type II neuron was almost always more than twice as large as that of type I and III neurons. Inhibitory postsynaptic potentials (IPSPs) were induced in all 3 types of PPN neurons following stimulation of SNr. SNr-induced IPSPs were usually followed by a slow depolarizing potential from which rebound spikes were triggered. These rebound excitations were found only in type I and II neurons. These data indicate that heterogeneous groups of neurons exist in the PPN in terms of morphology, transmitter phenotypes and electrical membrane properties.
Studies of the pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum have emphasized the organization and projections of the cholinergic neurons. We report here that exhibiting glutamate immunoreactivity are present in both the LDT and PPT. These glutamatergic neurons are interspersed among the cholinergic neurons within both nuclei with no apparent segregation. These data raise the possibility that excitatory amino acids contribute to the functions of the LDT and PPT.
A map of cholinergic cells of the human brainstem identified by immunohistochemistry of choline acetyltransferase (ChAT) is presented, along with a map of acetylcholinesterase (AChE)-containing cells and fibers. ChAT-positive structures belong to 4 brainstem systems: the cranial motor nuclei; the parabrachial complex; the reticular system; and the vestibular system. All motor nuclei of the cranial nerves, as well as the nucleus supraspinalis, are ChAT-positive. The positively staining structures of the parabrachial system include the nucleus tegmentali pedunculopontinus, and the nuclei parabrachialis medialis and lateralis. Nuclei of the reticular system containing some ChAT-positive cells include the nucleus reticularis pontis oralis and caudalis, the nucleus reticularis tegmenti pontis, the nucleus reticularis gigantocellularis, the nucleus reticularis lateralis and the formatio reticularis centralis (medulla). Structures of the vestibular and auditory systems which contain some ChAT-positive cells include the nucleus vestibularis lateralis, and the nuclei olivaris superioris medialis and lateralis. All ChAT-positive structures stain strongly for AChE. AChE-positive, ChAT-negative structures were noted in several sensory systems. The substantia nigra, locus coeruleus and raphe nuclei, known to contain non-cholinergic cells, also stain positively. The significance of the AChE-positive, ChAT-negative staining in most structures remains to be determined. A knowledge of the cholinergic systems of human brain may be important to an understanding of the pathology of a number of diseases.
Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte balance. Immunohistochemical studies have shown that large, multipolar atriopeptin-like immunoreactive (APir) neurons are present in areas of the midbrain corresponding to the large neurons of the pedunculopontine tegmental (PPT) and lateral dorsal tegmental (TLD) nuclei, all of which can be stained immunohistochemically for choline acetyltransferase-like immunoreactivity (ChATir). A subpopulation of these cholinergic PPT and TLD neurons are also known to contain substance P-like immunoreactivity (SPir). Using an immunofluorescent technique that allows simultaneous localization of two antigens, we have studied the relationship between APir, SPir and ChATir in the pontine tegmentum of the rat. We have found that the large multipolar APir neurons of the pontine tegmentum are identical to the ChATir neurons of the PT and TLD nuclei and a subpopulation of the APir neurons in PPT and TLD neurons are also SPir.
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.
The distribution and collaterlization of ascending and descending projections from neurons in the nucleus tegmenti pedunculopontinus (PPN) were studied in the rat by using retrograde transport of HRP, HRP/WGA, and fluorescent dyes. The PPN and its two subdivisions, the subnucleus compactus (PPNc) and subnucleus dissipatus (PPNd), were delineated on sagittal Nisslstained sections by using cytoarchitectural features as guidelines.
Large bilateral pressure injections of HRP and/or fluorescent dyes into the cervical cord retrogradely labeled moderate numbers of fusiform and polygonal PPN cells which ranged in size between 65 and 390 μm2. The labeled cells were scattered throughout the PPNd and were somewhat more numerous in the medial half of the subnucleus. The PPNc contained only occasional labeled cells in its ventralmost portion.
Following single unilateral HRP/WGA injections in the striatum, globus pallidus, entopeduncular nucleus, subthalamus, or the substantia nigra, the distribution of the labeled cells was similar to that of the spinal cord-projecting PPN neurons. Multiple HRP injections were then made bilaterally in the substantia nigra and the entopeduncular nucleus and/or subthalamus in order to label the entire population of PPN neurons projecting to the basal ganglia. In this case, not only the PPNd but also the PPNc contained a substantial number of retrogradely labeled cells. The rostrally projecting PPN cells outnumbered 5.4 times those projecting to the spinal cord, and their somata were somewhat larger, ranging between 114 and 472 μm2. While both fusiform and polygonal shapes were encountered, the polygonal cell somata were more numerous.
In the double-labeling experiments, Granular Blue and Diamidino Yellow Dihydrochloride were injected into the cervical cord and the entopeduncular nucleus or subthalamus. In general, these experiments confirmed the extensive overlap of forebrain- and spinal cord-projecting neurons within the PPNd and the quantitative preponderance of ascending neurons. They also demonstrated that these two projection systems originate largely from separate cell populations since the double-labeled cells always composed less than 5% of the labeled neurons.
The results confirm the existence of a direct PPN projection to the spinal cord. This pathway originates mainly in the PPNd and appears to be quantitatively weaker than the PPN projections to the forebrain. The spinal cord-projecting cells are not spatially segregated from the cells projecting to the basal ganglia, but they represent a separate population of the PPN projection neurons.
The superficial and intermediate gray layers of the superior colliculus are heavily innervated by fibers that utilize the neurotransmitter acetylcholine. The distribution, ultrastructure, and sources of the cholinergic innervation of these layers have been examined in the cat by using a combination of immunocytochemical and axonal transport methods. Putative cholinergic fibers and cells were localized by means of a monoclonal antibody to choline acetyltransferase (ChAT).
ChAT immunoreactive fibers are distributed throughout the depth of the superior colliculus, with particularly dense zones of innervation in the upper part of the superficial grey layer and in the intermediate grey layer. Within the superficial grey layer, the fibers form a continuous, dense band, whereas within the intermediate grey layer the fibers are arranged in clusters or patches. Although the patches are present throughout the rostrocaudal extent of the superior colliculus, they are most prominent in middle to caudal sections.
The structure of the ChAT immunoreactive terminals was examined electron microscopically. The appearance of the terminals is similar in the superficial and intermediate grey layers. They contain closely packed, mostly round vesicles, and form contacts with medium-sized dendrites that exhibit small, but prominent postsynaptic densities; a few of the terminals contact vesicle-containing profiles.
To identify the sources of the cholinergic input to the superior colliculus, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made in the superior colliculus and the sections were processed to demonstrate both the retrograde transport of WGA-HRP and ChAT immunoreactivity. Neurons containing both labels were found in the parabigeminal nucleus, and in the lateral dorsal and pedunculopontine tegmental nuclei of the pontomesencephalic reticular formation. Almost every cell in these nuclei that contained retrograde label was also immunoreactive for ChAT.
The similarities between the laminar distributions of the ChAT terminals and the terminations of the pathway from the parabigeminal nucleus (Graybiel: Brain Res. 145:365–374, '78) support the view that the latter nucleus is a source of the cholinergic fibers in the superficial grey layer. The possibility that the pedunculopontine tegmental nucleus is a source of cholinergic fibers in the deep layers was tested by examining the distribution of labeled fibers following injections of WGA-HRP into this region of the tegmentum. Patches of labeled terminals were found in the intermediate grey layer that resemble in distribution the patches of ChAT immunoreactive fibers in this layer. Only a sparse distribution of labeled terminals was found in the other layers. These results suggest that there are at least two distinct sources of the cholinergic innervation of the superior colliculus: the parabigeminal nucleus for the superficial grey layer and the pedunculopontine tegmental nucleus for the intermediate grey layer. Other potential sources of cholinergic input to the superior colliculus include ChAT immunoreactive neurons that were observed in the present study in the superficial layers of the superior colliculus itself and the cholinergic cells of the lateral dorsal tegmental nucleus that project to the superior colliculus.
Since the pontomesencephalic reticular formation is known to have extensive connections with efferent pathways of the basal ganglia, in a final series of experiments the relationships between the cholinergic pathways from the tegmentum to the superior colliculus and the projections of substantia nigra pars reticulata were explored. Injections of WGA-HRP were madeinto the substantia nigra pars reticulata and alternate sections were processed for either HRP histochemistry or ChAT immunocytochemistry. Within the superior colliculus, the nigrotectal terminals form patches that are approxi-mately equal in number and aligned with the patches of ChAT immunoreac-tive processes. Within the tegmentum, extensive overlap was found between the ChAT immunoreactive cells in the pedunculopontine tegmental nucleus and the terminal field of substantia nigra pars reticulata. These results suggest that there is a close association between the cholinergic innervation of the intermediate grey layer and the nigral outflow of the basal ganglia.
Choline acetyltransferase immunohistochemistry showed that the human rostra1 brainstem contained cholinergic neurons in the oculomotor, trochlear, and parabigeminal nuclei as well as within the reticular formation. The cholinergic neurons of the reticular formation were the most numerous and formed two intersecting constellations. One of these, designated Ch5, reached its peak density within the compact pedunculopontine nucleus but also extended into the regions through which the superior cerebellar peduncle and central tegmental tract course. The second constellation, designated Ch6, was centered around the laterodorsal tegmental nucleus and spread into the central gray and medial longitudinal fasciculus. There was considerable transmitterrelated heterogeneity within the regions containing Ch5 and Ch6. In particular, Ch6 neurons were intermingled with catecholaminergic neurons belonging to the locus coeruleus complex. The lack of confinement within specifiable cytoarchitectonic boundaries and the transmitter heterogeneity justified the transmitter‐specific Ch5 and Ch6 nomenclature for these two groups of cholinergic neurons.
The cholinergic neurons in the nucleus basalis (Ch4) and those of the Ch5‐Ch6 complex were both characterized by perikaryal heteromorphism and isodendritic arborizations. In addition to choline acetyltransferase, the cell bodies in both complexes also had high levels of acetylcholinesterase activity and nonphosphorylated neurofilament protein. However, there were also marked differences in cytochemical signature. For example, the Ch5‐Ch6 neurons had high levels of NADPHd activity, whereas Ch4 neurons did not. On the other hand, the Ch4 neurons had high levels of NGF receptor protein, whereas those of Ch5‐Ch6 did not.
On the basis of animal experiments, it can he assumed that the Ch5 and Ch6 neurons provide the major cholinergic innervation of the human thalamus and that they participate in the neural circuitry of the reticular activating, limbic, and perhaps also extrapyramidal systems.
The organization of the dopaminergic mesostriatal fibers and their patterns of innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus) were studied immunohistochemically with an antiserum raised against tyrosine hydroxylase (TH). Numerous fibers arose from midbrain TH-positive cell bodies of the substantia nigra pars compacta (group A9), the retrorubral area (group A8), and the lateral portion of the ventral tegmental area (group A10). These fibers accumulated dorsomedially to the rostral pole of the substantia nigra where they formed a massive bundle that coursed through the prerubral field and ascended along the laterodorsal aspect of the medial fore-brain bundle in the lateral hypothalamus. Some ventrally located fibers ran throughout the rostrocaudal extent of the lateral preopticohypothalamic area and could be followed up to the olfactory tubercle, whereas other fibers turned laterodorsally to invade the head of the caudate nucleus. At more dorsal levels in the lateral hypothalamus, many fiber fascicles detached themselves from the main bundle and swept laterally to reach the globus pallidus, the putamen, and the amygdala. Several TH-positive fibers coursed along the dorsal surface of the subthalamic nucleus, and some invaded the dorsomedial third of this structure. The remaining portion of the subthalamic nucleus contained relatively few TH-positive elements. In contrast, the globus pallidus received a dense dopaminergic innervation deriving mostly from two fascicles that coursed backward along the two major output pathways of the pallidum: the lenticular fasciculus caudodorsally and the ansa lenticularis rostroventrally. At the pallidal level, the labeled fibres merged within the medullary laminae and arborized profusely in the internal pallidal segment and less abundantly in the external pallidal segment. However, the caudoventral portion of the external pallidum displayed a dense field of TH-positive axonal varicosities. Other fibers ran through the dorsal two-thirds of the external pallidum en route to the putamen. The striatum contained a multitude of thin axonal varicosities among which a few long and varicosed fibers were scattered. These immunoreactive neuronal profiles were rather uniformly distributed along the rostrocaudal extent of the striatum but appeared slightly more numerous in the ventral striatum than in the dorsal striatum. The pattern of distribution of the TH-positive axonal varicosities in the dorsal striatum was markedly heterogeneous: it consisted of typical zones of poor TH immunoreactivity lying within a matrix of dense terminal labeling.(ABSTRACT TRUNCATED AT 400 WORDS)
The distribution of neurons, fibers and terminal fields in rat brainstem displaying positive immunoreactivity to a polyclonal antiserum to human placental choline acetyltransferase (ChAT) is described. The antiserum was used at the high dilution of 1:10,000 and was coupled with a sensitive detection system using the nickel ammonium sulfate intensification method. In addition to previously described ChAT immunopositive groups of large cells in the cranial motor nuclei, and the parabrachial and reticular complexes, many small or medium size, weakly immunopositive neurons were identified. Some of these appeared in structures in the region of the fourth ventricle, including the area postrema. Others were in structures associated with the superior olivary complex, including the lateral superior olive, and the medioventral, lateroventral and superior periolivary nuclei. Scattered, weakly positive cells were seen in numerous other structures, including the ventral tegmental area of Tsai, central gray, superior colliculus, spinal nucleus of nerve 5, dorsal cochlear nucleus and non-motor regions of the spinal cord. The prominent ascending fiber tract of the laterodorsal tegmental pathway was traceable from the parabrachial area to the subgeniculate region of the thalamus. Prominent terminal fields were seen in a number of brainstem structures, including the superior colliculus, pontine nuclei, anterior pretectal nucleus, interpeduncular nucleus and spinal nucleus of nerve 5. The association of small ChAT positive cells and terminal fields with many sensory structures suggests a significant cholinergic participation in the physiology of sensory function.
The pedunculopontine tegmental nucleus (PPTn) was originally defined on cytoarchitectonic grounds in humans. We have employed cytoarchitectonic, cytochemical, and connectional criteria to define a homologous cell group in the rat. A detailed cytoarchitectonic delineation of the mesopontine tegmentum, including the PPTn, was performed employing tissue stained for Nissl substance. Choline acetyltransferase (ChAT) immunostained tissue was then analyzed in order to investigate the relationship of cholinergic perikarya, dendritic arborizations, and axonal trajectories within this cytoarchitectonic scheme. To confirm some of our cytoarchitectonic delineations, the relationships between neuronal elements staining for ChAT and tyrosine hydroxylase were investigated on tissue stained immunohistochemically for the simultaneous demonstration of these two enzymes.
The PPTn consists of large, multipolar neurons, all of which stain immunohistochemically for ChAT. It is present within cross‐sections that also include the A‐6 through A‐9 catecholamine cell groups and is traversed by catecholaminergic axons within the dorsal tegmental bundle and central tegmental tract. The dendrites of PPTn neurons respect several nuclear boundaries and are oriented perpendicularly to several well‐defined fiber tracts. Cholinergic axons ascend from the mesopontine tegmentum through the dorsal tegmental bundle and a more lateral dorsal ascending pathway. A portion of the latter terminates within the lateral geniculate nucleus.
It has been widely believed that the PPTn is reciprocally connected with several extrapyramidal structures, including the globus pallidus and substantia nigra pars reticulata. Therefore, the relationships of pallidotegmental and nigrotegmental pathways to the PPTn were investigated employing the anterograde autoradiographic methodology. The reciprocity of tegmental connections with the substantia nigra and entopeduncular nucleus was investigated employing combined WGA‐HRP injections and ChAT immunohistochemistry.
The pallido‐ and nigrotegmental terminal fields did not coincide with the PPTn, but, rather, were located just medial and dorsomedial to it (the midbrain extrapyramidal area). The midbrain extrapyramidal area, but not the PPTn, was reciprocally connected with the substantia nigra and entope‐duncular nucleus. We discuss these results in light of other cytoarchitec‐tonic, cytochemical, connectional, and physiologic studies of the functional anatomy of the mesopontine tegmentum.
The distribution of catecholaminergic and cholinergic neurons in the upper brainstem of the ferret were mapped by staining immunohistochemically two adjacent series of sections of brainstem for tyrosine hydroxylase and choline acetyltransferase, respectively. As in other species, large numbers of tyrosine‐hydroxylase‐positive neurons are localized in the ventral tegmental area (A10), the substantia nigra (A9), and in A8. Tyrosine‐hydroxylase‐positive neurons in the dorsolateral pontine tegmentum (A4, A6, and A7‐the locus coeruleus complex) of the ferret are rather diffusely distributed, as has been observed in other carnivore species such as the cat and the dog, but unlike in the cat, these cells in the ferret display a relative uniformity in size and morphology.
Choline‐acetyltransferase‐positive neurons which extend in the ferret's pedunculopontine tegmental nucleus and ventral parabrachial area (Ch5) are relatively large cells that stain intensely for choline acetyltransferase, and their dendrites form prominent bundles in regions where unstained fibre tracts are prevalent. Choline‐acetyltransferase‐positive neurons distributed in the laterodorsal tegmental nucleus (Ch6) are smaller than the cholinergic cells of Ch5, and they stain less intensely for choline acetyltransferase.
Rostrally, there is little overlap between the catecholaminergic cell groups A8, A9, and A10 and the cholinergic cell groups of Ch5 and Ch6. Caudally, the Ch5 neurons extend some considerable extent into the locus coeruleus complex. In the region of overlap, no cells with staining for both tyrosine hydroxylase and choline acetyltransferase were observed, as was ascertained with a double staining method employing a combination of tyrosine hydroxylase immunofluorescence and choline acetyltransferase peroxidase‐antiperoxidase immunohistochemistry.
In conclusion, the ferret has a typically carnivore pattern for the distribution of catecholaminergic cells in the upper brainstem, and there is a significant overlap between the catecholaminergic and cholinergic cell groups in the dorsolateral pontine tegmentum.
The immunohistochemical localization of the neurotransmitter synthesizing enzymes choline acetyltransferase, tyrosine hydroxylase and dopamine-beta-hydroxylase was examined in the feline pontomesencephalic tegmentum. Examination of adjacent sections stained for either choline acetyltransferase, tyrosine hydroxylase or dopamine-beta-hydroxylase immunoreactivity, as well as individual sections doubly stained for both choline acetyltransferase and tyrosine hydroxylase immunoreactivity, unequivocally demonstrated that noradrenergic and cholinergic neurons were extensively intermingled in the brainstem tegmentum of the cat. This contrasts with the situation in various other species, where neurons utilizing these two neurotransmitters are discretely localized in distinct nuclei. Furthermore, the present studies demonstrate the existence of two types of choline acetyltransferase immunoreactive neurons in the feline tegmentum: the magnocellular neurons of the pedunculopontine and laterodorsal tegmental nuclei which stain histochemically for NADPH diaphorase, plus a population of small spindle-shaped neurons in the medial and lateral parabrachial nuclei which do not stain positively for NADPH diaphorase. The data are discussed with respect to several influential hypotheses of sleep cycle control.
The laterodorsal tegmental nucleus (ntdl) contains a cluster of cells located just medial to the locus coeruleus in the pontine brainstem. The ntdl has been shown to project both rostrally to the forebrain and diencephalon and caudally to the spinal cord. In an effort to characterize this region neurochemically, the present study was conducted to identify a variety of neurochemicals localized within perikarya and fibers of the ntdl and surrounding nuclei. Rats were perfused with formalin, and brain sections were processed for fluorescence immunocytochemistry and acetylcholinesterase (AChE). Of the neurochemicals screened, atrial natriuretic factor (ANF), choline acetyltransferase (ChAT), cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), dynorphin B (Dyn B), galanin, somatostatin, substance P, neurotensin (NT), neuropeptide Y (NPY), vasopressin, vasoactive intestinal polypeptide (VIP), serotonin (5HT), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were studied. AChE and ChAT staining revealed that the ntdl contains mostly cholinergic neurons. In addition, brightly reactive substance P and galanin and paler staining CRF, ANF, CGRP, NT, VIP, and Dyn B cell bodies were found within the ntdl. Varicose fibers in this nucleus also contained these peptides in addition to CCK, GAD, TH, 5HT, and NPY. The dorsal tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and the parabrachial region contained a dense and varied assortment of peptides with distinct positions and patterns. This multiplicity of neurochemicals within this area suggests a possible influence on a variety of functions modulated by the ntdl and other closely associated tegmental nuclei.
The coexistence of immunoreactivities for choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in some brain regions of the rat, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for ChAT and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the retina, cerebral cortex, basal forebrain and spinal cord, colocalization of ChAT-like and GAD-like or GABA-like immunoreactivities was observed in some cell types, whereas no such colocalization was observed in cells in the striatum or brainstem. In the retina, the majority of ChAT-like immunoreactive (ChAT-LI) amacrine cells contained GABA-like or GAD-like immunoreactivity. About half of the ChAT-LI neurons in the cerebral cortex showed GABA-like immunoreactivity. In the basal forebrain only a small proportion of ChAT-LI neurons (0.6%) contained GAD-like immunoreactivity. In the spinal cord, about one-third of ChAT-LI central canal cluster cells and about half of ChAT-LI dorsal horn cells showed GAD-like and/or GABA-like immunoreactivities. These observations indicate the possible coexistence of two classical transmitters, GABA and acetylcholine, in various brain regions and spinal cord of the rat.
Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase), which is specifically localized in neurons, has been histochemically demonstrated in human brain by using a perfusion-fixation procedure. With such fixed human brainstem, it was possible to study the topographic organization of NADPH-diaphorase-containing neurons that were visualized in fine detail for the first time. In the pontomesencephalic region, positive neurons were observed in nuclei around the decussation and arm of the superior cerebellar peduncle. These nuclei included the pedunculopontine tegmental, lateral parabrachial and oral pontine reticular nuclei. The positive somata were mainly multipolar in shape and medium to large in size. The positive neurons appeared to correspond to cholinergic neurons, at least partly in the brainstem, in terms of both the patterns of distribution and the cellular morphology.
The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.
The topographic distribution of central cholinergic and catecholaminergic neurons has been investigated in the baboon (Papio papio). The perikarya were mapped on an atlas through the brain and spinal cord employing sections processed for acetylcholinesterase (AChE) pharmacohistochemistry coupled with choline acetyltransferase (ChAT) immunohistochemistry or aqueous catecholamine-fluorescence histochemistry. Compared with subprimates, there is a remarkable increase in the volume occupied by and the number of cholinergic cells contained in the nucleus basalis and nucleus tegmenti pedunculopontinus (subnucleus compacta). The elaboration of these parts of the cholinergic system is accompanied by a large extension of catecholaminergic cell groups in the midbrain (groups A8-A10), particularly the substantia nigra (pars compacta), and in the dorsolateral pontine tegmentum (A5-A7 complex). Although cholinergic and catecholaminergic soma generally occupy distinctly different regions of the brain, a close apposition of cholinergic and noradrenergic neurons occurs in the dorsolateral pontine tegmentum. In the peripeduncular region ChAT-positive cells and green fluorescent neurons of the A6-A7 complex form parallel lines and do not intermingle as has previously been demonstrated in the cat. Two distribution patterns, aggregated or disseminated, are another common feature of central cholinergic and catecholaminergic perikarya. The cholinergic neurons in the nucleus tegmenti pedunculopontinus and the catecholaminergic neurons in A6-A7 complex display both patterns. This comparative study of three transmitter systems in the baboon suggests that the cholinergic as well as the catecholaminergic neurons that give rise to ascending telencephalic and dorsal diencephalic projections undergo phylogenetic development in terms of cell number and nuclear volume.
The morphological characteristics of cholinergic neurons in the central nervous system (CNS) of the baboon (Papio papio) were studied by choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. The distributions of central cholinergic neurons as visualized by these two histochemical techniques were similar in most, but not all regions of the brain and spinal cord. Based upon these observations, central cholinergic neurons that are immunoreactive to ChAT and intensely stained for AChE by the pharmacohistochemical procedure can be divided into four major groups: (1) those in the caudate nucleus, putamen, nucleus accumbens and anterior perforated substance. These ChAT-containing and AChE-intense neurons are large and multipolar, and are scattered throughout these structures. (2) The rostral cholinergic column, which consists of a continuous mass of cholinergic perikarya situated in the medial septal nucleus, nucleus of the diagonal band, and nucleus basalis (Meynert). The ChAT-immunoreactive and AChE-intense cell bodies of the nucleus basalis are a prominent feature in the basal forebrain of the baboon. The labeled neurons are large, multipolar, and hyperchromic and show a tendency to aggregate in cell clusters. These cells are distributed within the full extent of the substantia innominata, often being associated with subcortical fiber networks such as the medullary laminae of the globus pallidus. (3) The caudal cholinergic column, which consists of a continuous group of cholinergic neurons in the caudal midbrain and pontine tegmentum. The rostral component of this group of cells is the nucleus tegmenti pedunculopontinus (subnucleus compacta) and it extends caudally to include the laterodorsal tegmental nucleus. Compared to that in other species the nucleus tegmenti pedunculopontinus in the baboon appears to occupy a relatively greater volume and is composed of a greater number of cholinergic neurons. The cells of the caudal column are large and hyperchromic. (4) Nuclei of origin of somatic and visceral efferents of the cranial nerves (III, IV, V, VI, VII, IX, X, XI, XII) and spinal nerves. In addition to these major cholinergic cell groups, a small population of ChAT-positive and AChE-intense cell bodies can be observed at the floor of the fourth ventricle and in lamina VII and X of the cervical cord. The present findings indicate that although some differences exist, the overall distribution and morphological features of cholinergic cell bodies identified in the baboon brain and spinal cord are similar to those demonstrated previously in investigations of the rhesus monkey and nonprimates.
The fiber degenerations resulting from variously located lesions of the lentiform nucleus were studied in the rhesus monkey by the aid of the Nauta-Gygax and Albrecht-Fernstrom techniques. The following observations were made.(1)Putaminofugal connections. Thin fibers originating in the putamen and composing Wilson's ‘pencil’ bundles traverse the globus pallidus, converging toward the medial point of the lentiform nucleus. The mjority of these fibers terminate in both segments of the globus pallidus, but a considerable number continue caudalward, perforating the cerebral peduncle as ventral components of Edinger's comb system, and terminate in lateral parts of the substantia nigra, pars reticulata.(2)Pallidofugal connections. The ansa lenticularis as defined by von Monakow originates exclusively from the globus pallidus. Its middle division, composed of fibers of medium calibre, arises in the external pallidal segment and traverses the cerebral peduncle as the dorsal component of the comb system to end in the subthalamic nucleus. The thick-fibered dorsal and ventral ansal divisions arise in the internal pallidal segment and combine to form the fasciculus lenticularis which represents the only apparent direct connection of the globus pallidus with the thalamus and the mesencephalic tegmentum.(a)Pallidothalamic fibers follow successively the lenticular and thalamic fasciculi and are distributed to the nuclei ventralis lateralis (subnuclei medialis and oralis of Olszewski and Baxter; none to Zone X and subnucleus caudalis) and ventralis anterior (except subnucleus VAmc). A considerable number of thinner fibers, possibly collaterals of those to VL and VA, terminate in the ‘centre médian’; this connection appears to close a potential transthalamic circuit: putamen-globus pallidus-‘centre médian’-putamen.(b)There is suggestive evidence of pallidofugal fibers following the stratum zonale thalami to the habenula.(c)Pallidohypothalamic connections could not be identified. Most, and possibly all, of the ansal fibers composing the so-called pallidohypothalamic tract loop back into Forel's fields after a shorter or longer descent into the hypothalamus.(d)Fibers of the fasciculus lenticularis by-passing the thalamus are distributed to the nucleus of Forel's field H (prerubral field). Longer fibers of the same category pass caudalward lateral and ventral to the red nucleus and terminate in the nucleus tegmenti pedunculopontinus, particularly in the latter's caudal subnucleus compactus (terminology of Olszewski and Baxter). A few such pallidomesencephalic fibers appear to end in a small circumscript caudal area of the substantia nigra, pars compacta. No evidence was obtained of pallidotegmental fibers extending caudally beyond the mesencephalon.(e)Pallidal efferents to the zona incerta could not be identified. Only sporadic pallidofugal fibers could be followed to the red nucleus, nucleus interstitialis, and nucleus of Darkschewitsch.
Antisera were raised against γ‐aminobutyric acid (GABA) or glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde. After purification, these antisera reacted strongly with fixed GABA or Glu, but not significantly with other amino acids fixed with glutaraldehyde to brain macromolecules. The antisera were used to demonstrate the distributions of Glu‐like and GABA‐like immunoreactivities (Glu‐LI and GABA‐LI) in parts of the perfusion‐fixed mouse and rat brain, including the olfactory bulb, cerebral neocortex, thalamus, basal ganglia, lower brain stem, and cerebellum.
The level of GABA‐LI varied widely among brain regions, thus it was very high in the globus pallidus and substantia nigra and low in the bulk of the thalamus. The GABA antisera labeled nonpyramidal neurons of the neocortex, most cells of the reticular nucleus of the thalamus, medium‐sized cells of the caudatoputamen, and stellate, basket, Golgi, and Purkinje cells of the cerebellum. The distribution of GABA‐LI closely matched that of the GABA‐synthesizing enzyme, glutamic acid decarboxylase (GAD), as revealed in immunocytochemical studies by others. However, the GABA antisera seem to be better suited than GAD antisera for demonstrating putative GABA‐ergic axons. The results suggest that GABA‐LI, as displayed by the present method, is a good marker of neurons thought to use GABA as a transmitter.
Glutamate‐like immunoreactivity was much more evenly distributed among regions than GABA‐LI, but was particularly low in globus pallidus and substantia nigra and high in the cerebral cortex. Mitral cells of the olfactory bulb, pyramidal neocortical cells, and other cells assumed to use Glu or aspartate as transmitter were stained for Glu‐LI, but so also were neurons that are thought to use other transmitters, such as cells in the substantia nigra pars compacta, in the dorsal raphe nucleus, and in the brain stem motor nuclei. The Glu antisera seem to reveal the “transmitter pool” as well as the “metabolic pool” of Glu in perfusion‐fixed material.
This report shows that it is possible by means of immunocytochemistry to display reliably the tissue contents of GABA and Glu in material that has been fixed by perfusion with glutaraldehyde.
The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.
The neocortex receives a major cholinergic innervation from magnocellular neurones in the basal forebrain. However, an ascending cholinergic reticular system has also been postulated to arise from acetylcholinesterase (AChE)-containing neurones in the midbrain and pontine tegmentum. Lesions of this region decrease both AChE and choline acetyltransferase (ChAT) in various forebrain areas, and recent immunohistochemical studies have identified a group of ChAT-containing cell bodies in the midbrain reticular formation and dorsolateral pontine tegmentum. Here we have combined retrograde tracing with ChAT immunohistochemistry to demonstrate that this tegmental cholinergic cell group also directly innervates the cerebral cortex. Other immunohistochemical studies have indicated that the neuropeptide substance P is also present in certain cells in the laterodorsal tegmentum, and these too appear to project to the forebrain. We have therefore performed immunohistochemistry for both ChAT and substance P and have discovered that a subpopulation of the ascending cholinergic reticular neurones contains substance P. Thus, peptide-cholinergic coexistence, previously noted in peripheral neurones, also occurs in the brain.
Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Ch1 and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5-Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5-Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation. These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.
The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.
Choline acetyltransferase immunohistochemistry was used to map the cholinergic cell bodies in the forebrain and upper brainstem of the macaque brain. Neurons with choline acetyltransferase-like immunoreactivity were seen in the striatal complex, in the septal area, in the diagonal band region, in the substantia innominata, in the medial habenula, in the pontomecencephalic tegmentum and in the oculomotor and trochlear nuclei. The ventral striatum contained a higher density of cholinergic cell bodies than the dorsal striatum. All of the structures that contained the choline acetyltransferase positive neurons also had acetylcholinesterase-rich neurons. Choline acetyltransferase positive neurons were not encountered in the cortex. Some perikarya in the midline, intralaminar, reticular and limbic thalamic nuclei as well as in the hypothalamus were rich in acetylcholinesterase but did not give a positive choline acetyltransferase reaction. A similar dissociation was observed in the substantia nigra, the raphe nuclei and the nucleus locus coeruleus where acetylcholinesterase-rich neurons appeared to lack perikaryal choline acetyltransferase activity.
The topographical distribution of neurons containing acetylcholinesterase (AChE, EC 3.1.1.7) in the basal forebrain and upper brainstem of the squirrel monkey (Saimiri sciureus) was studied by means of Butcher's pharmacohistochemical technique which involves staining for AChE at various times after the systemic administration of the AChE inhibitor di-isopropylphosphorofluoridate (DFP). Only those neurons whose AChE staining was as intense as that of known cholinergic neurons present in the same material (e.g., neurons of cranial nerve nuclei) were examined and mapped. Three major collections of such strongly-stained AChE neurons were disclosed in squirrel monkey brain: one located in the striatum, the other lying along the ventralmost aspects of the basal forebrain, and a third one present within the midbrain-pontine tegmentum. The striatal AChE neurons vary in shape from fusiform with 2 thick processes to polygonal with 4-5 thinner processes. They are uniformly scattered throughout the caudate nucleus and putamen and represent only a small proportion of the total striatal cell population (4-6 cells/mm2). They most likely correspond to the aspiny type II cells described in Golgi material of monkey striatum. Similar neurons occur also in ventral striatal areas comprising nucleus accumbens septi and the deep polymorph layer of the olfactory tubercle. The second major AChE neuronal population is composed of the magnocellular neurons that form a somewhat continuous chain of neuronal aggregates extending rostrocaudally from the septal region to the caudal pole of the lentiform nucleus. It includes the neurons of the medial septal nucleus, the nucleus of the diagonal band of Broca and the nucleus basalis of Meynert, all displaying strikingly similar morphological and histochemical characteristics. The AChE neuronal population of nucleus basalis encroaches markedly upon the lateral hypothalamus laterally and the globus pallidus dorsally. The third important AChE cell collection occurs within the pedunculopontine nucleus area in upper brainstem. In that constellation, the AChE neurons are clustered in 2 continuous cell groups: one located dorsolaterally, the other lying ventromedially to the brachium conjunctivum. The thick processes of these neurons form impressive AChE neuronal networks that surround and pervade the brachium conjunctivum over long distances. This cell group, which is one of the most highly AChE reactive structures of the entire brain in the squirrel monkey, may provide a major cholinergic input to various basal ganglia structures, particularly the substantia nigra.
Topographic relationship between cholinergic and aminergic neuronal somata has been studied in the dorsolateral pontine tegmentum by using three histochemical techniques, acetylcholinesterase (AchE) histochemistry in rats having received systemic injection of diisopropylfluorophosphate, choline acetyltransferase (CAT) immunohistochemistry and catecholamine fluorescence histochemistry. Based on comparing the data obtained from these different techniques, it seems evident that all noradrenergic neurons contain AchE. The remaining population of AchE-containing somata appears to correspond with CAT-containing, therefore, cholinergic neuronal cell bodies. No AchE-positive perikarya were detected in neuronal structures other than cholinergic and aminergic neurons. In addition, coexistence of noradrenaline and acetylcholine in a single cell seems improbable, at least, in the dorsolateral pontine tegmentum.
The histochemical fluorescence technique for the demonstration of monoamines in the central nervous system was employed to assess the distribution of serotonin-containing neurons within the brain stem of the immature and adult stump-tailed macaque (Macaca arctoides). Microspectrofluorometric analysis was performed in order to verify the existence of serotonin within perikarya which contained yellow histofluorescence. Serotonin-containing perikarya were found with raphe nuclei including nucleus raphe-pallidus, -obscurus, -points, -magnus, -dorsalis, and -centralis superioralis. Serotoninergic perikarya did not appear confined exclusively to the raphe, but were observed in the reticular formation and other brain stem nuclei including the locus coeruleus and nucleus subcoeruleus. Serotoninergic cells were not seen within the brain stem at superior collicular levels. The localization of serotoninergic perikarya in regions other than the raphe nuclei presents certain dissimilarities in relation reported in other mammalian species.
An atlas of the distribution of cholinergic cell bodies, fibers, and terminals, as well as cholinoceptive cells, in the central nervous system of the cat (excluding the cerebellum) is presented from results obtained in immunohistochemical work on choline acetyltransferase. Cholinergic cell bodies are observed in more than forty areas, and cholinoceptive cells in sixty discrete areas of brain sections from the spinal cord to the olfactory bulb. The atlas is presented in seventy cross-sectional drawings of cat brain extending from the olfactory bulb to the upper cervical spinal cord.