Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses

Monoclonal antibodies to choline acetyltransferase (ChAT) were used in an immunocytochemical study to characterize putative cholinergic neurons and synaptic junctions in rat caudate-putamen. Light microscopy (LM) revealed that ChAT-positive neurons are distributed throughout the striatum. These cells have large oval or multipolar somata, and exhibit three to four primary dendrites that branch and extend long distances. Quantitative analysis of counterstained preparations indicated that ChAT-positive neurons constitute 1.7% of the total neuronal population. Electron microscopy (EM) of immunoreactive neurons initially studied by LM revealed somata characterized by deeply invaginated nuclei and by abundant amounts of organelle-rich cytoplasm. Surfaces of ChAT-positive neurons are frequently smooth, but occasional somatic protrusions and dendritic spines occur. Although infrequently observed, axons of ChAT-positive neurons branch, receive synapses, and become myelinated. Unlabeled boutons make both symmetrical and asymmetrical synapses with ChAT-positive somata and proximal dendrites, but are more numerous on distal dendrites. In addition, some unlabeled terminals form asymmetrical synapses with ChAT-positive somata and dendrites that are distinguished by prominent subsynaptic dense bodies. Light microscopy demonstrated a dense distribution of ChAT-positive fibers and punctate structures in the striatum, and these structures appear to correlate, respectively, with labeled preterminal axons and presynaptic boutons identified by EM. ChAT-positive boutons contain pleomorphic vesicles, and make symmetrical synapses primarily with unlabeled dendritic shafts. Furthermore, they establish synaptic contacts with somata, dendrites and axon initial segments of unlabeled neurons that ultrastructurally resemble medium spiny neurons. These observations, together with the results of other investigations, suggest that medium spiny GABAergic projection neurons receive a cholinergic innervation that is probably derived from ChAT-positive striatal cells. The results of this study also indicate that cholinergic neurons within caudate-putamen belong to a single population of cells that have large somata and extensive sparsely spined dendrites. Such neurons, in combination with dense concentrations of ChAT-positive fibers and terminals, are the likely basis for the large amounts of ChAT and acetylcholine detected biochemically within the neostriatum.     

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.     

Nigral innervation of cholinergic and glutamatergic cells in the rat mesopontine tegmentum: Light and electron microscopic anterograde tracing and immunohistochemical studies

The substantia nigra (SN) has long been known as an important source of afferents to the pedunculopontine tegmental nucleus (PPN). However, it has not been established which of the chemospecific cell populations receive this synaptic input. We sought to address this issue by a correlative light and electron microscopic approach that combines anterograde tracing of nigral efferents with pre-embedding choline acetyltransferase (ChAT) and/or glutamate (Glu) immunohistochemistry. Following large bilateral injections of Phaseolus vulgaris–leucoagglutinin (PHA-L) in the SN, the labeled nigrotegmental fibers were concentrated in a small area of the mesopontine tegmentum which contained very few ChAT-immunoreactive (ChAT-ir) cell bodies. However, strands of fine varicose fibers penetrated to adjacent regions of the PPN which harbored numerous cholinergic perikarya. The anterogradely labeled boutons were often seen in the proximity of ChAT-ir perikarya and dendrites, but the majority (82–93%) established symmetric synaptic junctions with noncholinergic profiles. In the pars dissipata of the PPN (PPNd), one-third of the labeled terminals synapsed onto noncholinergic perikarya and primary dendrites, while in the pars compacta of the PPN (PPNc) axosomatic synapses were rare. The possibility that the perikarya receiving a rich synaptic input from the SN are glutamatergic was tested in experiments combining anterograde transport of biotinylated tracers biocytin and dextran-amine (BDA) with glutamate immunohistochemistry. In double-labeled sections, Glu-ir perikarya within the terminal plexus of nigrotegmental fibers were surrounded by synaptic terminals. The PPNd also contained retrogradely BDA-labeled neurons which were contacted by anterogradely labeled terminals. These results indicate that although a small subpopulation of cholinergic neurons in the mesopontine tegmentum receive direct synaptic input from the SN, the primary target of nigrotegmental fibers are glutamatergic cells in the PPNd. Our results also provide ultrastructural evidence that some nigrotegmental fibers innervate pedunculonigral neurons. J. Comp. Neurol. 395:359–379, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural examination of enkephalin and substance P input to cholinergic neurons within the rat neostriatum.

Enkephalin and substance P-containing inputs to cholinergic perikarya were examined in the rat neostriatum using an ultrastructural immunocytochemical double-labeling protocol. Sections of rat neostriatum were double-labeled for either choline acetyltransferase (ChAT) and substance P or ChAT and enkephalin using silver intensified colloidal gold and peroxidase as labels. Regions containing both ChAT-positive neurons and peroxidase reaction product were identified in the light microscope prior to sectioning for electron microscopy. Substance P-containing terminals which contained round synaptic vesicles and made symmetrical synaptic contacts were commonly observed in the neostriatum. Substance P synapses onto ChAT-positive perikarya and dendrites were frequently observed: up to 5 synaptic contacts were observed onto a ChAT-positive dendrite. Enkephalin labeling was also seen in a population of axon terminals containing round synaptic vesicles and exhibiting symmetrical synaptic specializations. In contrast to substance P-containing terminals, relatively few synaptic contacts were observed onto ChAT-positive labeled perikarya and dendrites although enkephalin-labeled terminals were seen in frequent contact with perikarya and dendrites of unlabeled spiny neurons. Since enkephalin and substance P are contained within different populations of striatal spiny neurons, the results of the present study suggest that these two types of neurons differ in their intrinsic striatal connections.     

Postnatal development of neurons containing choline acetyltransferase in rat spinal cord: an immunocytochemical study

A monoclonal antibody to choline acetyltransferase (ChAT) has been used in an immunocytochemical study of the postnatal development of ChAT-containing neurons in cervical and thoracic spinal cord. Specimens from rat pups ranging in age from 1 to 28 days postnatal (dpn) were studied and compared with adult specimens (Barber et al., '84). The development of established cholinergic neurons, the somatic motoneurons and sympathetic preganglionic cells, has been described as has that of previously unidentified ChAT-positive neurons in the dorsal, intermediate, and central gray matter. Cell bodies of somatic and visceral motoneurons contained moderate amounts of ChAT-positive reaction product at birth that gradually increased in intensity until 14-21 dpn. The most intensely stained ChAT-positive neurons in 1-5-dpn specimens were named partition cells because this cell group extended from the central gray to an area dorsal to the lateral motoneurons, and thereby divided the spinal cord into dorsal and ventral halves. Partition cells were medium to large in size with 5-7 primary dendrites, and axons that, in fortuitous sections, could be traced into the ventrolateral motoneuron pools, the ventral funiculi, or the ventral commissure. Small ChAT-positive cells clustered around the central canal and scattered in laminae III-VI of the dorsal horn were detectable at birth. These neurons were moderately immunoreactive at 11-14 dpn and intensely ChAT positive by 21 dpn. The band of ChAT-positive terminal-like structures demonstrated in lamina III of adult specimens (Barber et al., '84) was first visible in 11-14-dpn specimens. By 28 dpn, both laminae I and III contained punctate bands that approximated the density of those observed in adult spinal cord. This investigation has demonstrated ChAT within individual neurons of developing spinal cord, and has identified a group of neurons, the partition cells, that exhibit intense ChAT-positive immunoreactivity earlier than any other putative cholinergic cells in spinal cord, including motoneurons. Another important observation has been that each ChAT-positive neuronal type achieves adult levels of staining intensity at different times during development. A likely explanation for this differential staining is that various groups of neurons acquire their mature concentration of ChAT molecules at different developmental stages. In turn, this may correlate with the maturation of cholinergic synaptic activity manifest by individual cells or groups of neurons.     

Pedunculopontine nucleus in the squirrel monkey: Distribution of cholinergic and monoaminergic neurons in the mesopontine tegmentum with evidence for the presence of glutamate in cholinergic neurons

The topographical relationships between cholinergic neurons, identified by their immuno-reactivity for choline acetyltransferase (ChAT) or their staining for β-nicotinamide ademine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, Nonadrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta(PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal toe the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occurnin these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are clolinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that (1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and (2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and Glutamate in primates. © 1994 Wiley-Liss, Inc.     

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.     

Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers

This study deals with two characteristic cell types in the rat septal complex i.e., cholinergic and GABAergic neurons, and their synaptic connections. Cholinergic elements were labeled with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antiserum against glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was employed to identify GABAergic perikarya and terminals, by using either the peroxidase-antiperoxidase (PAP) technique or a biotinylated second antiserum and avidinated gold or ferritin. With these contrasting immunolabels we have studied the cholinergic-GABAergic interconnections in double-labeled sections of intact septal regions and the GABAergic innervation of medial septal area cholinergic neurons in sections taken from animals 1 week following lateral septal area lesion. In other electron microscopic experiments we have studied cholinergic and GABAergic neurons in the septal complex for synaptic contacts with hippocamposeptal fibers, which were identified by anterograde degeneration following fimbria-fornix transection. Our results are summarized as follows: (1) GAD-positive terminals form synaptic contacts on ChAT-immunoreactive dendrites in the medial septum/diagonal band complex (MSDB), (2) surgical lesion of the lateral septal area resulted in a dramatic decrease of the number of GABAergic boutons on MSDB cholinergic neurons, (3) cholinergic terminals establish synaptic contacts with GAD immunoreactive cell bodies and proximal dendrites in the MSDB as well as in the lateral septum (LS), (4) degenerated terminals of hippocampo-septal fibers were mainly observed in the LS, where they formed asymmetric synaptic contacts on dendrites of GABAergic neurons and on nonimmunoreactive spines. We did not observe degenerated boutons in contact with ChAT-positive dendrites or cell bodies in the MSDB. From these results and from data in the literature we conclude that excitatory hippocampo-septal fibers activate GABAergic cells, and as yet unidentified spiny neurons in the LS, which may control the discharge of medial septal cholinergic neurons known to project back to the hippocampal formation.     

Synaptic organization of cholinergic neurons in the monkey neostriatum

Cholinergic neurons in the monkey neostriatum were examined at the light and electron microscopic level by immunohistochemical methods in order to localize choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. At the light microscopic level a sparse distribution of cholinergic neurons was identified throughout the caudate nucleus. Neurons had large (25-30 microns) somata, eccentric invaginated nuclei, primary dendrites of unequal diameters, and varicosities on distal dendritic branches. Ultrastructural study showed that the cholinergic cells had a cytoplasm abundant in organelles. Within dendritic branches, mitochondria and cisternae were localized primarily to varicosities. Synaptic inputs were distributed mostly to the dendrites and at least four types that formed symmetric or asymmetric synapses were observed. Immunoreactive fibers were relatively numerous within the neuropil and exhibited small diameters (0.1-0.15) micron) and swellings at frequent intervals. Cholinergic boutons that formed synapses were compared to unlabeled terminals making asymmetric synapses with dendritic spines. Results showed that ChAT-positive axons had significantly smaller cross-sectional areas, shorter synaptic junctions, and a higher density and surface area of mitochondria than the unlabeled boutons. Cholinergic axons formed symmetric synapses mostly with dendritic spines (53%) and the shafts of unlabeled primary and distal dendrites (37%). A relatively small proportion of the boutons contacted axon initial segments (1%) and cell bodies (9%) that included medium-sized neurons with unindented (spiny) and indented (aspiny) nuclei. The majority of dendritic spines contacted by cholinergic axons were also postsynaptic to unlabeled boutons forming asymmetric synapses. The results suggest that cholinergic neurons in the primary neostriatum belong to a single morphological class corresponding to the large aspiny (type II) interneuron identified in previous Golgi studies. Present results along with earlier Golgi-electron microscopic observations from this laboratory suggest that neostriatal cholinergic cells integrate many sources of intrinsic and extrinsic inputs. The observed convergence of ChAT-immunoreactive boutons and unlabeled axons onto the same dendritic spines suggests that intrinsic cholinergic axons modulate extrinsic inputs onto neostriatal spiny neurons at postsynaptic sites close to the site of afferent input.     

Organization and morphological characteristics of cholonergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase

Choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, has been localized immunocytochemically with a monoclonal antibody inlight and electron microscopic preparations of rat central nervous system (CNS). The antibody was an IgG₁ subclass immunoglobulin that removed ChAT activity from solution. The specificity of the antibody and immunocytochemical methods has been confirmed by the demonstration of ChAT-positive neurons in a number of well-characterized cholinergic systems. For examples, ChAT-positive reaction product was present in the cell bodies of spinal and cranial nerve motoneurons, as well as in their axons and terminations as motor end-plates in skeletal muscle. In addition, the somata of preganglionic sympathetic and parasympathetic neurons were ChAT-positive. The specificity of staining was further supported by a lack of reaction in several groups of neurons thought to use neuroactive substances other than acetylcholine. No specific staining was observed in control specimens.The findings indicated that ChAT had an extensive intraneuronal distribution in many cholinergic neurons, being present in cell bodies, dendrites, axons and axon terminals. ChAT-positive somata were found in the medial septum and diagonal band, the medial habenula, and the basal nucleus of, the forebrain, 3 regions that are sources of cholinergic afferents to the hippocampus, interpeduncular nucleus and cerebral cortex, respectively. In addition, positively stained cell bodies were present within the cerebral cortex. ChAT-positive punctate structures were observed in the ventral horn of the spinal cord, where electron microscopic studies demonstrated that some of these structures were synaptic terminals. Other regions containing numerous ChAT-positive puncta included the hippocampus, the interpeduncular nucleus and the cerebral cortex. The light microscopic appeaance of these putative cholinergic terminals varied among different brain regions. Large punctate structures related to well-defined postsynaptic elements were characteristic of some regions, such as the ventral horn of the spinal cord, while smaller punctate structures and varicose fibers with a diffuse pattern of organization distinguished otther regions, such as the cerebral cortex.     

Ultrastructural study of cholinergic and noncholinergic neurons in the pars compacta of the rat pedunculopontine tegmental nucleus

A group of medium-to-large cholinergic neurons situated in the dorsolateral mesopontine tegmentum comprises the pedunculopontine tegmental nucleus (PPT). The PPT pars compacta (PPT-pc), which occupies the lateral part of the caudal two-thirds of the nucleus, contains a dense aggregation of cholinergic neurons. In the present study, we have employed immunohistochemistry for choline acetyltransferase (ChAT) and electron microscopy to investigate the ultrastructure and synaptic organization of neuronal elements in the PPT-pc. Our results demonstrate that: (1) ChAT-immunoreactive (i.e., cholinergic) PPT-pc neurons are characterized by abundant cytoplasm and organelles, and have few axosomatic synapses (both asymmetric and symmetric); (2) ChAT-immunoreactive dendrites comprise 6-15% of total dendritic elements in the neuropil; the mean percentage of dendritic membrane covered by synaptic terminals is approximately 15%, and nearly all synapses with ChAT-immunoreactive dendrites are asymmetric; (3) within the boundaries described by cholinergic PPT-pc, there are noncholinergic neurons which, in contrast, exhibit a lucent cytoplasm and a higher frequency of axosomatic synapses (10.5% versus 3.7% for cholinergic neurons); and (4) noncholinergic neurons are morphologically heterogeneous with one subpopulation exhibiting a mean diameter that approximates that of cholinergic cells (i.e., >15 μm and <20 μm) and a very high frequency of axosomatic synapses (>20%). Only 0.2-0.7% of terminal elements in the neuropil were ChAT-immunoreactive and these were not observed to synapse with cholinergic dendrites or somata. This relative paucity of terminal labeling and lack of cholinergic-cholinergic interactions seems inconsistent with the recognized and prominent physiological actions of acetylcholine on cholinergic PPT-pc neurons, and suggests a methodological limitation and/or a potential paracrine-like action of nonsynaptically released acetylcholine in the PPT region. J. Comp. Neurol. 382:285-301, 1997. © 1997 Wiley-Liss, Inc.     

The substantia nigra of the rat: a Golgi study.

Three variants of the Golgi method were employed to examine the cell types, their dendritic fields and organization and axonal trajectories within the substantia nigra of albino and hooded rats. In both sagittal and coronal sections, large, medium and small neurons were classified on the basis of soma size, extent of dendritic fileds and dendritic caliber. In general nigral cells have three to five primary dendrites that branch relatively infrequently. Some dendrites of all cell types have thinly scattered spines or varicosities. Small cells, found in all areas of the nucleus, have thin dendrites and small, nondirectional dendritic fields. These are considered to be interneurons. The medium cells found in pars compacta, presumed to be the dopaminergic cells of the nigroneostriatal pathway, send long dendrites into pars reticulata perpendicular to the course of pars compacta. In addition, these cells have a number of dendrites which remain in pars compacta. These cells have axons that run medio-dorsally. No axon collaterals were detected. Both large and medium cells are found in pars reticulata. Cells in the dorso-medial aspect of pars reticulata orient rostro-caudally and roughly perpendicular to the course of pars compacta, while cells in the peripeduncular area show a strict orientation which is parallel to the crus cerebri. Some pars reticulata cells emit axon collaterals while others remain unbranced for their observable lenght. Both large and medium cells are also seen in pars lateralis. These cells send long dendrites ventrally into pars reticulata where they run parallel to the crus cerebri, while some shorter dendrites remain in pars lateralis. In total, the substantia nigra appears to have a layered organization: the superior layer is the cellular pars compacta, the second is the dorso-medial area of pars reticulata where both pars compacta and pars reticulata dendrites run rostro-caudally and dorso-ventrally and the third layer is the peripeduncular region where dendrites from all areas run parallel to the crus cerebri.     

Golgi study of the primate substantia nigra. II. Spatial organization of dendritic arborizations in relation to the cytoarchitectonic boundaries and to the striatonigral bundle

The spatial organization of Golgi-stained dendritic arborizations of the substantia nigra was studied in three dimensions by using a video computer system. Dendritic orientation was analyzed in relation to the cytoarchitectonic boundaries and to the direction of the axons of the striato-pallidonigral bundle. All the brains, humans and macaques, were sectioned according to the same ventricular planes. The striatal bundle is made up of dense fascicles of very thin parallel axons. Sixty neurons located in the pars reticulata, lateralis, and compacta were reconstructed from serial sections. In the anterior pars reticulata and lateralis, the dendritic arborizations spread in all directions inside the striatal bundle. Below the pars compacta fringes, the dendrites of pars reticulata neurons extend ventrolaterally in the bundle. Because one nigral arborization can cover the whole thickness of the striatal bundle, we are led to believe that nigral neurons exert a role of convergence of the corticostriatal information similar to that of pallidal neurons (Percheron et al., '84a,b). The pars reticulata neurons appear to receive information mainly from the associative striatal territory. The pars lateralis neurons, conversely, appear to receive information from the sensorimotor territory. The anterior pars compacta neurons are organized in such a way that their ventral dendrites, located inside the pars reticulata, are ventrolaterally oriented, perpendicular to the striatal bundle. Their dorsal dendrites remaining in the pars compacta can receive other input. At more caudal levels, the posterior pars compacta neurons have dendrites radiating outside the striatal bundle.     

GABAergic input to cholinergic forebrain neurons: an ultrastructural study using retrograde tracing of HRP and double immunolabeling

Amygdalopetal cholinergic neurons in the ventral pallidum were identified by combining choline acetyltransferase (ChAT) immunohistochemistry with retrograde tracing of horseradish peroxidase (HRP) following injections of the tracer in the basolateral amygdaloid nucleus. Although ChAT-positive terminals were identified in the ventral pallidum, they were never seen in contact with either immunonegative or ChAT-positive amygdalopetal neurons. In material, in which immunostaining against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA was combined with retrograde tracing of HRP from the basolateral amygdaloid nucleus, GAD-positive terminals were seen to contact immunonegative amygdalopetal neurons. In addition, when sections of the rostral forebrain were processed, first to preserve and identify the transported HRP, and then were sequentially tested for both ChAT and GAD immunohistochemistry with the immunoperoxidase reaction for both tissue antigens, GAD-immunopositive terminals were seen to make synaptic contacts with cholinergic amygdalopetal neurons. These results suggest that amygdalopetal, presumably cholinergic, neurons receive GAD-positive terminals. In separate experiments using immunoperoxidase for ChAT and ferritin-avidin for GAD labeling, we confirmed the presence of GAD-containing terminals on cholinergic neurons. In addition, cholinergic terminals were seen in synaptic contact with GAD-positive cell bodies. These morphological studies suggest that direct GABAergic-cholinergic and cholinergic-GABAergic interactions take place in the rostral forebrain.     

Electrophysiologically identified nigral dopaminergic neurons intracellularly labeled with HRP: light-microscopic analysis

Intracellular recordings were obtained in vivo from neurons of the rat substantia nigra, pars compacta. Neurons that were identified as dopaminergic by a variety of electrophysiological criteria, including antidromic activation from ipsilateral neostriatum or globus pallidus, were microiontophoretically injected with horseradish peroxidase and examined at the level of the light microscope. Dopaminergic neurons were of medium size and had ovoid, polygonal, or fusiform cell bodies that emitted from 3-6 primary dendrites. Much of the sparse and relatively unbranched dendritic arborization of these neurons remained within pars compacta, except for 1 or 2 large dendrites that were directed ventrally or ventrolaterally into pars reticulata, roughly perpendicular to the plane of the pars compacta. In coronal sections, the dendrites of ovoid- or polygonal-shaped pars compacta neurons were oriented mainly along the dorsoventral axis, whereas fusiform-shaped neurons had dendrites that were oriented primarily mediolaterally. Although some of the dendrites of dopaminergic neurons exhibited variations in diameter, most were not markedly varicose. Dendrites were sometimes sparsely invested with spinelike appendages or other dendritic extrusions, particularly along their distal portions. The axons of dopaminergic pars compacta neurons were emitted from primary or proximal secondary dendrites, and were extremely fine processes, 0.5 micron or less in diameter. No local axon collaterals were observed.     

Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson's disease

Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.     

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and lamina IX of the cervical spinal cord of the rat

The cholinergic structures in the cranial nerve motor nuclei and lamina IX of the cervical spinal cord of the rat were examined immunohistochemically with a monoclonal antibody to choline acetyltransferase (ChAT). The brainstem motor nuclei were classified into 3 groups according to the way of distribution of ChAT-positive structures in the neuropil of the nucleus. 1. The oculomotor, trochlear and abducent nuclei contained moderately ChAT-positive perikarya and dendrites. A small number of ChAT-positive bouton-like structures were found in the neuropil, but they were not in contact with ChAT-positive perikarya and dendrites. 2. Motoneurons in the facial, hypoglossal and trigeminal motor nuclei were moderately to strongly ChAT-positive. There were in the neuropil numerous ChAT-positive bouton-like structures and many of them were in contact with ChAT-positive perikarya and dendrites. The same pattern of organization of ChAT-positive structures was observed in lamina IX of the cervical spinal cord. In the nucleus ambiguus, the perikarya and proximal dendrites of about half population of motoneurons contacted with ChAT-positive bouton-like structures while the rest of motoneurons did not. ChAT-positive bouton-like structures in contact with motoneurons are interpreted as the cholinergic synaptic terminals, and this suggests that motoneurons in this group are cholinoceptive as well as cholinergic. 3. Neuronal perikarya and dendrites in the dorsal nucleus of the vagus showed weak to moderate positivity of ChAT. The neuropil of this nucleus was free from any distinctly ChAT-positive structures.     

The postnatal development of the substantia nigra: A light and electron microscopy study

The early postnatal development of neurons, dendrites, and synaptic connectivity in kitten substantia nigra (SN) was studied by light and electron microscopy. The compact and reticular divisions of the SN are present at birth but boundaries are indistinct. Most nigral neurons stain deeply in routine histological sections and their diameters increase slightly with age. Ultrastructurally, cell bodies are characterized by eccentrically located and often invaginated nuclei surrounded by cytoplasm rich in well-formed or-ganelles. Axosomatic synapses are infrequent and cell surfaces are enveloped by glial processes. Immature dendritic features, including growth cones and filiform processes, are commonly observed during the first 10 days. Gradually the dendritic profiles elongate and thicken and contours become smoother, retaining only scattered spinelike appendages. Clear examples of the three synaptic types described in cat are found in newborn kittens, but immature terminals contain fewer synaptic vesicles and mitochondria. Approximately 90% of synapses present at birth in both nigra subdivisions are Type I, which contain large pleomorphic vesicles and contact dendrites symmetrically. Asymmetrical contacts characterize most of the remaining definable synapses. The postnatal increase in synaptic connectivity, which was estimated from random photographs of pars reticulata neuropil, is twofold during the first 50 days of life. Initially young dendrites are enveloped by glia and then gradually become ensheathed by axon terminals. Synaptogenesis in pars reticulata reflects the postnatal increase of neostriatal inputs to this subdivision and can be correlated with functional changes in strionigral connectivity.     

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

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