Differential regulation of neocortical synapses by neuromodulators and activity.

Synapses are continually regulated by chemical modulators and by their own activity. We tested the specificity of regulation in two excitatory pathways of the neocortex: thalamocortical (TC) synapses, which mediate specific inputs, and intracortical (IC) synapses, which mediate the recombination of cortical information. Frequency-sensitive depression was much stronger in TC synapses than in IC synapses. The two synapse types were differentially sensitive to presynaptic neuromodulators: only IC synapses were suppressed by activation of GABA(B) receptors, only TC synapses were enhanced by nicotinic acetylcholine receptors, and muscarinic acetylcholine receptors suppressed both synapse types. Modulators also differentially altered the frequency sensitivity of the synapses. Our results suggest a mechanism by which the relative strength and dynamics of input and associational pathways of neocortex are regulated during changes in behavioral state.

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors.

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co-localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers.

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis.

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.

Neuronal nicotinic synapse assembly requires the adenomatous polyposis coli tumor suppressor protein.

Normal cognitive and autonomic functions require nicotinic synaptic signaling. Despite the physiological importance of these synapses, little is known about molecular mechanisms that direct their assembly during development. We show here that the tumor-suppressor protein adenomatous polyposis coli (APC) functions in localizing alpha3-nicotinic acetylcholine receptors (nAChRs) to neuronal postsynaptic sites. Our quantitative confocal microscopy studies indicate that APC is selectively enriched at cholinergic synapses; APC surface clusters are juxtaposed to synaptic vesicle clusters and colocalize with alpha3-nAChRs but not with the neighboring synaptic glycine receptors or perisynaptic alpha7-nAChRs on chick ciliary ganglion (CG) neurons. We identify PSD (postsynaptic density)-93, beta-catenin, and microtubule end binding protein EB1 as APC binding partners. PSD-93 and beta-catenin are also enriched at alpha3-nAChR postsynaptic sites. EB1 shows close proximity to and partial overlap with alpha3-nAChR and APC surface clusters. We tested the role of APC in neuronal nicotinic synapse assembly by using retroviral-mediated in vivo overexpression of an APC dominant-negative (APC-dn) peptide to block the interaction of endogenous APC with both EB1 and PSD-93 during synapse formation in CG neurons. The overexpressed APC-dn led to dramatic decreases in alpha3-nAChR surface levels and clusters. Effects were specific to alpha3-nAChR postsynaptic sites; synaptic glycine receptor and perisynaptic alpha7-nAChR clusters were not altered. In addition, APC-dn also reduced surface membrane-associated clusters of PSD-93 and EB1. The results show that APC plays a key role in organizing excitatory cholinergic postsynaptic specializations in CG neurons. We identify APC as the first nonreceptor protein to function in localizing nAChRs to neuronal synapses in vivo.

Vesicular localization and activity-dependent trafficking of presynaptic choline transporters.

Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.

Localization of cholinergic innervation in guinea pig heart by immunohistochemistry for high-affinity choline transporters.

OBJECTIVE: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. METHODS: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. RESULTS: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. CONCLUSIONS: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

Biosynthetic pathway for a new series of gangliosides, GT1a alpha and GQ1b alpha.

A new class of gangliosides, GT1a alpha and GQ1b alpha, were initially identified as cholinergic neuron-specific antigens in bovine brain. These gangliosides have in common alpha 2-6 NeuAc linked to the GalNAc residue in the gangliotetraose core structure. In this study, we have determined the biosynthetic pathways of GT1a alpha and GQ1b alpha using rat liver Golgi fraction. The results showed that GT1a alpha and GQ1b alpha were synthesized from GD1a and GT1b, respectively, by the action of a GalNAc alpha 2-6 sialyltransferase. It was also demonstrated that these two gangliosides were found to exist as extremely minor components in rat liver.

Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is single transmembrane glycoprotein that plays a critical role in the trafficking of lysosomal enzymes and the internalization of circulating IGF-II. At present, there is little information regarding the cellular distribution of the IGF-II/M6P receptor within the adult rat brain. With the use of immunoblotting and immunocytochemical methods, we found that the IGF-II/M6P receptor is widely but selectively expressed in all major brain areas, including the olfactory bulb, striatum, cortex, hippocampus, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. Intense IGF-II/M6P receptor immunoreactivity was apparent on neuronal cell bodies within the striatum, deeper layers (layers IV and V) of the cortex, pyramidal and granule cell layers of the hippocampal formation, selected thalamic nuclei, Purkinje cells of the cerebellum, pontine nucleus and motoneurons of the brainstem as well as in the spinal cord. Moderate neuronal labeling was evident in the olfactory bulb, basal forebrain areas, hypothalamus, superior colliculus, midbrain areas, granule cells of the cerebellum and in the intermediate regions of the spinal gray matter. We also observed dense neuropil labeling in many regions, suggesting that this receptor is localized in dendrites and/or axon terminals. Double-labeling studies further indicated that a subset of IGF-II/M6P receptor colocalizes with cholinergic cell bodies and fibers in the septum, striatum, diagonal band complex, nucleus basalis, cortex, hippocampus, and motoneurons of the brainstem and spinal cord. The observed widespread distribution and colocalization of IGF-II/M6P receptor in the adult rat brain provide an anatomic basis to suggest a multifunctional role for the receptor in a wide-spectrum of central nervous system neurons, including those expressing a cholinergic phenotype.

Organization of ascending hypothalamic projections to the rostral forebrain with special reference to the innervation of cholinergic projection neurons.

Axonal projections from hypothalamic nuclei to the basal forebrain, and their relation to cholinergic projection neurons in particular, were studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with choline acetyltransferase (ChAT) immunocytochemistry. Discrete iontophoretic PHA-L injections were delivered to different portions of the caudal lateral hypothalamus, as well as to various medial hypothalamic areas, including the ventromedial, dorsomedial, and paraventricular nuclei, and anterior hypothalamic and medial preoptic areas. The simultaneous detection of PHA-L-labeled fibers/terminals and ChAT-positive neurons was performed by using nickel-enhanced diaminobenzidine (DAB) and nonenhanced DAB as chromogens. Selected cases were investigated at the electron microscopic level. Ascending hypothalamic projections maintained an orderly lateromedial arrangement within the different components of the medial forebrain bundle, as well as with respect to their terminal projection fields (e.g., within the bed nucleus of the stria terminalis and lateral septal nucleus). The distribution pattern of hypothalamic inputs to cholinergic projection neurons corresponded to the topography of ascending hypothalamic axons. Axons originating from neurons in the far-lateral hypothalamus reached cholinergic neurons in a zone that extended from the dorsal part of the sublenticular substantia innominata (SI) caudolaterally, to the lateral portion of the bed nucleus of the stria terminalis rostromedially, encompassing a narrow band along the ventral part of the globus pallidus and medial portion of the internal capsule. Axons originating from cells in the medial portion of the lateral hypothalamus reached cholinergic cells primarily in more medial and ventral parts of the SI, and in the magnocellular preoptic nucleus and horizontal limb of the diagonal band nucleus (HDB). Axons from medial hypothalamic cells appeared to contact cholinergic neurons primarily in the medial part of the HDB, and in the medial septum/vertical limb of the diagonal band complex. Electron microscopic double-labeling experiments confirmed contacts between labeled terminals and cholinergic cells in the HDB and SI. Individual hypothalamic axons established synapses with both cholinergic and noncholinergic neuronal elements in the same regions. These findings have important implications for our understanding of the organization of afferents to the basal forebrain cholinergic projection system.

Chemical coding of the human gastrointestinal nervous system: cholinergic, VIPergic, and catecholaminergic phenotypes.

The aim of this investigation was to identify the proportional neurochemical codes of enteric neurons and to determine the specific terminal fields of chemically defined nerve fibers in all parts of the human gastrointestinal (GI) tract. For this purpose, antibodies against the vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), serotonin (5-HT), vasoactive intestinal peptide (VIP), and protein gene product 9.5 (PGP 9.5) were used. For in situ hybridization (35)S-labeled VMAT1, VMAT2, and VAChT riboprobes were used. In all regions of the human GI tract, 50-70% of the neurons were cholinergic, as judged by staining for VAChT. The human gut unlike the rodent gut exhibits a cholinergic innervation, which is characterized by an extensive overlap with VIPergic innervation. Neurons containing VMAT2 constituted 14-20% of all intrinsic neurons in the upper GI tract, and there was an equal number of TH-positive neurons. In contrast, DBH was absent from intrinsic neurons. Cholinergic and monoaminergic phenotypes proved to be completely distinct phenotypes. In conclusion, the chemical coding of human enteric neurons reveals some similarities with that of other mammalian species, but also significant differences. VIP is a cholinergic cotransmitter in the intrinsic innervation of the human gut. The substantial overlap between VMAT2 and TH in enteric neurons indicates that the intrinsic catecholaminergic innervation is a stable component of the human GI tract throughout life. The absence of DBH from intrinsic catecholaminergic neurons indicates that these neurons have a dopaminergic phenotype.

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain.

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization o NGF mRNA was examined by using a 35S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic innervation in adult rat cerebral cortex: a quantitative immunocytochemical description.

A method for determining the length of acetylcholine (ACh) axons and number of ACh axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase (ChAT) was used to estimate the areal and laminar densities of this innervation in the frontal (motor), parietal (somatosensory), and occipital (visual) cortex of adult rat. The number of ACh varicosities per length of axon (4 per 10 microm) appeared constant in the different layers and areas. The mean density of ACh axons was the highest in the frontal cortex (13.0 m/mm(3) vs. 9.9 and 11.0 m/mm(3) in the somatosensory and visual cortex, respectively), as was the mean density of ACh varicosities (5.4 x 10(6)/mm(3) vs. 3.8 and 4.6 x 10(6)/mm(3)). In all three areas, layer I displayed the highest laminar densities of ACh axons and varicosities (e.g., 13.5 m/mm(3) and 5.4 x 10(6)/mm(3) in frontal cortex). The lowest were those of layer IV in the parietal cortex (7.3 m/mm(3) and 2.9 x 10(6)/mm(3)). The lengths of ACh axons under a 1 mm(2) surface of cortex were 26.7, 19.7, and 15.3 m in the frontal, parietal, and occipital areas, respectively, for corresponding numbers of 11.1, 7.7, and 6.4 x 10(6) ACh varicosities. In the parietal cortex, this meant a total of 1.2 x 10(6) synaptic ACh varicosities under a 1 mm(2) surface, 48% of which in layer V alone, according to previous electron microscopic estimates of synaptic incidence. In keeping with the notion that the synaptic component of ACh transmission in cerebral cortex is preponderant in layer V, these quantitative data suggest a role for this innervation in the processing of cortical output as well as input. Extrapolation of particular features of this system in terms of total axon length and number of varicosities in whole cortex, length of axons and number of varicosities per cortically projecting neuron, and concentration of ACh per axon varicosity, should also help in arriving at a better definition of its roles and functional properties in cerebral cortex.

Distribution of acetylcholinesterase in the hippocampal region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and presubiculum.

The distribution of acetylcholinesterase (AChE) was examined in the multilayered posterior part of the hippocampal region of the adult mouse (Mus musculus domesticus), namely, the entorhinal area, the parasubiculum, the presubiculum, and those parts of the retrosplenial cortex that extend into the posterior hippocampal region (area retrosplenialis 29d and 29e). A modification of the Koelle copper thiocholine method was employed for the histochemical demonstration of AChE. The AChE staining resulted in a distinctly stratified pattern, which has been compared in detail with the fields and layers defined by cyto- and fibro-architecture. Most of the enzyme activity was located in the neuropil, but both moderately and intensely stained nerve cell bodies were observed too. In the entorhinal area two main subfields were identified, which have been designated pars medialis and pars lateralis. In pars medialis, the superficial two thirds of layer I, the interstices between the stellate cell bodies in layer II, and layers IV and VI showed moderate to high content of AChE, whereas layer V and, especially, layer III were poor in enzyme activity. A particular feature was the occurrence of cone-shaped, darkly stained areas within layer II and, occasionally, the deep part of layer I. The staining of pars laterais differed in several respects from that of pars medialis, the most prominent feature being a less conspicuous stratification. In addition, intensely stained somata occurred more frequently than in pars medialis, although they still constituted only a very small minority of the total number of nerve cell bodies. In the parasubiculum, a clear cytoarchitectural subdivision into a posterolateral parasubiculum a and an anteromedial parasubiculum b was observed. These subfields showed, however, only minor differences in AChE staining. Thus, in both subfields, layers I and IV stained intensely, whereas layers II and III showed moderate to intense staining. Layers V and VI did not differ in appearance from the corresponding layers of the entorhinal area. The retrosplenial areas 29d and 29e appeared very light in the AChE pattern, area 29e being the better stained. The presubiculum was very rich in AChE, with layers, I, III and IV being particularly intensely stained. The small nerve cell bodies of layer II were unstained, whereas the intervening neuropil was intensely stained. The distribution of AChE in the mouse was compared with that in the rat, guinea pig, and rabbit, described previously. The staining pattern is largely similar in all four species, but striking species-specific differences do exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic innervation of the chick basilar papilla.

Antibodies directed against choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker of cholinergic neurons, were used to label axons and nerve terminals of efferent fibers that innervate the chick basilar papilla (BP). Two morphologically distinct populations of cholinergic fibers were labeled and classified according to the region of the BP they innervated. The inferior efferent system was composed of thick fibers that coursed radially across the basilar membrane in small fascicles, gave off small branches that innervated short hair cells with large cup-like endings, and continued past the inferior edge of the BP to ramify extensively in the hyaline cell area. The superior efferent system was made up of a group of thin fibers that remained in the superior half of the epithelium and innervated tall hair cells with bouton endings. Both inferior and superior efferent fibers richly innervated the basal two thirds of the BP. However, the apical quarter of the chick BP was virtually devoid of efferent innervation except for a few fibers that gave off bouton endings around the peripheral edges. The distribution of ChAT-positive efferent endings appeared very similar to the population of efferent endings that labeled with synapsin antisera. Double labeling with ChAT and synapsin antibodies showed that the two markers colocalized in all nerve terminals that were identified in BP whole-mounts and frozen sections. These results strongly suggest that all of the efferent fibers that innervate the chick BP are cholinergic.

Synaptic connections of cholinergic antennal lobe relay neurons innervating the lateral horn neuropile in the brain of Drosophila melanogaster.

Presumed cholinergic projection neurons (PNs) in the brain of the fruit fly Drosophila melanogaster, immunoreactive to choline acetyltransferase (ChAT), convey olfactory information between the primary sensory antennal lobe neuropile and the mushroom body calyces, and finally terminate in the lateral horn (LH) neuropile. The texture and synaptic connections of ChAT PNs in the LH and, comparatively, in the smaller mushroom body calyces were investigated by immuno light and electron microscopy. The ChAT PN fibers of the massive inner antennocerebral tract (iACT) extend into all portions of the LH, distributing in a nonrandom fashion. Immunoreactive boutons accumulate in the lateral margins of the LH, whereas the more proximal LH exhibits less intense immunolabeling. Boutons with divergent presynaptic sites, unlabeled as well as ChAT-immunoreactive, appear to be the preponderant mode of synaptic input throughout the LH. Synapses of ChAT-labeled fibers appear predominantly as divergent synaptic boutons (diameters 1-3 microm), connected to unlabeled postsynaptic profiles, or alternatively as a minority of tiny postsynaptic spines (diameters 0.05-0.5 microm) among unlabeled profiles. Together these spines encircle unidentified presynaptic boutons of interneurons which occupy large areas of the LH. Thus, synaptic circuits in the LH differ profoundly from those of the PNs in the mushroom body calyx, where ChAT spines have not been encountered. Synaptic contacts between LH ChAT elements were not observed. The synaptic LH neuropile may serve as an output area for terminals of the ChAT PNs, their presynaptic boutons providing input to noncholinergic relay neurons. The significance of the postsynaptic neurites of the ChAT PNs is discussed; either local or other interneurons might connect the ChAT PNs within the LH, or PNs might receive inputs arising from outside the LH.

Neurokinin 1 receptor distribution in cholinergic neurons and targets of substance P terminals in the rat nucleus accumbens.

Substance P (SP) is the major endogenous ligand for neurokinin 1 (NK1) receptors and, together with acetylcholine, has an important role in motivated behaviors involving the limbic shell and motor core of the nucleus accumbens (NAc). To determine the functional sites for SP activation of NK-1 receptors and potential interactions with cholinergic neurons in these regions, the authors examined the electron microscopic immunocytochemical localization either of antisera against the NK1 receptor or of the NK1 receptor and either 1) SP or 2) the vesicular acetylcholine transporter (VAchT) in rat NAc. In both the NAc shell and core, NK1 receptor labeling was localized mainly to somatic and dendritic plasma membranes and nearby endosomal organelles in aspiny neurons. In sections through the ventromedial shell that were processed for NK1/SP labeling, 46% of the NK1-immunoreactive dendrites (n = 603 dendrites) showed symmetric or appositional contacts with SP-containing terminals. These terminals and several others that formed symmetric synapses also occasionally were immunoreactive for NK1 receptors. Analysis of the shell region for NK1/VAchT labeling showed that 61% of the total immunoreactive dendrites (n = 534 dendrites) contained NK1 receptors without VAchT, 29% contained both products, and 10% contained VAchT only. Many of the labeled somata and dendrites also received synaptic contact from VAchT-containing terminals. These findings suggest that, in the NAc, NK1 receptors are recycled through endosomal compartments and play a role in modulating mainly the postsynaptic responses, but also the presynaptic release, of SP and/or inhibitory neurotransmitters onto aspiny interneurons, some of which are cholinergic.

Chemical heterogeneity of the striosomal compartment in the human striatum.

The neurochemical organization of the striosomal compartment in the human striatum was analyzed by histochemical and immunohistochemical techniques applied to postmortem tissue from normal individuals. The striosomes were delineated by using the following markers: acetylcholinesterase (AChE), enkephalin (ENK), substance P (SP), calbindin-D28k (CB), parvalbumin (PV), calretinin (CR), limbic system-associated membrane protein (LAMP), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and NADPH-diaphorase. Comparisons were made between striosomal boundaries, as outlined by each marker applied on adjacent sections, and particular attention was paid to possible variations in the chemical features of striosomes along the rostrocaudal extent of the striatum. The main findings of this study are as follows: 1) the striosomal compartment is composed of two chemically distinct domains: a core and a peripheral region; 2) the core is largely devoid of CB and displays a less intense staining for ENK and LAMP than the peripheral region; 3) although striosomes are largely devoid of AChE, the activity of this enzyme is slightly higher in the core than in the peripheral region; 4) the core and peripheral regions are weakly stained for PV and intensely stained for SP; 5) ChAT-, CR- and NADPH-diaphorase-positive neurons are preferentially distributed in the peripheral region; 6) at rostral striatal levels, striosomes are largely devoid of TH, whereas the inverse is true caudally; and 7) at caudal striatal levels, the peripheral region of striosomes is intensely stained for CB and ChAT. These results demonstrate that the striosomes in human display a strikingly complex and heterogeneous chemical architecture.

Large cholinergic nerve terminals on subsets of motoneurons and their relation to muscarinic receptor type 2.

The cholinergic C-bouton is a large nerve terminal found exclusively apposing motoneuron cell somata and proximal dendrites. The origin and function of the C-bouton is not known. An antiserum against the vesicular acetylcholine transporter was used to identify large cholinergic nerve terminals putatively of the C-type in close apposition to motoneuron cell somata. This type of nerve terminal was present in the rat spinal cord ventral horn, but only in some cranial motor nuclei. Fluoro-Gold tracing showed that subsets of spinal motoneuron cell somata were contacted by different numbers of putative C-boutons. Thus, motoneurons innervating an intrinsic foot muscle were contacted by about half the number of cholinergic terminals found on motoneurons of the predominantly fast-twitch gastrocnemius muscle. Slow-twitch soleus motoneurons showed an intermediate innervation. There was a strong correlation between the presence of putative C-boutons and muscarinic receptor 2 (m2)-like immunoreactivity (-LI) within a motor nucleus. By using confocal laser microscopy, the m2-LI appeared to be confined to the motoneuron cell membrane and strongly enriched beneath the C-type nerve terminal. Thus, our results suggested a differential distribution of large cholinergic C-boutons, depending on motoneuron type, and that the presence of this nerve terminal type is associated with m2-LI in the postsynaptic membrane.

Mesopontine organization of cholinergic and catecholaminergic cell groups in the normal and narcoleptic dog.

Canine narcolepsy is a unique experimental model of a human sleep disorder characterized by excessive daytime sleepiness and cataplexy. There is a consensus recognition of an imbalance between cholinergic and catecholaminergic systems in narcolepsy although the underlying mechanisms remain poorly understood. Possible substrates could be an abnormal organization, numbers and/or ratio of cholinergic to catecholaminergic cells in the brain of narcoleptic dogs. Therefore, we sought to characterize the corresponding neuronal populations in normal and narcoleptic dogs (Doberman Pinscher) by using choline acetyltransferase (ChAT), nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase, tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH). Cholinergic cell groups were found in an area extending from the central to the gigantocellular tegmental field and the periventricular gray corresponding to the pedunculopontine tegmental nucleus (PPT), the laterodorsal tegmental nucleus (LDT), and the parabrachial nucleus. An almost perfect co-localization of ChAT and NADPH-diaphorase was also observed. Catecholaminergic cell groups detected included the ventral tegmental area, the substantia nigra, and the locus coeruleus nucleus (LC). The anatomical distribution of catecholaminergic neurons was unusual in the dog in two important aspects: i) TH- and/or DBH-immunoreactive neurons of the LC were found almost exclusively in the reticular formation and not within the periventricular gray, ii) very few, if any TH-positive neurons were found in the central gray and dorsal raphe. Quantitative analysis did not reveal any significant differences in the organization and the number of cells identified in the LDT, PPT, and LC of normal and narcoleptic dogs. Moreover, the cholinergic to catecholaminergic ratio was found identical in the two groups. In conclusion, the present results do not support the hypothesis that the neurochemical imbalance in narcolepsy could result from abnormal organization, numbers, or ratio of the corresponding neuronal populations.