Distribution Of Cholinergic Neurons Research Articles

Simultaneous Acquisition of Multicolor Information From Neural Circuits in Resin-Embedded Samples.

Resin embedding has been widely used for precise imaging of fluorescently labeled biological samples with optical and electron microscopy. The low preservation rate of fluorescence, especially for red fluorescent proteins, has limited the application of resin embedding in multifluorescent protein-labeled samples. Here, we optimized the embedding method to retain the intensity of multiple fluorescent proteins during resin embedding. By reducing the polymerization temperature from 50 to 35°C and adding a fluorescent protein protection reagent during the embedding process, we successfully increased the fluorescence preservation rate by nearly twofold for red fluorescent proteins, including tdTomato, mCherry, and DsRed. Meanwhile, the background fluorescence decreased significantly in the optimized embedding method. This method is suitable not only for red fluorescent protein-labeled samples but also for blue (BFP) and green fluorescent protein (GFP)-labeled samples. We embedded brains labeled with BFP, DsRed, and GFP via AAV and rabies virus and acquired the distribution of input neurons to different cortical areas. With GFP/tdTomato double-labeled samples in resin, we obtained the cholinergic projectome of the pedunculopontine tegmental nucleus (PPTg) and the distribution of cholinergic neurons at single-neuron resolution in the whole brain simultaneously. Input cholinergic terminals from the PPTg were found to innervate the cholinergic soma and fiber in the neocortex, basal forebrain and brainstem, indicating that local cholinergic neurons received long-range cholinergic modulation from the midbrain. Our optimized method is useful for embedding multicolor fluorescent protein-labeled samples to acquire multidimensional structural information on neural circuits at single-neuron resolution in the whole brain.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

The cholinergic basal forebrain in the ferret and its inputs to the auditory cortex.

Cholinergic inputs to the auditory cortex can modulate sensory processing and regulate stimulus-specific plasticity according to the behavioural state of the subject. In order to understand how acetylcholine achieves this, it is essential to elucidate the circuitry by which cholinergic inputs influence the cortex. In this study, we described the distribution of cholinergic neurons in the basal forebrain and their inputs to the auditory cortex of the ferret, a species used increasingly in studies of auditory learning and plasticity. Cholinergic neurons in the basal forebrain, visualized by choline acetyltransferase and p75 neurotrophin receptor immunocytochemistry, were distributed through the medial septum, diagonal band of Broca, and nucleus basalis magnocellularis. Epipial tracer deposits and injections of the immunotoxin ME20.4-SAP (monoclonal antibody specific for the p75 neurotrophin receptor conjugated to saporin) in the auditory cortex showed that cholinergic inputs originate almost exclusively in the ipsilateral nucleus basalis. Moreover, tracer injections in the nucleus basalis revealed a pattern of labelled fibres and terminal fields that resembled acetylcholinesterase fibre staining in the auditory cortex, with the heaviest labelling in layers II/III and in the infragranular layers. Labelled fibres with small en-passant varicosities and simple terminal swellings were observed throughout all auditory cortical regions. The widespread distribution of cholinergic inputs from the nucleus basalis to both primary and higher level areas of the auditory cortex suggests that acetylcholine is likely to be involved in modulating many aspects of auditory processing.

Cholinergic Neurotransmission from Mechanosensory Afferents to Giant Interneurons in the Terminal Abdominal Ganglion of the Cricket Gryllus bimaculatus

Crickets respond to air currents with quick avoidance behavior. The terminal abdominal ganglion (TAG) has a neuronal circuit for a wind-detection system to elicit this behavior. We investigated neuronal transmission from cercal sensory afferent neurons to ascending giant interneurons (GIs). Pharmacological treatment with 500 muM acetylcholine (ACh) increased neuronal activities of ascending interneurons with cell bodies located in the TAG. The effects of ACh antagonists on the activities of identified GIs were examined. The muscarinic ACh antagonist atropine at 3-mM concentration had no obvious effect on the activities of GIs 10-3, 10-2, or 9-3. On the other hand, a 3-mM concentration of the nicotinic ACh antagonist mecamylamine decreased spike firing of these interneurons. Immunohistochemistry using a polyclonal anti-conjugated acetylcholine antibody revealed the distribution of cholinergic neurons in the TAG. The cercal sensory afferent neurons running through the cercal nerve root showed cholinergic immunoreactivity, and the cholinergic immunoreactive region in the neuropil overlapped with the terminal arborizations of the cercal sensory afferent neurons. Cell bodies in the median region of the TAG also showed cholinergic immunoreactivity. This indicates that not only sensory afferent neurons but also other neurons that have cell bodies in the TAG could use ACh as a neurotransmitter.

Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain.

The distribution of cholinergic neurons and fibers was studied immunohistochemically in the brain of two species of lampreys (Petromyzon marinus and Lampetra fluviatilis), by using an antiserum against choline acetyltransferase (ChAT). The results obtained in both species were similar, but there appeared some interspecies differences. In the forebrain, cholinergic cells were present in the striatum, preoptic region, paraventricular nucleus, pineal and parapineal organs, habenula, and pretectum. The cranial nerve motoneurons (III, IV, V, VI, VII, IX, and X), the first and second spino-occipital nerves (so), and the ventral horn of the spinal cord showed a strong ChAT immunoreactivity. Additional cholinergic neurons were observed: the mesencephalic M5 nucleus of Schober, two different cell populations in the isthmic region, the efferent component of the eighth nerve, putative preganglionic parasympathetic cells, cells in the solitary tract nucleus, and the rhombencephalic reticular formation. Cholinergic fibers were widely distributed in the brain. Comparison with previous studies in other vertebrates suggests that major cholinergic pathways, like tectal innervation from the isthmic region, are also present in lampreys. Of particular interest was the prominent projection to the neurohypophysis from cholinergic neurons in the preoptic region and paraventricular nucleus. Present data were analyzed within the segmental paradigm, as was previously done in other vertebrates. Our results reveal that the organization of many cholinergic systems in the lamprey as, for example, in the striatal, preoptic, and isthmic regions, comprises features of the anamniote brain that remain common to all living amniotes studied so far, thus being conservative to a surprisingly high degree. Therefore, the distribution of ChAT-immunoreactive structures in the lamprey brain is, in general, comparable to that previously described in other vertebrate species.

Mice Lacking Specific Nicotinic Acetylcholine Receptor Subunits Exhibit Dramatically Altered Spontaneous Activity Patterns and Reveal a Limited Role for Retinal Waves in Forming ON and OFF Circuits in the Inner Retina

Before phototransduction, spontaneous activity in the developing mammalian retina is required for the appropriate patterning of retinothalamic connections, and there is growing evidence that this activity influences the development of circuits within the retina itself. We demonstrate here that the neural substrate that generates waves in the mouse retina develops through three distinct stages. First, between embryonic day 16 and birth [postnatal day 0 (P0)], we observed both large, propagating waves inhibited by nicotinic acetylcholine receptor (nAChR) antagonists and small clusters of cells displaying nonpropagating, correlated calcium increases that were independent of nAChR activation. Second, between P0 and P11, we observed only larger propagating waves that were abolished by toxins specific to alpha3 and beta2 subunit-containing nAChRs. Third, between P11 and P14 (eye opening) we observed propagating activity that was abolished by ionotropic glutamate receptor antagonists. The time course of this developmental shift was dramatically altered in retinas from mice lacking the beta2 nAChR subunit or the beta2 and beta4 subunits. These retinas exhibited a novel circuit at P0, no spontaneous correlated activity between P1 and P8, and the premature induction at P8 of an ionotropic glutamate receptor-based circuit. Retinas from postnatal mice lacking the alpha3 nAChR subunit exhibited spontaneous, correlated activity patterns that were similar to those observed in embryonic wild-type mice. In alpha3-/- and beta2-/- mice, the development and distribution of cholinergic neurons and processes and the density of retinal ganglion cells (RGCs) and the gross segregation of their dendrites into ON and OFF sublaminae were normal. However, the refinement of individual RGC dendrites is delayed. These results indicate that retinal waves mediated by nAChRs are involved in, but not required for, the development of neural circuits that define the ON and OFF sublamina of the inner plexiform layer.

The distribution of cholinergic neurons in the human central nervous system.

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the adult trout and tract-tracing observations on the connections of the nuclei of the isthmus.

The distribution of cholinergic neurons and fibers was studied in the brain and rostral spinal cord of the brown trout and rainbow trout by using an antiserum against the enzyme choline acetyltransferase (ChAT). Cholinergic neurons were observed in the ventral telencephalon, preoptic region, habenula, thalamus, hypothalamus, magnocellular superficial pretectal nucleus, optic tectum, isthmus, cranial nerve motor nuclei, and spinal cord. In addition, new cholinergic groups were detected in the vascular organ of the lamina terminalis, the parvocellular and magnocellular parts of the preoptic nucleus, the anterior tuberal nucleus, and a mesencephalic tegmental nucleus. The presence of ChAT in the magnocellular neurosecretory system of trout suggests that acetylcholine is involved in control of hormone release by neurosecretory terminals. In order to characterize the several cholinergic nuclei observed in the isthmus of trout, their projections were studied by application of 1,1;-dioctadecyl-3,3,3;, 3;-tetramethylindocarbocyanine perchlorate (DiI) to selected structures of the brain. The secondary gustatory nucleus projected mainly to the lateral hypothalamic lobes, whereas the nucleus isthmi projected to the optic tectum and parvocellular superficial pretectal nucleus, as previously described in other teleost groups. In addition, other isthmic cholinergic nuclei of trout may be homologs of the mesopontine system of mammals. We conclude that the cholinergic systems of teleosts show many primitive features that have been preserved during evolution, together with characteristics exclusive to the group.

Distribution of cholinergic neurons in cell group k of the rabbit brainstem

The cell bodies of efferent neurons supplying the masseter and digastric muscles of the rabbit are located in two brainstem nuclei: the trigeminal motor nucleus and cell group k. The latter also contains neurons innervating muscles of the middle ear and Eustachian tube, as well as neurons that project to the cerebellum and the oculomotor complex. As part of an attempt to identify the functional subpopulations within the three cell divisions (k1–k3) that make up cell group k, we have investigated the distribution of neurons containing choline acetyltransferase, because these are likely to be motoneurons. Five rabbits anaesthetized with sodium pentobarbital (90 mg/kg, i.v.) were used in this study. They were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). Two animals were used for preliminary studies. In the other three cases, serial Vibratome coronal sections of the brainstem were cut at 50 μm and two series of alternating sections were collected. The first was stained with a monoclonal antibody (code AB8, Incstar) directed against choline acetyltransferase, using the avidin–biotin–peroxidase method. The other was stained with Cresyl Violet. Cell counts and three-dimensional reconstructions were made for both series to determine positions and ratios of cholinergic and non-cholinergic neurons within the trigeminal motor nucleus and the subdivisions of cell group k. The results showed that the numbers of choline acetyltransferase- and Nissl-stained neurons within the trigeminal motor nucleus were almost identical. In cell group k, significantly fewer choline acetyltransferase-stained cells were counted in all three animals (ratios of choline acetyltransferase/Nissl=0.53–0.71). In addition, the distribution of cholinergic neurons was not uniform throughout cell group k. Subdivisions k1 and k3 contained proportionately less choline acetyltransferase-positive cells (ratios of choline acetyltransferase/Nissl=0.23–0.64) than did k2 (ratios choline acetyltransferase/Nissl=0.75–0.88). Within each subdivision, there were significant differences in the spatial coordinates of Nissl- and choline acetyltransferase-positive neurons. We conclude that cell group k contains at least two populations of neurons which are unevenly distributed between and within the three subdivisions. While the majority of neurons in subgroup k2 contain choline acetyltransferase and thus are likely to be motoneurons, more than half of the neurons in subgroups k1 and k3 are not cholinergic. It remains to be determined whether these are the neurons that project to the cerebellum and to other CNS regions.

The distribution of cholinergic neurons in the central nervous system of turtles.

The distribution of cholinergic neurons was studied in painted and red-eared turtles using antisera against choline acetyltransferase (ChAT). The results showed that the organization of cholinergic systems in the central nervous system of turtles was remarkably similar to that previously described in diverse other species. For example, the present studies revealed the presence of cholinergic neurons in the striatum, the basal telencephalon, the isthmo-tectal system, and a region of cells near the cerebellar peduncle. The motoneurons of the III, IV, V, VI, VII, X and XII cranial nerves and the spinal ventral horn were also shown to be cholinergic. Additional cholinergic neurons were observed in the hypothalamus and rhombencephalic reticular formation. These results help to clarify the precise extent and location of the major cholinergic cell groups of the turtle brain, thereby adding to our understanding of the organization of the turtle nervous system. The results also reveal that the cholinergic systems of the striatum, basal forebrain, isthmic region, and cerebellar peduncular region are ancestral features of the amniote brain and are common to all living amniotes that have been studied. Thus, the organization and, presumably, the function of the cholinergic systems appear to be conservative.

Distribution of cholinergic neurons in the chick spinal cord during embryonic development. Comparison of ChAT immunocytochemistry with AChE histochemistry

The location of cholinergic neurons was studied during the development of the chick embryo spinal cord. A comparison between choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry was performed. ChAT-positive neurons could be detected only from embryonic day 9 (E9) onwards by the FITC technique and from E12 onwards by the PAP technique. These neurons were located mainly in the medial and lateral motor columns in the ventral horn of the gray matter and some of them were observed in the intermediate region of the spinal cord. AChE-containing cell bodies were much more numerous than the ChAT immunoreactive ones and were distributed in the ventral horn of the gray matter, the intermediate gray region and mostly off the apical part of the dorsal horn. ChAT should provide a reliable and specific marker for cholinergic neurons.

Morphogenesis of adrenergic cells in a frog parasympathetic ganglion

The development of the parasympathetic cardiac ganglion of the frog Xenopus laevis was marked by the differentiation of a population of adrenergic small intensely fluorescent (SIF) cells. The neural crest contributes precursors for both SIF cells and the well-studied cholinergic cardiac ganglion neurons; this situation provided an opportunity to determine whether morphogenesis of the two cell types was correlated. Accordingly, we examined the initial differentiation, developmental regulation, and territorial domain of cardiac SIF cells for comparison with their cholinergic neuron neighbors. Adrenergic SIF cells were present during the time when the first cholinergic precursors were becoming postmitotic. Although SIF cells were present first, cholinergic neurons differentiated almost 16 times faster during the first week of embryonic and larval development and outnumbered SIF cells at all subsequent stages. Nonetheless, the accumulation of both cell types were correlated, since the ratio of cholinergic neurons to SIF cells remained at approximately 10 to 1 up to adult life. Early in development, SIF cells and cholinergic neurons were clustered in the sinus venosus portion of the atrium. The asymmetric distribution of cholinergic neurons within the atrium was lost but that of the SIF cells was maintained throughout life. These results identify relationships between the morphogenesis of two cell types in an autonomic ganglion and place constraints upon the cellular mechanisms that could produce the cells from their neural crest precursors.

Transient increase in the number of cholinergic neurons in the developing rat dentate gyrus.

Using a monoclonal antibody against choline acetyltransferase (ChAT), we have examined the distribution of cholinergic neurons in the rat dentate gyrus during development. ChAT-positive neurons were occasionally detected in the hilus on postnatal day 2 (P2). There was a transient abrupt increase in the number and density of ChAT-positive neurons between P15 and P20 and then a decline to the adult level with few ChAT-immunoreactive neurons. A few ChAT-positive varicose fibers and punctae were first seen at P5. They increased in number and density until P20 when they reached the adult level and distribution. These observations suggest the occurrence of a transient expression of cholinergic markers in the hippocampus.

Choline acetyltransferase immunocytochemistry of Edinger-Westphal and ciliary ganglion afferent neurons in the cat

The distribution of cholinergic neurons in the region of the cat Edinger-Westphal nucleus (EW) was determined by immunocytochemical localization of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT). Neurons containing ChAT-like immunoreactivity (ChAT-LI) were densely distributed within EW, the anteromedian nucleus (AM), and the oculomotor nucleus (III), and were also present in immediately adjacent regions of the periaqueductal gray and ventral tegmental region. The majority of labelled neurons in EW and AM showed a markedly lower intensity of ChAT-LI than the labelled neurons in III and adjacent regions. To determine the relationship of cells with ChAT-LI to the distribution of ciliary ganglion afferent neurons, a double labelling immunocytochemistry/retrograde transport technique was also used. These experiments showed that many of the cells located outside of III that stained intensely for ChAT-LI project to ciliary ganglion. Very few ciliary ganglion afferent neurons were found in EW or AM itself; instead, the distribution of lightly labelled ChAT-LI-positive neurons in EW and AM more closely matched the known distribution of peptide-containing cells that have descending, central projections.

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammilary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammilary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.

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

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

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase.

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.

Intra- and extraganglionic peripheral cholinergic neurons in the urogenital organs of the cat.

The distribution of cholinergic neurons in the urinary tract and male genital organs of the cat was studied by a histochemical method for acetylcholinesterase. In addition to cell clusters in autonomic ganglia (intraganglionic cells), isolated extraganglionic cholinergic cells were found within the innervated tissues, usually in association with nerve trunks and blood vessels. Smaller neural cells with multiple axonal processes, identical to Cajal's “interstitial cells”, were found in the meshes of the terminal nerve plexus in smooth muscle, lamina propria and vascular wall.