A monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine (ACh)-synthesizing enzyme, has been used to localize ChAT within neurons in immunocytochemical preparations of adult rat spinal cord. Morphological details of known cholinergic spinal neurons are presented in this study, and previously unidentified ChAT-containing neurons are also described. Immunoreaction product was present within cell bodies, dendrites, axons, and axon terminals, thereby allowing comprehensive descriptions of the distribution of ChAT-positive neurons and the interrelationships of their processes. In the ventral horn, ChAT-positive motoneurons were located in the medial, central, and lateral motor columns, and their dendrites formed elaborate longitudinal and transverse ChAT-positive bundles. These bundles were present throughout the rostrocaudal extent of the spinal cord. In the central gray matter, small ChAT-positive cell bodies were clustered around the central canal. Small longitudinal fascicles of immunoreactive processes were also observed in this region adjacent to the ependymal layer. The intermediate gray matter of virtually the entire spinal cord was spanned by medium to large ChAT-positive multipolar cells termed partition neurons. At autonomic spinal levels, partition neurons were intermingled with other immunoreactive cells that were identified as preganglionic sympathetic or parasympathetic neurons because of their locations and morphological characteristics. In the sympathetic system, four groups of ChAT-positive neurons were observed; the principal intermediolateral nucleus (ILp) in the lateral horn, the central autonomic cell column (CA) dorsal to the central canal, the intercalated nucleus (IC) located between ILp and CA, and the funicular intermediolateral neurons (ILf) in the white matter lateral to the ILp. The dendrites of ILp and CA neurons formed substantial longitudinal bundles within each group, as well as transverse bundles between the groups that resembled the rungs of a ladder. ChAT-positive cell bodies were also present in the dorsal horn, and those located in laminae III-V extended dendrites dorsally into a longitudinal plexus within lamina III.

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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 11-VI, but were most concentrated in layers 11-111. The somata were small in size, and the majority of ChAT-t 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.

A dopaminergic neuroblastoma was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell line N18TG2. The resulting interspecies hybrid, named MES23.5, has retained a stable phenotype and karyotype for a continuous culture period of 1 year. The hybrid exhibits several properties that suggest that the parent primary neurons originated in the substantia nigra. The cell line contains tyrosine hydroxylase, which is identifiable both by biochemical and immunological methods and synthesizes dopamine, but no other catecholamine. Additionally, the cell line expresses apparent voltage-gated CA2+ channels as measured by high-affinity omega-conotoxin binding. The MES23.5 omega-conotoxin receptors are of similar affinity class to those found in adult rat mesencephalon. No dihydropyridine receptors, as measured by PN200-100 ligand binding, are present. None of these properties are found in the N18TG2 parent. At least three neuronal features, namely, tyrosine hydroxylase, dopamine synthesis, and omega-conotoxin receptor expression, are quantitatively elevated after sustained treatment with cAMP analogs. The cell line expresses a complex range of neural properties found in the dopaminergic neurons of the substantia nigra, and may therefore be useful elucidating further details of their cell biology.

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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.

Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) MATERIALS AND METHODSCell Culture. Parental embryonic day 15 rat ventral spinal neuron preparation, mouse N18TG2 neuroblastoma culture, somatic cell fusion, and hybrid selection were performed as described (25,26). VSC 4.1 hybrid cells were maintained in logarithmic-phase growth on poly(L-ornithine)-precoated T-75 flasks (Coming) in Dulbecco's modified Eagle's medium/F-12 growth medium (GIBCO) containing Sato's components (Sigma) and 2% heat inactivated newborn calf serum (HyClone). VSC 4.1 cells undergoing cAMP-induced differentiation were plated at 1.2-1.5 x 106 cells per 75-mm2 flask and treated for 7 days with 1 mM dibutyryl cAMP or 1 mM 8-bromo-cAMP before use. Aphidicolin (0.4 pg/ml) (27) (Sigma) was added for 2 days after 24 hr in cAMP, and after 2 additional days of aphidicolin-free growth surviving cells were maintained in medium containing 1 mM cAMP and 0.025-0.05 ,ug of aphidicolin per ml. Differentiated hybrids were harvested by trituration after 1-hr incubation at 370C in Ca2+/Mg2+-free Hanks' balanced salt solution containing 1 mM EDTA and transferred into 24-well plates (Coming) at 2.0 x 104 cells per well [for direct cell count and lactate dehydrogenase (LDH) assays] or per 11-mm-diameter glass coverslip (for fluorescence assays). Pharmacologic agents and immunoglobulins were added to cells 24 hr after replating and were maintained in growth medium for the entire experAbbreviations: ALS, amyotrophic lateral sclerosis; LEMS, Lam-bert-Eaton myasthenic syndrome; VGCC, voltage-gated calcium channel; FDA, fluorescein diacetate; PI, propidium iodide; LDH, lactate dehydrogenase; AMPA, DL-a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.tPresent address:

Choline acetyltransferase (EC 2.3