Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

THE UPTAKE, METABOLISM AND RELEASE OF HOMOCHOLINE: STUDIES WITH RAT BRAIN SYNAPTOSOMES AND CAT SUPERIOR CERVICAL GANGLION

The accumulation of [³H]homocholine (3-trimethylamino-propan-1-01) by isolated synaptosomes prepared from rat brain was resolved kinetically into a high (K_T= 3.0 μM) and a low (K_T= 14.5 μM) affinity system. Although homocholine was not acetylated by solubilized choline acetyltransferase, 64% of the homocholine accumulated by intact synaptosomes via the high affinity uptake process was acetylated. Homocholine was also acetylated in the superior cervical ganglion of the cat, and the amount of acetylhomocholine formed was increased (12-fold) by preganglionic nerve stimulation. In ganglia, acetylhomocholine was available for release by preganglionic nerve impulses, and its release was Ca²⁺-dependent, It is concluded that homocholine can form a cholinergic false transmitter, and that the substrate specificity of choline acetyltransferase in vitro might be different from that in situ.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

HIGH AFFINITY CHOLINE TRANSPORT IN GUINEA PIG BRAIN AND THE EFFECT OF NOREPINEPHRINE

—The influence of 1-norepinephrine on the accumulation of [¹⁴C]choline by nuclei-free homogenates and synaptosomes of guinea-pig brain was studied. Kinetic analysis of choline accumulation by guinea-pig brain resulted in both high and low affinity Michaelis constants. Norepinephrine stimulated the high affinity choline transport process but not the low and the magnitude of its stimulation in 3 different brain regions was correlated with the choline acetyltransferase activity of those regions. Depletion of norepinephrine from the brainstem by pretreatment with the catecholamine depleter alpha-methyl-para-tyrosine significantly decreased the maximal velocity of choline transport. Both the alpha adrenergic receptor blocker phentolamine and the beta adrenergic receptor blocker propranalol inhibited norepinephrine induced stimulation of choline transport. Cocaine stimulated choline transport at low concentrations and pretreatment of animals with reserpine significantly antagonized cocaine's stimulation of choline transport. The results suggest that endogenous norepinephrine may modify the high affinity choline transport process in guinea-pig brain.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Modulation of synaptosomal high affinity choline transport.

Depolarization of synaptosomes produced by incubation in 35mMK⁺ Krebs Ringer phosphate buffer results in an increased Vmax and no change in K_T of the high affinity transport of [³H]-choline as determined upon re-incubation in normal K⁺ Krebs Ringer phosphate buffer. The high K⁺ induced increase in the uptake of choline appears to be independent of transmitter release. The K⁺ stimulated increase in the Vmax of the high affinity transport of choline is totally blocked by high, 11mM, Mg⁺². The proportion of choline converted to acetylcholine in synaptosomes previously depolarized is the same as those incubated in normal K⁺ Krebs Ringer; thus the absolute rate of acetylcholine synthesis in nerve terminals is increased as a result of prior depolarization.