Choline transport and de novo choline synthesis support acetylcholine biosynthesis in Caenorhabditis elegans cholinergic neurons

The cho-1 gene in Caenorhabditis elegans encodes a high-affinity plasma-membrane choline transporter believed to be rate limiting for acetylcholine (ACh) synthesis in cholinergic nerve terminals. We found that CHO-1 is expressed in most, but not all cholinergic neurons in C. elegans. cho-1 null mutants are viable and exhibit mild deficits in cholinergic behavior; they are slightly resistant to the acetylcholinesterase inhibitor aldicarb, and they exhibit reduced swimming rates in liquid. cho-1 mutants also fail to sustain swimming behavior; over a 33-min time course, cho-1 mutants slow down or stop swimming, whereas wild-type animals sustain the initial rate of swimming over the duration of the experiment. A functional CHO-1GFP fusion protein rescues these cho-1 mutant phenotypes and is enriched at cholinergic synapses. Although cho-1 mutants clearly exhibit defects in cholinergic behaviors, the loss of cho-1 function has surprisingly mild effects on cholinergic neurotransmission. However, reducing endogenous choline synthesis strongly enhances the phenotype of cho-1 mutants, giving rise to a synthetic uncoordinated phenotype. Our results indicate that both choline transport and de novo synthesis provide choline for ACh synthesis in C. elegans cholinergic neurons.     

Minireview. Thyrotropin releasing hormone and CNS cholinergic neurons

The centrally mediated pharmacological effects of thyrotropin releasing hormone (TRH), their mechanistic basis and therapeutic implications, along with the possible physiological significance of extrahypothalamic TRH, have been the subject of numerous investigations for over a decade. Despite this effort a holistic perspective on these issues and considerations does not exist. However, with continued research employing multiple and diverse experimental approaches, many interactions of TRH and related peptides with central cholinergic mechanisms have been revealed. These interactions are documented in this review and it is proposed that they can account for several of the more prominent pharmacological actions of these peptides. Additionally, it is suggested that a function of endogenous YHR, throughout the neuroaxis, may be to regulate the excitability of central cholinergic neurons.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

ATP citrate lyase in cholinergic nerve endings

The activity of ATP-citrate lyase in homogenates of five selected rat brain regions varied from 2.93 to 6.90 nmol/min/mg of protein in the following order: cerebellum < hippocampus < parietal cortex < striatum < medulla oblongata and that of the choline acetyltransferase from 0.15 to 2.08 nmol/min/mg of protein in cerebellum < parietal cortex < hippocampus=medulla oblongata < striatum. No substantial differences were found in regional activities of lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase or acetyl-CoA synthase. High values of relative specific activities for both choline acetyltransferase and ATP-citrate lyase were found in synaptosomal and synaptoplasmic fractions from regions with a high content of cholinergic nerve endings. There are significant correlations between these two enzyme activities in general cytocol (S₃), synaptosomal (B) and synaptoplasmic (B_s) fractions from the different regions (r=0.92–0.99). These data indicate that activity of ATP-citrate lyase in cholinergic neurons is several times higher than that present in glial and noncholinergic neuronal cells.     

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.     

Evidence for the regulatory function of synaptoplasmic acetyl-CoA in acetylcholine synthesis in nerve endings.

Isolated synaptosomes maintained a relatively stable level of acetyl-CoA during their incubation in the presence of 30 mM-KCl. Addition of Ca2+ resulted in inhibition of pyruvate oxidation and slight activation of acetylcholine synthesis. The cation decreased acetyl-CoA in intrasynaptosomal mitochondria, but did not alter its content in synaptoplasm. Verapamil did not affect pyruvate oxidation, but decreased acetyl-CoA in synaptoplasm and inhibited acetylcholine synthesis in synaptosomes. It indicates that Ca2+ might regulate acetylcholine synthesis through changes in the direct transfer of acetyl-CoA from mitochondria to synaptoplasm.     

AChE synthesis in cholinergic neurons: electron histochemistry of enzyme translocation

Summary Endoplasmic reticulum of cholinergic nerve cells exhibits acetylcholinesterase activity. In central neurons that exert an ephemeric acetylcholinesterase activity only during some ontogenetical states, enzyme reaction product is present also in the Golgi system. Neurotubular and neurofilamentar structures exert an acetylcholinesterase in terminal axons of young animals. From these electron histochemical studies it is concluded that enzyme protein molecules, synthesized in the endoplasmic reticulum, are translocated to the active sites (surface membranes) via axonal filaments (or tubules), or will be extruded from the neuron via the Golgi system.Our thanks are due to Dr. P. Röhlich, head of the Budapest Electron Microscope Laboratory, for his generous help in various aspects of the electron microscopic work.     

Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.     

Mouse brain distribution of a carbon-11 labeled vesamicol derivative: presynaptic marker of cholinergic neurons

The regional mouse brain distribution of a new carbon-11 labeled derivative of vesamicol, [11C]-5-(N-methylamino)benzovesamicol [( 11C]MABV) is reported. Radiotracer concentrations in vivo are in the rank order of striatum greater than cortex greater than hippocampus greater than hypothalamus greater than cerebellum, consistent with reported distributions of other presynaptic cholinergic neuronal markers. In time course studies, striatum/cerebellum and cortex/cerebellum ratios for (-)-[11C]MABV continue to increase to values of 13 and 5, respectively, 75 min after i.v. injection of [11C]MABV. The specific binding in striatum and cortex is lowered by pretreatment with (+/-)-vesamicol, and shows stereoselectivity with lower uptake and lower ratios for the (+)-enantiomer. (-)-enantiomer. (-)-[11C]MABV is proposed as a positron-emitting radioligand for the in vivo study of presynaptic cholinergic neurons.     

Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes

Acetylcholine is an important neurotransmitter, and the habenulo-interpeduncular projection is a major cholinergic pathway in the brain. To study the physiological properties of cholinergic transmission in the interpeduncular nucleus (IPN), we used a transgenic mouse line in which the light-gated cation channel ChannelRhodopsin-2 is selectively expressed in cholinergic neurons. Cholinergic axonal terminals were activated by light pulses, and postsynaptic responses were recorded from IPN neurons. Surprisingly, brief photostimulation produces fast excitatory postsynaptic currents that are mediated by ionotropic glutamate receptors, suggesting wired transmission of glutamate. By contrast, tetanic photostimulation generates slow inward currents that are largely mediated by nicotinic acetylcholine receptors, suggesting volume transmission of acetylcholine. Finally, vesicular transporters for glutamate and acetylcholine are coexpressed on the same axonal terminals in the IPN. These results strongly suggest that adult brain "cholinergic" neurons can corelease glutamate and acetylcholine, but these two neurotransmitters activate postsynaptic neurons via different transmission modes.     

Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: Regulation by NGF

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.