Acetylcholine Formation Research Articles

Gut production of GABA by a probiotic formula: an in vitro study.

The objective of this study was to evaluate the influence of a probiotic formula on the production of neuroactive compounds in different parts of the colon in batch culture and in vitro gut simulator experiments. Thirteen lactic acid bacterial strains, belonging to the species Levilactobacillus brevis, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Ligilactobacillus salivarius, Streptococcus thermophilus, were characterised for their in vitro ability to produce neurotransmitters. L. brevis P30021 and L. plantarum P30025 were selected based on their capability to produce γ-aminobutyric acid (GABA) and acetylcholine in vitro. A probiotic formulation with potential psychobiotic activity was prepared and tested in a batch culture of human microbiota monitoring the formation of GABA and acetylcholine. Samples of the three colonic tracts were taken from the Simulator of the Human Intestinal Microbiota (SHIME®) evaluating the production of GABA and other neurotransmitters by LC-MS. Short chain fatty acids (SCFA) by GC and microbiota composition by 16S rRNA gene sequencing were also determined. Probiotic supplementation led to the formation of GABA and acetylcholine with a decrease in glutamate concentrations in the in vitro batch fermentation. Production of GABA after the treatment with probiotics was confirmed in the SHIME® short-term experiment. No differences in short-chain fatty acids were observed up to 72h of fermentation. Different microbiota composition was found in the three different parts of the colon, with a higher abundance of Veillonellaceae in the ascending colon vessels. The probiotic-exposed microbiota showed higher levels of Bacteroides, a gut microbe associated with anti-inflammatory activities and a potential GABA producer. Results demonstrate the impact of the tested probiotic formula on gut microbiota structure and GABA production. In conclusion, the probiotic treatment changed the microbiota composition and increased neuroactive metabolites production, indicating promising potential as psychobiotics, even if further clinical evidence is needed to confirm the effectiveness of these probiotics in improving mental health.

The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.

Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy favors amyloid-β (Aβ) aggregation. Citrate inhibits Aβ25-35 and Aβ1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aβ aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need clinical studies if citrate reverses Aβ deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

Recovery of the Functional Activity of K<sub>ATP</sub>-Channels of Pial Arteries after Ischemia/Reperfusion Using Cell Therapy

This study aims to examine the efficiency of intravenous transplantation of human mesenchymal stem cells (hMSCs) performed 7 days after cerebral ischemia/reperfusion (I/R) for recovery of the functional activity of KATP-channels of cerebral arteries. Using a device for intravital visualization of pial vessels, the reaction of arteries to the KATP-channel blocker glibenclamide (GB), the activator of the same channels of pinacidil (PI), acetylcholine (ACh), and ACh against a background of GB action (ACh/GB) 14 and 21 days after I/R and intravenous hMSC transplantation performed 7 days after ischemic exposure. On exposure to GB 14 days after I/R, 1.5–1.8 times fewer arteries narrowed than in the sham–operated (SO) rats. By day 21 after I/R, the constriction reaction was completely restored, except for arteries with a diameter more 40 μm. In the cell–therapy group, the constrictor response to GB was completely recovered to the level of SO animals in arteries with a diameter less than 40 μm by 14 day after I/R exposure; in arteries with a diameter of more than 40 μm, the constriction reaction did not recover until 21 days. The number of dilations per ACh/GB compared to a clear ACh in SO rats was reduced in 1.6–1.8 times on 14 day after I/R and in 1.6–6.6 after 21 days. In I/R animals on 14 day, the number of dilatations per ACh/GB compared to clear ACh was significantly increased in arteries with a diameter of more than 20 μm by 1.5–1.7 times, and after 21 days in arteries with a diameter of more than 40 μm by 1.2 times. After the introduction of hMSC, GB blocked ACh–mediated dilation in arteries less than 40 μm in diameter both on days 14 and 21 after I/R. In arteries with a diameter of more than 40 μm the functional activity of KATP-channels did not recover until 21 days. Conclusion. I/R of the rat cerebral cortex reduces the contribution of KATP-channels to maintaining the basal tone of the pial arteries and almost completely excludes these channels from the formation of ACh–mediated dilation during 21 days of the postischemic period. Practically did not participate in the dilatory response. Intravenous transplantation of hMSC, performed 7 days after I/R, results in restoration of participation of SMC KATP-channels in maintaining the basal tone and ACh–mediated dilatation of pial arteries with a diameter less than 40 μm already 14 days after I/R.

Brain CoA and acetyl CoA system in mechanisms of neurodegeneration

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, the key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, the main substrate of acetylation processes, including the formation of N-acetylaspartate and acetylcholine, post-translational modification of histones, determines the protection of neurons from degenerative signals and cholinergic neurotransmission. The biochemical mechanisms of neurodegenerative syndromes in PANK and CoASY defects and the possibility of correcting the development of CoA biosynthesis in knockout models for these enzymes are described. The data of a post-mortem study of the brain of patients with Huntington’s and Alzheimer’s diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in pathognomonic neurostructures. In the frontal cortex of patients with Parkinson’s disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. The redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro on synaptosomal membranes and mitochondria in the modeling of aluminum neurotoxicosis, accompanied by a decrease in the level of CoA in the CNS. The ability of CoA biosynthesis precursors to stabilize the glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism when exposed to neurotoxins, the development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA - redox-dependent processes of CoAlation of proteins, the possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology, the study of the bioavailability and biotransformation of Pan derivatives, in particular, D-panthenol, 4′-phospho-pantetheine, its acylated derivatives and compositions with redox pharmacological compounds are promising as potential etiopathogenetic agents.

Brain CoA and Acetyl CoA Metabolism in Mechanisms of Neurodegeneration

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, which is the main substrate of acetylation processes including formation of N-acetyl aspartate and acetylcholine, post-translational modification of histones, predetermines protection of the neurons against degenerative signals and cholinergic neurotransmission. Biochemical mechanisms of neurodegenerative syndromes in the cases of PANK and CoASY defects, and the possibility of correcting of CoA biosynthesis in the models with knockouts of these enzymes have been described. The data of a post-mortem study of the brains from the patients with Huntington's and Alzheimer's diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in the pathognomonic neurostructures. In the frontal cortex of the patients with Parkinson's disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. Redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro with synaptosomal membranes and mitochondria during modeling of aluminum neurotoxicity accompanied by the decrease in the level of CoA in CNS. The ability of CoA biosynthesis precursors to stabilize glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism during exposure to neurotoxins, development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA (redox-dependent processes of CoAlation of proteins, possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology), it seems promising to study bioavailability and biotransformation of Pan derivatives, in particular of D-panthenol, 4'-phospho-pantetheine, its acylated derivatives, and compositions with redox pharmacological compounds, are promising for their potential use as etiopathogenetic agents.

Genetic Editing and Pharmacogenetics in Current And Future Therapy Of Neurocognitive Disorders.

Dementia is an important issue in western societies, and in the following years, this problem will also rise in the developing regions, such as Africa and Asia. The most common types of dementia in adults are Alzheimer's Disease (AD), Dementia with Lewy Bodies (DLB), Frontotemporal Dementia (FTD) and Vascular Dementia (VaD), of which, AD accounts for more than half of the cases. The most prominent symptom of AD is cognitive impairment, currently treated with four drugs: Donepezil, rivastigmine, and galantamine, enhancing cholinergic transmission; as well as memantine, protecting neurons against glutamate excitotoxicity. Despite ongoing efforts, no new drugs in the treatment of AD have been registered for the last ten years, thus multiple studies have been conducted on genetic factors affecting the efficacy of antidementia pharmacotherapy. The researchers investigate the effects of variants in multiple genes, such as ABCB1, ACE, CHAT, CHRNA7, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, CYP3A7, NR1I2, NR1I3, POR, PPAR, RXR, SLC22A1/2/5, SLC47A1, UGT1A6, UGT1A9 and UGT2B7, associated with numerous pathways: the development of pathological proteins, formation and metabolism of acetylcholine, transport, metabolism and excretion of antidementia drugs and transcription factors regulating the expression of genes responsible for metabolism and transport of drugs. The most promising results have been demonstrated for APOE E4, dementia risk variant, BCHE-K, reduced butyrylcholinesterase activity variant, and CYP2D6 UM, ultrarapid hepatic metabolism. Further studies investigate the possibilities of the development of emerging drugs or genetic editing by CRISPR/Cas9 for causative treatment. In conclusion, the pharmacogenetic studies on dementia diseases may improve the efficacy of pharmacotherapy in some patients with beneficial genetic variants, at the same time, identifying the carriers of unfavorable alleles, the potential group of novel approaches to the treatment and prevention of dementia.

Autocrine control of angiogenesis by endogenous acetylcholine in an in vitro model using human endothelial cells: evidence for an autocrine cholinergic system in endothelial cells.

We wanted to elucidate whether acetylcholine as the endogenous ligand at cholinoceptors (ChRs) may have effects on angiogenesis and whether they are transduced through muscarinic or nicotinic ChRs. Human umbilical vein endothelial cells were cultured until confluence and thereafter seeded in Matrigel in vitro angiogenesis assays for 18 hours. During the entire cell culture and angiogenesis period, cells were treated with vehicle, eserine (1 μM), in the absence or presence of additional atropine (1 μM) or mecamylamine (1 μM). Finally, the resulting angiogenetic network was investigated histologically. Eserine significantly enhanced acetylcholine formation. When acetylcholine acted through muscarinic ChRs (eserine + mecamylamine), we observed enhanced complexity of the angiogenic network pattern with increased tube length and cell number. In contrast, when acting through nicotinic ChRs (eserine + atropine), we found reduced complexity of pattern with less branches, shorter tubes, and reduced cell number. If acting on both types of ChRs (eserine alone), there were only very small effects. Using α-bungarotoxin, lobeline, and dihydro-β-erythroidine, we also could show that these effects to various degrees involve α7, α3/β2, and α4/β2 n-ChRs. In conclusion, our results support the hypothesis that human umbilical vein endothelial cells possess an autocrine nonneuronal cholinergic system regulating angiogenesic branch formation through the partially opposing effects of n-ChRs and m-ChRs.

Signalling molecules in the urothelium.

The urothelium was long considered to be a silent barrier protecting the body from the toxic effects of urine. However, today a number of dynamic abilities of the urothelium are well recognized, including its ability to act as a sensor of the intravesical environment. During recent years several pathways of these urothelial abilities have been proposed and a major part of these pathways includes release of signalling molecules. It is now evident that the urothelium represents only one part of the sensory web. Urinary bladder signalling is finely tuned machinery of signalling molecules, acting in autocrine and paracrine manner, and their receptors are specifically distributed among different types of cells in the urinary bladder. In the present review the current knowledge of the formation, release, and signalling effects of urothelial acetylcholine, ATP, adenosine, and nitric oxide in health and disease is discussed.

High‐Affinity Choline Uptake (HACU) and Choline Acetyltransferase (ChAT) Activity in Neuronal Cultures for Mechanistic and Drug Discovery Studies

Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

ACR-26: A novel nicotinic receptor subunit of parasitic nematodes

Nematode nicotinic acetylcholine receptors are the targets for many effective anthelmintics, including those recently introduced into the market. We have identified a novel nicotinic receptor subunit sequence, acr-26, that is expressed in all the animal parasitic nematodes we examined from clades III, IV and V, but is not present in the genomes of Trichinella spiralis, Caenorhabditis elegans, Pristionchus pacificus and Meloidogyne spp. In Ascaris suum, ACR-26 is expressed on muscle cells isolated from the head, but not from the mid-body region. Sequence comparisons with other vertebrate and nematode subunits suggested that ACR-26 may be capable of forming a functional homomeric receptor; when acr-26 cRNA was injected into Xenopus oocytes along with Xenopus laevis ric-3 cRNA we occasionally observed the formation of acetylcholine- and nicotine-sensitive channels. The unreliable expression of ACR-26 in vitro may suggest that additional subunits or chaperones may be required for efficient formation of the functional receptors. ACR-26 may represent a novel target for the development of cholinergic anthelmintics specific for animal parasites.

The butanol fraction of Eclipta prostrata (Linn) increases the formation of brain acetylcholine and decreases oxidative stress in the brain and serum of cesarean-derived rats

Eclipta prostrata has been used as a traditional medicinal plant to prevent dementia and to enhance memory in Asia. Its potential as a nootropic and as an antioxidant have been reported in mice. We hypothesized that Eclipta may affect the formation of neurotransmitters and the inhibition of oxidative stress. Charles River cesarean-derived rats (male, 180 ± 10 g) were fed experimental diets supplemented with 0 mg (control), 25 mg (E25), 50 mg (E50), or 100 mg (E100) of a freeze-dried butanol fraction of E prostrata per kilogram of diet for 6 weeks. The acetylcholine level was significantly increased by 9.6% and 12.1% in the brains of E50 and E100 groups, respectively, as compared with the control group that was fed standard diet alone. The acetylcholine esterase activity was significantly increased by 13.1% and 19.7% in the brains of E50 and E100 groups, respectively, compared with the control group. Monoamine oxidase-B activity was significantly decreased by 10.5% in the brains of the E100 group, and the superoxide radical level was significantly reduced by 9.4% in the serum of the E100 group compared with the control group. Superoxide dismutase activity was significantly increased by 9.6% and 11.6% in the serum of E50 and E100 groups, respectively, compared with the control group. These results clearly demonstrate the effects of E prostrata on the formation of acetylcholine in the brain and the inhibition of oxidative stress in the brain and serum of rats. These findings may have implications for preventing dementia and enhancing memory function in humans.

Redesign of Cosubstrate Specificity and Identification of Important Residues for Substrate Binding to hChAT

In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.

Determination of high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in the same population of cultured cells

Cholinergic neurons are a major constituent of the mammalian central nervous system. Acetylcholine, the neurotransmitter used by cholinergic neurons, is synthesized from choline and acetyl CoA by the enzymatic action of choline acetyltransferase (ChAT). The transport of choline into the cholinergic neurons, which results in synthesis of ACh, is hemicholinium-sensitive and is referred to as high-affinity choline uptake (HACU). Thus, the formation of acetylcholine in cholinergic neurons largely depends on both the levels of choline being transported into the cells from the extracellular space and the activity of ChAT. Several methods were described previously to measure HACU and ChAT simultaneously in synaptosomes, but the same for cultured cells is lacking. We describe a procedure to measure HACU and ChAT at the same time in cultured cells by simple techniques employing radionuclides. In this procedure, we determined quantitatively hemicholinium-sensitive choline uptake and ChAT enzyme activity in a small number of differentiated human neuroblastoma (SK-N-SH) cells. We also determined the kinetics of choline uptake in the SK-N-SH cells. We believe that these simple methods can be used for neurochemical and drug discovery studies in several models of neurodegenerative disorders including Alzheimer's disease.

Treatment of the Spasticity in Children with Cerebral Palsy

Botulinum toxin is a natural purified protein and one of the strongest biological poisons--neurotoxin. It is produced by the bacterium Clostridium botulinum. Its medical usage started in USA in 1981 and in Europe in 1992. There are seven different immune types of the toxin: A, B, C1, D, E, F and G. Toxin types A and B are used to decrease muscular spasticity. Botulinum toxin prevents the formation of acetylcholine from cholinergic nerve tissues in muscles, which in the end irreversibly destroys neuromuscular synapses. It is called temporary local chemodenervation. It does not affect the synthesis of acetylcholine. As it affects neuromuscular bond it also affects one of the symptoms of cerebral palsy--spasticity. Decreasing the spasticity of children with cerebral palsy leads to the improvement of conscious movements, muscles are less toned, passive mobility is improved, orthosis tolerance is also improved, and the child is enabled to perform easier and better motor functions such as crawling, standing and walking. Since the action of Botulinum toxin is limited to 2-6 months, new neural collaterals are formed and neuromuscular conductivity is reestablished which in the end once again develops a muscular spasm. This leads to a conclusion that botulinum toxin should again be applied into spastic muscles. It is very important for good effect of Botulinum toxin to set the goals of the therapy in advance. The goals include improvement of a function, prevention of contractions and deformities, ease of care and decrease of pain for children with cerebral palsy. After application of botulinum toxin, it is necessary to perform adequate and intensive physical treatment with regular monitoring of effects. This work shows a case of a boy with spastic form of cerebral palsy. After being rehabilitated using Vojta therapy and Bobath concept and the conduct of certain physical procedures, botulinum toxin is administered into his lower limbs' muscles and kinesiotherapy is intensified. After the administration of botulinum toxin significant functional improvement is noted.

High-affinity choline uptake and acetylcholine-metabolizing enzymes in CNS white matter. A quantitative study

The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25–31% of that in hippocampus. White matter choline acetyltransferase activity was 10–50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20–25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8–4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.

Different temporal processing of sensory inputs in the rat thalamus during quiescent and information processing states in vivo

Sensory inputs from the whiskers reach the primary somatosensory thalamus through the medial lemniscus tract. The main role of the thalamus is to relay these sensory inputs to the neocortex according to the regulations dictated by behavioural state. Intracellular recordings in urethane-anaesthetized rats show that whisker stimulation evokes EPSP-IPSP sequences in thalamic neurons. Both EPSPs and IPSPs depress with repetitive whisker stimulation at frequencies above 2 Hz. Single-unit recordings reveal that during quiescent states thalamic responses to repetitive whisker stimulation are suppressed at frequencies above 2 Hz, so that only low-frequency sensory stimulation is relayed to the neocortex. In contrast, during activated states, induced by stimulation of the brainstem reticular formation or application of acetylcholine in the thalamus, high-frequency whisker stimulation at up to 40 Hz is relayed to the neocortex. Sensory suppression is caused by the depression of lemniscal EPSPs in relatively hyperpolarized thalamocortical neurons. Sensory suppression is abolished during activated states because thalamocortical neurons depolarize and the depressed lemniscal EPSPs are able to reach firing threshold. Strong IPSPs may also contribute to sensory suppression by hyperpolarizing thalamocortical neurons, but during activated states IPSPs are strongly reduced altogether. The results indicate that the synaptic depression of lemniscal EPSPs and the level of depolarization of thalamocortical neurons work together in thalamic primary sensory pathways to suppress high-frequency sensory inputs during non-activated (quiescent) states while permitting the faithful relay of high-frequency sensory information during activated (processing) states.

Regulation of the activity of choline acetyl transferase by lipoic acid.

The observations reported in this article demonstrate that lipoic acid strongly influences the activity of a purified preparation of choline acetyl transferase. The reduced form, dihydrolipoic acid, is a powerful activator of the enzyme while lipoic acid itself has an inhibitory effect and counteracts the stimulatory effect of dihydrolipoic acid. It is proposed that dihydrolipoic acid serves an essential function in the action of this enzyme and that the ratio of reduced to oxidized lipoic acid in the cell may play an important role in the regulation of the activity of the enzyme. The implications of these findings for cell function and acetyl choline formation are discussed.

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release

It is well established that extracellular choline is transported into central cholinergic nerve terminals by `high' and `low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1- 14C]acetate (0.1–100 μM) and the uptake of [1- 14C]acetate and the amount of [ 14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1- 14C]acetate was temperature-dependent and saturable having an apparent Michaelis constant ( K m) of 22 μM. The formation of [ 14C]ACh in the tissue as a function of extracellular [1- 14C]acetate appeared to occur by both `high' and `low' affinity processes with apparent K m values of 0.5 and 19.6 μM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1- 14C]acetate uptake and the amount of [ 14C]ACh formed from that [1- 14C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [ 14C]ACh formed from extracellular [1- 14C]acetate. High K + depolarization of hippocampal tissue loaded with extracellular [1- 14C]acetate not only increased the synthesis but also the release of [ 14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow. © 1997 Elsevier Science B.V. All rights reserved.

Drug interactions with neuromuscular blockers.

Drugs administered to patients undergoing anaesthesia may complicate the use of the neuromuscular blockers that are given to provide good surgical conditions. The various sites of interaction include actions on motor nerve conduction and spinal reflexes, acetylcholine (ACh) synthesis, mobilisation and release, sensitivity of the motor end plate to ACh and the ease of propagation of the motor action potential. In addition, many drugs affect the pharmacokinetics of neuromuscular blockers, especially as most drugs depend to a greater or lesser extent upon renal excretion. The clinically significant interaction between nondepolarisers and depolarisers may be due to blockade of the pre-synaptic nicotinic receptors by the depolarisers, leading to decreased ACh mobilisation and release. Synergism between nondepolarisers probably results from post-synaptic receptor mechanisms. Volatile anaesthetic agents affect the sensitivity of the motor end-plate (post-synaptic receptor blockade) in addition to having effects on pre-synaptic nicotinic function. The effects of nondepolarisers are likely to be potentiated and their action prolonged by large doses of local anaesthetics due to depression of nerve conduction, depression of ACh formation, mobilisation and release, decreases in post-synaptic receptor channel opening times and reductions in muscular contraction. Most antibacterials have effects on pre-synaptic mechanisms. Procainamide and quinidine principally block nicotinic receptor channels. Magnesium has a marked inhibitory effect on ACh release. Calcium antagonists could theoretically interfere with neurotransmitter release and muscle contractility. Phenytoin and lithium decrease ACh release, whilst corticosteroids and furosemide (frusemide) tend to increase the release of the transmitter. Ecothiopate, tacrine, organophosphates, propanidid, metoclopramide and bambuterol depress cholinesterase activity and prolong the duration of the neuromuscular block. The probability of clinically significant interactions increases in patients receiving several drugs with possible effects on neuromuscular transmission and muscle contraction.

Metabolism and actions of CDP-choline as an endogenous compound and administered exogenously as citicoline.

CDP-choline, supplied exogenously as citicoline, has beneficial physiological actions on cellular function that have been extensively studied and characterized in numerous model systems. As the product of the rate-limiting step in the synthesis of phosphatidylcholine from choline, CDP-choline and its hydrolysis products (cytidine and choline) play important roles in generation of phospholipids involved in membrane formation and repair. They also contribute to such critical metabolic functions as formation of nucleic acids, proteins, and acetylcholine. Orally-administered citicoline is hydrolyzed in the intestine, absorbed rapidly as choline and cytidine, resynthesized in liver and other tissues, and subsequently mobilized in CDP-choline synthetic pathways. Citicoline is efficiently utilized in brain cells for membrane lipid synthesis where it not only increases phospholipid synthesis but also inhibits phospholipid degradation. Exogenously administered citicoline prevents, reduces, or reverses effects of ischemia and/or hypoxia in most animal and cellular models studied, and acts in head trauma models to decrease and limit nerve cell membrane damage, restore intracellular regulatory enzyme sensitivity and function, and limit edema. Thus, considerable accumulated evidence supports use of citicoline to enhance membrane maintenance, membrane repair, and neuronal function in conditions such as ischemic and traumatic injuries. Beneficial effects of exogenous citicoline also have been postulated and/or reported in experimental models for dyskinesia, Parkinson's disease, cardiovascular disease, aging, Alzheimer's disease, learning and memory, and cholinergic stimulation.