High-affinity Choline Uptake Research Articles

Regulated recycling and plasma membrane recruitment of the high‐affinity choline transporter

The high-affinity choline transporter (CHT1) is responsible for uptake of choline from the synaptic cleft and supplying choline for acetylcholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K(+)-depolarization promotes Ca(2+)-dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K(+)-depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular transporters to the recycling pool.

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421–1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 μM in AChE-deficient mice and 4.36 μM in wild-type mice. Addition of choline (10 μM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 μM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.

Inhibition of choline transport by redox-active cholinomimetic bis-catechol reagents

Both N, N′-(2,3-dihydroxybenzyl)- N, N, N′, N′-tetramethyl-1,6-hexanediamine dibromide (DTH, 6) and N, N′-(2,3-dihydroxybenzyl)- N, N, N′, N′-tetramethyl-1,10-decanediamine dibromide (DTD, 7), which are symmetrical bis-catechol substituted hexamethonium and decamethonium analogues, respectively, were found to inhibit high-affinity choline transport in mouse brain synaptosomes. Inhibitory properties were evaluated using an extraordinarily sensitive capillary electrophoresis method employing electrochemical detection at an enzyme-modified microelectrode. Dose–response curves were generated for each inhibitor and IC 50 values were determined to be 76 μM for 6 and 21 μM for 7. Lineweaver–Burk analysis revealed that both molecules inhibit high-affinity choline uptake by a mixed inhibition mechanism. The K I values for 6 and 7 were determined to be 73 ± 1 and 31 ± 2 μM, respectively. The inhibition properties were further compared to a series of mono-catechol analogues, 3-[(trimethylammonio)methyl]catechol ( 1), N, N-dimethylepinephrine ( 4) and 6-hydroxy- N, N-dimethylepinephrine ( 5), as well as the well-characterized hemicholinium inhibitors, hemicholinium-15 (HC-15, 8) and hemicholinum-3 (HC-3, 9).

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na + channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66 kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na +/K +)ATPase

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na +/K +)ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl 3 and ouabain, the foremost utilized selective inhibitor of (Na +/K +)ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na +/K +)ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl 3, whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K + depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl 3 induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na +/K +)ATPase activity was slight when compared to its inhibitory action on mammalian (Na +/K +)ATPase. In conclusion, inhibition of (Na +/K +)ATPase by AlCl 3 and ouabain jeopardized the high-affinity (Na +-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca 2+-dependent, K + depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl 3 on choline uptake and acetylcholine release only resembled those of ouabain when rat synaptosomes were assayed. Therefore, important differences were found between the species regarding the cholinotoxic action of aluminum. The variability of (Na +/K +)ATPase sensitivity to aluminum of cholinergic neurons might contribute to their differential susceptibility to this neurotoxic agent.

Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain

Chronic dichlorvos exposure (6 mg/kg b.wt/day) for a period of 8 weeks resulted in significant reduction in body weight gain of the male Wistar rats. However, the dietary intake remained unchanged in experimental animals following dichlorvos treatment. Activity of the synthesizing enzyme of acetylcholine (ACh) ie, choline acetyltransferase, was found to be significantly increased and the activity of hydrolyzing enzyme, acetyl cholinesterase (AChE), was inhibited in all the three brain regions studied. Chronic dichlorvos treatment also caused significant reduction in both high affinity (HA) and low affinity (LA) choline uptake (CU), with maximal effect being observed in the brain stem followed by cerebellum and cerebrum. Muscarinic receptor binding was significantly decreased in brain stem and cerebellum as reflected in the decreased receptor number (B(max)), without any change in the binding affinity (K(d)) of the receptors. Dichlorvos treatment caused marked inhibition in cAMP synthesis as indicated by decreased adenylate cyclase activity as well as cAMP levels in cerebrum, cerebellum and brain stem. Our study shows that organophosphates may interact with muscarinic receptor-linked second messenger system and this could be a potential mechanism for the neurotoxic effects observed after repeated exposure to low levels of organophosphates, which are unexplainable on the basis of cholinergic hyperactivity.

Reduction in CHT1-mediated choline uptake in primary neurons from presenilin-1 M146V mutant knock-in mice

The memory loss in Alzheimer's disease (AD) has been linked to cholinergic hypoactivity. Mutations in presenilin-1 (PS-1) may regulate cholinergic signaling, although their precise roles in cholinergic neurotransmission in AD are unsettled. Neuronal uptake of choline via the high affinity choline transporter (CHT1) is essential for cholinergic neurotransmission. CHT1 is a Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter. Although cholinergic neurons in the nucleus basalis of Meynert are a major source of cholinergic projections for the cerebral cortex, it is unclear whether cortical neurons exhibit intrinsic CHT1 activity that is altered in AD. We now report that primary cortical neurons express intrinsic and biologically active CHT1, and that, in these neurons, CHT1-mediated choline uptake activity is significantly reduced in PS-1 M146V mutant knock-in mice. Further kinetic studies using HC-3 binding and cell surface biotinylation assays showed that the PS-1 mutation inhibits CHT1 mediated choline uptake by reducing the ligand binding affinity of CHT1 without significantly altering levels of CHT1 expression in the plasma membrane. Since human neocortex has recently been shown to possess intrinsic cholinergic innervation, our results indicate that alterations in CHT1-mediated high affinity choline uptake in cortical neurons may contribute to Alzheimer's dementia.

Evaluation of [ 11C]hemicholinium-15 and [ 18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[ 11C]Hemicholinium-15 ([ 11C]HC-15) and [ 18F]hemicholinium-15 ([ 18F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [ 11C]HC-15 was prepared by N-[ 11C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [ 11C]CH 3OTf in 55–70% radiochemical yield decay corrected to end of bombardment (EOB) and 2–3 Ci/μmol specific activity at end of synthesis (EOS). [ 18F]HC-15 was prepared by N-[ 18F]fluoromethylation of the precursor using [ 18F]FCH 2OTf in 20–30% radiochemical yield decay corrected to EOB and >1.0 Ci/μmol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20 min post-intravenous injection, and the results show the heart region uptakes 1.32 ± 0.75%ID/g in R-ventricle for [ 11C]HC-15 and 1.28 ± 0.81%ID/g in L-ventricle for [ 18F]HC-15, respectively. The dynamic PET imaging studies of [ 11C]HC-15 in rats were acquired 60 min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0 mg/kg of unlabeled HC-15 prior to [ 11C]HC-15 injection. [ 11C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [ 13N]NH 3, [ 11C]HC-15, and [ 18F]HC-15. PET studies in dogs of both [ 11C]HC-15 and [ 18F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [ 11C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.

Opposite effects of a single versus repeated doses of gabapentin on retention performance of an inhibitory avoidance response in mice

CF-1 male mice were trained in an inhibitory avoidance (IA) task. A single gabapentin (GBP) administration (50 mg/kg, ip) immediately after training enhanced retention performance when mice were tested 8 days after training. On the contrary, when the same dose of the anticonvulsant drug was given twice a day for 7 days (repeated treatment), a significant impairment on retention performance 12 h after the last injection of GBP was observed. When the retention test was delayed 7 days after the end of the repeated treatment, the retention performance was not significant different from the control group, whereas if the retention test was delayed 14 days, retention performance was higher than control group but similar to that observed when GBP was administered once immediately after training. The impairment on retention performance was correlated with a significant decrease in the high affinity choline uptake in the hippocampus at the end of the retention test. The pretest administration of the direct muscarinic cholinergic agonist oxotremorine (50 μg/kg, ip) reversed the impairment on retention performance. This reversion was prevented by the muscarinic cholinergic antagonist scopolamine (0.5 mg/kg, ip). Taken together, these results suggest that the impairment on retention performance of an IA task in mice induced by repeated administration of GBP affected memory retrieval but not memory consolidation and that this impairment may be attributable to a reduction on central cholinergic activity.

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.

The antinociceptive effects of centrally administered CDP-choline on acute pain models in rats: The involvement of cholinergic system

This study investigates the antinociceptive effect of intracerebroventricular (i.c.v.) injection of cytidine-5′-diphosphate choline (CDP-choline; citicoline) and the involvement of cholinergic mechanisms in rats. Three different pain models were utilized: thermal paw withdrawal test, mechanical paw pressure test and acetic acid writhing test. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 μmol) produced dose and time-dependent antinociception. Equimolar dose of choline (1 μmol; i.c.v.) produced antinociceptive response similar to the one observed in CDP-choline given animals. On the other hand, cytidine (1 μmol; i.c.v.) failed to produce response in the thermal paw withdrawal test and the mechanical paw pressure test but in the writhing test in which it produced significant antinociceptive effect. CDP-choline-induced antinociception was prevented by the neuronal high affinity choline uptake inhibitor HC-3 (1 μg; i.c.v.), the nonselective nicotinic receptor antagonist mecamylamine (50 μg; i.c.v.) and by the α 7-selective nicotinic receptor antagonist, MLA (25 μg; i.c.v.). However, it was not changed by the nonselective muscarinic receptor antagonist atropine (10 μg; i.c.v.) in the thermal paw withdrawal test and mechanical paw pressure test. In the writhing test, all antagonist pretreatments produced blockade similar to that obtained from CDP-choline injected animals. CDP-choline did not impair the motor performance of rats as evaluated by a rota-rod test. Therefore, it can be postulated that CDP-choline exerts an antinociceptive effect mediated by a central cholinergic mechanism. Activation of specific α 7-nicotinic cholinergic receptors through the activation of presynaptic cholinergic mechanisms appears to be involved in the antinociceptive effect of this drug.

The Caenorhabditis elegans Choline Transporter CHO-1 Sustains Acetylcholine Synthesis and Motor Function in an Activity-Dependent Manner

Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.

Inhibitory effect of hemicholinium-3 on presynaptic nicotinic acetylcholine receptors located on the terminal region of myenteric motoneurons

Previously we have demonstrated the presence of presynaptic nicotinic acetylcholine receptors on the terminals of myenteric neurons in Auerbach's plexus of guinea-pig ileum. During these studies we observed, that the presence of hemicholinium-3, an inhibitor of the high affinity choline uptake significantly influences the contraction of the longitudinal muscle strip preparation. Our aim was to investigate the neurochemical background of this effect and quantitatively characterize the action of HC-3. We studied the effect of HC-3 on epibatidine- and electrical stimulation-evoked contraction and release of [ 3H]acetylcholine from the guinea-pig longitudinal muscle strip preparation. We found that in the presence of tetrodotoxin, when the contribution of somatodendritic nicotinic acetylcholine receptors to the response was prevented due to the inhibition of axonal conduction, HC-3 inhibited the epibatidine-evoked contraction and [ 3H]acetylcholine release in the submicromolar range (IC 50 = 897 nM and IC 50 = 693 nM, respectively), whereas the electrical stimulation-evoked contraction was not affected by HC-3, and the release of [ 3H]acetylcholine was apparently enhanced. Our data indicate that HC-3 inhibits the presynaptic nicotinic acetylcholine receptors of myenteric neurons. Since these receptors play an important role in the regulation of cholinergic neurotransmission in the enteric nervous system, the use of HC-3 in [ 3H]acetylcholine release experiments might bias the interpretation of data.

Cholinergic dysfunction in a mouse model of Alzheimer disease is reversed by an anti-A antibody

Disruption of cholinergic neurotransmission contributes to the memory impairment that characterizes Alzheimer disease (AD). Since the amyloid cascade hypothesis of AD pathogenesis postulates that amyloid beta (A beta) peptide accumulation in critical brain regions also contributes to memory impairment, we assessed cholinergic function in transgenic mice where the human A beta peptide is overexpressed. We first measured hippocampal acetylcholine (ACh) release in young, freely moving PDAPP mice, a well-characterized transgenic mouse model of AD, and found marked A beta-dependent alterations in both basal and evoked ACh release compared with WT controls. We also found that A beta could directly interact with the high-affinity choline transporter which may impair steady-state and on-demand ACh release. Treatment of PDAPP mice with the anti-A beta antibody m266 rapidly and completely restored hippocampal ACh release and high-affinity choline uptake while greatly reducing impaired habituation learning that is characteristic of these mice. Thus, soluble "cholinotoxic" species of the A beta peptide can directly impair cholinergic neurotransmission in PDAPP mice leading to memory impairment in the absence of overt neurodegeneration. Treatment with certain anti-A beta antibodies may therefore rapidly reverse this cholinergic dysfunction and relieve memory deficits associated with early AD.

S.04.03 Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines

In order to test the hypothesis that α-noradrenergic receptors in the septum 1) play an important functional role in the mediation of trans-synaptic control of the neurones of the cholinergic septo-hippocampal pathway and 2) produce resultant modulation of working memory performance, we have investigated the effects in vivo of the acute intraseptal injection of an α-antagonist, phenoxybenzamine, in mice. Neurochemical analysis was performed using measures of the kinetics of sodium-dependent high-affinity choline uptake in samples of hippocampus from injected mice and their relevant controls in both quiet conditions and immediately following selective working memory testing in an 8-arm radial maze. Results show that whereas the injection of phenoxybenzamine produces no significant alteration of the activity of the cholinergic septo-hippocampal neurones in quiet conditions, the pretrial (20 min) administration of this drug almost totally abolished the usually observed increase in hippocampal cholinergic activity induced by testing. This inhibition of cholinergic activation was associated with a parallel working memory deficit. The results provide further direct support for the hypothesis that septal noradrenergic afferents via α-receptors mediate a phasic and net excitatory trans-synaptic influence on the cholinergic septohippocampal pathway during working memory testing and thereby significantly contribute to the modulation of the level of working memory performance.

Chronic treatment with amyloid β 1–42 inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling

We investigated the influence of the amyloid-β-peptide 1–42 on hemicholinum-3-sensitive high-affinity choline uptake in NG108-15 cells. RT-PCR analysis revealed the presence of mRNA for a choline transporter-like protein but not for cholinergic high-affinity choline transporter. Differentiation of cells increased both hemicholinum-3-sensitive choline uptake and high-affinity hemicholinium-3 binding. This transport was not influenced by tenfold excess of carnitine. Continuous presence of submicromolar concentrations of amyloid-β-peptide 1–42 during differentiation resulted in a decrease of both choline uptake and hemicholinium-3 binding. These effects were not present when amyloid-β-peptide 1–42 was added 5 min prior to measurements. Neither differentiation nor amyloid-β-peptide 1–42 treatment changed levels of choline transporter-like protein mRNA. Protein kinase C inhibition by staurosporine or its inactivation by continuous presence of tetradecanoyl phorbol acetate prevented the inhibitory effect of amyloid-β-peptide 1–42 treatment on choline uptake. Activation of protein kinase C by tetradecanoyl phorbol acetate during measurement had inhibitory effect on choline uptake in control but not amyloid-β-peptide 1–42-treated cells. The concentration of amyloid-β-peptide 1–42 maximally effective on hemicholinium-3-sensitive choline uptake had no effect on cell growth, oxidative activity, membrane integrity, number of surface muscarinic receptors, caspase-3 and -8 activities, or uptake of deoxyglucose. Results demonstrate that long-term treatment with non-toxic concentrations of amyloid-β-peptide 1–42 downregulates choline uptake presumably mediated by a choline transporter-like protein through activation of protein kinase C signaling. The decrease of choline uptake may have relevance to the pathogenesis of Alzheimer's disease.

Increased Capacity and Density of Choline Transporters Situated in Synaptic Membranes of the Right Medial Prefrontal Cortex of Attentional Task-Performing Rats

Cholinergic neurons innervating the cortex have been conceptualized as a major component of the attention system of the brain. Because of recent evidence indicating plastic mechanisms regulating choline transporter (CHT)-mediated high-affinity choline uptake, which is the rate-limiting step of acetylcholine synthesis, the present experiment determined the capacity of cholinergic terminals to transport choline, and the proportion of choline transporters localized in the membrane of synaptic terminals, in several brain regions of rats performing a cognitive vigilance task (CVT) and a simple reaction time task (SRTT) and nonperforming (NP) rats. Compared with evidence from NP rats, increased choline transporter capacity [as indicated by maximum transporter velocity (Vmax)] and an increased density of CHTs situated in synaptic plasma membrane, relative to intracellular locations, were observed in the medial prefrontal cortex of the right but not left hemisphere of CVT-performing animals. Furthermore, right medial prefrontal Vmax values of CVT-performing rats correlated positively and left medial Vmax values correlated negatively with the animals' performance in signal trials. Measures of CHT function in the brains of SRTT-performing animals did not differ significantly from those in NP rats. The present data support the hypothesis that an increased capacity of choline transporters in the right medial prefrontal cortex, primarily attributable to increased trafficking of transporters from intracellular compartments to the terminal membrane, represents a cellular mechanism contributing to the mediation of attentional performance.

Neuromechanical effects of pyrethroids, allethrin, cyhalothrin and deltamethrin on the cholinergic processes in rat brain

Our previous microdialysis study of freely moving rats demonstrated that 3 pyrethroids, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) differentially modulate acetylcholine (ACh) release in the hippocampus. To better understand the mechanisms of their modulatory effects and also other effects on the cholinergic system in the brain, the activities of ACh hydrolyzing enzyme acetylcholinesterase (AChE), ACh synthesizing enzyme choline acetyltrasferase (ChAT) and ACh synthesizing rate-limiting step, high-affinity choline uptake (HACU) were examined in the present study. The pyrethroids studied had no effect on AChE activity in the cortex, hippocampus and striatum. These pyrethroids had no significant effect on ChAT in the cortex and hippocampus, but striatal ChAT was increased at higher dosage (60 mg/kg) by all three compounds. Lineweaver-Burk analysis of hippocampal HACU revealed that the pyrethroids did not alter the Michaelis-Menten constant (Km) value but caused alteration of maximal velocity (Vmax). Allethrin (60 mg/kg) and cyhalothrin (20 and 60 mg/kg) decreased while deltamethrin (60 mg/kg) increased the Vmax for HACU. In vitro study showed that at higher concentrations (≥ 10 − 6 M) allethrin and cyhalothrin reduced the hippocampal HACU but deltamethrin increased it. These results suggest that mechanisms of ACh synthesis are involved in the modulatory effects of the pyrethroids on ACh release and other cholinergic activities.

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy- N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.

Exposure of Postnatal Rats to a Static Magnetic Field of 0.14 T Influences Functional Laterality of the Hippocampal High-Affinity Choline Uptake System in Adulthood; In vitro Test with Magnetic Nanoparticles

Our previous experiments indicated an age- and sex-dependent functional lateralization of a high-affinity choline uptake system in hippocampi of Wistar rats. The system is connected with acetylcholine synthesis and also plays a role in spatial navigation. The current study demonstrates that a single in vivo exposure of 7- or 14-day-old males to a static magnetic field of 0.14 T for 60-120 min evokes asymmetric alterations in the activity of carriers in adulthood. Namely, the negative field (antiparallel orientation with a vertical component of the geomagnetic field) mediated a more marked decrease in the right hippocampus. The positive field (parallel orientation) was ineffective. Moreover, differences between the carriers from the right and the left hippocampi were observed on synaptosomes pretreated with superparamagnetic nanoparticles and exposed for 30 min in vitro. The positive field enhanced more markedly the activity of carriers from the right hippocampus, the negative that from the left hippocampus, on the contrary. Our results demonstrate functionally teratogenic risks of the alterations in the orientation of the strong static magnetic field for postnatal brain development and suggest functional specialization of both hippocampi in rats. Choline carriers could be involved as secondary receptors in magnetoreception through direct effects of geomagnetic field on intracellular magnetite crystals and nanoparticles applied in vivo should be a useful tool to evaluate magnetoreception in future research.