Hemicholinium-3 Binding Research Articles

Paternal Δ9-Tetrahydrocannabinol Exposure Prior to Mating Elicits Deficits in Cholinergic Synaptic Function in the Offspring.

Little attention has been paid to the potential impact of paternal marijuana use on offspring brain development. We administered Δ9-tetrahydrocannabinol (THC, 0, 2, or 4 mg/kg/day) to male rats for 28 days. Two days after the last THC treatment, the males were mated to drug-naïve females. We then assessed the impact on development of acetylcholine (ACh) systems in the offspring, encompassing the period from the onset of adolescence (postnatal day 30) through middle age (postnatal day 150), and including brain regions encompassing the majority of ACh terminals and cell bodies. Δ9-Tetrahydrocannabinol produced a dose-dependent deficit in hemicholinium-3 binding, an index of presynaptic ACh activity, superimposed on regionally selective increases in choline acetyltransferase activity, a biomarker for numbers of ACh terminals. The combined effects produced a persistent decrement in the hemicholinium-3/choline acetyltransferase ratio, an index of impulse activity per nerve terminal. At the low THC dose, the decreased presynaptic activity was partially compensated by upregulation of nicotinic ACh receptors, whereas at the high dose, receptors were subnormal, an effect that would exacerbate the presynaptic defect. Superimposed on these effects, either dose of THC also accelerated the age-related decline in nicotinic ACh receptors. Our studies provide evidence for adverse effects of paternal THC administration on neurodevelopment in the offspring and further demonstrate that adverse impacts of drug exposure on brain development are not limited to effects mediated by the embryonic or fetal chemical environment, but rather that vulnerability is engendered by exposures occurring prior to conception, involving the father as well as the mother.

Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.

Prenatal nicotine alters the developmental neurotoxicity of postnatal chlorpyrifos directed toward cholinergic systems: better, worse, or just "different?".

This study examines whether prenatal nicotine exposure sensitizes the developing brain to subsequent developmental neurotoxicity evoked by chlorpyrifos, a commonly-used insecticide. We gave nicotine to pregnant rats throughout gestation at a dose (3mg/kg/day) producing plasma levels typical of smokers; offspring were then given chlorpyrifos on postnatal days 1-4, at a dose (1mg/kg) that produces minimally-detectable inhibition of brain cholinesterase activity. We evaluated indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, in brain regions possessing the majority of ACh projections and cell bodies; we measured nicotinic ACh receptor binding, hemicholinium-3 binding to the presynaptic choline transporter and choline acetyltransferase activity, all known targets for the adverse developmental effects of nicotine and chlorpyrifos given individually. By itself nicotine elicited overall upregulation of the ACh markers, albeit with selective differences by sex, region and age. Likewise, chlorpyrifos alone had highly sex-selective effects. Importantly, all the effects showed temporal progression between adolescence and adulthood, pointing to ongoing synaptic changes rather than just persistence after an initial injury. Prenatal nicotine administration altered the responses to chlorpyrifos in a consistent pattern for all three markers, lowering values relative to those of the individual treatments or to those expected from simple additive effects of nicotine and chlorpyrifos. The combination produced global interference with emergence of the ACh phenotype, an effect not seen with nicotine or chlorpyrifos alone. Given that human exposures to nicotine and chlorpyrifos are widespread, our results point to the creation of a subpopulation with heightened vulnerability.

Letters to the Editor: Cardiomyocyte‐secreted acetylcholine

I read the paper, “Cardiomyocyte-secreted acetylcholine is required for maintenance of homeostasis in the heart” (1), on acetylcholine (ACh) originating in cardiac myocytes, with great interest. The significance of this phenomenon continues to fascinate since it was first described over 40 years ago. The authors have overlooked reports and experimental limits from the earlier papers that deserve careful consideration. Edouard Coraboeuf and colleagues (2) reported in 1970 that cardiac myocytes could synthesize and release ACh. Of note, they obtained data from embryonic hearts before innervation occurred. They also observed that electrical stimulation of the embryonic chick heart caused a transient slowing of heart rate and an increased release of ACh (2). The transient slowing was opposed by atropine. So far, more recent reports (1, 3) have not asked whether stimuli can regulate ACh secretion. Divergent results on the age of animals whose heart cells can secrete ACh are another unsettled issue. You report that myocytes from neonatal mice secrete ACh. This is opposite the report by Rana et al. (3), who state that “… adult, but not neonatal cardiomyocytes are able to synthesize, transport and excrete acetylcholine in the rat heart.” This discrepancy calls for an explanation, inasmuch as rats and mice develop similarly after nearly equal gestation periods. The immunofluorescent detection of choline acetyltransferase (ChAT) requires validation of specificity. Some years ago (4), it was reported that the method to detect ChAT must carefully distinguish ChAT from carnitine acetyltransferase, which is found in cardiac muscle. Thus, Roskoski et al. (4) showed that embryonic chick heart cells, per se, had no ChAT activity and no neurons. Neonatal rat heart cells in culture had no ChAT, but carnitine acetyltransferase was present. Thus, in the absence of cholinergic neurons, heart cells did not synthesize ACh (4). Does the immunofluorescent method you use distinguish between ChAT and carnitine acetyltransferase? The experiments with carbachol and pyridostigmine overlook important drug properties. It is reported that addition of carbachol caused ACh release from cardiac myocytes, as indicated by increased NO production, a downstream fluorescent signal (1). There are concerns with this observation. First, cholinergic neurons have muscarinic receptors whose occupancy by ACh reduces ACh release (5). This negative-feedback mechanism, a physiological hallmark of homeostasis, seems lacking in your system. Second, “… pyridostigmine also augmented the fluorescent signal (Supplemental Fig. S2F).” Pyridostigmine, like neostigmine (and carbachol), has a quaternary N that not only renders it impermeable to cell membranes but also makes it an agonist acting directly at cholinoceptive sites, just like carbachol. Thus, pyridostigmine and neostigmine can activate receptors, in addition to inhibiting acetylcholinesterase. Similarly, hemicholinium-3 (HC-3) is another quaternary N compound whose actions against pyridostigmine can be explained by HC-3 binding to the same site as pyridostigmine and occluding its effect. Indeed, one should test vesamicol and HC-3 against carbachol, as was done against pyridostigmine in Supplemental Fig. 2SF. Such testing should clarify some essential elements of the hypothesis. The current state of the problem seems not to have evolved significantly from that described by earlier investigators. This is a consequence, in part, of a failure to attend to precedent.

Transgenic overexpression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance

The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC–CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [3H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC–CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC–CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC–CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC–CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC–CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.

Prenatal dexamethasone augments the sex-selective developmental neurotoxicity of chlorpyrifos: Implications for vulnerability after pharmacotherapy for preterm labor

Glucocorticoids are routinely given in preterm labor and are also elevated by maternal stress; organophosphate exposures are virtually ubiquitous, so coexposures to these two agents are pervasive. We administered dexamethasone to pregnant rats on gestational days 17–19 at a standard therapeutic dose (0.2mg/kg); offspring were then given chlorpyrifos on postnatal days 1–4, at a dose (1mg/kg) that produces barely-detectable (<10%) inhibition of brain cholinesterase activity. We evaluated indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, in brain regions possessing the majority of ACh projections and cell bodies; we measured nicotinic ACh receptor binding, hemicholinium-3 binding to the presynaptic choline transporter and choline acetyltransferase activity, all known targets for the adverse developmental effects of dexamethasone and chlorpyrifos given individually. Dexamethasone did not enhance the systemic toxicity of chlorpyrifos, as evidenced by weight gain and measurements of cholinesterase inhibition during chlorpyrifos treatment. Nevertheless, it enhanced the loss of presynaptic ACh function selectively in females, who ordinarily show sparing of organophosphate developmental neurotoxicity relative to males. Females receiving the combined treatment showed decrements in choline transporter binding and choline acetyltransferase activity that were unique (not found with either treatment alone), as well as additive decrements in nicotinic receptor binding. On the other hand, males given dexamethasone showed no augmentation of the effects of chlorpyrifos. Our findings indicate that prior dexamethasone exposure could create a subpopulation that is especially vulnerable to the adverse effects of organophosphates or other developmental neurotoxicants.

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

Nicotine Exposure during the Third Trimester Equivalent of Human Gestation: Time Course of Effects on the Central Cholinergic System of Rats

Up to 22% of pregnant women smoke, which constitutes a major health concern. Nicotine, a cholinergic agonist, causes deleterious effects on brain development. However, most studies investigate its effects during rodents' gestation, which corresponds, in terms of neural development, to the first two trimesters of human gestation. Here, we focused on effects of nicotine on the brain cholinergic system during the third trimester equivalent of human gestation. From the 2nd to the 19th day of lactation, dams were exposed either to nicotine (6 mg/kg/day) or to saline via sc osmotic minipumps. Offspring were sacrificed during exposure (PN15, PN, postnatal) or at 2 days (PN21), 11 days (PN30), or 10 weeks (PN90) of withdrawal. In the cerebral cortex, midbrain, and hippocampus, we assessed nicotinic acetylcholine receptor (nAChR) binding, [(3)H]hemicholinium-3 (HC-3) binding to the high-affinity choline transporter, choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) activities. Nicotine-exposed offspring presented nAChR upregulation during exposure in all brain regions, reduced HC-3 binding during and 11 days postexposure, and increased HC-3 binding on PN90. Effects on ChAT and AChE were dependent on the brain region and restricted to the withdrawal period: There were increased activities in the midbrain on PN30. In the hippocampus, AChE as reduced on PN30, whereas, for ChAT, the decrease was followed by late-emergent increased activity. These data indicate that maternal nicotine exposure during the third trimester equivalent of human gestation promotes cholinergic system alterations in the offspring's brain. In addition, detrimental effects are observable even long after the exposure has been interrupted.

A novel choline cotransporter sequestration compartment in cholinergic neurons revealed by selective endosomal ablation

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT. Selective ablation of early sorting and recycling endosomes resulted in a decrease of approximately 75% of [3H]choline uptake and approximately 70% of [3H]hemicholinium-3 binding. Western blot analysis showed that ablation produced a similar decrease in ChCoTs in the plasma membrane subcellular fraction. The time frame for this loss was approximately 2 h which has been shown to be the constitutive cycling time for ChCoTs in this tissue. Ablation appears to be dependent on the intracellular cycling of transferrin-conjugated horseradish peroxidase and the selective deposition of transferrin-conjugated horseradish peroxidase in early endosomes, both sorting and recycling. Ablated brain slices retained their capacity to recruit via regulated trafficking ChCoTs to the plasma membrane. This recruitment of ChCoTs suggests that the recruitable compartment is distinct from the early endosomes. It will be necessary to do further studies to identify the novel sequestration compartment supportive of the ChCoT regulated trafficking.

Early postnatal parathion exposure in rats causes sex-selective cognitive impairment and neurotransmitter defects which emerge in aging

Developmental exposure of rats to the organophosphate (OP) pesticides leads to altered neurobehavioral function in juvenile and young adult stages. The current study was conducted to determine whether effects of neonatal parathion exposure on cognitive performance persist in older adult and aged rats, and the relationship of behavioral changes to underlying cholinergic and serotonergic mechanisms. We administered parathion to rat pups on postnatal days 1–4, at doses spanning the threshold for the initial signs of systemic toxicity and for barely detectable cholinesterase inhibition (0.1 or 0.2 mg/kg/day). Beginning at 14 months of age and continuing until 19 months, the rats were trained in the 16-arm radial maze. Controls showed the normal sex difference in this spatial learning and memory task, with the males committing significantly fewer working memory errors than females. Neonatal parathion exposure eliminated the sex difference primarily by causing impairment in males. In association with the effects on cognitive performance, neonatal parathion exposure elicited widespread abnormalities in indices of serotonergic (5HT) and cholinergic synaptic function, characterized by upregulation of 5HT 2 receptors and the 5HT transporter, deficits in choline acetyltransferase activity and nicotinic cholinergic receptors, and increases in hemicholinium-3 binding to the presynaptic choline transporter. Within-animal correlations between behavior and neurochemistry indicated a specific correlation between working memory performance and hippocampal hemicholinium-3 binding; parathion exposure eliminated this relationship. Like the behavioral effects, males showed greater effects of parathion on neurochemical parameters. This study demonstrates the sex-selective, long-term behavioral alterations caused by otherwise nontoxic neonatal exposure to parathion, with effects increasingly expressed with aging.

Additive and synergistic effects of fetal nicotine and dexamethasone exposure on cholinergic synaptic function in adolescence and adulthood: Implications for the adverse consequences of maternal smoking and pharmacotherapy of preterm delivery

Maternal smoking contributes to preterm delivery; glucocorticoids are the consensus treatment for prematurity, thus producing fetal coexposure to nicotine and dexamethasone. We administered nicotine to pregnant rats throughout gestation at a dose (3 mg/kg/day) producing plasma levels typical of smokers. Later in gestation, animals received dexamethasone (0.2 mg/kg). We assessed developmental indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, evaluating brain regions possessing major ACh projections and cell bodies; we measured choline acetyltransferase activity, hemicholinium-3 binding to the presynaptic choline transporter and nicotinic ACh receptor binding. In general, nicotine and dexamethasone, alone or in combination, produced regionally-selective increases or decreases in choline acetyltransferase activity but larger, consistent elevations in hemicholinium-3 and nicotinic ACh receptor binding; the patterns were indicative of ACh synaptic hyperactivity. Superimposed on these overall effects, there were significant disparities in temporal and regional relationships among the different treatments, notably involving effects that emerged later in life, after a period of apparent normality. This indicates that nicotine and dexamethasone do not simply produce an initial ACh neuronal injury that then persists throughout the lifespan but rather, they alter the developmental trajectory of ACh function. Most importantly, the combined exposure to nicotine + dexamethasone elicited greater changes than either of the individual exposures, involving both additive and synergistic effects. Our results thus point to potentially worse neurobehavioral outcomes of the pharmacotherapy of preterm labor in the offspring of smokers.

Fat Chance? A High-Fat Diet May Offset the Effects of Developmental Neurotoxicity

Widespread exposure to a variety of neurotoxic chemicals has been posited as one potential factor behind what has been called a “silent pandemic” of autism spectrum disorders, learning disabilities, and other neurodevelopmental disorders. Exposure to organophosphate insecticides is of particular concern because these widely used compounds have been shown in rodents to induce persistent synaptic abnormalities in neural acetylcholine (ACh) systems at doses too low to cause symptoms of systemic exposure. Pilot studies have reported some evidence of improvement when a “ketogenic” diet—high in fat and low in carbohydrates—was used to treat certain neurologic disorders. Drawing from this preliminary clinical research, researchers have now demonstrated that many of the abnormalities in ACh systems produced by neonatal organophosphate exposure were not evident in adult rats fed a high-fat diet [EHP 117:916–922; Slotkin et al.]. Rats were injected with the organophosphate parathion on each of postnatal days 1–4, at doses of 0.1 or 0.2 mg/kg/day—these dosages straddle the threshold at which cholinesterase inhibition is first detectable. In adulthood, half the animals were switched to a high-fat diet for 8 weeks. The investigators then examined brain regions of the rats to assess specific aspects of ACh synaptic function, including nicotinic ACh receptor binding, choline acetyltransferase activity, and hemicholinium-3 binding to the presynaptic choline transporter. Adult rats on a standard lab chow diet showed multiple abnormalities in regional ACh synaptic markers following parathion exposure. All seven abnormalities observed in parathion-exposed females on the standard diet were absent in exposed females on the high-fat diet, and eight of ten abnormalities observed in parathion-exposed males on the standard diet were absent in exposed males on the high-fat diet. The results suggest that diet may offer a way to ameliorate the effects of developmental neurotoxicant exposure. However, the authors offer several caveats. Their earlier work showed that neonatal exposure to organophosphates produced long-term changes in metabolic function that have been linked with obesity, prediabetes, and cardiovascular risk factors such as elevated serum lipids. Because these metabolic abnormalities could be exacerbated by a high-fat diet, future studies should seek to uncover whether and how specific aspects of the diet influence abnormalities in ACh systems. Moreover, although this study showed that dietary modifications may offset synaptic changes, future studies will need to determine whether such modifications can actually lead to improved neurobehavioral outcomes. The authors also highlight a potential connection between early-life toxicant exposure and subsequent diet-related disease. If a high-fat diet can indeed ameliorate the impact of developmental neurotoxicants, then this might serve as an underlying, subconscious reinforcement to consume a high-fat diet as a way of self-remediating underlying neurobehavioral deficits—potentially expanding the public health implications of the developmental effects of neurotoxicant exposure.

Combined exposure to nicotine and ethanol in adolescent mice: effects on the central cholinergic systems during short and long term withdrawal

Relapse to drug use is a major public health problem. In this sense, understanding the biological substrates that are affected during withdrawal may provide information to prevent relapse. Both smoking and alcoholic beverage consumption usually begin during adolescence, however, little is known about the basic neurobiology of the combined adolescent exposure, particularly during withdrawal. Since nicotine is a cholinergic agonist and it has been shown that ethanol interferes with nicotinic acetylcholine receptors (nAChRs), the current study focused on the effects of drug withdrawal on the central cholinergic system. From the 30th to the 45th postnatal day (PN), C57BL/6 male and female mice were exposed to nicotine free base (NIC) and/or ethanol (ETOH). Four groups were analyzed: (1) concomitant NIC (50 μg/ml in 2% saccharin to drink) and ETOH (25%, 2 g/kg i.p. injected every other day) exposure; (2) NIC exposure; (3) ETOH exposure; (4) vehicle. We assessed nAChR binding, choline acetyltransferase (ChAT) activity and [3H]hemicholinium-3 (HC-3) binding in the cerebral cortex and midbrain of mice at short (PN50) and long term (PN75) withdrawal. NIC and NIC+ETOH promoted nAChR upregulation during a short-term withdrawal. NIC short-term withdrawal elicited an increase in ChAT activity that was reversed by ETOH withdrawal. In addition, NIC+ETOH elicited a decrease in ChAT activity at long term withdrawal. Regarding HC-3, ETOH and NIC+ETOH promoted a decrease that persisted at long-term withdrawal. The present study provides experimental evidence that nicotine and ethanol during adolescence interact resulting in cholinergic system alterations during withdrawal.

Consumption of a High-Fat Diet in Adulthood Ameliorates the Effects of Neonatal Parathion Exposure on Acetylcholine Systems in Rat Brain Regions

BackgroundDevelopmental exposure to a wide variety of developmental neurotoxicants, including organophosphate pesticides, evokes late-emerging and persistent abnormalities in acetylcholine (ACh) systems. We are seeking interventions that can ameliorate or reverse the effects later in life.ObjectivesWe administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could reverse the effects on ACh systems.MethodsNeonatal rats received parathion on postnatal days 1–4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals were switched to a high-fat diet for 8 weeks. We assessed three indices of ACh synaptic function: nicotinic ACh receptor binding, choline acetyltransferase activity, and hemicholinium-3 binding. Determinations were performed in brain regions comprising all the major ACh projections and cell bodies.ResultsNeonatal parathion exposure evoked widespread abnormalities in ACh synaptic markers, encompassing effects in brain regions possessing ACh projections and ACh cell bodies. In general, males were affected more than females. Of 17 regional ACh marker abnormalities (10 male, 7 female), 15 were reversed by the high-fat diet.ConclusionsA high-fat diet reverses neurodevelopmental effects of neonatal parathion exposure on ACh systems. This points to the potential for nonpharmacologic interventions to offset the effects of developmental neurotoxicants. Further, cryptic neurodevelopmental deficits evoked by environmental exposures may thus engender a later preference for a high-fat diet to maintain normal ACh function, ultimately contributing to obesity.

Regional changes in the cholinergic system in mice lacking monoamine oxidase A

Elevated brain monoamine concentrations resulting from monoamine oxidase A genetic ablation (MAOA knock-out mice) lead to changes in other neurotransmitter systems. To investigate the consequences of MAOA deficiency on the cholinergic system, we measured ligand binding to the high-affinity choline transporter (CHT1) and to muscarinic and nicotinic receptors in brain sections of MAOA knock-out (KO) and wild-type mice. A twofold increase in [ 3H]-hemicholinium-3 ([ 3H]-HC-3) binding to CHT1 was observed in the caudate putamen, nucleus accumbens, and motor cortex in MAOA KO mice as compared with wild-type (WT) mice. There was no difference in [ 3H]-HC-3 labeling in the hippocampus (dentate gyrus) between the two genotypes. Binding of [ 125I]-epibatidine ([ 125I]-Epi), [ 125I]-α-bungarotoxin ([ 125I]-BGT), [ 3H]-pirenzepine ([ 3H]-PZR), and [ 3H]-AFDX-384 ([ 3H]-AFX), which respectively label high- and low-affinity nicotinic receptors, M1 and M2 muscarinic cholinergic receptors, was not modified in the caudate putamen, nucleus accumbens, and motor cortex. A small but significant decrease of 19% in M1 binding densities was observed in the hippocampus (CA1 field) of KO mice. Next, we tested acetylcholinesterase activity and found that it was decreased by 25% in the striatum of KO mice as compared with WT mice. Our data suggest that genetic deficiency in MAOA enzyme is associated with changes in cholinergic activity, which may account for some of the behavioral alterations observed in mice and humans lacking MAOA.

Nicotine and ethanol interact during adolescence: Effects on the central cholinergic systems

Co-occurrence of tobacco smoking and alcohol consumption during adolescence is frequent and well documented. However, little is known about the basic neurobiology of the combined exposure in the adolescent brain. Since nicotine is a cholinergic agonist and it has been shown that ethanol interferes with nicotinic acetylcholine receptors (nAChRs), the current work focused on cholinergic systems. From the 30th to the 45th postnatal day (PN), C57BL/6 male and female mice were exposed to nicotine free base (NIC) and/or ethanol (ETOH). Four groups were analyzed: 1) concomitant NIC (50 μg/ml in 2% saccharin to drink) and ETOH (25%, 2 g/kg i.p. injected every other day) exposure; 2) NIC exposure; 3) ETOH exposure; 4) vehicle. We assessed nAChR binding, choline acetyltransferase (ChAT) activity and [ 3H]hemicholinium-3 (HC-3) binding in the cerebral cortex and midbrain of mice on PN45. In the cortex, ETOH had no effect on nAChRs. In contrast, NIC produced nAChR upregulation while NIC + ETOH elicited a more pronounced effect. In the midbrain, neither ETOH nor NIC had effects on nAChRs. NIC + ETOH, however, elicited a robust nAChR upregulation. Regarding ChAT activity, treatment effects differed between males and females in the cortex. Male NIC mice presented an increase in ChAT. However, ETOH reversed this effect. In contrast, female NIC mice presented decreased ChAT activity. In the midbrain, ETOH increased ChAT. HC-3 binding was not affected. These results indicate that the central cholinergic system is a site at which nicotine and ethanol interact. This interaction might underlie the association between tobacco and alcohol consumption during adolescence.

Developmental neurotoxic effects of chlorpyrifos on acetylcholine and serotonin pathways in an avian model

The developmental neurotoxicity of organophosphates such as chlorpyrifos (CPF) involves multiple mechanisms that ultimately compromise the function of specific neurotransmitter systems, notably acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT). Studies in mammalian models incorporate both direct effects on brain development and indirect effects mediated through maternal physiology and maternal/neonatal interactions. We examined the effects of CPF in an avian model, which does not share these potential confounds. Chick eggs were injected with CPF (10 or 20 mg/kg) on incubation days 2 and 6 and markers of ACh and 5HT systems were examined at hatching. The higher dose caused a reduction in cholinesterase activity but there was no consistent downregulation of m 2-muscarinic ACh receptors as would have been expected from ACh hyperstimulation. Both doses evoked significant reductions in the presynaptic high-affinity choline transporter, the rate-limiting factor in ACh biosynthesis, as monitored by binding of hemicholinium-3. Choline acetyltransferase, a constitutive marker for ACh terminals, was unaffected. This suggests that CPF reduces ACh presynaptic activity rather than compromising the development of ACh projections per se. CPF exposure also reduced the expression of cerebrocortical 5HT 1A receptors. These effects in the chick model recapitulate many of the actions of early gestational CPF exposure in rats, and thus suggest that CPF exerts direct actions on the immature brain to compromise the development of ACh and 5HT pathways.

Adolescent nicotine treatment changes the response of acetylcholine systems to subsequent nicotine administration in adulthood

Nicotine alters the developmental trajectory of acetylcholine (ACh) systems in the immature brain, with vulnerability extending from fetal stages through adolescence. We administered nicotine to adolescent rats (postnatal days PN30–47) and then examined the subsequent response to nicotine given in adulthood (PN90–107), simulating plasma levels in smokers, and performing evaluations during nicotine treatment (PN105) and withdrawal (PN110, PN120 and PN130), as well as assessing persistent changes at 6 months of age (PN180). We measured nicotinic acetylcholine receptor (nAChR) binding, choline acetyltransferase (ChAT) activity, a marker for ACh terminals, and hemicholinium-3 (HC3) binding to the choline transporter, an index of ACh presynaptic activity. By itself, adolescent nicotine exposure evoked sex-selective deficits in cerebrocortical HC3 binding while elevating ChAT in young adulthood in striatum and midbrain. Nicotine given in adulthood produced profound nAChR upregulation lasting 2 weeks after discontinuing treatment, and decrements in cerebrocortical and striatal HC3 binding emerged during withdrawal, indicative of reduced ACh synaptic activity. For all three parameters, adolescent nicotine altered the responses to nicotine given in adulthood, producing both sensitization and desensitization that depended on sex and brain region, effects that parallel the disparate behavioral outcomes reported for these treatments. The interaction seen here for the impact of adolescent nicotine exposure on adult nicotine responses was substantially greater than that found previously for the effects of prenatal nicotine exposure on adult responses. Our findings thus reinforce the importance of adolescence as a critical period in which the future responsiveness to nicotine is programmed.

Activity and Subcellular Trafficking of the Sodium-Coupled Choline Transporter CHT Is Regulated Acutely by Peroxynitrite

Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.

Separate or sequential exposure to nicotine prenatally and in adulthood: Persistent effects on acetylcholine systems in rat brain regions

Nicotine is a developmental neurotoxicant but the proposed “sensitization-homeostasis” model postulates that even in adulthood nicotine permanently reprograms synaptic function. We administered nicotine to rats throughout gestation or in adulthood (postnatal days PN90–107), simulating plasma levels in smokers, with evaluations on PN105, PN110, PN120, PN130 and PN180. We assessed nicotinic acetylcholine receptor (nAChR) binding, choline acetyltransferase activity, a marker for acetylcholine (ACh) terminals, and hemicholinium-3 (HC3) binding to the choline transporter, an index of ACh presynaptic activity. Prenatal nicotine exposure elicited persistent deficits in HC3 binding in male cerebral cortex and female striatum, but little change in other parameters. Nicotine given in adulthood produced profound nAChR upregulation lasting 2 weeks after discontinuing treatment. Decrements in cerebrocortical and striatal HC3 binding emerged during withdrawal and persisted through PN180, indicative of reduced ACh synaptic activity. Prenatal nicotine did not evoke any major alterations in the response to nicotine given in adulthood. The effects seen here are substantially different from those found previously for nicotine given to adolescent rats, which showed more prolonged nAChR upregulation and profound, widespread and persistent deficits in markers of ACh synaptic function; for adolescents, prenatal nicotine exposure desensitized nAChR responses, exacerbated withdrawal-induced ACh functional deficits, and worsened the long-term outcome. Our results indicate that the effects of nicotine during prenatal or adolescent stages are indeed distinct from the effects in adults, but that even adults show persistent changes after nicotine exposure, commensurate with the sensitization-homeostasis model. These effects may contribute to lifelong vulnerability to readdiction.