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In rodent studies, elevated cholinergic neurotransmission in right prefrontal cortex (PFC) is essential for maintaining attentional performance, especially in challenging conditions. Apparently paralleling the rises in acetylcholine seen in rodent studies, fMRI studies in humans reveal right PFC activation at or near Brodmann's areas 9 (BA 9) increases in response to elevated attentional demand. In the present study, we leveraged human genetic variability in the cholinergic system to test the hypothesis that the cholinergic system contributes to the BA 9 response to attentional demand. Specifically, we scanned (BOLD fMRI) participants with a polymorphism of the choline transporter gene that is thought to limit choline transport capacity (Ile89Val variant of the choline transporter gene SLC5A7, rs1013940) and matched controls while they completed a task previously used to demonstrate demand-related increases in right PFC cholinergic transmission in rats and right PFC activation in humans. As hypothesized, we found that although controls showed the typical pattern of robust BA 9 responses to increased attentional demand, Ile89Val participants did not. Further, pattern analysis of activation within this region significantly predicted participant genotype. Additional exploratory pattern classification analyses suggested that Ile89Val participants differentially recruited orbitofrontal cortex and parahippocampal gyrus to maintain attentional performance to the level of controls. These results contribute to a growing body of translational research clarifying the role of cholinergic signaling in human attention and functional neural measures, and begin to outline the risk and resiliency factors associated with potentially suboptimal cholinergic function with implications for disorders characterized by cholinergic dysregulation.
Copyright © 2015 Elsevier Inc. All rights reserved.
Nerves are a common feature of the microenvironment, but their role in tumor growth and progression remains unclear. We found that the formation of autonomic nerve fibers in the prostate gland regulates prostate cancer development and dissemination in mouse models. The early phases of tumor development were prevented by chemical or surgical sympathectomy and by genetic deletion of stromal β2- and β3-adrenergic receptors. Tumors were also infiltrated by parasympathetic cholinergic fibers that promoted cancer dissemination. Cholinergic-induced tumor invasion and metastasis were inhibited by pharmacological blockade or genetic disruption of the stromal type 1 muscarinic receptor, leading to improved survival of the mice. A retrospective blinded analysis of prostate adenocarcinoma specimens from 43 patients revealed that the densities of sympathetic and parasympathetic nerve fibers in tumor and surrounding normal tissue, respectively, were associated with poor clinical outcomes. These findings may lead to novel therapeutic approaches for prostate cancer.
Estradiol has been shown to affect cholinergic modulation of cognition in human and nonhuman animal models. This study examined the brain-based interaction of estradiol treatment and anticholinergic challenge in postmenopausal women during the performance of a working memory task and functional MRI. Twenty-four postmenopausal women were randomly and blindly placed on 1mg oral 17-β estradiol or matching placebo pills for three months after which they participated in three anticholinergic challenge sessions. During the challenge sessions, subjects were administered the antimuscarinic drug scopolamine, the antinicotinic drug mecamylamine, or placebo. After drug administration, subjects completed an fMRI session during which time they performed a visual verbal N-back test of working memory. Results showed that scopolamine increased activation in the left medial frontal gyrus (BA 10) and mecamylamine increased activation in the left inferior frontal gyrus (BA 46). Estradiol treatment compared to placebo treatment significantly reduced the activation in this left medial frontal region during scopolamine challenge. Estradiol treatment also increased activation in the precuneus (BA 31) during mecamylamine challenge. These data are the first to show that estradiol modulated antimuscarinic- and anitnicotinic-induced brain activity and suggest that estradiol affected cholinergic system regulation of cognition-related brain activation in humans.
Copyright Â© 2012 Elsevier Inc. All rights reserved.
The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.
The cholinergic system has consistently been implicated in Pavlovian fear conditioning. Considerable work has been done to localize specific nicotinic receptor subtypes in the hippocampus and determine their functional importance; however, the specific function of many of these subtypes has yet to be determined. An alpha7 nicotinic antagonist methyllycaconitine (MLA) (35 microg), and a broad spectrum non-alpha7 nicotinic antagonist mecamylamine (35 microg) was injected directly into the dorsal hippocampus or overlying cortex either 15 min pre-, 1 min post-, or 6h post-fear conditioning. One week after conditioning, retention of contextual and cue (tone) conditioning were assessed. A significant impairment in retention of contextual fear was observed when mecamylamine was injected 15 min pre- and 1 min post-conditioning. No significant impairment was observed when mecamylamine was injected 6h post-conditioning. Likewise, a significant impairment in retention of contextual fear was observed when MLA was injected 1 min post-conditioning; however, in contrast, MLA did not show any significant impairments when injected 15 min pre-conditioning, but did show a significant impairment when injected 6h post-conditioning. There were no significant impairments observed when either drug was injected into overlying cortex. No significant impairments were observed in cue conditioning for either drug. In general, specific temporal dynamics involved in nicotinic receptor function were found relative to time of receptor dysfunction. The results indicate that the greatest deficits in long-term retention (1 week) of contextual fear are produced by central infusion of MLA minutes to hours post-conditioning or mecamylamine within minutes of conditioning.
Neural and endocrine factors (i.e., Ach and GLP-1) restore defective glucose-stimulated insulin release in pancreatic islets lacking sulfonylurea type 1 receptors (SUR1(-/-)) (Doliba NM, Qin W, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, and Matschinsky FM. Am J Physiol Endocrinol Metab 286: E834-E843, 2004). The goal of the present study was to assess fuel-induced respiration in SUR1(-/-) islets and to correlate it with changes in intracellular Ca(2+), insulin, and glucagon secretion. By use of a method based on O(2) quenching of phosphorescence, the O(2) consumption rate (OCR) of isolated islets was measured online in a perifusion system. Basal insulin release (IR) was 7-10 times higher in SUR1(-/-) compared with control (CON) islets, but the OCR was comparable. The effect of high glucose (16.7 mM) on IR and OCR was markedly reduced in SUR1(-/-) islets compared with CON. Ach (0.5 microM) in the presence of 16.7 mM glucose caused a large burst of IR in CON and SUR1(-/-) islets with minor changes in OCR in both groups of islets. In SUR1(-/-) islets, high glucose failed to inhibit glucagon secretion during stimulation with amino acids or Ach. We conclude that 1) reduced glucose responsiveness of SUR1(-/-) islets may be in part due to impaired energetics, as evidenced by significant decrease in glucose-stimulated OCR; 2) elevated intracellular Ca(2+) levels may contribute to altered insulin and glucagon secretion in SUR1(-/-) islets; and 3) The amplitudes of the changes in OCR during glucose and Ach stimulation do not correlate with IR in normal and SUR1(-/-) islets suggesting that the energy requirements for exocytosis are minor compared with other ATP-consuming reactions.
OBJECTIVE - Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves.
METHODS - Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison.
RESULTS - Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node.
CONCLUSIONS - Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.
Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.
We have utilized a guinea pig in vitro brain slice preparation of the medial septum (MS) and the vertical and horizontal limbs of the nucleus of the diagonal band of Broca (nDBB) to identify and classify different cell types within cholinergic nuclei. Utilizing a double-labeling technique which pairs intracellular injection of the fluorescent dye Lucifer yellow with acetylcholinesterase (AChE) histochemistry, we were able to correlate electrophysiological characteristics with a specific cholinergic cell marker. We report that at least two cell groups can be identified electrophysiologically within the MS/nDBB complex, and one population of neurons demonstrates distinct electrophysiological characteristics that are highly correlated with positive AChE-staining.