PubMed · 14958753
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1952-06-01. [Acetylcholine].. https://pubmed.ncbi.nlm.nih.gov/14958753/
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The modulation of ACh on delayed rectifier-like potassium currents (I(K)) was studied in freshly dissociated cerebral cortical neurons using the whole-cell patch-clamp technique. Wistar rats between 10- and 14-day old of both sexes were used. After rats were decapitated, their brains were quickly removed, iced, and then manually cut into 400 mum slices. Slices were then incubated for 0.5 h at 32 degrees C in a buffered artificial cerebrospinal fluid (ACSF) bubbled with 95% O2, 5% CO2. Slices were then removed into buffered ACSF containing protease (0.5 mg/ml) at 32 degrees C. After 30 min of enzyme digestion, tissue was rinsed three times in the buffered saline. Then the enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35 mm dish and placed on the stage of a Olympus inverted microscope. For whole-cell recordings of currents, standard voltage-clamp techniques were used. Neurons were held at -80 mV, and the I(K) was evoked by 2 000 ms depolarizing voltage commands to potential between -40 mV and +60 mV in 10 mV steps applied at a frequency of 0.5 Hz. It was found that the inhibitory effect of ACh (0.1, 1, 10, 100 mumol/L) on I(K) was dose-dependent. It was also found that ACh affected the activation process of I(K) significantly, i.e., the activation curve of I(K) was characterized by half-activation potential of (-41.8+/-9.7) mV and a slope factor of (30.7+/-7.2) mV in the cortical neurons and they were changed to (-122.4+/-38.6) mV and (42.4+/-7.0) mV, respectively, after giving ACh (10 mumol/L). Tubocurarine (100 mumol/L) antagonized the inhibitory effect of ACh on I(K), and the drop of currents varied from the control value of (36.5+/-7..8)% to (16.9+/-13.8)% (n=8, P<0.01). 4-DAMP (10 mumol/L) blocked the inhibitory effect of ACh on I(K), and the currents reduced from the control value of (36.5+/-7.8)% to (26.8+/-4.7) % (n=6, P<0.05). Pirenzepin did not antagonize the inhibition of ACh on I(K) (n=7, P>0.05). Chelerythrine (20 mumol/L) blocked the inhibitory effect of ACh on I(K) and the currents reduced from the control value of (36.5+/-7.8)% to (11.7+/-17.3)% (n=6, P<0.05). On the contrary, PDBu (10 mumol/L) strengthened the inhibition of ACh on I(K) and the drop of currents changed from the control value of (36.5+/-7.8)% to (59.2+/-14.0)% (n=5, P<0.05). PDBu abolished the antagonism of chelerythrine on ACh in cortical neurons. It is suggested that the ACh-induced depolarization of neurons in the cortex is attributed to the inhibition of I(K) that is most likely evoked by the activation of nicotinic ACh receptors and muscarinic M3 receptor via protein kinase C (PKC) signal transduction pathway.
Cholinergic afferents to the ventral tegmental area (VTA) contribute substantially to the regulation of motivated behaviors and the rewarding properties of nicotine. These actions are believed to involve connections with dopamine (DA) neurons projecting to the nucleus accumbens (NAc). However, this direct synaptic link has never been investigated, nor is it known whether cholinergic inputs innervate other populations of DA and gamma-aminobutyric acid (GABA) neurons, including those projecting to the prefrontal cortex (PFC). We addressed these questions by using electron microscopic analysis of retrograde tract-tracing and immunocytochemistry for the vesicular acetylcholine transporter (VAChT) and for tyrosine hydroxylase (TH) and GABA. In tissue labeled for TH, VAChT(+) terminals frequently synapsed onto DA mesoaccumbens neurons but only seldom contacted DA mesoprefrontal cells. In tissue labeled for GABA, one-third of VAChT(+) terminals innervated GABA-labeled dendrites, including both mesoaccumbens and mesoprefrontal populations. VAChT(+) synapses onto DA and mesoaccumbens neurons were more commonly of the asymmetric (presumed excitatory) morphological type, whereas VAChT(+) synapses onto GABA cells were more frequently symmetric (presumed inhibitory or modulatory). These findings suggest that cholinergic inputs to the VTA mediate complex synaptic actions, with a major portion of this effect likely to involve an excitatory influence on DA mesoaccumbens neurons. As such, the results suggest that natural and drug rewards operating through cholinergic afferents to the VTA have a direct synaptic link to the mesoaccumbens DA neurons that modulate approach behaviors.
Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.