Neural Subtype-dependent Cholinergic Modulation of Neural Activities by Activation of Muscarinic 2 Receptors and G Protein-activated Inwardly Rectifying Potassium Channel in Rat Periaqueductal Gray Neurons

Article

Author: Kaneko, Ryosuke ; Saito, Yasuhiko ; Matsumura, Sachie ; Kobayashi, Masayuki ; Hirose, Kensuke ; Nakaya, Yuka ; Sugawara, Shiori

Acetylcholine plays a pivotal role in the regulation of functions such as pain and the sleep and wake cycle by modulating neural activities of the ventrolateral periaqueductal gray (vlPAG). Electrophysiological studies have shown that cholinergic effects are inconsistent among recorded neurons, particularly in the depolarization and hyperpolarization of the resting membrane potential (RMP). This discrepancy may be due to the neural subtype-dependent cholinergic modulation of the RMP. To examine this possibility, we performed whole-cell patch-clamp recordings from subtype-identified neurons using vesicular GABA transporter (VGAT)-Venus × ChAT-TdTomato rats and elucidated cellular mechanisms of cholinergic effects on the RMP. The application of carbachol hyperpolarized the RMP of cholinergic neurons in a dose-dependent manner but had much less of an effect on other neural subtypes, including GABAergic/glycinergic and glutamatergic neurons. Cholinergic hyperpolarization was accompanied by a decrease in input resistance. These cholinergic effects were blocked by AF-DX384 or gallamine and were mimicked by arecaidine but-2-ynyl ester tosylate, suggesting that the carbachol-induced hyperpolarization of the RMP in cholinergic neurons is mediated via M₂ receptors. Tertiapin suppressed the carbachol-induced G protein-activated inwardly rectifying potassium channel (GIRK) currents and hyperpolarization of the RMP in cholinergic neurons. Intracellular application of GDP-β-S blocked the carbachol-induced hyperpolarization of the RMP. Neostigmine slowly hyperpolarized the RMP in cholinergic neurons. These results suggest that neural firing of vlPAG cholinergic neurons is suppressed by GIRK currents induced via M₂ receptor activation, and this negative feedback regulation of cholinergic neuronal activities can be induced by acetylcholine, which is intrinsically released in the vlPAG.

01 Dec 2021·Pflügers Archiv - European Journal of PhysiologyQ4 · MEDICINE

Differential regulation of medium spiny and cholinergic neurons in the nucleus accumbens core by the insular and medial prefrontal cortices in the rat

Q4 · MEDICINE

Article

Author: Yanagawa, Yuchio ; Kaneko, Ryosuke ; Saito, Yasuhiko ; Kitano, Kohei ; Shirakawa, Tetsuo ; Kobayashi, Masayuki ; Yamamoto, Kiyofumi ; Hirose, Kensuke ; Nakaya, Yuka

The nucleus accumbens (NAc) receives cortical projections principally from the insular cortex (IC) and medial prefrontal cortex (mPFC). Among NAc neurons, cholinergic interneurons (ChNs) regulate the activities of medium spiny neurons (MSNs), which make up ~ 95% of NAc neurons, by modulating their firing and synaptic properties. However, little is known about the synaptic mechanisms, including their cell-type-dependent corticoaccumbal projection properties and cholinergic effects on the NAc core. Here, we performed whole-cell patch-clamp recordings from NAc MSNs and ChNs in acute brain slice preparations obtained from rats that received an AAV5-hSyn-ChR2(H134R)-mCherry injection into the IC or mPFC. Light stimulation of IC or mPFC axons induced comparable phase-locked excitatory postsynaptic currents (EPSCs) in MSNs. On the other hand, ChNs showed consistent EPSCs evoked by light stimulation of mPFC axons, whereas light stimulation of IC axons evoked much smaller EPSCs, which often showed failure in ChNs. Light-evoked EPSCs were abolished by tetrodotoxin and were recovered by 4-aminopyridine, suggesting that corticoaccumbal projections monosynaptically induce EPSCs in MSNs and ChNs. Carbachol effectively suppressed the amplitude of EPSCs in MSNs and ChNs evoked by light stimulation of IC or mPFC axons and in ChNs evoked by stimulating mPFC axons. The carbachol-induced suppression was recovered by atropine or pirenzepine, while preapplication of gallamine, J104129, PD102807, or AF-DX384 did not block the carbachol-induced EPSC suppression. These results suggest that NAc MSNs and ChNs are differentially regulated by excitatory projections from the IC and mPFC and that these corticoaccumbal excitatory inputs are modulated by M₁ receptor activation.

01 Dec 2018·Nature Chemical BiologyQ1 · BIOLOGY

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor

Q1 · BIOLOGY

Article

Author: Kinoshita, Masahiro ; Iwata, So ; Maeda, Shoji ; Horita, Shoichiro ; Kobilka, Brian K ; Vaidehi, Nagarajan ; Hirata, Kunio ; Kobayashi, Takuya ; Yasuda, Satoshi ; Yamamoto, Masaki ; Yamashita, Keitaro ; Suno, Ryoji ; Tsujimoto, Hirokazu ; Murata, Takeshi ; Tawaramoto, Maki S ; Lee, Sangbae

Human muscarinic receptor M₂ is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M₂ receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M₂ was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M₂ receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M₂ receptors compared to M₃ receptors leads to subtype selectivity of AF-DX 384.

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Indication	Highest Phase	Country/Location	Organization	Date
Dementia	Preclinical	-	C.H. Boehringer Sohn AG & Co. KG	-

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