Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1-mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligandbinding experiments with cell-permeable and -impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1-mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic-binding sites were abolished in M1-mAChR-gene-knockout mice. Activation of cell surface M1-mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long-term potentiation. On the other hand, activation of intracellular M1-mAChRs phosphorylated extracellular-regulated kinase 1/2 and gradually enhanced long-term potentiation. Our data thus demonstrate that M1-mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system. Keywords: cell surface and intracellular GPCR, ERK1/2, LTP, M1-muscarinic receptor, synaptic plasticity. Among the five muscarinic acetylcholine receptor (mAChR) subtypes, M1-mAChRs predominantly exist in the CNS and are involved in cognitive enhancement (Wess 2004;

Functional acetylcholine receptors (AChRs) were recently demonstrated to exist not only in the plasma membrane but also intracellularly in brain tissues. In order to activate intracellular AChRs, endogenous hydrophilic ACh must cross the plasma membrane. Here, we examined the pharmacological characteristics of this process, including whether it is mediated by active ACh uptake. When ACh esterase (AChE) was suppressed by diisopropylfluorophosphate, [ H]ACh was effectively taken up into segments of rat cerebral cortex and other brain regions, in contrast to peripheral tissues such as liver and kidney. The uptake of [ H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [ H]choline. However, [ H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [ H]choline. Uptake of [ H]ACh was also inhibited by several organic cations, including choline, hemicholinium-3 (HC-3), quinidine, decynium 22, clonidine, diphenhydramine, but was little affected by some amino acids and biogenic amines, corticosterone, spermine, atropine, and tetrodotoxin. Unlike diisopropylfluorophosphate, several ACh esterase inhibitors, including drugs for Alzheimer's disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [ H]ACh, but not [ H]choline. These results indicate that in the brain, ACh is specifically taken up through a unique transport system with different pharmacological properties from known organic cation transporters (OCTs), and suggest that this mechanism may be involved in intracellular cholinergic transmission in the brain.

The effects of Polygala tenuifolia root fractions and the acyl groups of its constituents on the retrieval process of spatial cognition in rats were studied using an eight-arm radial maze task. Oral administration of a precipitate fraction (PTB) obtained by concentration of the n-BuOH-soluble portion from the extract of the roots significantly decreased the number of total errors (TEs) and that of working memory errors (WMEs) at doses of 100 mg/kg and 200 mg/kg. However, it caused no significant decrease in the number of reference memory errors (RMEs). In addition, the saponin-rich fraction (PTBM) obtained by purification of PTB also showed significant decreases in TEs and WMEs at a dose of 100 mg/kg. Among the cinnamic acid derivatives present as the acyl groups in the P. tenuifolia constituents, sinapic acid (SNPA) significantly decreased TEs and WMEs at doses of 10 to 100 mg/kg. These results indicated that P. tenuifolia extracts, PTB and PTBM, and SNPA had a beneficial effect on the memory impairment induced by dysfunction of the cholinergic system in the brain. The memory improvement in the scopolamine-induced memory impairment seen in the radial maze performance was due to improvement in the short-term memory. A contribution of some constituents other than SNPA to the memory improvement was also suggested.

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.

Kava-kava extract is an herbal medicine having not only hypnotic effects, but also sleep quality-enhancement effects.

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 μM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 μM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of −70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

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