2018 Fiscal Year Research-status Report
Molecular targets of volatile anesthetics isoflurane at the calyx of Held synapse.
| Project/Area Number |
18K16467
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
WANG Han・Ying 沖縄科学技術大学院大学, 細胞分子シナプス機能ユニット, 研究員 (70814333)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | Isoflurane / Calyx of Held / exocytic machinery / neurotransmission / Action potential |
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| Outline of Annual Research Achievements |
Volatile anesthetics are widely utilized for surgery since the nineteenth century. At inhibitory synapses, volatile anesthetics prolong postsynaptic responses and, at both excitatory and inhibitory synapses, they inhibit neurotransmitter release. Various mechanisms are proposed for presynaptic inhibitory effects of volatile anesthetics. Of all potential mechanisms, it remains unidentified which mechanism operates primarily for inhibiting transmitter release. Baumgart et al (2015) have shown that isoflurane inhibits presynaptic Ca2+ influx induced by a single action potential (AP). However, it remains open whether this mechanism operates for inhibiting repetitive transmission. It also remains unidentified by what mechanism isoflurane inhibits Ca2+ influx.
Hence, using the calyx of Held in rat brainstem slices, we first addressed the presynaptic targets of isoflurane. Our results indicated that isoflurane inhibits presynaptic voltage-gated Ca2+ channels and exocytic machinery downstream of Ca2+ influx, with the former and latter mechanisms predominantly operating for inhibiting brief and prolonged exocytosis, respectively. Simultaneous pre- and postsynaptic AP recording at physiological temperature revealed that isoflurane blocks high-frequency neurotransmission but has little effect on low-frequency transmission. We propose that isoflurane directly inhibits transmitter release machinery, thereby inhibiting high-frequency excitatory transmission that underlies consciousness, while preserving life-support neurotransmission at low frequency.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We have almost finished slice recording experiments including dose-response of isoflurane in EPSC at calyx of Held synapse, variance-mean analysis for estimation of quantal parameters with or without isoflurane, presynaptic action potential waveform and ionic currents recordings before and after isoflurane application, and presynaptic capacitance and calcium current charge analysis with and without isoflurane. We found isoflurane significantly reduces excitatory neurotransmission at calyx of Held synapse by inhibiting exocytic machinery downstream of Ca2+ influx. As excitatory neurotransmission requires postsynaptic AP generation and it is blocked when the peak of excitatory postsynaptic potential declines below firing threshold, we are currently performing simultaneous pre- and post-recording to clarify the effect of isoflurane on excitatory neurotransmission. Our preliminary paired-recording data shows that isoflurane reduced the fidelity of postsynaptic APs followed high frequency presynaptic APs in a dose-dependent and frequency-dependent manners, suggesting direct inhibition of exocytic machinery seems the primary mechanism for isoflurane anesthesia.
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| Strategy for Future Research Activity |
We will address the effect of isoflurane on central synaptic transmission at cortico-cortical excitatory synapses in awake head-restrained mice. Whisker primary motor cortex (wM1) is anatomically and functionally connected to whisker primary somatosensory cortex (wS1) via mutual monosynaptic excitatory connections (Welker et al., 1988; Ferezou et al., 2007; Aronoff et al., 2010; Mao et al., 2011; Zagha et al., 2013; Sreenivasan et al., 2016). Using a layer 5 (L5)-specific Cre-expressing mouse line crossed with Cre-dependent channelrhodopsin-2-expressing mouse line, we will evok APs at different frequency in wS1 neurons by optical stimulation (1-ms blue light pulse) of L5 pyramidal neurons in wM1. Optically evoked APs will be recorded extracellularly from wS1 neurons using a 32-channel silicone probe. In awake states, before isoflurane inhalation, 0.2-Hz and 2-Hz photo-pulse (1 ms) stimulation will be delivered to evoke APs. These APs (including spontaneous and evoked) will be monitored in wS1 neurons as control. When isoflurane be inhaled at 1.4% (~1 Mac), both spontaneous and evoked APs, will expect to be inhibited. The inhibitory effect of isoflurane on the rate of evoked AP within the 25-ms window will be compared whether it is frequency-dependent or not between 0.2-Hz and 2-Hz stimulation. These in vivo experiments will give us a hint that isoflurane not only attenuates global excitability of cortical neurons, but also preferentially inhibits high-frequency excitatory neurotransmission.
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| Causes of Carryover |
Consumables cost is less than expected. The remaining budget will be used for in vivo recording experiment.
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Research Products
(2 results)
