| [Purpose]Propofol, an emulsion formulation of 2,6-diisopropylphenol, which is a rapid-acting sedative-hypnotic medication, has been widely used for the induction and maintenance of anesthesia, as well as for sedation in intensive care units. Propofol is a global central nervous system depressant, and is also most common side effects are dose-dependent cardiorespiratory depression. It is known that propofol activates γ-aminobutyric acid (GABAa) receptors directly, inhibits the N-methyl-D-aspartate (NMDA) receptor and modulates calcium influx through slow calcium-ion channels. Under in vitro conditions, propofol inhibits neuronal activities of spinal cord, hipocampus and hypothalamus neurons via activation of GABAA and glycine receptors. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. We aim to understand the mechanisms of propofol affect the spontaneous simple spike and complex spike activities of cerebellar PCs in living animals.[Methods]Adult (6-8 weeks old) ICR mice were anesthetized by intraperitoneal injection of urethane (1.3 g/kg body weight)。The mice tracheotomized to avoid respiratory obstruction, and fixed on a custom-made stereotaxic frame. After a watertight chamber was created, a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Crus II (inferior semilunar lobule). The dura mater was carefully removed, and the brain surface was constantly superfused with oxygenated artificial cerebrospinal fluid (ACSF) with a peristaltic pump. Cell-attached recordings from PCs were performed with an Axopatch-200B amplifier (Molecular Devices, Foster City, CA). The PC spontaneous activities were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampfit 10.3 software. Recording electrodes were filled with ACSF and with resistances of 3-5 MΩ. PCs were identified by the presence of both simple and complex spikes under cell-attached recording conditions. Spontaneous activity was calculated from a train of interspike intervals recorded for 100 sec. Electrophysiological data were analyzed using Clampfit 10.3 software. All values are expressed as the mean±SEM, and differences were evaluated by the Student’s paired t-test or one-way ANOVA using SPSS 17.0 software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results](1) Cerebellar PCs express regular spontaneous SS firing accompanied with irregular CS activity. Cerebellar surface perfusion of propofol (10-1000 μM) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner. The IC50 of propofol for inhibiting SS firing of PCs was 144.5 μM.(2) High concentration of propofol (500μM) induced completely inhibition of the PC simple spike (SS) firing rate, but without significantly changing the frequency and properties of complex spikes (CS).(3) Application of GABAA receptor antagonist, SR95531 (40 μM) or GABAB receptor antagonist, saclofen (20 μM), as well as glycine receptor antagonist, strychnine (10μM) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity.(4) Application the mixture of SR95531 (40 μM) and strychnine (10μM) completely blocked the propofol-induced inhibition of PC SS firing.[Conclusions](1) These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions.(2) The functional glycine receptor exits in cerebellar PCs and contributes to the anesthetic action of propofol in cerebellar cortex.[Purpose]Propofol is an intravenous sedative-hypnotic agent with emnestic properties, which causes rapid and reliable loss of consciousness. The molecular mechanisms of propofol anesthetic action are related to activation of GABAA, glycine receptors directly, and inhibition of the N-methyl-D-aspartate (NMDA) receptor, as well as modulation of calcium influx. Under in vivo conditions, propofol decreases information processing in the neocortex in a dose-dependent manner. However, the effects of propofol on the sensory information processing in cerebellar cortex under in vivo conditions are currently unknown. Therefore, we here examined the effects of propofol on sensory stimulation-evoked responses in the cerebellar PCs of urethane-anesthetized mice, by electrophysiological and pharmacological methods. We aim to understand the mechanisms of propofol affect cerebellar PCs to transfer the sensory information in living animals.[Methods]Adult (6-8 weeks old) ICR mice were anesthetized by intraperitoneal injection of urethane (1.3 g/kg body weight), then tracheotomized to avoid respiratory obstruction, and fixed on a custom-made stereotaxic frame. After a watertight chamber was created, a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Crus Ⅱ (inferior semilunar lobule). The dura mater was carefully removed, and the brain surface was constantly superfused with oxygenated artificial cerebrospinal fluid (ACSF) with a peristaltic pump. Cell-attached recordings from PCs were performed with an Axopatch-200B amplifier (Molecular Devices, Foster City, CA). The potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.3 software. Recording electrodes were filled with ACSF and with resistances of 3-5 MΩ. PCs were identified by the presence of both simple and complex spikes under cell-attached recording conditions. Spontaneous activity was calculated from a train of interspike intervals recorded for 100 sec. Electrophysiological data were analyzed using Clampfit 10.3 software. Facial stimulation was performed by air-puff (60 ms, 50-60 psi) of ipsilateral whisker pad. The stimulation of air-puff was controlled by master 8 and a personal computer. The air-puff stimulations were also synchrinized with the electrophysiologial recordings. All values are expressed as the mean±SEM, and differences were evaluated by the Student’s paired t-test or one-way ANOVA using SPSS 17.0 software. P values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results](1) Air-puff stimulation on ipsilateral whisker pad evoked a sequence of negative component (N1) followed by a positive component (P1), accompanied with a pause of SS firing.(2) Administration of propofol induced a decrease in amplitude of P1 in a dose-dependent manner. The IC50 of propofol induced-inhibition of P1 was 360 μM.(3) Perfusion of propofol inhibited spontaneous activity and sensory stimulation evoked responses PI, but induced a significant increase in amplitude of N1.(4) Application of GABAA receptor antagonist, SR95531 (40 μM) not only blocked the sensory stimulation evoked P1, but also revealed the sensory stimulation evoked SS firing of the PCs.(5) In the presence of GABAA receptor antagonist, propofol (300μM) completely inhibited the spontaneous SS firing, but induced an increase in the sensory stimulation evoked SS firing rate.[Conclusions](1) These data indicated that propofol significant inhibited sensory stimulation evoked molecular layer interneuron-PC GABAergic synaptic transmission, but enhanced the sensory stimulation evoked parallel fiber -PC excitatory synaptic inputs via modulation of GABAA receptors activity in adult cerebellar PCs under in vivo conditions.(2) Cerebellar cortex may also be involved in the mechanism of the central anesthesia of propofol. |
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