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Perioperative Medicine  |   July 2014
Fast-spiking Cell to Pyramidal Cell Connections Are the Most Sensitive to Propofol-induced Facilitation of GABAergic Currents in Rat Insular Cortex
Author Affiliations & Notes
  • Yuko Koyanagi, D.D.S., Ph.D.
    From the Department of Anesthesiology (Y.K., Y.O.), and Department of Pharmacology (K.Y., N.K., M.K.), Nihon University School of Dentistry, Tokyo, Japan; Division of Immunology and Pathobiology (Y.K., Y.O.), and Division of Oral and Craniomaxillofacial Research (K.Y., N.K., M.K.), Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan; and Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Kobe, Japan (M.K.).
  • Yoshiyuki Oi, M.D., Ph.D.
    From the Department of Anesthesiology (Y.K., Y.O.), and Department of Pharmacology (K.Y., N.K., M.K.), Nihon University School of Dentistry, Tokyo, Japan; Division of Immunology and Pathobiology (Y.K., Y.O.), and Division of Oral and Craniomaxillofacial Research (K.Y., N.K., M.K.), Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan; and Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Kobe, Japan (M.K.).
  • Kiyofumi Yamamoto, Ph.D.
    From the Department of Anesthesiology (Y.K., Y.O.), and Department of Pharmacology (K.Y., N.K., M.K.), Nihon University School of Dentistry, Tokyo, Japan; Division of Immunology and Pathobiology (Y.K., Y.O.), and Division of Oral and Craniomaxillofacial Research (K.Y., N.K., M.K.), Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan; and Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Kobe, Japan (M.K.).
  • Noriaki Koshikawa, D.D.S., Ph.D.
    From the Department of Anesthesiology (Y.K., Y.O.), and Department of Pharmacology (K.Y., N.K., M.K.), Nihon University School of Dentistry, Tokyo, Japan; Division of Immunology and Pathobiology (Y.K., Y.O.), and Division of Oral and Craniomaxillofacial Research (K.Y., N.K., M.K.), Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan; and Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Kobe, Japan (M.K.).
  • Masayuki Kobayashi, D.D.S., Ph.D.
    From the Department of Anesthesiology (Y.K., Y.O.), and Department of Pharmacology (K.Y., N.K., M.K.), Nihon University School of Dentistry, Tokyo, Japan; Division of Immunology and Pathobiology (Y.K., Y.O.), and Division of Oral and Craniomaxillofacial Research (K.Y., N.K., M.K.), Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan; and Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Kobe, Japan (M.K.).
  • Presented at the 59th Annual Meeting of the Japanese Society of Anesthesiologists, Kobe, Japan, June 7–9, 2012, the 40th Annual Meeting of the Japanese Dental Society of Anesthesiology, Fukuoka, Japan, October 4–6, 2012, the 60th Annual Meeting of the Japanese Society of Anesthesiologists, Sapporo, Japan, May 23–25, 2013, and the 41th Annual Meeting of the Japanese Dental Society of Anesthesiology, Yokohama, Japan, October 2–4, 2013.
    Presented at the 59th Annual Meeting of the Japanese Society of Anesthesiologists, Kobe, Japan, June 7–9, 2012, the 40th Annual Meeting of the Japanese Dental Society of Anesthesiology, Fukuoka, Japan, October 4–6, 2012, the 60th Annual Meeting of the Japanese Society of Anesthesiologists, Sapporo, Japan, May 23–25, 2013, and the 41th Annual Meeting of the Japanese Dental Society of Anesthesiology, Yokohama, Japan, October 2–4, 2013.×
  • Submitted for publication September 3, 2013. Accepted for publication January 17, 2014.
    Submitted for publication September 3, 2013. Accepted for publication January 17, 2014.×
  • Address correspondence to Dr. Kobayashi: Department of Pharmacology, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101–8310, Japan. kobayashi.masayuki@nihon-u.ac.jp. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Basic Science / Pharmacology
Perioperative Medicine   |   July 2014
Fast-spiking Cell to Pyramidal Cell Connections Are the Most Sensitive to Propofol-induced Facilitation of GABAergic Currents in Rat Insular Cortex
Anesthesiology 07 2014, Vol.121, 68-78. doi:10.1097/ALN.0000000000000183
Anesthesiology 07 2014, Vol.121, 68-78. doi:10.1097/ALN.0000000000000183
Abstract

Background:: Propofol facilitates γ-aminobutyric acid–mediated inhibitory synaptic transmission. In the cerebral cortex, γ-aminobutyric acidergic interneurons target both excitatory pyramidal cells (Pyr) and fast-spiking (FS) and non-FS interneurons. Therefore, the propofol-induced facilitation of inhibitory transmission results in a change in the balance of excitatory and inhibitory inputs to Pyr. However, it is still unknown how propofol modulates γ-aminobutyric acidergic synaptic transmission in each combination of Pyr and interneurons.

Methods:: The authors examined whether propofol differentially regulates inhibitory postsynaptic currents (IPSCs) depending on the presynaptic and postsynaptic cell subtypes using multiple whole cell patch clamp recording from γ-aminobutyric acidergic interneurons and Pyr in rat insular cortex.

Results:: Propofol (10 μM) consistently prolonged decay kinetics of unitary IPSCs (uIPSCs) in all types of inhibitory connections without changing paired-pulse ratio of the second to first uIPSC amplitude or failure rate. The FS→Pyr connections exhibited greater enhancement of uIPSC charge transfer (2.2 ± 0.5 pC, n = 36) compared with that of FS→FS/non-FS connections (0.9 ± 0.2 pC, n = 37), whereas the enhancement of charge transfer in non-FS→Pyr (0.3 ± 0.1 pC, n = 15) and non-FS→FS/non-FS connections (0.2 ± 0.1 pC, n = 36) was smaller to those in FS→Pyr/FS/non-FS. Electrical synapses between FS pairs were not affected by propofol.

Conclusions:: The principal inhibitory connections (FS→Pyr) are the most sensitive to propofol-induced facilitation of uIPSCs, which is likely mediated by postsynaptic mechanisms. This preferential uIPSC enhancement in FS→Pyr connections may result in suppressed neural activities of projection neurons, which in turn reduces excitatory outputs from cortical local circuits.

In brain slices from the insular cortex, propofol preferentially enhanced fast spiking GABAergic interneuron connections to pyramidal neurons. This suppresses excitatory output from the cortex.

What We Already Know about This Topic
  • Propofol facilitates γ-aminobutyric acid–mediated inhibitory synaptic transmission and reduces excitatory output from cortical circuits. How excitatory output is reduced is not clear.

What This Article Tells Us That Is New
  • In brain slices from the insular cortex, propofol preferentially enhanced fast-spiking γ-aminobutyric acidergic interneuron connections to pyramidal neurons. This suppresses excitatory output from the cortex.

PROPOFOL modulates several ionic channel activities such as γ-aminobutyric acid type A (GABAA) receptors, glycine receptors, voltage-dependent potassium channels, sodium channels, and L-type calcium channels.1  Among these ionic channels, GABAA receptors, which involve Cl channels, are considered to be the principal target of propofol.2,3  Nelson et al.4  reported that systemic administration of the GABAA receptor antagonist gabazine abolishes the propofol-induced loss-of-righting reflex in rats, indicating that the sedative effect induced by propofol is mediated by GABAA receptors. In vitro electrophysiological studies have revealed that propofol potentiates GABAA receptor–mediated Cl currents recorded from cultured rat cortical neurons5  and oocytes expressing human GABAA receptors.6,7  In addition, propofol slows the decay time constant of spontaneous inhibitory postsynaptic currents (IPSCs) without changing the IPSC amplitude in cultured rat cortical neurons.8  The facilitative effects of propofol on GABAA receptors are caused by increasing the open probability of GABA-gated Cl channels.5,9 
Cortical local circuits consist of excitatory and inhibitory neurons, both of which receive GABAergic inhibitory inputs. GABAergic inputs to excitatory neurons are potentiated by propofol, which reduces excitatory outputs from cortical local circuits. However, propofol-induced facilitation of GABAergic currents in inhibitory neurons may result in disinhibition of excitatory neurons. Although anesthesia does not necessarily require selective inhibition of the excitatory transmission,10  examining the type of connection most sensitive to propofol, that is, inhibitory neurons to either excitatory neurons or inhibitory neurons, is crucial for better understanding the mechanisms of propofol-induced anesthesia.
Multiple subtypes of GABAergic interneurons11,12  induce heterogeneous modulations of neural functions. For example, chandelier cells, a part of fast-spiking (FS) cells, are axo-axonic cells that may be crucial in modulating the generation of action potentials of cortical cells,13  whereas regular-spiking, low-threshold-spike (LTS), and late-spiking cells make symmetrical synapses onto dendrites and somata.14  Moreover, FS and LTS show different spontaneous IPSC characteristics depending on the subunit composition of their GABAA receptors.15  Therefore, propofol may differentially modulate inhibitory synaptic transmission based on the subtypes of projecting and targeting neurons. However, little is known about the effects of propofol on different types of GABAergic synapses. In addition, no information is available about the effect of propofol on electrical synapses in the cerebral cortex that are frequently observed between inhibitory neurons of the same type, especially FS,16,17  although several studies demonstrate the propofol-induced suppression of gap junction in rat-cultured hippocampal18  and P19 cells.19 
The current study tested the hypothesis that inhibitory synaptic transmission mediated by GABA is differentially regulated by propofol depending on the presynaptic and postsynaptic cell subtypes in the insular cortex, where multimodal sensation including nociception, gustation, and visceral sensation are processed.20–23  Furthermore, an effect of propofol on electrical synapses was examined in FS pairs. We found that connections between presynaptic FS and postsynaptic pyramidal cells (Pyr) have the largest propofol-induced increases in unitary IPSC (uIPSC) charge transfer and that propofol did not change junctional conductance of electrical synapses.
Materials and Methods
All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Nihon University, Tokyo, Japan. All efforts were made to minimize the number and suffering of animals used in experiments.
Slice Preparations
The techniques for preparing and maintaining rat cortical slices in vitro were similar to those previously described.24–26  In brief, vesicular GABA transporter Venus line A transgenic rats11,27  of either sex, ranging in age from 14 to 34 days, were deeply anesthetized with sodium pentobarbital (100 mg/kg, intraperitoneal) and decapitated. Tissue blocks including the insular cortex were rapidly removed and stored for 3 min in ice-cold modified artificial cerebrospinal fluid (ACSF) (230 mM of sucrose, 2.5 mM of KCl, 10 mM of MgSO4, 1.25 mM of NaH2PO4, 26 mM of NaHCO3, 0.5 mM of CaCl2, and 10 mM of d-glucose). Coronal slices were cut at 350-μm thickness using a microslicer (Linearslicer Pro 7; Dosaka EM, Kyoto, Japan). Slices were incubated at 32°C for 40 min in a submersion-type holding chamber that contained 50% modified ACSF and 50% normal ACSF (pH, 7.35 to 7.40). Normal ACSF contained: 126 mM of NaCl, 3 mM of KCl, 2 mM of MgSO4, 1.25 mM of NaH2PO4, 26 mM of NaHCO3, 2 mM of CaCl2, and 10 mM of d-glucose. Modified and normal ACSF were continuously aerated with a mixture of 95% O2–5% CO2. Slices were then placed in normal ACSF at 32°C for 1 h and were maintained thereafter at room temperature until used for recording.
Cell Identification and Paired Whole Cell Patch Clamp Recording
Slices were transferred to a recording chamber that was continuously perfused with normal ACSF at a rate of 2.0 ml/min. Dual or triple whole cell patch clamp recordings were obtained from Venus-positive fluorescent interneurons and Venus-negative Pyr identified in layer V by a fluorescence microscope equipped with Nomarski optics (×40; Olympus BX51W1, Tokyo, Japan) and an infrared-sensitive video camera (C3077-78; Hamamatsu Photonics, Hamamatsu, Japan; fig. 1, A and B). The distance between recorded cells was less than 100 μm. Electrical signals were recorded by amplifiers (Multiclamp 700B; Molecular Devices, Sunnyvale, CA), digitized (Digidata 1440A; Molecular Devices), observed on-line, and stored on a computer hard disk using Clampex (pClamp 10; Molecular Devices).
Fig. 1.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
Fig. 1.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
×
The composition of the pipette solution for recordings was: 70 mM of potassium gluconate, 70 mM of KCl, 10 mM of HEPES, 2 mM of MgCl2, 2 mM of magnesium adenosine triphosphate, 0.3 mM of sodium guanosine triphosphate, 0.5 mM of EGTA, and 15 mM of biocytin. The pipette solution had a pH of 7.3 and an osmolarity of 300 mOsm. The liquid junction potentials for current-clamp and voltage-clamp recordings were −9 mV, and the voltage was not corrected. Thin-wall borosilicate patch electrodes (2 to 5 MΩ) were pulled on a micropipette puller (P-97; Sutter Instruments, Novato, CA).
Recordings were obtained at 30° ± 1°C. The seal resistance was greater than 5 GΩ, and the only data obtained from electrodes with an access resistance of 6 to 20 MΩ and less than 20% change during recordings were included in this study. Before the uIPSC recordings, the voltage responses of pre- and postsynaptic cells were recorded by the application of long hyperpolarizing and depolarizing current pulse (300 to 1,000 ms) injections to examine basic electrophysiological properties, including the input resistance, single-spike kinetics, voltage–current relationship, and repetitive firing patterns (fig. 1C). Because some cell pairs had mutual or two or more connections, all cells were recorded under voltage clamp conditions (holding potential = −60 mV) during uIPSC recording. Short depolarizing voltage-step pulses (2 ms, 80 mV) were applied to the presynaptic cells to induce action currents, and postsynaptic responses (uIPSCs) were simultaneously recorded (fig. 1D). For evaluation of electrical coupling among FS, hyperpolarizing voltage pulses from −60 to −100 mV (300 ms) were applied to one of the potentially coupled FS, and current responses in the other FS were recorded under voltage clamp mode (fig. 1E).
2,6-Diisopropylphenol (propofol; Sigma-Aldrich, St. Louis, MO) was dissolved in dimethyl sulfoxide at a concentration of 10 mM and diluted to 10 μM in the perfusate. In part, 0.1 to 100 μM propofol was applied to examine the dose-dependency. The membrane currents and potentials were low-pass filtered at 5 to 10 kHz and digitized at 20 kHz.
Data Analysis
Clampfit (pClamp 10; Molecular Devices) was used for analyses of electrophysiological data. The uIPSC amplitudes were measured as the difference between the peak postsynaptic currents and the baseline currents taken from a 2- to 3-ms time window close to the onset of the uIPSCs. An uIPSC amplitude in the range of synaptic noise was considered a failure. The uIPSC amplitude, 20 to 80% rise time, 80 to 20% decay time, decay time constant, and charge transfer were measured from single uIPSC-averaged traces, which were obtained from 10 to 20 consecutive traces and aligned to the negative peak of presynaptic action currents. Because the non-FS→non-FS connections showed high failure rate, uIPSCs with less than 5 pA were excluded from the averaged traces. The paired-pulse ratio (PPR) of the second to the first uIPSC amplitude and the failure rate were calculated from 10 to 20 consecutive sweeps. The amplitude of the electrical coupling conductance was measured by dividing the current evoked in an FS in response to the voltage-step pulses (40 mV) in the coupled FS. The dose–response curve of uIPSC charge transfer in response to propofol was fitted by logistic function by using a software (Origin 9; OriginLab, Northampton, MA).
Histology
To visualize biocytin-labeled neurons after whole cell patch clamp recording, slices were fixed and cryoprotected. Sections were processed by the ABC method (Vector Laboratories, Burlingame, CA), and fluorescence was visualized with Alexa 488–conjugated streptavidin (Molecular Probes, Eugene, OR). Slices were examined and imaged with a fluorescent microscope (BZ-9000; Keyence, Osaka, Japan). All chemicals were purchased from Sigma-Aldrich unless otherwise specified.
Statistical Analysis
The data are presented as the mean ± SEM. Comparisons of the uIPSC amplitude, PPR, 20 to 80% rise time, 80 to 20% decay time, and decay time constant between the control and propofol application were conducted using a two-tailed paired t test. The effect of propofol on the amplitude of the electrical coupling conductance was also analyzed with the two-tailed paired t test. The Wilcoxon signed-rank test was used for comparison of the failure rate of uIPSCs between control and drug application. Comparison of the drug-induced changes in the uIPSC charge transfer between pre- and postsynaptic cell pairs was conducted by the Mann–Whitney U test or two-tailed Student t test. The statistical analyses were performed using IBM SPSS Statistics (Ver. 21.0; IBM, Chicago, IL), and values of P less than 0.05 were defined as significant.
Results
Multiple whole cell patch clamp recordings from GABAergic interneurons and Pyr in the insular cortex layer V were used to examine the propofol-induced modulation of GABAergic synaptic transmission (fig. 1). GABAergic interneurons were identified as the Venus-positive cells (Fig. 1Ab), which were further classified into FS, LTS, late-spiking, and regular-spiking based on their electrical membrane and firing properties.12,24–26  FS were characterized by their low-input resistance, short duration of action potential and afterhyperpolarization, and high frequency of repetitive firing (fig. 1C). In contrast, LTS, late-spiking, and regular-spiking showed a wide variation of passive membrane and spike-firing properties. Because the propofol-induced modulation of uIPSCs was not significantly different among the LTS, late-spiking, and regular-spiking, the current study categorized them as non-FS. According to this categorization, the inhibitory synaptic connections were classified into six groups: (1) FS to Pyr (FS→Pyr), (2) FS to FS (FS→FS), (3) FS to non-FS (FS→non-FS), (4) non-FS to Pyr (non-FS→Pyr), (5) non-FS to FS (non-FS→FS), and (6) non-FS to non-FS (non-FS→non-FS). We recorded uIPSCs during application of normal ACSF with dimethyl sulfoxide (0.1%, vol/vol) for 5 to 10 min as a control. Next, propofol dissolved in dimethyl sulfoxide (0.1%, vol/vol) was applied for 7.5 to 10 min.
Propofol-induced Enhancement of uIPSCs in FS→Pyr/FS/Non-FS Connections
Figure 2 shows a typical example of uIPSC modulation by propofol in the FS→Pyr/FS/non-FS connections. As previously reported in the miniature excitatory postsynaptic currents/IPSC analysis,8  propofol (10 μM) increased the rise and decay kinetics of uIPSCs. Figure 2E showed the dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The effect of propofol was consistently observed at 10 μM, and therefore, propofol was applied at this concentration in the current study.
Fig. 2.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
Fig. 2.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
×
Propofol (10 μM) increased the following uIPSC kinetics: 20 to 80% rise time from 0.7 ± 0.0 to 0.9 ± 0.1 ms, n = 36 (fig. 3A); 80 to 20% decay time from 12.6 ± 0.6 to 25.1 ± 1.5 ms, n = 36 (fig. 3B); and a decay time constant from 11.1 ± 0.9 to 21.5 ± 1.7 ms, n = 34 (fig. 3C). Contrary to the miniature study,8  the uIPSC amplitude was significantly increased by propofol (104.4 ± 17.9 to 125.7 ± 21.2 pA, n = 36; fig. 3D). In addition to these changes in the uIPSC kinetics, our uIPSC recordings revealed that the PPR and failure rate of the first uIPSCs were not significantly changed by propofol (fig. 3, E and F). These results suggest that propofol-induced facilitation of uIPSCs is mediated by postsynaptic mechanisms that include an increase in the open probability of GABAA receptors.5,9 
Fig. 3.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Fig. 3.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
×
Similar to the FS→Pyr connections, FS→FS connections showed the propofol-induced enhancement of the 20 to 80% rise time (0.4 ± 0.0 to 0.5 ± 0.1 ms, n = 22; fig. 3A), 80 to 20% decay time (6.3 ± 0.5 to 12.9 ± 1.5 ms, n = 22; fig. 3B), decay time constant (4.8 ± 0.4 to 11.2 ± 1.4 ms, n = 22; fig. 3C), and amplitude (169.7 ± 34.3 to 201.4 ± 37.9 pA, n = 22; fig. 3D). In the FS→non-FS connections, propofol increased the 80 to 20% decay time (12.2 ± 0.8 to 18.9 ± 2.0 ms, n = 16; fig. 3B) and decay time constant (9.8 ± 0.7 to 15.2 ± 1.9 ms, n = 16; fig. 3C) but had little effect on the 20 to 80% rise time (0.7 ± 0.1 to 0.6 ± 0.1 ms, n = 16; fig. 3A) and amplitude (51.5 ± 13.3 to 70.5 ± 26.1 pA, n = 16; fig. 3D). In FS→FS and FS→non-FS connections, the PPR and failure rate were not significantly changed by propofol (fig. 3, E and F). Thus, the uIPSC recordings from FS→Pyr/FS/non-FS demonstrated that propofol facilitates uIPSCs by postsynaptic mechanisms.
Little Effect of Propofol on Electrical Synapses between FS Pairs
In agreement with the previous studies, paired whole cell recordings from FS frequently exhibited electrical synapses (figs. 1E and 4, A and B).16,17  To examine whether propofol modulate the strength of electrical coupling conductance between FS, evoked currents responding to the hyperpolarizing voltage-step pulses from −60 to −100 mV were recorded before and during propofol application. The amplitude of electrical coupling conductance was not significantly changed by propofol (463.8 ± 51.8 to 453.9 ± 49.8 pS, n = 25; P > 0.54) (fig. 4, C and D).
Fig. 4.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
Fig. 4.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
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Propofol-induced Enhancement of uIPSCs in Non-FS→FS/Non-FS/Pyr Connections
Figure 5 shows a typical example of uIPSC modulation by propofol in the non-FS→Pyr/FS/non-FS connections. The 20 to 80% rise time was significantly increased by propofol (1.4 ± 0.3 to 2.1 ± 0.4 ms [n = 15] in non-FS→Pyr, 0.6 ± 0.0 to 0.8 ± 0.1 ms [n = 29] in non-FS→FS, and 0.6 ± 0.1 to 0.8 ± 0.1 ms [n = 7] in non-FS→non-FS connections) (fig. 6A). Propofol increased the 80 to 20% decay time from 11.3 ± 0.9 to 19.5 ± 1.7 ms (n = 15) in non-FS→Pyr, from 7.8 ± 0.7 to 12.3 ± 1.4 ms (n = 29) in non-FS→FS, and from 11.2 ± 2.0 to 20.8 ± 3.1 ms (n = 6) in non-FS→non-FS connections (fig. 6B). The decay time constant of uIPSCs was also increased by propofol (11.8 ± 2.0 to 18.6 ± 2.0 ms [n = 13] in non-FS→Pyr, 5.8 ± 0.4 to 10.2 ± 1.1 ms [n = 29] in non-FS→FS, and 8.1 ± 1.3 to 16.3 ± 3.3 ms [n = 7] in non-FS→non-FS connections) (fig. 6C). In contrast, the uIPSC amplitude was not changed by propofol (45.7 ± 39.0 to 43.1 ± 11.2 pA [n = 15] in non-FS→Pyr, 58.3 ± 10.1 to 59.7 ± 9.3 pA [n = 29] in non-FS→FS, and 53.2 ± 16.5 to 50.4 ± 18.9 pA [n = 7] in non-FS→non-FS connections) (fig. 6D). Similar to the connections of presynaptic FS, the presynaptic non-FS connections showed little change in the PPR and failure rate in response to propofol (fig. 6, E and F). These results suggest that the propofol-induced facilitation of uIPSCs is mediated by postsynaptic mechanisms.
Fig. 5.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in AC. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in A–C. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
Fig. 5.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in AC. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
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Fig. 6.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Fig. 6.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
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Comparison of Propofol-induced uIPSC Enhancement between FS→ and Non-FS→ Connections
In agreement with previous studies,28  the current study revealed the different uIPSC kinetics between FS and non-FS presynaptic connections. The presynaptic FS connections induced the uIPSCs with larger amplitudes in comparison with the non-FS presynaptic connections. The different kinetics of uIPSCs may affect the impact of propofol-induced uIPSC facilitation. To clarify this possibility, we quantified the increases in the uIPSC charge transfer, which was defined as the area under the curve of the uIPSCs.
In the connections where the postsynaptic cells were Pyr, the presynaptic FS connections showed much larger increases in uIPSC charge transfer (2.2 ± 0.5 pC, n = 36) than that in the presynaptic non-FS connections (0.3 ± 0.1 pC, n = 15; P < 0.01, Mann–Whitney U test; fig. 7A). This tendency was also observed in the cases where the postsynaptic cells were FS or non-FS: FS→interneuron connections (0.9 ± 0.2 pC, n = 37) showed a larger increase in the uIPSC charge transfer than that in the presynaptic non-FS connections (0.2 ± 0.1 pC, n = 36; P < 0.01, Mann–Whitney U test; fig. 7B). These results suggest that propofol potently facilitates the inhibitory connections of the presynaptic FS in comparison with the connections of the presynaptic non-FS. Notably, the FS→Pyr connections showed the most prominent propofol-induced increases in uIPSC charge transfer among the inhibitory connections, which suggests that the principal inhibitory regulation of excitatory cells is most sensitive to propofol.
Fig. 7.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
Fig. 7.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
×
However, there is a possibility that propofol-induced enhancement of uIPSC charge transfer might not depend on presynaptic cell type but depend on the uIPSC kinetics including the amplitude and half duration. To examine this possibility, uIPSCs with the similar kinetics (<100 pA of amplitude and 5 to 10 ms of half duration) were selected from FS→Pyr and non-FS→Pyr connections, and the increases in these uIPSC charge transfer were compared (fig. 7C). The average of the uIPSC amplitude of the selected FS→Pyr connections (39.1 ± 3.6 pA, n = 17) was not significantly different from that of the selected non-FS→Pyr connections (29.5 ± 7.3 pA, n = 11; P > 0.11, Student t test). The half duration of uIPSCs obtained from the selected FS→Pyr (7.1 ± 0.3 ms, n = 17) and non-FS→Pyr connections (7.3 ± 0.4 ms, n = 11) was also comparable (P > 0.11, Student t test). The increase in charge transfer of these uIPSCs was significantly larger in FS→Pyr connections than in non-FS→Pyr connections (P < 0.05, Student t test). This finding supports the dominant effects of propofol in the FS→Pyr connections as described above.
Discussion
The current study focused on the differential impact of propofol-induced uIPSC enhancement in the cortical local circuits. We found that FS→Pyr connections show the largest enhancement of uIPSC charge transfer. Considering the evidence that FS and Pyr are the major sources of inhibition and excitation, respectively, our results indicate that propofol effectively suppresses excitatory output from the cortex.
Postsynaptic Cell-type–dependent Enhancement of uIPSCs by Propofol
Propofol-induced uIPSC enhancement depended on the cell subtypes of postsynaptic neurons, that is, postsynaptic Pyr showed larger increases in charge transfer than that in postsynaptic GABAergic interneurons. The most likely mechanism underlying this observation may be due to the differences in the subtypes of GABAA receptor subunits, especially β subunits that are expressed in the postsynaptic Pyr and GABAergic interneurons.
The main target of propofol is considered to be the β subunits of GABAA receptors.1  Jurd et al.29  reported that the propofol-induced hypnotic and immobilizing response occurs via β3 subunits. It is known that the combination of GABAA receptor subunits varies among cell subtypes; Pyr invariably express β3 subunits,30  whereas only 70% of GABAergic interneurons express β3 subunits in the visual cortex.31  Layer V of the cerebral cortex densely expresses β1 subunits,32  though only 17% of Pyr express β1 subunits,30  suggesting that GABAergic interneurons may also densely express β1 subunits. This difference in β-subunit composition may influence the propofol-induced enhancement of uIPSCs.
Among the postsynaptic interneuron connections, uIPSC decay kinetics recorded in non-FS were slower than those recorded in FS. This observation suggests that the subtypes of GABAA receptor subunits expressed on non-FS may differ from those on FS. The LTS, a major population of non-FS, have a lower expression of functional β2/β3-containing GABAA receptors, suggesting that β1 expression may be an important factor in shaping IPSC decay in LTS.15  Therefore, FS may contain more β2/β3 subunits than that in non-FS. Taken together with the finding that approximately 70% of interneurons express β3 subunits, the composition of β subunits in FS may be similar to that in Pyr. If the difference in β2/β3 subunit expression is a part of the mechanisms that mediate postsynaptic cell-type–dependent enhancement of uIPSCs by propofol, Pyr and FS might show similar enhancement of propofol-induced uIPSCs. The prolongation rate of the uIPSC decay time by propofol was nearly identical between FS and Pyr. However, the increase in the charge transfer of FS was smaller than that of Pyr. This difference may be caused by the fast decay kinetics of FS, which is due to α1-subunit expression.33  Consistent with the idea described above, the neurons expressing more β2/β3 subunits (FS in this case) showed greater enhancement of uIPSCs than that of LTS (figs. 3 and 6).
Presynaptic Cell-type–dependent Enhancement of uIPSCs by Propofol
Previous studies have reported that propofol has little effect on the amplitude of miniature IPSCs and spontaneous IPSCs in cultured cortical cells8  or on spontaneous IPSCs of isolated neurons in the solitary tract nucleus.34  These findings differ from our current results that demonstrate propofol-induced uIPSC enhancement in presynaptic FS connections. This discrepancy may be due to the differences in recording methodologies (i.e., spontaneous/miniature recordings vs. paired recordings). Although the former recordings cannot discriminate the sources of synaptic events, our paired recording technique can classify the presynaptic inhibitory neurons. In addition, our study showed propofol-induced increases in the uIPSC amplitude of presynaptic FS but not non-FS, which may mask the increase in the uIPSC amplitude in spontaneous/miniature recordings.
FS→Pyr connections showed a more prominent enhancement of uIPSCs than that in non-FS→Pyr connections. Similarly, FS→interneuron connections showed larger increases in the charge transfer of uIPSCs than that in non-FS→interneuron connections. Importantly, the decay kinetics of uIPSCs recorded from postsynaptic Pyr did not differ from the presynaptic neural subtypes (FS and non-FS). This observation suggests that there is a similar GABAA receptor subunit composition in both FS→Pyr and non-FS→Pyr synapses. However, this hypothesis seems to contradict the current results of the functional difference between FS→Pyr and non-FS→Pyr synapses induced by propofol.
Fast-spiking cells are anatomically categorized into at least two groups: basket cells and chandelier cells.12,35,36  The axon targets of both cells are the soma, proximal dendrites, and axon hillock.12,36,37  Non-FS, however, tend to project to the distal dendrites.12,14  Such anatomical profiles generate differences in uIPSC kinetics, including rise time and amplitude.28  It is possible that the decay profiles of uIPSCs may depend on the sites of synaptic inputs; the amplitude does not linearly decay during conduction from the distal dendrites to the somata. In addition, smaller amplitudes with smaller increases may be difficult to detect at the somata. Such nonlinear decay of uIPSCs may generate the difference in the propofol-induced enhancement observed between presynaptic FS and non-FS.
Propofol Has Little Effect on Electrical Synapses
In contrast to the facilitative effects of propofol on GABAergic currents, the electrical coupling conductance was not changed by propofol. Wentlandt et al.18,19  have shown the propofol-induced attenuation of gap junction coupling in P19 cell and hippocampal slice cultures. The discrepancy may be due to the differences in cell lines and techniques for electrical coupling measurement. We consider that the acute slice preparation maintains intrinsic cell properties rather than cultured cells. In addition, paired whole cell recordings have an advantage in direct measurement of electrical coupling conductance in comparison to dye coupling, recovery of fluorescence photo bleaching technique, or extracellular recording.
Functional Implication
Propofol mainly acts to suppress excitatory principal neurons by potentiating inhibitory inputs from FS and non-FS. We also observed that propofol induces the potentiation of inhibitory inputs to GABAergic interneurons. Such potentiation of inhibition to inhibitory neurons seems to contradict the function of excitatory neurons. Interestingly, several studies have reported that the roles of FS→FS connections are to inhibit inhibitory interneurons to increase the synchronization of their spike firing.16,17,38  Because FS have electrical synapses between other FS, their activities may propagate to the adjacent cortical areas.16,17  The current findings, therefore, reveal not only the dominant enhancement of inhibition on Pyr by propofol but also the possibility of propofol-induced facilitation of synchronization by potentiating inhibitory connections among interneurons. This interneuronal synchronization may promote the synchronous Pyr spike firing, which would appear as the predominance of α frequency band (8 to 13 Hz) on an electroencephalogram, which are associated with propofol-induced loss of consciousness.39–43  The mechanisms of synchronization by propofol should be further explored in the future.
Acknowledgments
The authors thank Yuchio Yanagawa, M.D., Ph.D., Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; Masumi Hirabayashi, Ph.D., Section of Mammalian Transgenesis, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan; and Yasuo Kawaguchi, M.D., Ph.D., Division of Cerebral Circuitry, National Institute for Physiological Sciences, Okazaki, Aichi, Japan, for generating VGAT-Venus transgenic rats by using pCS2-Venus which is provided by Atsushi Miyawaki, M.D., Ph.D., Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, Wako, Saitama, Japan.
This work was supported by JSPS KAKENHI 24792257, 22592264, 25463150, and 25293379 from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, Tokyo, Japan, and Research Grants from Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
Competing Interests
The authors declare no competing interests.
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Fig. 1.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
Fig. 1.
Triple whole cell patch clamp recordings from Venus-positive γ-aminobutyric acidergic and Venus-negative pyramidal cells (Pyr) in layer V insular cortex. (A) An example of recorded neurons under Nomarski observation (a). The neurons indicated by single and double arrowheads were Venus-positive neurons, whereas the neuron with an arrow was Venus negative (b). The recorded neurons were stained with the fluorescent dye Alexa 568 (c). (B) A schematic of connections among the recorded neurons shown in A. Venus-positive fast-spiking cells (FS) indicated by an arrowhead (FS1) and double arrowheads (FS2) were mutually connected. In addition, FS1 and FS2 were electrically coupled. (C) Repetitive spike-firing properties of neurons shown in A and B in response to intracellular current pulse injections. The Venus-negative neuron showed a typical Pyr-like firing pattern. The other two neurons (FS1 and FS2) showed FS firing. The resting membrane potentials were shown on the left of the traces. (Da) A depolarizing voltage pulse injection to FS1 triggered action currents. The action currents in FS1 induced unitary inhibitory postsynaptic currents in FS2. Ten consecutive traces and their average are shown in thin gray lines and a thick black line, respectively. (Db) The action currents in FS2 also induced unitary inhibitory postsynaptic currents in FS1, indicating the mutual synaptic connections between FS1 and FS2. (E) The voltage responses to hyperpolarizing currents in FS1 induced smaller hyperpolarizing responses in FS2 (arrowhead) and vice versa (double arrowheads), indicating that FS1 and FS2 were electrically coupled.
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Fig. 2.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
Fig. 2.
Effects of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. (A) A schematic of an FS→Pyr connection with repetitive spike-firing properties of each neuron. Resting membrane potentials are shown on the left of the traces. (B) Effects of 10 μM of propofol on uIPSCs of the FS→Pyr connection. Presynaptic action currents in FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the FS→Pyr connection shown in A–C. (E) A dose–response curve of propofol-induced enhancement of uIPSC charge transfer in FS→Pyr connections. The data were fitted by logistic function. The number of cell pairs was shown in the parentheses. (F, G) Effects of 10 μM of propofol on uIPSCs obtained from FS→FS (F) and FS→non-FS connections (G). Note the enhancement of uIPSCs in both connections.
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Fig. 3.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Fig. 3.
Summary of the effects of 10 μM of propofol on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in fast-spiking cell (FS)→pyramidal cell (Pyr)/FS/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
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Fig. 4.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
Fig. 4.
Effects of propofol on electrical synapses between fast-spiking cells (FS). (A) A schematic of an electrical and reciprocal chemical synapses between FS with repetitive spike-firing properties of each neuron. (B) A hyperpolarizing current pulse injection to FS1 induced a voltage response in FS2 (arrowhead) and vice versa (double arrowhead). (C) Hyperpolarizing voltage pulses were applied to FS1 and FS2. Current responses in the coupling FS in the control and propofol application were shown in the lower panels. Ten consecutive traces are shown in gray lines, and their average is shown in black. (D) Time courses of the amplitude of electrical coupling conductance before and during propofol application in the FS shown in A–C.
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Fig. 5.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in AC. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in A–C. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
Fig. 5.
Effects of 10 μM of propofol on unitary inhibitory postsynaptic currents (uIPSCs) recorded from non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. (A) A schematic of a non-FS→Pyr connection with firing properties of each neuron. (B) Effects of 10 μM of propofol on uIPSCs of the non-FS→Pyr connection. Presynaptic action currents in non-FS induced uIPSCs in Pyr. Propofol enhanced uIPSCs. Ten consecutive traces are shown in gray lines, and their average is shown in black. (C) Superimposed uIPSC traces in B. Traces during control conditions and propofol application are shown as thin and thick lines, respectively. Note that propofol increased the rise and decay kinetics of uIPSCs. (D) Time courses of the uIPSC amplitude, 80 to 20% decay time, and charge transfer before, during, and after propofol application in the non-FS→Pyr connection shown in AC. (E, F) Effects of propofol on uIPSCs of non-FS→FS (E) and non-FS→non-FS connections (F). Note the enhancement of uIPSCs in both connections.
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Fig. 6.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
Fig. 6.
Summary of the propofol (10 μM)-induced effects on the 20–80% rise time (A), 80 to 20% decay time (B), decay time constant (C), amplitude (D), paired-pulse ratio (E), and failure rate of unitary inhibitory postsynaptic currents (F) in non–fast-spiking cell (non-FS)→pyramidal cell (Pyr)/fast-spiking cell (FS)/non-FS connections. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired t test.
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Fig. 7.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
Fig. 7.
Comparison of the propofol-induced increases in the charge transfer of unitary inhibitory postsynaptic currents (uIPSCs) between fast-spiking cell (FS)→pyramidal cell (Pyr) and non-FS→Pyr connections (A), and FS→interneuron (FS/non-FS) and non-FS→interneuron connections (B). Note that presynaptic FS connections showed larger increases in the uIPSC charge transfer than that in the presynaptic non-FS connections. (C) Selected uIPSCs with the similar kinetics (<100 pA of amplitude and 5–10 ms of half duration) were compared between FS→Pyr and non-FS→Pyr connections. Note that the increase in charge transfer was larger in FS→Pyr connections than that in non-FS→Pyr connections. **P < 0.01, Mann–Whitney U test. †P < 0.05, two-tailed Student t test.
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