Brain Research Bulletin 93 (2013) 69–79
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Review
The effects of volatile anesthetics on synaptic and extrasynaptic GABA-induced neurotransmission Naoki Kotani a,∗ , Norio Akaike a,b a b
Research Division of Neurophysiology, Kitamoto Hospital, 3-7-6 Kawarasone, Koshigaya 343-0821, Japan Research Division for Life Science, Kumamoto Health Science University, 325 Izumi-machi, Kumamoto 861-5598, Japan
a r t i c l e
i n f o
Article history: Received 17 May 2012 Received in revised form 17 July 2012 Accepted 1 August 2012 Available online 17 August 2012 Keywords: Volatile anesthetics Extrasynaptic and synaptic GABAA receptors Presynaptic and postsynaptic transmission Spontaneous, miniature, and evoked inhibitory postsynaptic currents ‘Synaptic bouton’ preparation
a b s t r a c t Examination of volatile anesthetic actions at single synapses provides more direct information by reducing interference by surrounding tissue and extrasynaptic modulation. We examined how volatile anesthetics modulate GABA release by measuring spontaneous or miniature GABA-induced inhibitory postsynaptic currents (mIPSCs, sIPSCs) or by measuring action potential-evoked IPSCs (eIPSCs) at individual synapses. Halothane increased both the amplitude and frequency of sIPSCs. Isoflurane and enflurane increased mIPSC frequency while sevoflurane had no effect. These anesthetics did not alter mIPSC amplitudes. Halothane increased the amplitude of eIPSCs, with a decrease in failure rate (Rf) and paired-pulse ratio. In contrast, isoflurane and enflurane decreased the eIPSC amplitude and increased Rf, while sevoflurane decreased the eIPSC amplitude without affecting Rf. Volatile anesthetics did not change kinetics except for sevoflurane, suggesting that presynaptic mechanisms dominate changes in neurotransmission. Each anesthetic showed somewhat different GABA-induced response and these results suggest that GABA-induced synaptic transmission cannot have a uniformly common site of action as suggested for volatile anesthetics. In contrast, all volatile anesthetics concentration-dependently enhanced the GABAinduced extrasynaptic currents. Extrasynaptic receptors containing ␣4 and ␣5 subunits are reported to have high sensitivities to volatile anesthetics. Also, inhibition of GABA uptake by volatile anesthetics results in higher extracellular GABA concentration, which may lead to prolonged activation of extrasynaptic GABAA receptors. The extrasynaptic GABA-induced receptors may be major site of volatile anesthetic-induced neurotransmission. This article is part of a Special Issue entitled ‘Extrasynaptic ionotropic receptors’. © 2012 Elsevier Inc. All rights reserved.
Contents 1. 2.
3.
4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A new method for evaluation of single synaptic transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Acute isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Spontaneous, miniature and action potential-evoked postsynaptic currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile anesthetic actions on synaptic and extrasynaptic currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Effects of volatile anesthetics on extrasynaptic currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Effects of volatile anesthetics on miniature or spontaneous IPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Effects of volatile anesthetics on evoked IPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Different mechanisms between evoked and spontaneous or miniature IPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Presynaptic-dominant volatile anesthetic-induced GABA transmissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The reported effects of volatile anesthetics on extrasynaptic GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The possibility of other modulating factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +81 48 969 6050; fax: +81 48 969 6051. E-mail address:
[email protected] (N. Kotani). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.08.001
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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The pharmacologic actions of volatile anesthetics were long considered as a perturbation of the fluidity of the membrane lipid bilayer. This concept derives from the correlation between the olive oil/water partition coefficient for the anesthetic and its anesthetic potency (Meyer–Oveton correlation) but was upset by the demonstration of chemically specific binding to anesthetic recognition sites (Campagna et al., 2003). However recent electrophysiological studies on interaction with membrane channels of the central nervous system suggest that general anesthetics act thorough hydrophilic regions of receptors in neurons. The most common candidate for target of general anesthetics is the GABAA receptor, a ligand-gated chloride receptor-channel complex for the GABA, which usually inhibits neuronal excitability. In fact, halothane (Mihic et al., 1997; Wakamori et al., 1991), enflurane (Mihic et al., 1997; Siegwart et al., 2003; Wakamori et al., 1991), isoflurane (Mihic et al., 1997), and sevoflurane (Sebel et al., 2006; Wu et al., 1994, 1996), all potentiated GABA-induced postsynaptic currents at their clinically relevant concentration. However, growing evidence suggesting that GABAA receptors are not main site of volatile anesthetic action has been also reported. For example, volatile anesthetic-induced anesthesia cannot be antagonized by GABA antagonists (Pittson et al., 2004). GABA-mediated neurons change to excitatory neurotransmission in immature animals (Jang et al., 2001; Kahraman et al., 2008; Kakazu et al., 1999). The changes of chloride cotransporters from sodium-potassium-chloride cotransporter to potassium-chloride cotransporter decrease the intracellular chloride concentration and the switch from the chloride influx to efflux (Jang et al., 2001; Kakazu et al., 1999). Although molecular cloning studies have identified the ligand-binding molecules of the GABAA receptors, each volatile anesthetic has different sensitivity to various target protein (McCracken et al., 2010; Olsen and Li, 2011). The interpretation of activation of GABAA -induced postsynaptic currents by exposure to volatile anesthetics is also very problematic. A major limitation is that previous patch clump studies were performed on either slice preparations or cultured cells and that the electric responses in these studies were influenced by extrasynaptic modulations or by other surrounding structures including neuronal and glia cells. In fact, extrasynaptic GABAA receptor-mediated currents represent a much greater proportion of the total GABAA receptor-mediated current than that mediated by synaptic activity (Walker and Semyanov, 2008). It thus remains unknown how volatile anesthetics exactly act on GABA-induced neurotransmission at the single synapse level. We established a method for isolation of single nerveadherent-synaptic-bouton using enzyme-free mechanical dissociation (Akaike and Moorhouse, 2003; Akaike et al., 2002). This ‘synaptic bouton’ preparation has the advantages of single neurons that are isolated from extrasynaptic responses and surrounding neurons and glia but retain adherent function terminals. This new technique enables us to evaluate better how volatile anesthetics act on synaptic transmission at a single synapse. This approach allows use to simultaneously quantify the presynaptic and postsynaptic contributions to GABA-induced neurotransmission by measuring the amplitude and frequency of spontaneous or miniature as well as directly evoked inhibitory postsynaptic currents (sIPSC, mIPSC and eIPSC). The responses measured during volatile anesthetic exposures using the synaptic bouton preparation give new insights that
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are completely different from those reported previously (Kotani et al., 2012; Ogawa et al., 2011). One of the aims in this review is to show how this new isolation method reveals synaptic function at individual synaptic boutons. Then we review our results of volatile anesthetic-induced changes in sIPSCs or mIPSCs followed by changes in IPSCs evoked from single synapses along with comparisons to previous studies including extrasynaptic GABA-induced responses. Finally, we will reconcile such results with possible mechanisms for these differences. 2. A new method for evaluation of single synaptic transmission 2.1. Experimental procedures Our new method isolates single neurons from the central nervous system using mechanical dissociation without any enzymatic treatment while retaining adherent and functioning excitatory and inhibitory synaptic nerve terminals (boutons). The isolated neurons are free from confounding effects from extrasynaptic modulation and other neuronal and glial cells. The acute mechanical dissociation avoids possible changes in tissue and/or its function as result of either enzyme treatment or in vitro culture. This synaptic bouton preparation offers improvements to inquiries into the mechanisms of synaptic transmission in the central nervous system. 2.2. Acute isolation The experimental procedure was described in detail previously (Akaike and Moorhouse, 2003; Akaike et al., 2002). Briefly, the procedure begins with slices from various mammalian brain or spinal cord slice prepared by the standard method. For mechanical dissociation, the brain slice was transferred to a culture dish containing standard external solution, and fixed to the bottom of the dish by an anchor made from a platinum frame and nylon thread (Fig. 1A). The target region for harvesting neurons was identified under a binocular microscope and the tip of a fire-polished glass pipette was lightly placed on the slice surface above the target neurons and vibrated horizontally (0.2–2 mm displacement) at 50–60 Hz using a custom assembled vibration device (Fig. 1B). The dish is typically moved horizontally along the target region by hand to harvest neurons from sub region of interest within the slice. Then, the slices were removed from the dish and the mechanically dissociated neurons left to settle and adhere to the bottom of the dish for at least 15 min before electrophysiological measurements. Electron microscopic and fluorescent studies confirm that boutons remain attached to the dissociated neurons (Fig. 1C and D). To date, we have used this approach successfully on neurons from various brain regions including the Meynert’s nuclei (Arima et al., 2001), the basolateral amygdala (Koyama et al., 1999), the hippocampal CA1 (Matsumoto et al., 2002), and CA3 (Yamamoto et al., 2011), periaqueductal gray (Kishimoto et al., 2001), the ventromedial hypothalamus (Jang et al., 2001), and the spinal sacral dorsal commissural nucleus (Akaike et al., 2010). 2.3. Spontaneous, miniature and action potential-evoked postsynaptic currents In the synaptic bouton preparation, neurotransmitter is released from the adherent terminals that give rise to spontaneous synaptic
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Fig. 1. Mechanical isolation of single neurons with attached synaptic boutons from central nervous system slices. (A and B) Schematic illustration of the mechanical dissociation of rat hippocampal CA1 pyramidal neuron. A fire-polished glass pipette is vibrated horizontally across the surface of the CA 1 area at 50–60 Hz. Successful liberation of viable neurons results in a fine mist originating from the dissociation site. The treated slice is then removed and the harvested neurons are left to adhere to the base of the culture dish. (C) Electron microscopy image showing two presynaptic boutons (arrow head) adherent to a dissociated hippocampal CA 1 neuron. (D) A dissociated hippocampal CA 1 neuron with presynaptic boutons stained by FM1-43 fluorescence (arrowhead), demonstrating synaptic membrane turnover at a functioning synapse. The intensity of the fluorescent signal in the boutons disappeared after adding of 15 mM K+ to external solution, suggesting activity dependent synaptic membrane cycling. Part C and D was used with permission from Akaike et al. (2002).
potentials. The frequency of spontaneous inhibitory or excitatory postsynaptic currents (sIPSCs and sEPSCs) recorded from brain or spinal cord is between 1 and 10 Hz and this range of activity presumably reflects the differences in the number and excitability of adherent boutons. The spontaneous synaptic currents consist of both action potential-dependent and -independent (tetrodotoxin resistant) components. The addition of tetrodotoxin, a selective Na channel blocker, decreases the frequency of GABAinduced or glycine-induced spontaneous inhibitory postsynaptic currents (sIPSCs) (Rhee et al., 1999) and glutamate-induced spontaneous excitatory postsynaptic currents (sEPSCs) (Jang et al., 2001) by about 50% from in situ slice recordings. Tetrodotoxinresistant currents are termed miniature (m) IPSCs or mEPSCs. Including results from studies in the presence of Ca channel blockers (Rhee et al., 1999) or in Ca2+ -free external solutions (Maeda et al., 2009), the tetrodotoxin-resistant synaptic currents are independent from Ca2+ influx. The ability to dissect transmitter release into such spontaneous and miniature postsynaptic currents and whether these are Ca2+ influx resistant or sensitive, help to define the locus of action of presynaptic neuromodulators. In these isolated neurons, a single presynaptic nerve terminal can be visualized and the synaptic bouton focally stimulated with electrical pulses to evoke inhibitory or excitatory postsynaptic currents. (eIPSCs or eEPSCs) (Akaike and Moorhouse, 2003; Akaike et al., 2002; Murakami et al., 2002) Briefly, the stimulating pipette for focal electrical stimulation of a single bouton was made from glass tube. The partition of glass separate two adjacent compartments so that current can be passed from one barrel returning through the adjacent barrel for a compact
stimulus delivery. Both compartments were filled with external solution with electrical wire inputs, thus acting as a bipolar electrode. The pipette was placed close as possible to the postsynaptic soma membrane of a single neuron during a whole cell patch recording (Fig. 2A). The stimulating pipette was then carefully moved along the surface membrane of the soma while applying stimulation pulses and monitoring evoked responses. Paired pulses were typically used when assessing presynaptic mechanisms. To determine whether eIPSCs or eEPSCs were evoked from a single bouton, the stimulus-amplitude and stimulus-distance relationships were examined. When eIPSCs or eEPSCs were identified, they appeared in all-or-none fashion as stimulus strength with a sharp threshold for activation (Fig. 2B), which indicates that the stimulating pipette was positioned just above a single bouton. Furthermore, when the stimulus pipette was moved horizontally along the surface of a dissociated neuron, the eIPSCs or eEPSCs again appeared or disappeared in all-or-none fashion. With shifts in distance of less than 0.4 m, the eIPSCs or eEPSCs were unaffected in most boutons. The shift in the electrode did not affect the amplitude or shape of synaptic events but increased their failure rate (Rf) of eIPSCs or eEPSCs. These activation protocols successfully detected evoked release from either glutamate (Akaike et al., 2010; Yamamoto et al., 2011), GABA (Akaike et al., 2010, 2002; Kotani et al., 2012; Murakami et al., 2002; Ogawa et al., 2011) or glycine (Akaike et al., 2010; Nonaka et al., 2010) nerve terminals using the synaptic bouton preparation and focal electric stimulation techniques. Test drugs were applied by a pressure application system allowing for exchange of the external solution surrounding the cell within 20 ms (Murase et al., 1989; Nakagawa et al., 1990; Shirasaki et al., 1991) (Fig. 3).
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Fig. 2. Focal electrical stimulation of a single bouton. (A) Schematic representation of a synaptic bouton preparation and focal electrical stimulation of a single nerve ending (bouton). (B) The relationship between stimulus strength and the amplitude of GABA-induced evoked inhibitory postsynaptic currents, which appeared in an all-or-none nature as stimulus strength varied. Inset shows two failure responses and tow successfully evoked responses.
In the experiments using the synaptic bouton preparation we discovered several novel aspects of roles of voltage-dependent Ca channel subtypes on GABA or glycine synaptic release (Murakami et al., 2002; Nonaka et al., 2010), GABAA receptor-mediated autoinhibition and presynaptic inhibition (Jang et al., 2006; Jeong et al., 2003; Shin et al., 2011; Yamamoto et al., 2011), modulation of 5hydroxytryptophan on GABA release (Katsurabayashi et al., 2003;
Koyama et al., 1999, 2000, 2002), and excitatory and inhibitory presynaptic modulation by A type botulinum (A1LL, A1NTX, A2NTX) and scorpion toxin (Akaike et al., 2009, 2010). 3. Volatile anesthetic actions on synaptic and extrasynaptic currents 3.1. Effects of volatile anesthetics on extrasynaptic currents
Fig. 3. A schematic illustration of the ‘Y-tube’ method. The solution was ejected from the tip of the Y-tube. The solution in the tube was changed rapidly through suction pump with electromagnetic valve allowing complete exchange of the solution within 20 ms. Rec. Elec: recording electrode; Ref Elec.: reference electrode; Stim Elec: stimulating electrode.
It is well known that volatile anesthetics enhance extrasynaptic currents. In fact, halothane (Mihic et al., 1997; Wakamori et al., 1991), enflurane (Mihic et al., 1997; Siegwart et al., 2003; Wakamori et al., 1991), isoflurane (Mihic et al., 1997), and sevoflurane (Sebel et al., 2006; Wu et al., 1994, 1996), all potentiated GABA-induced neurotransmission at clinically relevant concentration. We re-evaluated the effects of these anesthetics on extrasynaptic currents in dose dependent fashion using a directly focal flow from our Y-tube which accomplished rapid application and high volumes of solution exchange surrounding the isolated neurons (Kotani et al., 2012; Ogawa et al., 2011). First, at a holding potential of 0 mV, exogenous application of 1 M GABA rapidly and reversibly induced outward postsynaptic currents in mechanically dissociated hippocampal CA1 and CA3 neurons. Co-application of increasing concentration of all anesthetics produced concentration-dependent facilitation of GABA (1 M) responses measured as extrasynaptic currents. The increases in GABA-induced extrasynaptic currents were minimal at subclinical concentration of volatile anesthetics. The GABA-induced extrasynaptic responses increased approximately 1.5 times greater than controls in the presence of each anesthetic at its clinically relevant concentration (Fig. 4). This enhancement was similar to many previous studies (Drexler et al., 2006; Joksovic et al., 2009; Kitamura et al., 2003; McDougall et al., 2008; Nishikawa and MacIver, 2001; Peters et al., 2008). These responses are considered to be extrasynaptic and the contribution of synaptically released GABA must be minimal. In using the ‘synaptic bouton’ preparation, any GABA diffusing from the synaptic cleft was immediately washed out of the extrasynaptic space. Therefore, the open access to our substantial flow of external superfusion of the isolated neurons means that the amount of GABA existing in extrasynaptic spaces was minimized
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enflurane, and enflurane did not change mIPSCs at their subclinical concentration (100 M). 3.3. Effects of volatile anesthetics on evoked IPSCs
Fig. 4. Concentration-response relationship for modulation of 1 M GABA-induced responses by isoflurane (), enflurane (♦), sevoflurane (), or halothane () at various concentration. All current amplitudes were normalized to the respective control currents induced by 1 M GABA alone. Each point is the average ± SEM (n = 4–7). ***p < 0.001. This figure was used with permission from Kotani et al. (2012) and Ogawa et al. (2011).
especially as compared with slice preparations. Also the optimal concentration of GABA for extrasynaptic and synaptic transmission is markedly different in neurons in situ compared to isolated neurons. The optimal concentration for GABA-induced synaptic activation is 1.5–3 mM (Mozrzymas et al., 2003), whereas reaction 1000 fold lower concentrations (1 M) activate extrasynaptic GABAA receptors in our isolated neurons (Kotani et al., 2012; Ogawa et al., 2011). 3.2. Effects of volatile anesthetics on miniature or spontaneous IPSCs In our study (Kotani et al., 2012) halothane increased the amplitude of sIPSCs significantly without affecting current kinetics in contrast to previous studies (Kitamura et al., 2003; Nishikawa and MacIver, 2000, 2001) that showed halothane decreased the amplitude and prolonged current kinetics of sIPSCs. The frequency of sIPSCs increased in our studies consistent with previous work (Nishikawa and MacIver, 2000). However, Kitamura et al. (2003) reported that frequency of mIPSCs did not change in cultured cortical neuron at clinically relevant concentration and decreased at higher concentration of halothane (Fig. 5 and Table 1). Isoflurane and enflurane, structural isomers, significantly increased the frequency of mIPSCs without affecting amplitudes (Ogawa et al., 2011) as is consistent with previous reports in the inhibitory glycine-induced mIPSC frequency (Cheng and Kendig, 2002; Yamashita et al., 2001) (Fig. 5 and Table 1). Nishikawa and MacIver (2001) and Drexler et al. (2006) reported that enflurane decreased amplitude but increased frequency of sIPSCs. The results of frequency and amplitude in mIPSCs or sIPSCs of isoflurane were different among studies (Joksovic et al., 2009; Nishikawa and MacIver, 2001; Peters et al., 2008). Although isoflurane and enflurane did not alter the current kinetic of mIPSCs or sIPSCs in our study, all previous studies that evaluated enflurane and isoflurane showed prolonged current kinetics of mIPSCs or sIPSCs (Drexler et al., 2006; Joksovic et al., 2009; McDougall et al., 2008; Nishikawa and MacIver, 2001; Pearce, 1996; Peters et al., 2008) (Table 1). Sevoflurane did not change the frequency and amplitude of mIPSCs but prolonged kinetics (Ogawa et al., 2011) (Fig. 5 and Table 1). Similarly, Nishikawa et al. (2005) and Nishikawa and MacIver (2001) reported that frequency of sIPSCs was increased but amplitude was decreased by sevoflurane exposure. Isoflurane,
Enflurane and isoflurane at clinically relevant concentration rapidly and strongly decreased the amplitude of eIPSCs evoked from single boutons but increased their Rf for these focal activations (Ogawa et al., 2011) (Fig. 6). Sevoflurane rapidly decreased the amplitude of single terminal eIPSCs but did not change their Rf (Ogawa et al., 2011) (Fig. 6). In contrast, halothane increased the amplitude of eIPSCs and decreased the Rf (Kotani et al., 2012) (Fig. 6). Also halothane decreased the paired pulse ratio. Although all anesthetics prolonged the kinetics of eIPSCs (Drexler et al., 2006; Joksovic et al., 2009; Kitamura et al., 2003; McDougall et al., 2008; Nishikawa et al., 2005; Nishikawa and MacIver, 2000, 2001; Pearce, 1996; Peters et al., 2008), this parameter was not changed by anesthetics except sevoflurane in our study (Fig. 6 and Table 1). Our present studies clearly shows that each anesthetic has quite different evoked responses of eIPSCs on the GABA synapse (Fig. 6 and Table 1). Therefore, GABA-mediated synaptic transmission is not consistent with the conventional view of the main site of action of volatile anesthetic anesthesia. On the other hand, the extrasynaptic GABAA receptors mainly contribute to the enhancement of the inhibitory GABA-induced responses in the presence of volatile anesthetics at clinically relevant concentrations. 3.4. Different mechanisms between evoked and spontaneous or miniature IPSCs Recently, there is growing evidence that spontaneous and evoked neurotransmitter release is regulated by different presynaptic mechanisms. For example labeling with two different fluorescent tags with and without tetrodotoxin demonstrated that spontaneous and action potential dependent GABA releases were drawn from separate pools of synaptic vesicles (Fredj and Burrone, 2009). Our previous reports also indicated that intracellular cyclic AMP controls spontaneous glycine release from presynaptic terminals projecting the rat spinal sacral dorsal commissural nucleus neurons while action potential dependent glycine release is mediated via activation of phosphokinase A (Katsurabayashi et al., 2004). Furthermore, the eIPSCs from a single glycine-induced bouton of rat spinal sacral dorsal commissural nucleus neuron were abolished immediately by calcium-free bath solution whereas the mIPSC was reduced but not abolished even with extended calcium-free exposures. Moreover, divalent cations such as Ca, Ba, and Sr ions differently affected on mIPSCs and eIPSCs consistent with differentially controlled release mechanisms (Maeda et al., 2009). 4. Presynaptic-dominant volatile anesthetic-induced GABA transmissions Studies aimed at quantifying the presynaptic and postsynaptic effects of anesthetics have demonstrated actions on both the release of neurotransmitters and the function of neurotransmitter receptors, with latter having a dominant role. The predominance of focus on volatile anesthetic action on neurotransmission as postsynaptic mechanisms derives mainly from changes in current kinetics of mIPSCs or sIPSCs and eIPSCs being prolonged approximately 2–4 times of control in previous slice preparations or cultured cells (Drexler et al., 2006; Joksovic et al., 2009; Kitamura et al., 2003; McDougall et al., 2008; Nishikawa and MacIver, 2000, 2001; Pearce, 1996; Peters et al., 2008). These previous methods, however, lacked the quantitative resolution fro presynaptic contributions inherent in our synaptic
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Fig. 5. Amplitude (Amp, left panel) and frequency (Freq, right panel) of spontaneous (halothane) or miniature (enflurane, isoflurane and sevoflurane) inhibitory postsynaptic currents at clinically relevant concentration (halothane, isoflurane, sevoflurane 300 M; enflurane, 600 M). All responses were normalized to the respective control values. Each column is the mean value from 4 to 7 neurons and error bars represent ± SEM. ns not significant, *p < 0.05 and ***p < 0.001. This figure was used with permission from Kotani et al. (2012) and Ogawa et al. (2011).
Table 1 Effects of volatile anesthetics on spontaneous or miniature, and evoked GABA-induced inhibitory postsynaptic currents. Anesthetics
Comparison
Halothane
Previous (Kitamura et al., 2003; Nishikawa and MacIver, 2000, 2001; Pearce, 1996) Our study (Kotani et al., 2012) Previous (Drexler et al., 2006; Nishikawa and MacIver, 2001; Pearce, 1996) Our study (Ogawa et al., 2011) Previous (Joksovic et al., 2009; McDougall et al., 2008; Nishikawa and MacIver, 2001; Peters et al., 2008) Our study (Ogawa et al., 2011) Previous (Nishikawa et al., 2005; Nishikawa and MacIver, 2001) Our study (Ogawa et al., 2011)
Enflurane
Isoflurane
Sevoflurane
mIPSCs or sIPSCs
eIPSCs
Frequency
Amplitude
Kinetics
Amplitude
Kinetics
↓a ∼ ↑
↓
↑
↓
↑
↑
↑
→
↑
→
↑
↓
↑
↓
↑
↑
→
→
↓
→
↓ (Joksovic et al., 2009) → (Peters et al., 2008) ↑ (Nishikawa and MacIver, 2001)
↓ (Nishikawa and MacIver, 2001) → (Joksovic et al., 2009) ↑ (Nishikawa and MacIver, 2001)
↑
↓
↑
↑
→
→
↓
→
↑
↑
↓
↑
↓
→
→
↑
↓
↑
→
Paired pulse
Failure rate ↑
↓
↓
↑
mIPSCs, sIPSCs and eIPSCs mean miniature, spontaneous, and evoked GABA-induced inhibitory postsynaptic currents. a The frequency decreased at higher than clinically relevant concentration. We evaluated sIPSCs in halothane and mIPSCs in other volatile anesthetics.
Fig. 6. Relative amplitude (Amp, left panel) and failure rate (Rf, right panel) of evoked inhibitory postsynaptic currents with and without halothane, enflurane, isoflurane and sevoflurane at clinically relevant concentration (halothane, isoflurane, sevoflurane 300 M; enflurane, 600 M). Each column is mean value from 4 to 7 neurons. Error bars represent ± SEM. ns not significant, *p < 0.05 and **p < 0.01. This figure was used with permission from Kotani et al. (2012) and Ogawa et al. (2011).
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Fig. 7. The spontaneous (halothane) and miniature (enflurane, isoflurane, sevoflurane) inhibitory postsynaptic currents were averaged before and during application of volatile anesthetics at clinically relevant concentration (halothane, isoflurane, sevoflurane 300 M; enflurane, 600 M). Representative averaged current trace was normalized to the control amplitude (control, black trace; volatile anesthetics, grey trace). This figure was used with permission from Kotani et al. (2012) and Ogawa et al. (2011).
bouton preparation. All anesthetics except sevoflurane did not change current kinetics of mIPSC or sIPSCs (Fig. 7) and eIPSCs (Fig. 8) at the clinical concentrations, suggesting that postsynaptic GABAA receptors were little affected. Also halothane decreased paired pulse rate (Fig. 9). The paired pulse rate is considered to be a presynaptic phenomenon that is regulated by intracellular Ca2+ of presynaptic terminals (Zucker and Regehr, 2002). Our results testing single GABA terminal responses using the synaptic bouton preparation clearly indicates that volatile anesthetics except sevoflurane act at the presynaptic site but not at the synaptic GABAA receptors assumed to be the main modulatory site of volatile anesthetics. 5. The reported effects of volatile anesthetics on extrasynaptic GABAA receptors Synaptic GABAA receptors mediate fast synaptic inhibition by generating transient inhibitory postsynaptic currents. In addition to this phasic inhibition, a tonic or persistent inhibitory conductance has been identified that involves the activation of extrasynaptic GABAA receptors (Isaacson et al., 1993; Rossi and Hamann, 1998; Semyanov et al., 2004). The extrasynaptic receptors have a much greater affinity for GABA and are activated by low concentrations of ambient GABA (Ebert et al., 1997; Glykys and Mody, 2007; Ruiz et al., 2003; Vizi, 2000). Typically GABA spilled outside the synapse activates extrasynaptic GABAA receptors, resulting in persistent inhibition (Farrant and Nusser, 2005; Semyanov et al., 2004). In our study, however, all volatile anesthetics failed to increase GABA-induced extrasynaptic current at their subclinical concentration (Kotani et al., 2012; Ogawa et al., 2011).
Similarly, isoflurane, enflurane and sevoflurane did not change the amplitude of mIPSCs at relatively low concentration (100 M) (Ogawa et al., 2011). The GABAA receptor is composed of pentameric subunits, and the subunit composition importantly determines their subcellular distribution pattern as well as pharmacological properties. It is now clear that there are 19 genes for GABAA receptors and that theses include 16 subunits (Olsen and Sieghart, 2008; Vizi et al., 2010). More than 20 different types of subunit combination have been reported (Olsen and Sieghart, 2008; Vizi et al., 2010) and only limited number of researches is available concerning to the relationship between subunit combination and anesthetic sensitivity. ␦ subunit can substitute for the ␥ subunit. ␦ subunit containing receptors are extrasynaptic, but not all extrasynaptic GABAA receptors contain ␦ subunit. ␥2 subunit is a major component of synaptic GABAA receptors and drives receptor clustering as the synapse (Fritschy and Brunig, 2003). By a study of recombinant receptors, ␦ subunit-containing (extrasynaptic) receptors are 10 times more sensitive to GABAA agonists than ␥2 subunit-containing receptors (Mortensen et al., 2010). At present, tonic inhibition by extrasynaptic GABAA receptors has been implicated in multiple pathophysiologic conditions including fragile X mental retardation (Curia et al., 2009), ␥hydroxybutyric aciduria (Drasbek et al., 2008), stress (Maguire et al., 2005), and idiopathic generalized and temporal lobe epilepsies (Dibbens et al., 2004; Feng et al., 2006; Naylor et al., 2005; Scimemi et al., 2006; Zhang et al., 2007). Two ␦ subunit variants (E177A and R220H) have been identified as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy (Dibbens et al., 2004).
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Fig. 8. The evoked inhibitory postsynaptic currents were averaged before and during application of volatile anesthetics at clinically relevant concentration (halothane, isoflurane, sevoflurane 300 M; enflurane, 600 M). Representative averaged current trace was normalized to the control amplitude (control, black trace; volatile anesthetics, grey trace). This figure was used with permission from Kotani et al. (2012) and Ogawa et al. (2011).
The thalamus has an important role for regulation of sleep–wake state. It is well known that thalamocortical neuron of the ventrobasal thalamus exhibit tonic inhibition by extrasynaptic GABAA receptors (Cope et al., 2005; Jia et al., 2005). As much as 30% of the total GABAA receptor population in thalamocortical neurons contains the ␣4 subunit (Sur et al., 1999). ␣4 and ␦ subunits often drive receptor clustering and are found predominantly at extrasynaptic sites (Jia et al., 2005). This finding is confirmed by the loss of tonic inhibition in thalamocortical neuron of ␣4 subunit knockout mouse (Chandra et al., 2006). Even low concentration (25–85 M) of isoflurane elicited a sustained current in thalamocortical neurons of the ventrobasal thalamus, which was associated with increase in conductance (Jia et al., 2008). This current was very close to Cl− potential, and was blocked by gabazine, a selective GABAA antagonist. This response was negated in ␣4 subunit knockout mice even at clinically relevant concentrations (250 M) of isoflurane. Hippocampal neurons have been extensively studied for analysis of volatile anesthetic-induced GABA responses. As in ␦ subunit containing GABAA receptors, ␣5-containing GABAA receptors show typical extrasynaptic current; higher sensitivity to GABA and slow desensitization (Caraiscos et al., 2004a). A tonic inhibitory conductance in hippocampal pyramidal neurons generated by ␣5 subunit-containing GABAA receptors is highly sensitive to very low concentrations (25–83 M) of the isoflurane (Caraiscos et al., 2004b). Even though the effects of volatile anesthetics may be mediated by multiple mechanisms in the brain, these results
suggest that extrasynaptic GABAA receptors can be a major player in volatile anesthetic-induced GABA-induced responses. 6. The possibility of other modulating factors Clinically relevant concentrations of volatile anesthetics did not enhance the action potential dependent synaptic GABA responses in our present single synapse preparation, but did increase the extra- and multisynaptic GABA responses such as prolongation of current kinetics. One factor that might afferent response is the contribution of other structures surrounding neurons. For example, astrocytes as well as presynaptic nerve terminals contain the GABA transporter that could alter GABA distribution and concentration (Madsen et al., 2008). The concentration and rate of clearance of transmitters determine extrasynaptic GABA concentration and therefore function. In fact, GABA uptake blockade prolongs IPSCs n rat hippocampal slices (Roepstorff and Lambert, 1992). The clearance of GABA relies on passive diffusion and reuptake into glia and neurons by transporters rather than on enzymatic inactivation (Kwan et al., 2001; Robinson et al., 1991). Sugimura et al. (2001) reported that isoflurane significantly inhibited GABA uptake in cultured cell expressed GABA transporters. Similarly, according to Westphalen and Hemmings (2003), isoflurane produced inhibition of GABA uptake in adult rat cerebral cortex. The extrasynaptic GABAA receptors are activated by low ambient concentration of GABA (Ebert et al., 1997; Glykys and Mody, 2007; Ruiz et al., 2003). The extrasynaptic tonic GABA
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synaptic transmission is not the major and commonly suggested site of action for volatile anesthetic clinical actions. These anesthetics (except sevoflurane) do not change current kinetics concurrent with decreases in paired pulse rate in halothane. These results clearly indicate that many volatile anesthetics act at the presynaptic site. In contrast, all volatile anesthetics concentration-dependently enhanced the GABA-induced extrasynaptic currents. The extrasynaptic GABA-induced receptors may be the major site of volatile anesthetic-induced GABA transmission. Acknowledgements We thank Dr. M. Andresen for his valuable comments and critical reading of the manuscript. We also acknowledge Dr. Wakita for graphical assistance. This work was supported by Grant-in-Aid from Kitamoto Hospital, Koshigaya for N. Kotani and N. Akaike. References
Fig. 9. (a) Representative GABA-induced evoked inhibitory postsynaptic currents by paired-pulse focal stimulation with and without 100 M halothane. Inset shows typical eIPSCs before (1) and during (2) 100 M halothane. The numbers 1 and 2 were obtained from 1 to 2 in lower panel. (b) Change in paired pulse rate (PPR) by halothane. This figure was used with permission from Kotani et al. (2012).
current is highly sensitive to low concentration of isoflurane in hippocampal pyramidal neurons (Caraiscos et al., 2004b). Also low concentration of isoflurane elicited an extrasynaptic tonic current in thalamocortical neurons of the ventrobasal thalamus, which was associated with increase in conductance (Jia et al., 2008). These results support the following hypothesis that volatile anesthetics may modulate not only GABA release from presynaptic nerve terminals but also GABA uptake by astrocytes and/or nerve terminals, resulting in increased GABA amounts at the extrasynaptic region as well as synaptic cleft. 7. Conclusion We evaluated and contrasted how four volatile anesthetics (halothane, enflurane, isoflurane, and sevoflurane) modulate GABA release at the single synaptic level by measuring frequency and amplitude of sIPSCs or mIPSCs and the failure rate and amplitude of eIPSCs. Each anesthetic has quite different synaptic responses especially in eIPSCs. Halothane increased the amplitude and decreased the Rf of eIPSCs. However, enflurane and isoflurane markedly decreased the amplitude and increased Rf of eIPSCs. Sevoflurane also decreased the amplitude of eIPSCs, but also prolonged the decay phase of eIPSCs. These results suggest that GABA-induced
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