Modulation of hyperpolarization-activated cation current Ih by volatile anesthetic sevoflurane in the mouse striatum during postnatal development

Accepted Manuscript Title: Modulation of hyperpolarization-activated cation current Ih by volatile anesthetic sevoflurane in the mouse striatum during postnatal development Authors: Yusuke Sugasawa, Masataka Fukuda, Nozomi Ando, Ritsuko Inoue, Sakura Nakauchi, Masami Miura, Kinya Nishimura PII: DOI: Reference:

S0168-0102(17)30402-9 https://doi.org/10.1016/j.neures.2017.09.009 NSR 4098

To appear in:

Neuroscience Research

Received date: Revised date: Accepted date:

8-3-2017 18-9-2017 26-9-2017

Please cite this article as: Sugasawa, Yusuke, Fukuda, Masataka, Ando, Nozomi, Inoue, Ritsuko, Nakauchi, Sakura, Miura, Masami, Nishimura, Kinya, Modulation of hyperpolarization-activated cation current Ih by volatile anesthetic sevoflurane in the mouse striatum during postnatal development.Neuroscience Research https://doi.org/10.1016/j.neures.2017.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Modulation of hyperpolarization-activated cation current Ih by volatile anesthetic sevoflurane in the mouse striatum during postnatal development

Yusuke Sugasawa,1,2 Masataka Fukuda,1,2 Nozomi Ando,1,2 Ritsuko Inoue,2 Sakura Nakauchi,2 Masami Miura,2 and Kinya Nishimura1,2

1

Department of Anesthesiology and Pain Medicine, Juntendo University Faculty of

Medicine and Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 2

Neurophysiology Research Group, Tokyo Metropolitan Institute of Gerontology, 35-2

Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan

Correspondence should be addressed to Masami Miura: 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Tel: +81-3-3964-3241; Fax: +81-3-3579-4776; E-mail: [email protected]

E-mail: Yusuke Sugasawa, [email protected] Masataka Fukuda, [email protected] Nozomi Ando, [email protected] Ritsuko Inoue, [email protected] Sakura Nakauchi, [email protected] 1

Kinya Nishimura, [email protected]

34 pages, 5 figures, 4 supplementary files

Highlights 

Neonate striatal cholinergic neurons already possessed Ih.



Sevoflurane decreased Ih of striatal cholinergic neurons in a dose-dependent manner.



Sevoflurane apparently slowed Ih activation in the cholinergic neurons.



Sevoflurane decreased spike firing during the rebound activation.

Abstract Volatile anesthetics have been reported to inhibit hyperpolarization-activated cyclic-nucleotide gated channels underlying the hyperpolarization-activated cation current (Ih) that contributes to generation of synchronized oscillatory neural rhythms. Meanwhile, the developmental change of Ih has been speculated to play a pivotal role during maturation. In this study, we examined the effect of the volatile anesthetic 2

sevoflurane, which is widely used in pediatric surgery, on Ih and on functional Ih activation kinetics of cholinergic interneurons in developing striatum. Our analyses showed that the changes in Ih of cholinergic interneurons occurred in conjunction with maturation. Sevoflurane application (1 to 4%) caused significant inhibition of Ih in a dose-dependent manner, and apparently slowed Ih activation. In current-clamp recordings, sevoflurane significantly decreased spike firing during the rebound activation, which is essential for responses to the sensory inputs from the cortex and thalamus. The sevoflurane-induced inhibition of Ih in striatal cholinergic interneurons may lead to alterations of the acetylcholine-dopamine balance in the neural circuits during the early postnatal period.

Keywords: cholinergic interneuron, HCN channel, rebound excitation, firing rate.

Introduction Volatile anesthetics have a variety of effects on the developing brain in animal models (Patel and Honore, 2001; Ishiwa et al., 2004; Ishizeki et al., 2008; Chae et al., 2010; Eckle et al., 2012; Blain-Moraes et al., 2015). Recent studies have postulated that general anesthetics cause the inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels (Chen et al., 2005, 2009). These channels are expressed in both

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the heart and the nervous system (Biel et al., 2009). In the nervous system, the HCN channels underlie the hyperpolarization-activated cation current (Ih) that contributes to the intrinsic and integrative neuronal properties, in addition to generating synchronized oscillatory neural rhythms (Biel et al., 2009). The absolute Ih amplitude increased and the functional Ih kinetics changed from P1 to P20 in the hippocampal pyramidal neurons. The Ih alteration suggests that Ih plays a role during the maturation process (Vasilyev and Barish, 2002), while misregulation of the Ih may lead to epilepsy, chronic pain, and other neurological disorders (Lewis and Chetkovich, 2011). Sevoflurane is one of the major volatile anesthetics that are commonly used during pediatric surgeries due to its fast action, short recovery time, and the fact that it exhibits less airway irritability (Anderson et al., 2013). However, it has recently been reported that there are undesirable effects associated with pediatric anesthetic exposure. Retrospective cohort studies demonstrated that childhood exposure to volatile anesthetics was related to lower neurodevelopmental outcome scores at 12 months of age (Andropoulos et al., 2014) and to an increased risk for developmental and behavioral disorders in later childhood (DiMaggio et al., 2009). Currently, although there is inconclusive clinical data demonstrating lasting effects of anesthetics on human cognitive function, accumulating evidence suggests that morpho-functional changes

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lead to the long-term effects of anesthetic exposure (Vutskits and Xie, 2016). A number of preclinical studies have shown neurotoxicity and apoptosis after exposure to volatile anesthetics, including sevoflurane (Hudson and Hemmings, 2011; Takaenoki et al., 2014; Makaryus et al., 2015; Raper et al., 2015). While the neurotoxic processes leading to neuronal degeneration have been extensively studied, the impact of the anesthetic on spiking activities in the developing brain, especially in the striatum, has yet to be definitively clarified. The striatal cholinergic interneurons also possess the Ih, with this current contributing to the generation of the pacemaker activity for these cells (Bennett et al., 2000). Spontaneous tonic firing of cholinergic neurons maintains the basal level of acetylcholine, which is indispensably important for motor and cognitive functions of the striatum (Wilson, 2005). In addition, cholinergic interneurons fired phasically in response to sensory stimuli that were associated with reward-related learning (Schulz and Reynolds, 2013). A conditioned sensory stimulus leads to initial excitation followed by a transient suppression of firing and a rebound excitation (Aosaki et al., 1994a, 1994b, 1995; Bennett and Wilson, 1999). Afterhyperpolarization terminates the initial spike firings and then induces Ih that shapes and amplifies the rebound excitation (Wilson, 2005). However, developmental aspects of Ih are not well characterized in the

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cholinergic interneurons. When considering the involvement of frontostriatal system in the pathophysiology of developmental disorders, disturbance of the striatal cholinergic system in early life might be a potential risk factor that can contribute to psychiatric and neurologic abnormalities later in life. Based on these situations, we attempted to determine if the effect of sevoflurane on Ih might disturb the physiological spiking of the cholinergic interneurons. If so, this could provide experimental evidence that would help in our understanding of the pathological alteration of neuronal activities in pediatric anesthesia. Thus, our current study examined (1) the developmental changes in Ih in the cholinergic interneurons during the postnatal period, (2) the effects of sevoflurane on the functional Ih kinetics and (3) the alteration of spike firing after the application of sevoflurane.

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Materials and methods Slice preparation All experimental procedures were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology, and carried out in accordance with the Guidelines for Animal Experimentation of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the guidelines of the NIH in the USA. C57BL/6J mice (male, n = 96; P7–P35; CLEA Japan, Tokyo, Japan) were anesthetized with ether and decapitated. The brains were rapidly removed and put into ice-cold artificial cerebrospinal fluid (ACSF) containing: 124 mM NaCl, 3 mM KCl, 1 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2 and 10 mM glucose, buffered to pH 7.4 with NaHCO3 (26 mM) and saturated with 95% O2 and 5% CO2. Coronal brain slices (300 μm thick), including the striatum, were prepared with a Pro 7 Liner Microslicer (Dosaka, Kyoto, Japan) and incubated in ACSF at 32°C for 30 min. Subsequently, the slices were maintained in ACSF at room temperature (21–25°C).

Electrophysiology All slices were placed in a recording chamber, which was perfused with ACSF at a rate of 1–2 mL/min at 30°C. The patch pipettes used in the current-clamp and

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voltage-clamp recordings contained a solution of the following composition: 129 mM K-gluconate, 11 mM KCl, 2 mM MgCl2, 10 mM HEPES, 4 mM Na2-ATP, 0.3 mM GTP, and 0.5% biocytin (brought to pH 7.3 with KOH; osmolarity, 280 mOsm). A whole-cell patch-clamp technique was performed using an EPC9/2 amplifier (HEKA Elektronik, Lamrecht/Pfalz, Germany). Patch pipettes (4–6 Ω) were made from borosilicate glass capillaries (1.5 mm outer diameter, 1.17 mm inner diameter; Harvard Apparatus, Holliston, MA, USA) on a PC-10 puller (Narishige, Tokyo, Japan). The pipette was attached to the cell, and suctioned after the sealing resistance increased over 1 GΩ and the whole-cell condition was achieved. Immediately after breaking into the whole-cell configuration, the cells were held at −70 mV, with the input resistance measured every 2 s by a hyperpolarizing pulse (−10 mV for 100 ms). At 5 min after the break in, the average resistances were obtained and then pooled and analyzed. Resting membrane potential (RMP), threshold, and the depolarizing “sag” during the rectangular current steps were measured during further current-clamp analysis. For the off-line analysis, RMP was measured as the average of the fluctuated membrane potential after the elimination of action potentials using a low-pass filter. Subsequently, we then switched to the voltage-clamp mode in order to obtain the Ih. Series resistance was compensated at 70% using the amplifier circuit. Measurements were not corrected for

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junction potentials of 10 mV. Experiments were discarded if the resistance changed by more than 15%. Signals were filtered at 5 kHz, digitized at 20 kHz and acquired using PULSE (HEKA Elektronik) and PowerLab (AD Instruments, Castle Hill, Australia) instruments. For the Ih recordings, the neurons were held at 0 mV in the presence of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid (25 µM), and the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist 6-cyano-7-nitroquinoxa-line-2,3-dione (10 µM) in order to block the glutamate receptors. GABAA receptors were blocked by adding picrotoxin (100 µM) to the ACSF. The amplitude of Ih was measured as a time-dependent current. This was calculated immediately after the capacitive transient by determining the difference between the current at the end of the voltage step and the current at the beginning of the step. Measurements of the total amount of Ih were performed at the end of each experiment after applying 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288; 40 µM). Subtraction of the ZD7288-resistant current did not change the I-V relationship for the Ih (Supplemental Fig. 1). Without sevoflurane application, ZD7288 application inhibited the time-dependent current by 91.1% ± 3.1% (n = 4). The reversal potential of Ih (−37.6 ± 2.8 mV, n = 21) was estimated from the extrapolation of the I-V

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curves. Tail currents were normalized, plotted as a function of the preceding hyperpolarization step voltage, and fitted with Boltzmann curves for the derivation of the half-activation voltage via the use of the Prism software (Prism version 6.0; GraphPad Software, La Jolla, CA, USA). Time constants were determined by fitting currents evoked during the hyperpolarizing steps to an exponential function of the PULSE program. All drugs were bath-applied.

Morphological and electrophysiological identification of cholinergic interneurons Experiments were performed on cholinergic interneurons in the striatum at different postnatal time points (P7, P14, P21, P28). Cholinergic interneurons in the striatum were identified according to their large somata and thick primary dendrites (Kawaguchi, 1992, 1993; Bennett and Wilson, 1999) using an infrared-differential interference contrast (IR-DIC) video microscope (BX50WI; Olympus, Tokyo, Japan, Dage-MTI; Michigan City, IN, USA). Electrical responses to current injections were used to further confirm that the targeted neurons were cholinergic cells (Kawaguchi, 1992, 1993; Bennett and Wilson, 1999). Negative current pulses produced an initial hyperpolarization followed by a depolarizing sag in the membrane potential. In the absence of current injection, many of the cells also fired spontaneously (Bennett and

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Wilson, 1999). The neurons were intracellularly injected with biocytin in order to confirm the cell types by histochemical procedures (see below) after electrical recordings.

Histochemical procedures Slices containing biocytin-filled cells were stained as previously described (Suzuki et al., 2001; Miura et al., 2007). Briefly, the slices were fixed in 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer overnight at 4°C, rinsed in phosphate buffer for 30 min, and incubated in phosphate buffer containing 0.5% H2O2 for 15 min to suppress the endogenous peroxidase activity. The slices were incubated with avidin-biotin-peroxidase complex for 12 h at room temperature, and then stained using a 3,3’-diaminobenzidine tetrahydrochloride reagent kit following the manufacturer’s protocol (VECTASTAIN Elite ABC kit PK-6100, SK4100; Vector Laboratories, Burlingame, CA, USA). In line with a previous study (Van Vulpen and Van Der Kooy, 1996), our data similarly showed a tendency for increases in the soma areas of the cholinergic interneurons, and extended dendritic trees in conjunction with development (Supplemental Table 1). For the immunohistochemistry procedure for the choline acetyltransferase (ChAT), slices were incubated overnight at 4°C with a rabbit

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antibody against ChAT (1:100, AB144P; Millipore, Billerica, MA, USA) and Alexa647-conjugated streptavidin (1:300, Invitrogen, Carlsbad, CA, USA). The slices were incubated at room temperature for 1 h with secondary anti-rabbit antibodies conjugated to Alexa555 (1:500, A21429; Invitrogen). Images were obtained through the use of a fluorescence microscope (FV10i; Olympus, Tokyo, Japan).

Drugs Sevoflurane was manufactured by the Maruishi Pharmaceutical Co., Ltd. (Osaka, Japan). ZD7288 and 2-amino-5-phosphonovaleric acid were obtained from Tocris Bioscience (Bristol, UK). Picrotoxin, 6-cyano-7-nitroquinoxa-line-2,3-dione and all of the other drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Volatile anesthetic sevoflurane application and measurement Concentrations of the volatile anesthetic sevoflurane ranging from 1.0–4.0% were added to the ACSF along with 95% O2 and 5% CO2 using a Meratec vaporizer (Senko Medical Instrument, Tokyo, Japan). During the experimental recordings, the concentration was continuously monitored using a Life Scope (BSM-5132; Nihon Kohden, Tokyo, Japan) (Oose et al., 2012; Ando et al., 2014).

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Statistics All data are shown as mean ± SEM. The statistical significance of the difference was assessed using a Student’s paired or unpaired t-test, one-way ANOVA and two-way ANOVA with post hoc Bonferroni correction (Prism).

Results Sevoflurane attenuates Ih in developing cholinergic interneurons For whole-cell recordings between P7 to P28, cholinergic interneurons were identified as cells with large somata and thick primary dendrites under an IR-DIC microscope (Song et al., 1998). After the electrical recordings, immunolabeling for ChAT confirmed that the examined cells were cholinergic interneurons (Fig. 1). All immunostained cells were ChAT positive (P7, n = 11; P14, n = 6; P21, n = 7; P28, n = 6). As seen in Figure 2A, sevoflurane (4.0%) caused inhibition of the maximal current amplitude, with ZD7288 providing further inhibition of the current. Moreover, sevoflurane significantly decreased the current amplitude in the hyperpolarization voltage steps between −130 and −80 mV (Fig. 2B) (two-way ANOVA with post hoc

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Bonferroni correction, F1,15 = 17.20, P < 0.01). Figure 2C shows the effects of the 4.0% concentration of sevoflurane on the ZD7288 sensitive currents calculated by current subtraction of the post ZD7288 administration. In this and all later experiments, the ZD7288 insensitive currents were subtracted from the control and sevoflurane traces. Clinically used concentrations of sevoflurane (1.0, 2.0 and 4.0%) reduced the Ih in a dose-dependent manner. Figure 2D shows the % of the inhibition that occurred for each concentration (1.0%, 7.33 ± 1.72%; 2.0%, 30.83 ± 3.90%; 4.0%, 39.69 ± 5.71%; ANOVA, F2,35 = 6.706, P < 0.01). There was a significant increase in the absolute amplitude of the Ih from P7 to P28 (Fig. 3A) (P7, −96.6 ± 20.8 pA; P14, −183.2 ± 22.0 pA; P21, −320.2 ± 36.3 pA; P28, −406.1 ± 45.1 pA; ANOVA, F3,41 = 16.53, P < 0.01). At the same time, the sag responses to the hyperpolarizing currents that were observed from P7 gradually became prominent during the postnatal maturation (Supplemental Fig. 2). Although sevoflurane decreased the absolute amplitude of the Ih (Fig. 2C), the inhibitory effects were unchanged from P7 to P28 (Fig. 3B) (P7, 33.94 ± 6.01%; P14, 41.41 ± 7.86%; P21, 34.56 ± 9.70%; P28, 37.44 ± 9.42%; ANOVA, F3,22 = 0.1811, NS).

Effect of sevoflurane on the functional Ih activation kinetics

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We evaluated the effect of sevoflurane on the functional Ih activation kinetics based on the hypothesis that a change of time constants induced by sevoflurane might possibly affect the spontaneous firing of the cholinergic interneurons. Figure 4A1 shows representative overlaid traces of the Ih from the striatal cholinergic interneurons. As seen in the figure, there was a reduction observed after the administration of sevoflurane at a concentration of 4.0%. Figure 4A2 shows the same traces when scaled to the maximum amplitude. As seen in these scaled traces, sevoflurane apparently slows the Ih activation. Furthermore, this slower Ih activation led to significantly increased time constants (Fig. 4B) (control, 533.7 ± 18.7 msec; sevoflurane, 655.6 ± 21.5 msec; paired t-test, P < 0.01). The native time constants did not differ between P7 to P28 (Fig. 4C) (P7, 464.7 ± 24.8 msec; P14, 580.3 ± 38.8 msec; P21, 541.8 ± 36.3 msec; P28, 542.1 ± 41.3 msec; ANOVA, F3,43 = 1.694, NS). Normalized tail currents were obtained following incremental hyperpolarizing steps, averaged, and then fitted with Boltzmann curves in the controls and in the presence of sevoflurane. Sevoflurane induced a shift in the half-activation voltage of the tail current (Fig. 4D) (control, −89.5 ± 1.9 mV; sevoflurane, −93.6 ± 2.5 mV; paired t-test, P < 0.05). The half-activation voltages of the control tail currents did not differ between P7 to P28 (Fig. 4E) (P7, −79.1 ± 5.4 mV; P14, −88.5 ± 3.4 mV; P21, −89.2 ±

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3.0 mV; P28, −88.4 ± 2.3 mV; ANOVA, F3,23 = 1.770, NS).

Effect of sevoflurane on the spike firing during the rebound depolarization Rebound spike activation is a salient feature of the cholinergic interneurons. The rebound depolarization provides a dramatic increase in the spike firing of the cholinergic interneurons, which is significant for responses to the sensory inputs from the cortex and thalamus (Schulz and Reynolds, 2013). Whole-cell current-clamp recordings were performed between P7 to P28 for the purpose of evaluating the effect of sevoflurane on the rebound spike firing of the cholinergic interneurons. Sevoflurane at a concentration of 4.0% decreased the rebound depolarization and the number of spikes (Fig. 5A). Furthermore, ZD7288 also strongly inhibited the rebound depolarization (Fig. 5B). Figures 5C1 and 5D1 show that the 4.0% sevoflurane significantly decreased the depolarizing sag (control, −12.48 ± 1.38 mV; sevoflurane, −10.89 ± 1.57 mV, paired t-test, P < 0.01) and spike firing rate (control, 3.18 ± 0.44 Hz; sevoflurane, 1.48 ± 0.51 Hz, paired t-test, P < 0.01), respectively. However, we found no correlation between the age and the sevoflurane-induced inhibition (Fig. 5C2, 5D2).

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Discussion Previous studies have demonstrated that volatile anesthetics induced inhibition of the neuronal Ih mediated by the HCN channels at clinically relevant concentrations in the brainstem, thalamus and cortex (Chen et al., 2005, 2009). While little is known about the effects of volatile anesthetics on the neuronal Ih in the striatum, Ih has been shown to be engaged in spontaneous rhythmic bursting, which is a characteristic feature of the striatal cholinergic interneurons (Wilson, 2005). Our current study investigated the developmental changes in the membrane properties of cholinergic interneurons in mice that were 7–28 days old, as this age bracket in mice is considered to be an important stage for the maturation of the striatal neural circuits. Our results confirmed that the cholinergic interneurons could be reliably identified by their morphology and electrophysiological properties at P7 in mice. Furthermore, the neonatal cholinergic interneurons (P7) possessed Ih and there was an increase in the amplitude between P7 and P28. The volatile anesthetic sevoflurane decreased the Ih amplitude and increased the time constant of the Ih during the early postnatal period, in addition to negatively shifting the half-activation voltage of the tail current. Thus, the inhibitory effects of sevoflurane on the Ih and its functional activation kinetics would be expected to affect the neural activities of the striatal cholinergic interneurons.

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Developmental changes in the Ih and the electrophysiological properties of the striatal cholinergic interneurons Aznavour et al. (2003) utilized immunocytochemistry with a highly sensitive antibody against ChAT for the purpose of investigating the acetylcholine innervations in the developing neostriatum of rats. Their findings showed that the maturation of the cholinergic innervation achieved between P16 to P32. Although membrane properties of the striatal MSN mature until P21 to P28 in mice (Ando et al., 2014) and rats (Tepper et al., 1998), the developing cholinergic interneurons have yet to be extensively studied due to the difficulty in identifying the cell type of the immature neurons. In our experiments, we visually identified the cholinergic interneurons by their large somata and thick dendrites, in addition to confirming the expression of ChAT in the recorded cells. All of the immunostained neonate cells (P7) were ChAT-positive, which indicates that the whole-cell recordings were successfully made from the cholinergic interneurons. Furthermore, the recorded cells exhibited characteristic features of cholinergic interneurons (Kawaguchi, 1992). The electrical responses to the current injection gradually changed from P7, with the responses becoming stable at P21 to P28. This time course indicates that maturation of the membrane properties continues to some

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extent until P21 to P28 in both the cholinergic interneurons and the MSN. In the hippocampal pyramidal neurons, there was an increase in the absolute Ih amplitude from P1 to P20, with the Ih activation becoming progressively more rapid during the P1 to P20 interval (Vasilyev and Barish, 2002). Genetic depletion of the HCN1 subunit prolongs the time constant of the Ih in the cortical pyramidal neurons from 80 ms to 500 ms, and negatively shifts the half-activation voltage, which indicates that the activation time course of the Ih is dependent on the composition of the HCN channels (Chen et al., 2009). Kanyshkova et al. (2009) indicated that the developmental increase in the Ih density was associated with the increased expression of HCN channel isoforms, and that the isoform composition determines the Ih properties to enable the progressive maturation of the rhythmic patterns in the thalamic neurons. In our current study, there was an increase in the absolute amplitude of the Ih in conjunction with the development, whereas there were no developmental changes in the time constants of the Ih (460–580 ms) and in the half activation voltages during the period from P7 to P28 in the striatal cholinergic interneurons. During this time period, we could not find any apparent kinetic changes of the Ih that indicated there were alternations of the HCN subunits.

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Inhibitory effects of sevoflurane on the Ih in the developing cholinergic interneurons The inhalational anesthetics, halothane and isoflurane, have been reported to decrease the neuronal Ih due to their specific effects on a few HCN subunits in the mouse cortical pyramidal neurons (Chen et al., 2005, 2009). Our current study confirmed that the Ih in the developing striatal cholinergic interneurons was inhibited after the application of sevoflurane at concentrations of 1.0–4.0%, which are clinically relevant

concentrations

of

sevoflurane

(Anderson

et

al.,

2013).

This

sevoflurane-induced inhibitory effect was dose-dependent. Even though the amount of the Ih increased in conjunction with the development, the percentage of the block by the sevoflurane remained unchanged. This indicates that the sensitivity of the inhibitory effect did not differ between P7 to P28. Previous studies have shown that halothane and isoflurane produced remarkable shifts (−9.8 mV in the hypoglossal nucleus motoneurons, and −11.7 mV in the cortical pyramidal neurons, respectively) in the half-activation voltage of the tail current (Chen et al., 2005, 2009). In our current study, we found that the sevoflurane also induced a shift (−4.1 mV) in the half-activation voltage of the tail current, in addition to significantly increasing the Ih time constants, suggesting that the sevoflurane slowed the Ih activation in the striatal cholinergic

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interneurons. These results raise the possibility that sevoflurane acts as an Ih channel modulator rather than as a simple channel blocker. However, elucidation of the effects on Ih will require performing a detailed study with suitable experimental methods, such as a heterologous expression system. At least four HCN subunits (HCN1–4) of the mammalian Ih channels have been previously identified (Clapham, 1998). Furthermore, while it has been reported that these HCN subunits are present in many neurons, significant differences exist in the distribution of each of these HCN subunits between the neurons. For example, the HCN1 and HCN2 subunits are mainly distributed in the cerebral cortex and the hippocampus (Santoro et al., 2000). In dopaminergic substantia nigra neurons, the HCN2–4 mRNA was identified as the distributed constituents, while the HCN1 mRNA was not detected at all (Franz et al., 2000). Dopaminergic neurons of the substantia nigra were reported to have an Ih that was similar to the striatal cholinergic interneurons (Mercuri et al., 1995). Likewise, the HCN2 and HCN4 subunits were reported to be the main constituents that were distributed in the striatum, with the HCN4 subunits mainly expressed in the cholinergic interneurons (Santoro et al., 2000). Considering the sparse distribution of the HCN1 subunit in the striatum, we speculate that sevoflurane-induced striatal Ih inhibition and the disturbance of the Ih activation are related to the HCN2 and

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HCN4 subunits.

Sevoflurane attenuates the rebound activation of the cholinergic interneurons HCN conductance increases and decreases slowly in conjunction with the voltage change, with the reversal potential of the HCN channel occurring at around −40 mV under physiological conditions. Since the cholinergic interneurons fire spontaneously and their potential fluctuates around the RMP (−40 mV to −50 mV), this may induce the HCN conductance to some extent. Our experiment showed that the spontaneous spiking of cholinergic interneurons was not regularly changed by the administration of the sevoflurane during the cell-attached recordings (Supplemental Fig. 3). When activating the HCN channels, however, large hyperpolarization would be expected to be more effective. After adjusting the current injection to achieve a membrane potential of −90 mV, we examined the effect of sevoflurane on the spike firing rate during the rebound depolarization of the cholinergic interneurons following the accentuated HCN conductance. In this recording configuration, sevoflurane significantly decreased the depolarizing sag and the number of spikes during the rebound depolarization, which is essential for the mediation of the behavioral response to the sensory stimulus (Schulz and Reynolds, 2013). Since the application of ZD7288 also reduced the rebound

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excitation, this suggested that the sevoflurane-induced inhibition of Ih would decrease the rebound spiking, thereby affecting the sensory responses in the cholinergic interneurons. Previous studies indicated that early postnatal exposure to volatile anesthetics might cause neurodevelopmental deficits (Hudson and Hemmings, 2011; Takaenoki et al., 2014; Andropoulos et al., 2014; Makaryus et al., 2015; Raper et al., 2015; Vutskits and Xie, 2016). Our present study demonstrated that neonate cholinergic interneurons (P7) already possessed Ih. Developmental changes in the amplitude and the kinetics of Ih likely contribute to the firing activity of the cholinergic interneurons. Based on our current findings, the sevoflurane-induced inhibition of Ih may lead to alterations of the acetylcholine-dopamine balance and the sensory responses in the developing striatum. The neurodevelopmental effect of volatile anesthetics with regard to the Ih will need to be further examined in future studies.

Grants This study was supported by Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Numbers 25462415 to KN, 26861249 to YS, and 15K20059 to NA), and the Smoking Research Foundation of Japan (to MM).

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References Anderson BJ, Lerman J, Coté CJ., 2013. Pharmacokinetics and Pharmacology of Drugs Used in Children. In: Coté CJ, Lerman J, Anderson BJ. (Eds.), Coté and Lerman’s A Practice of Anesthesia for Infants and Children, 5th ed, Elsevier Science, Saunders, pp. 77–149. Ando N, Sugasawa Y, Inoue R, Aosaki T, Miura M, Nishimura K., 2014. Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development. Eur. J. Neurosci. 40, 3147–3157. Andropoulos DB, Ahmad HB, Haq T, Brady K, Stayer SA, Meador MR, Hunter JV, Rivera C, Voigt RG, Turcich M, He CQ, Shekerdemian LS, Dickerson HA, Fraser CD, Dean McKenzie E, Heinle JS, Blaine Easley R., 2014. The association between brain injury, perioperative anesthetic exposure, and 12-month neurodevelopmental outcomes after neonatal cardiac surgery: a retrospective cohort study. Paediatr. Anaesth. 24, 266–274. Aosaki T, Graybiel AM, Kimura M., 1994a. Effect of the nigrostriatal dopamine system on acquired neural responses in the striatum of behaving monkeys. Science 265, 412–415. Aosaki T, Kimura M, Graybiel AM., 1995. Temporal and spatial characteristics of

24

tonically active neurons of the primate's striatum. J. Neurophysiol. 73, 1234–1252. Aosaki

T,

Miura

M,

Suzuki

T,

Nishimura

K,

Masuda

M.,

2010.

Acetylcholine-dopamine balance hypothesis in the striatum: an update. Geriatr. Gerontol. Int. 10 Suppl 1, S148–157. Aosaki T, Tsubokawa H, Ishida A, Watanabe K, Graybiel AM, Kimura M., 1994b. Responses of tonically active neurons in the primate's striatum undergo systematic changes during behavioral sensorimotor conditioning. J. Neurosci. 14, 3969–3984. Aznavour N, Mechawar N, Watkins KC, Descarries L., 2003. Fine structural features of the acetylcholine innervation in the developing neostriatum of rat. J. Comp. Neurol. 460, 280–291. Bennett BD, Callaway JC, Wilson CJ., 2000. Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons. J. Neurosci. 20, 8493–8503. Bennett BD, Wilson CJ., 1999. Spontaneous activity of neostriatal cholinergic interneurons in vitro. J. Neurosci. 19, 5586–5596. Biel M, Wahl-Schott C, Michalakis S, Zong X., 2009. Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89, 847–885. Blain-Moraes S, Tarnal V, Vanini G, Alexander A, Rosen D, Shortal B, Janke E,

25

Mashour GA., 2015. Neurophysiological correlates of sevoflurane-induced unconsciousness. Anesthesiology 122, 307–316. Chae YJ, Zhang J, Au P, Sabbadini M, Xie GX, Yost CS., 2010. Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK. Anesthesiology 113, 1326–1337. Chen X, Shu S, Kennedy DP, Willcox SC, Bayliss DA., 2009. Subunit-specific effects of isoflurane on neuronal Ih in HCN1 knockout mice. J. Neurophysiol. 101, 129–140. Chen X, Sirois JE, Lei Q, Talley EM, Lynch C3rd, Bayliss DA., 2005. HCN subunit-specific and cAMP-modulated effects of anesthetics on neuronal pacemaker currents. J. Neurosci. 25, 5803–5814. Clapham DE., 1998. Not so funny anymore: pacing channels are cloned. Neuron 21, 5–7. DiMaggio C, Sun LS, Li G., 2011. Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth. Analg. 113, 1143–1151. Eckle VS, Digruccio MR, Uebele VN, Renger JJ, Todorovic SM., 2012. Inhibition of T-type

calcium

current

in

rat

thalamocortical

26

neurons

by

isoflurane.

Neuropharmacology 63, 266–273. Franz O, Liss B, Neu A, Roeper J., 2000. Single-cell mRNA expression of HCN1 correlates with a fast gating phenotype of hyperpolarization-activated cyclic nucleotide-gated ion channels (Ih) in central neurons. Eur. J. Neurosci. 12, 2685–2693. Hudson AE, Hemmings HC Jr., 2011. Are anaesthetics toxic to the brain? Br. J. Anaesth. 107, 30–37. Ishiwa D, Kamiya Y, Itoh H, Saito Y, Ohtsuka T, Yamada Y, Andoh T., 2004. Effects of isoflurane and ketamine on ATP-sensitive K channels in rat substantia nigra. Neuropharmacology 46, 1201–1212. Ishizeki J, Nishikawa K, Kubo K, Saito S, Goto F., 2008. Amnestic concentrations of sevoflurane inhibit synaptic plasticity of hippocampal CA1 neurons through gamma-aminobutyric acid-mediated mechanisms. Anesthesiology 108, 447–456. Kanyshkova T, Pawlowski M, Meuth P, Dube C, Bender RA, Brewster AL, Baumann A, Baram TZ, Pape HC, Budde T., 2009. Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations. J. Neurosci. 29, 8847–8857. Kawaguchi Y., 1992. Large aspiny cells in the matrix of the rat neostriatum in vitro:

27

physiological identification, relation to the compartments and excitatory postsynaptic currents. J. Neurophysiol. 67, 1669–1682. Kawaguchi Y., 1993. Physiological, morphological, and histochemical characterization of three classes of interneurons in rat neostriatum. J. Neurosci. 13, 4908–4923. Lewis AS, Chetkovich DM., 2011. HCN channels in behavior and neurological disease: too hyper or not active enough? Mol. Cell. Neurosci. 46, 357–367. Makaryus R, Lee H, Feng T, Park JH, Nedergaard M, Jacob Z, Enikolopov G, Benveniste H., 2015. Brain maturation in neonatal rodents is impeded by sevoflurane anesthesia. Anesthesiology 123, 557–568. Mercuri NB, Bonci A, Calabresi P, Stefani A, Bernardi G., 1995. Properties of the hyperpolarization-activated cation current Ih in rat midbrain dopaminergic neurons. Eur. J. Neurosci. 7, 462–469. Miura

M,

Saino-Saito

S,

Masuda

M,

Kobayashi

K,

Aosaki

T.,

2007.

Compartment-specific modulation of GABAergic synaptic transmission by µ-opioid receptor in the mouse striatum with green fluorescent protein-expressing dopamine islands. J. Neurosci. 27, 9721–9728. Oose Y, Miura M, Inoue R, Andou N, Aosaki T, Nishimura K., 2012. Imbalanced suppression of excitatory and inhibitory synaptic transmission onto mouse striatal

28

projection neurons during induction of anesthesia with sevoflurane in vitro. Eur. J. Neurosci. 35, 1396–1405. Patel AJ, Honore E., 2001. Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci. 24, 339–346. Raper J, Alvarado MC, Murphy KL, Baxter MG., 2015. Multiple Anesthetic Exposure in Infant Monkeys Alters Emotional Reactivity to an Acute Stressor. Anesthesiology 123, 1084–1092. Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, Siegelbaum SA., 2000. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J. Neurosci. 20, 5264–5275. Schulz JM, Reynolds JN., 2013. Pause and rebound: sensory control of cholinergic signaling in the striatum. Trends Neurosci. 36, 41–50. Song WJ, Tkatch T, Baranauskas G, Ichinohe N, Kitai ST, Surmeier DJ., 1998. Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominantly of the A type and attributable to coexpression of Kv4.2 and Kv4.1 subunits. J. Neurosci. 18, 3124–3137. Suzuki T, Miura M, Nishimura K, Aosaki T., 2001. Dopamine-dependent synaptic plasticity in the striatal cholinergic interneurons. J. Neurosci. 21, 6492–6501.

29

Takaenoki Y, Satoh Y, Araki Y, Kodama M, Yonamine R, Yufune S, Kazama T., 2014. Neonatal exposure to sevoflurane in mice causes deficits in maternal behavior later in adulthood. Anesthesiology 120, 403–415. Tepper JM, Sharpe NA, Koos TZ, Trent F., 1998. Postnatal development of the rat neostriatum: electrophysiological, light- and electron-microscopic studies. Dev. Neurosci. 20, 125–145. Van Vulpen EH, Van Der Kooy D., 1996. Differential maturation of cholinergic interneurons in the striatal patch versus matrix compartments. J. Comp. Neurol. 365, 683–691. Vasilyev

DV,

Barish

ME.,

2002.

Postnatal

development

of

the

hyperpolarization-activated excitatory current Ih in mouse hippocampal pyramidal neurons. J. Neurosci. 22, 8992–9004. Vutskits L, Xie Z., 2016. Lasting impact of general anaesthesia on the brain: mechanisms and relevance. Nat. Rev. Neurosci. 17, 705-717. Wilson CJ., 2005. The mechanism of intrinsic amplification of hyperpolarizations and spontaneous bursting in striatal cholinergic interneurons. Neuron 45, 575–585.

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Figure legends Fig. 1. Expression of ChAT in the recorded biocytin-filled cells. Fluorescence labelling for biocytin (blue) and ChAT (red) at the age of P7 (A, B, C), P14 (D, E, F), P21 (G, H, I) and P28 (J, K, L). Merged images indicate colocalization of biocytin and ChAT in the recorded cells. Electrophysiologically identified medium-spiny neuron (MSN, P21, arrows) is negative for ChAT (M, N, O). Note the neighboring ChAT-positive cholinergic cell (arrowhead) in the merged image. Scale bars 20 μm.

Fig. 2. Sevoflurane decreases the Ih of the cholinergic interneurons in a dose-dependent manner. (A) Representative traces of the current response in the striatal cholinergic interneurons elicited by hyperpolarization voltage steps from −30 to −130 mV (P21). Sevoflurane (4%, 10 min) caused inhibition of the maximal current amplitude, and ZD7288, the Ih channel blocker, provided further inhibition. (B) I-V relationship in the striatal cholinergic interneurons during the voltage steps (P7–P28). Sevoflurane significantly decreased the current amplitude including the Ih during the hyperpolarization voltage steps between −130 and −80 mV (n = 16, two-way ANOVA with Bonferroni’s post hoc test, **P < 0.01 vs. control). (C) Representative subtracted traces of the Ih response in the striatal cholinergic interneurons evoked by a

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hyperpolarization voltage of −130 mV (P21). ZD7288 traces were subtracted from the control and sevoflurane traces. When the traces for the control and the post sevoflurane administration were overlaid, the results show sevoflurane caused inhibition of the Ih amplitude (arrow). (D) Averaged sevoflurane-induced inhibition of the Ih amplitude at −120 mV in the striatal cholinergic interneurons (P7–P28). Clinically used concentrations of sevoflurane (1.0, 2.0 and 4.0%) reduced the Ih in a dose-dependent manner.

P < 0.01 vs. 1.0%, ANOVA followed by Bonferroni’s post hoc test. The

**

number of experiments is indicated in the parentheses.

Fig. 3. Developmental changes in the absolute Ih amplitude and in the sevoflurane-induced inhibitory effect on the Ih. (A) The amplitude of Ih measured as the time-dependent ZD7288-sensitive current elicited by hyperpolarization from -30 to -120 mV for 2 sec. There was a significant increase in the absolute amplitude of the Ih **

under control conditions from P7 to P28.

P < 0.01 vs. P7,

##

P < 0.01 vs. P14, #P <

0.05 vs. P14, ANOVA followed by Bonferroni’s post hoc test. (B) The ratio of sevoflurane (4%)-induced inhibition of Ih elicited by hyperpolarization from -30 to -120 mV for 2 sec. There was no developmental change with age (ANOVA). The number of experiments is indicated in the parentheses.

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Fig. 4. Effect of sevoflurane on the Ih activation kinetics. (A) Sample traces for the Ih activation recorded at −130 mV in the striatal cholinergic interneurons (P28). In A1, the control and post sevoflurane (4%) administration traces are overlaid, while in A2 the same traces were normalized to the maximum amplitude. (B) Sevoflurane (4%) significantly increased the time constants for the Ih activation kinetics (n = 47; P7–P28; paired t-test, **P < 0.01 vs. control). (C) There was no developmental change in the control time constants (ANOVA). (D) Activation curves were determined by measuring the peak amplitude of tail currents elicited after a repolarization to -60 mV and fitting with a Boltzmann function. The half-activation voltage (V1/2) of the tail current was shifted significantly after the administration of sevoflurane (n = 17; P7–P28; paired t-test, *P < 0.05 vs. control). (E) There was no developmental change in the control half-activation voltage of the tail current (ANOVA). The number of experiments is indicated in the parentheses.

Fig. 5. Sevoflurane decreases the spike firing rate during the rebound depolarization. (A) Representative traces of the voltage response in the striatal cholinergic interneurons (P28). Hyperpolarization was adjusted to achieve a membrane

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potential of −90 mV in order to lead to subsequent rebound depolarization. Sevoflurane (4%) decreased the rebound depolarization and the number of spikes. The numbers of spikes were counted within the 400 ms time window from the end of the current injection. (B) Representative traces of the voltage response under the same settings that were described in A (P21). ZD7288 strongly inhibited the rebound depolarization. (C1, D1) Averaged sevoflurane-induced inhibition of the depolarizing sag potential (n = 16; P7–P28; paired t-test, **P < 0.01 vs. control) and the spike firing rate (n = 15; P7–P28; paired t-test, **P < 0.01 vs. control). Sevoflurane (4%) significantly reduced the rebound responses when using the same settings that were described in A. (C2, D2) Sevoflurane-induced inhibition of the sag potential and the rebound spiking were plotted versus the age.

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