Prelim Epub

Articles in PresS. J Neurophysiol (June 10, 2009). doi:10.1152/jn.00252.2009

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Title:

Isoflurane inhibits the neurotransmitter release machinery

Bruce E. Herring1, Zheng Xie2, Jeremy Marks3 and Aaron P. Fox1

Affiliation: Departments of 1Neurobiology, Pharmacology and Physiology, 2Anesthesia and Critical Care and 3Pediatrics, University of Chicago, 5835 S. Cottage Grove Ave, Chicago, Illinois 60637, USA.

Running Head: Isoflurane interacts with syntaxin 1A

Correspondence should be addressed to: Aaron P. Fox, The University of Chicago, 5835 S. Cottage Grove Ave, Abbott Hall, Ab131, Chicago, Illinois 60637 Email: [email protected].

Number of Figures: 5 Number of Tables: 0

Copyright © 2009 by the American Physiological Society.

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Abstract

46

poorly understood. Facilitation of inhibitory GABAA receptors plays an important role in

47

anesthesia but other targets have also been linked to anesthetic actions. Anesthetics are

48

known to suppress excitatory synaptic transmission, but it has been difficult to determine

49

whether they act on the neurotransmitter release machinery itself. By directly elevating

50

[Ca2+]i at neurotransmitter release sites without altering plasma membrane channels or

51

receptors, we show that the commonly used inhalational general anesthetic, isoflurane,

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inhibits neurotransmitter release at clinically relevant concentrations, in a dose-dependent

53

fashion in PC12 cells and hippocampal neurons. We hypothesized that a SNARE and/or

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SNARE-associated protein represents an important target(s) for isoflurane. Overexpression

55

of a syntaxin 1A mutant, previously shown in C. elegans to block the behavioral effects of

56

isoflurane, completely eliminated the reduction in neurotransmitter release produced by

57

isoflurane, without affecting release itself, thereby establishing the possibility that syntaxin

58

1A is an intermediary in isoflurane’s ability to inhibit neurotransmitter release.

Despite their importance, the mechanism of action of general anesthetics is still

59 60 61 62 63 64 65

Key words: syntaxin 1A, anesthesia, SNARE, synaptic vesicle release, amperometry.

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Introduction

67

Most, but not all, anesthetics are known to facilitate GABAA receptor activity

68

thereby enhancing inhibitory synaptic transmission. Modulation of GABAA receptors in

69

this manner is known to be an important part of the mechanism of action for many

70

anesthetics. More recent studies, however, have shown that general anesthetics can

71

influence a number of voltage and ligand gated ion channels (Campagna et al. 2003; Chen

72

et al. 2007; Hemmings et al. 2005; Krasowski and Harrison 1999; Petrenko et al. 2007).

73

Although not extensive, some work has investigated the effects of inhalational

74

anesthetics on neurotransmitter release. For instance, Richards and colleagues showed that

75

low concentrations of anesthetic affected chemical transmission but neither impulse

76

conduction nor cellular electrical properties were affected, raising the possibility of direct

77

modulation of the release machinery (Pocock and Richards 1988; Richards 1972; Richards

78

and White 1975). But the significant number of proteins targeted by general anesthetics has

79

made it difficult to isolate the release machinery in many of these experiments.

80

In the present study we set out to determine if the clinically used volatile anesthetic,

81

isoflurane, directly influences the mammalian neurotransmitter release machinery. To

82

accomplish this, it is necessary to observe the effect of isoflurane on evoked

83

neurotransmitter release independent of anesthetic modulation of channels and receptors.

84

To prevent actions of anesthetics on channels or receptors from altering neurotransmitter

85

release, we used experimental paradigms that kept membrane potential constant, but which

86

allowed [Ca2+]i to be elevated by a known amount. These paradigms allowed us to probe

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interactions between anesthetics and the release machinery directly. We observed that

4

88

clinically relevant concentrations of isoflurane dramatically inhibited the neurotransmitter

89

release machinery of PC12 cells and cultured rat hippocampal neurons. The robust nature

90

of the suppression suggests that inhibition of release machinery may represent an important

91

component of the anesthetic state. The hydrophobic nature of isoflurane suggests that it

92

might interact with proteins within the plane of lipid membranes. Thus, we sought to

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examine release machinery proteins with alterations in their transmembrane domains. The

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C. elegans md130 syntaxin 1A point mutation results in a truncated form of syntaxin 1A.

95

This mutant syntaxin is missing part of the H3 domain and the entire transmembrane

96

domain; the mutant syntaxin also includes 10 novel amino acids on the carboxy-terminus.

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The md130A mutation blocked the behavioral effects of isoflurane in C. elegans (van

98

Swinderen et al. 1999). Overexpression of md130A in PC12 cells completely blocked

99

isoflurane’s ability to inhibit the neurotransmitter release machinery. This data suggests a

100

possible role for syntaxin 1A as an intermediary in isoflurane’s ability to inhibit

101

neurotransmitter release.

102

5

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Materials and Methods

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PC12 and neuronal cell culture

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PC12 cells were grown on collagen-coated 10 cm Petri dishes in culture medium

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that consisted of RPMI-1640, 10% heat-inactivated horse serum, 5% fetal bovine serum, 2

107

mM glutamine, and 10 µg/ml gentamicin in a humidified 7% CO2 incubator at 37ºC.

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Culture medium was replaced every other day and cells were passaged once per week.

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Cells were replated on poly-lysine coated glass coverslips 24 hours prior to recording.

110

Hippocampal neuron cultures were prepared from E18 Sprague-Dawley rats as previously

111

described (Wang et al. 2006).

112 113

Amperometric measurement of catecholamine release

114

Carbon fiber electrodes were fabricated and used as previously described by

115

Grabner et al. 2005 (Grabner et al. 2005). The detection threshold for amperometric events

116

was set at 5 times the baseline root mean squared noise, and the spikes were automatically

117

detected. Amperometric spike features, quantal size, and kinetic parameters were analyzed

118

with a series of macros written in Igor Pro (Wavemetrics Inc.) and kindly supplied to us by

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Dr. Eugene Mosharov. On each day of recording amperometric measurements were made

120

from a similar number of experimental and control cells. This strategy reduces cell-to-cell

121

variation. A student’s t-test was used to assess differences between populations of cells.

122 123

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PC12 cell permeabilization and stimulation

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An amperometric electrode was placed gently against a cell. Following 2 min in a

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Ca2+-free solution (1), the cell was permeabilized with 20 µM digitonin (Ca2+-free) for 25

127

sec (2), and then stimulated for 2-3 min with a solution containing 100 µM Ca2+ (3). The

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cell was allowed to recover for 2 min in Ca2+-free media (4), and the cycle began again at

129

step (2). Cells were stimulated 4-5 times in this way. In drug treated cells isoflurane or the

130

non-immobilizer, F6, was introduced into the bath 25 sec prior to stimulation and was

131

present throughout the recording. This was done in order to maximize drug exposure time.

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The stimulation step (3) producing the greatest amount of release was analyzed. The

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recording solutions had standard compositions previously described in (Grabner et al.

134

2005).

135 136

Optical measurement of evoked RH414 release

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Coverslips containing live rat hippocampal neurons were briefly rinsed in HBSS

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before being placed in a 60 mM KCl loading solution containing 10 μM RH414 (Molecular

139

probes, Eugene, OR) for 75 sec. The coverslips were then put back into HBSS for 1-5 min.

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RH414 loaded synapses were observed using an Olympus IX81 inverted microscope

141

through a U Plan APO 60x water objective (0.512 um/pixel). 530-550 nm light from a high

142

power 100W Hg arc lamp was used for excitation, and emitted light was filtered through a

143

590LP filter. Images were captured using MetaMorph. Time-lapse sequences of synaptic

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fluorescence prior to and following evoked synaptic vesicle exocytosis were made with an

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acquisition rate of one image every 2 sec. Prior to stimulation neurons were washed for 4

7

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min with HBSS+0.5mM isoflurane or HBSS alone. Neurons were then exposed to 5 µM

147

ionomycin in HBSS+0.5 mM isoflurane or 5 µM ionomycin in HBSS alone. Fluorescent

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synapses were monitored 40 sec before and ~2 min after ionomycin treatment.

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Quantitative analysis of evoked RH414 release

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Fluorescent nerve terminals found to undergo de-staining following ionomycin

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treatment were marked as regions of interest in ImageJ (http://rsb.info.nih.gov/ij/). Circular

153

regions of interest were selected to include the largest portion of the fluorescent spot and

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include as little background as possible. ImageJ was then used to determine the pixel

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intensities of each region, which were then averaged together to produce values of local

156

fluorescence intensity in nerve terminals over time. Background fluorescence was

157

subsequently subtracted. The fluorescence intensity of each nerve terminal was then

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normalized to its average fluorescence value 40 sec prior to ionomycin exposure. The

159

percentage of de-staining following 2 min of ionomycin exposure as well as the time

160

constant of fluorescent decay were determined for each nerve terminal in the control

161

condition and compared to that of nerve terminals exposed to isoflurane. A student’s t-test

162

was again used to assess differences between the two conditions. Tau values were

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determined by fitting fluorescent intensity plots with a second-order exponential decay

164

function, y=y0+A1e-x/t1+A2e-x/t2. The fluorescence traces for all nerve terminals in a given

165

condition were then aligned at the initial point of de-staining and averaged.

166 167

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168 169

[Ca2+]i measurements using Fura-2

170

Hippocampal neurons were loaded with Fura-2 and imaged as previously described in

171

chromaffin cells (Xie et al. 2006). For these experiments, peak [Ca2+]i was measured.

172 173

Measurement of drug concentrations

174

Isoflurane solutions were prepared and measured as previously described (Jones et

175

al. 1992; Jones and Harrison 1993). Isoflurane was prepared in sealed plastic I-V bags. We

176

have previously found that isoflurane concentrations in the bags and in the bath are

177

remarkably constant for up to 1.5 hours when measured in representative experiments using

178

gas chromatography (GC) (Xie et al. 2006). All isoflurane concentrations in this manuscript

179

are provided in mM. The MAC (minimum alveolar concentration required for immobility

180

in response to a noxious stimulus in 50% of trials (Eger et al. 1965)) equivalents of

181

isoflurane have been reported to be in the range of ~0.3 mM (Franks and Lieb 1996) to ~0.5

182

mM (Franks and Lieb 1996; Jones and Harrison 1993) at 25°C. The equivalent of 2 MAC

183

was employed for the nonimobilizer, F6. This concentration was estimated to be ~36 μM

184

(Mihic et al. 1994).

185 186

Whole-cell patch clamp stimulation protocol

187

Whole-cell patch electrodes were pulled from microhematocrit capillary tubes

188

(Drummond Scientific Co., Broomall, PA), fire-polished, and filled with an internal

189

solution that contained 100 µM Ca2+, 145 mM NaCl, 2.0 mM KCl, 10 mM HEPES, 1 mM

9

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Na2ATP and 1.0 mM MgCl2, pH 7.2 NaOH, osmolarity 300 mOsm/kg. A single PC12 cell

191

was then selected and a gigaseal was obtained with a patch pipette connected to an

192

Axopatch-1C amplifier (Axon Instruments, Foster City, CA). An amperometric electrode

193

was then gently placed against the opposite side of the cell. This preparation was then

194

washed with either HBSS (Hank’s Balanced Saline Solution) or HBSS containing

195

isoflurane (1 mM) for 4 min. Using suction the cell was then patch clamped in the whole-

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cell configuration and held at -65 mV throughout the duration of the recording.

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Amperometric data was collected for 2.5 min. Data analysis started 15 sec after breaking

198

the membrane with the patch pipette to allow [Ca2+]i equilibration. This 15 sec delay

199

ensured a uniform concentration of the 100 µM Ca2+ pipette solution inside the cell prior to

200

data acquisition. Cells were continuously washed with HBSS or HBSS+isoflurane (1 mM)

201

throughout the duration of the recording.

202 203

md130A cloning and expression

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A pGMHE vector containing rat syntaxin 1A cDNA was provided by Dr. Richard

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Tsien. PCR cloning was used to obtain DNA encoding md130A and wild type syntaxin 1A

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from

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GAGAATTCCATGAAGGACCGAACCCA

208

GATCTAGACTCAACCATCTCTCCTTGTAATATCAAAAATTCCACAAATCTGGCT

209

CTCCACCAG.

210

GAGAATTCCATGAAGGACCGAACCCA

211

GATCTAGACTATCCAAAGATGCCCC. The resulting PCR products were cloned into

this

vector.

The

The

primers

primers

used

used

to

to

produce

md130A

were and

produce

wild

type

syntaxin

were and

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pcDNA3.1/Neo (Invitrogen), sequenced and purified. Cells were co-transfected with either

213

the md130A or wild type syntaxin plasmid and pEGFP-N1 (BD biosciences) using

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Lipofectamine 2000. A syntaxin plasmid:pEGFP ratio of 7:1 was used to ensure green cells

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expressed the desired form of syntaxin. Recordings were made from these cells 48 to 72

216

hours post transfection.

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Immunoblotting

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Levels of syntaxin and actin in PC12 cells were assessed using the following

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antibodies: syntaxin (#573831, Calbiochem), β-actin (JLA20; Developmental Systems

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Hybridoma Bank, University of Iowa) and horseradish peroxidase-labeled anti-mouse

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(Jackson ImmunoResearch). ECL Advance reagents (Amersham/GE Healthcare) were used

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for detection of the horseradish peroxidase-labeled secondary antibodies.

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Results

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Isoflurane dose-dependently inhibits the neurotransmitter release machinery

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of PC12 cells

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Exocytosis was elicited in digitonin-permeabilized cells in the presence and absence

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of isoflurane (0.5 mM). Basal (Ca2+-independent) neurotransmitter release is virtually non-

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existent in permeablized PC12 cells in Ca2+-free conditions, but robust release is observed

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upon exposing cells to Ca2+-containing solutions (Graham et al. 2002; Jankowski et al.

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1992). Fig. 1A plots a representative amperometric current observed in a PC12 cell upon

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stimulation. Physiologically, release is evoked by the activation of voltage-gated Ca2+

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channels. The proximity of Ca2+ channels to synaptic release sites suggests that [Ca2+]i may

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rise to levels above 100 μM at the vesicle (Llinas et al. 1992). To mimic these levels in our

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experiments, evoked neurotransmitter release was elicited by exposing digitonin-

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permeabilized cells to 100 μM Ca2+, for 2 min (as indicated), in the absence of isoflurane

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(Fig. 1A) or in the presence of isoflurane (0.5 mM; Fig. 1B). The amperometric trace in the

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presence of isoflurane contained many fewer amperometric events. Control cells had an

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average of 94 ± 13 (n = 25) amperometric events during a 2 min stimulation while cells

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exposed to isoflurane (0.5 mM) had 58 ± 11 (n = 24) events (mean ± SEM; fig. 1C). This

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38% reduction in the number of amperometric events was significant (P = 0.049). These

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data suggest that isoflurane inhibits the neurotransmitter release machinery at a clinically

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relevant concentration by reducing the number of vesicles released.

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[On each day of recording, amperometric measurements were made from a similar number

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of experimental and control cells. This strategy reduces cell-to-cell variation].

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Isoflurane was also found to inhibit the neurotransmitter release machinery in a

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dose-dependent fashion over a range of concentrations (0.3, 0.5, 1, 2, and 3 mM) that

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include the clinically relevant range (0.3 – 1 mM). Isoflurane at 0.3, 1, 2, and 3 mM

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reduced the number of amperometric events per 2 min stimulation by 27, 52, 64 and 54%,

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respectively. Fig. 1D plots the number of amperometric events observed as a function of

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isoflurane concentration. These data were fit with a standard dose-response equation (see

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legend). The best possible fit of the data suggests the effects of isoflurane on the

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neurotransmitter release machinery saturate at concentrations > 1 mM, which maximally

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reduce neurotransmitter release by ~70%. The inset plots the same data on a linear scale to

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better illustrate the saturation of the response to isoflurane. 1 mM, 2 mM and 3 mM

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isoflurane inhibited neurotransmitter release to the same extent. The EC50 provided by the

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fitting function was 0.38 mM isoflurane. Here, the EC50 refers to the concentration at which

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isoflurane reached 50% of its maximal inhibition (~70%). The MAC (minimum alveolar

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concentration required for immobility in response to a noxious stimulus in 50% of trials

269

(Eger et al. 1965)) equivalent of the isoflurane used in this study has been reported to be

270

~0.3 mM (Franks and Lieb 1996) at 25°C. Dose-dependent effects on quantal amplitude or

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kinetics were not observed (data not shown). Together, these data indicate that isoflurane

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has a statistical and biologically important dose-dependent effect on the release machinery

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at concentrations spanning this anesthetic’s clinically effective range.

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To ensure that isoflurane inhibited the neurotransmitter release machinery and not

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digitonin-permeabilization a patch pipette was used to dialyze cells with a 100 µM Ca2+

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solution in order to stimulate catecholamine release. Cells were patch clamped in whole-

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cell configuration and held at -65 mV, which precluded activation of voltage-gated

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channels. Fig. 2A plots a representative amperometric current observed in a patch-clamped

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cell perfused with a 100 µM Ca2+ solution for 2.5 min (as indicated by the bar in the

280

figure), in the absence of isoflurane while Fig. 2B plots a representative amperometric

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current observed from a cell exposed to isoflurane (1 mM). Isoflurane (1 mM) reduced the

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number of amperometric events during each 2.5 min stimulation period by 59% (P = 0.014,

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fig. 2C), a value similar to the inhibition observed with digitonin-permeabilized cells (see

284

fig. 1D). A box chart plots the range of these data in fig. 2D. Effects on quantal amplitude

285

or kinetics were not observed (data not shown). These data indicate that isoflurane’s

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influence on neurotransmitter release stems from an inhibition of the neurotransmitter

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release machinery.

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To further eliminate the possibility of non-specific effects, experiments were carried

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out with a non-immobilizing anesthetic analog, 1, 2-dichlorohexaflurocyclobutane (F6). F6,

290

although similar to volatile anesthetics in terms of its composition and hydrophobicity, does

291

not produce anesthesia. This experiment was conducted to determine whether inhibition of

292

the neurotransmitter release machinery is specific to agents capable of producing

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anesthesia. Inhibition of neurotransmitter release was not observed when cells were

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exposed to F6 (36 μM; predicted ~2X MAC, see methods). If anything, F6 increased the

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number of amperometric events observed in permeabilized cells by ~25% (fig. 3), but this

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difference was not significant (P = 0.29). Further studies in a larger pool of cells will be

297

required to determine whether this small augmentation is significant. Taken together, these

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data demonstrate the selective inhibitory actions of isoflurane on the neurotransmitter

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release machinery.

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Isoflurane inhibits the neurotransmitter release machinery of hippocampal

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neurons

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Neurotransmitter release mechanisms are strongly conserved between neurons and

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secretory cells (Rettig and Neher 2002). To assess whether isoflurane inhibited

304

neurotransmitter release in central neurons, we studied exocytosis in cultured embryonic

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hippocampal neurons. In order to monitor neurotransmitter release synaptic vesicles were

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loaded with the fluorescent dye RH414 prior to stimulation (fig. 4A). RH414 de-staining

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was monitored over time using time-lapse florescence imaging. Exposing neurons to a

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solution containing the ionophore ionomycin (5 µM), as indicated, evoked exocytosis.

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Synapses treated with isoflurane (0.5 mM) showed a significant reduction in exocytosis

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during ionomycin exposure (fig. 4B). After 2 min of ionomycin exposure the fluorescence

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of 58 control nerve terminals was reduced an average of 51 ± 3%, while the fluorescence of

312

98 isoflurane (0.5 mM)-treated nerve terminals was reduced an average of 37 ± 2% (P <

313

0.0001, fig. 4C). These results suggest that isoflurane (0.5 mM) inhibits synaptic release, as

314

measured by RH414 de-staining, by 27.5%. The time course of neurotransmitter release

315

was unaffected by isoflurane (both curves were well fit by single exponentials; τcontrol ~36

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ms and τisoflurane ~39 ms). Ionomycin was found to produce identical increases in [Ca2+]i in

317

the presence and absence of isoflurane in fura-2 loaded neurons (fig. 4D), a result which

318

suggests that the inhibition of neurotransmitter release results from interaction with the

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release machinery. Taken together, these data strongly suggest that clinically relevant

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concentrations of isoflurane inhibit the exocytotic machinery of neurons within the

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mammalian CNS.

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Isoflurane interacts with syntaxin 1A

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It is reasonable to assume that isoflurane, a strongly lipophilic molecule, might

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interact with release machinery proteins within the plane of the plasma membrane. We

325

examined the md130A syntaxin 1A mutant which is missing the entire transmembrane

326

domain (van Swinderen et al. 1999).

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reduced in C. elegans heterozygous for the md130 mutation (van Swinderen et al. 1999). In

328

addition, isoflurane has been shown to bind to syntaxin 1A (Nagele et al. 2005). To

329

determine if md130A influences the isoflurane sensitivity of the mammalian

330

neurotransmitter release machinery, PC12 cells were transfected with an md130A

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expression plasmid. Western blot analysis was used to confirm expression of the mutant

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(fig. 5A). Permeablized PC12 cells expressing the mutant syntaxin 1A in addition to

333

endogenous syntaxin 1A showed no inhibition upon exposure to isoflurane (1 mM; fig.

334

5B). Rather, it appears that isoflurane might augment release in cells expressing the

335

md130A mutant, but this difference was not significant. Further studies in a larger pool of

336

cells will be required to determine whether this augmentation is significant. Cells

337

transfected with wild-type syntaxin 1A showed a ~60% reduction in the number of release

338

events per stimulation (P = 0.032; fig. 5C) in the presence of isoflurane. Overexpression of

339

md130A and wild-type syntaxin 1A alone did not affect release rates in permeablized PC12

340

cells. Release from md130A-transfected PC12 cells was 95% of that in wild-type PC12

341

cells (P = 0.87). The mean rate of release from cells transfected with unmodified syntaxin

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1A was 119% of wild-type controls (P = 0.68) (data not shown). Taken together, these data

Behavioral sensitivity to isoflurane is significantly

16

343

demonstrate the direct involvement of syntaxin 1A in isoflurane’s ability to influence

344

neurotransmitter release.

345

[Please note that a high concentration of isoflurane (1 mM) was tested to ensure that the

346

md130A mutation completely suppressed the response to isoflurane.]

347 348 349 350 351 352 353 354 355 356 357 358 359 360

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Discussion

362

In this study, three different experimental protocols were used to stimulate

363

neurotransmitter release while bypassing confounding anesthetic effects on channels and

364

receptors. In PC12 cells, isoflurane strongly suppressed neurotransmitter release in

365

digitonin-permeablized cells perfused with elevated Ca2+ as well as in cells dialyzed with

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elevated levels of Ca2+, using a patch pipette. The amount of inhibition observed using the

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two different protocols was very similar. Isoflurane also suppressed neurotransmitter

368

release in cultured hippocampal neurons. The fact that clinically relevant concentrations of

369

isoflurane inhibited exocytosis in all three of these paradigms indicates that isoflurane

370

inhibits the neurotransmitter release machinery in different kinds of cells including those

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found within the mammalian CNS. Furthermore, the failure of F6 to inhibit

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neurotransmitter release from permeablized PC12 cells suggests that inhibition of

373

neurotransmitter release machinery is specific to agents that produce anesthesia. Finally,

374

overexpression of the syntaxin 1A mutant, md130A, completely eliminated the PC12 cell

375

response to isoflurane, suggesting that this t-SNARE may be involved in isoflurane’s

376

ability to inhibit the mammalian neurotransmitter release machinery. This final result,

377

though, does not preclude the involvement of other release machinery proteins in the

378

response to isoflurane.

379

It is widely held that general anesthetics influence glutamate neurotransmission via

380

presynaptic sites of action (Maclver et al. 1996; Perouansky et al. 1995; Schlame and

381

Hemmings 1995; Wu et al. 2004). However, the literature is unclear regarding the ability of

382

anesthetics to influence the neurotransmitter release machinery itself. Richards and

18

383

colleagues found that low concentrations of anesthetics affected chemical transmission but

384

not impulse conduction or cellular electrical properties in hippocampal neurons (Pocock

385

and Richards 1988; Richards 1972; Richards and White 1975). These observations led them

386

to raise the possibility of direct modulation of the release machinery by general anesthetics.

387

This group later dismissed the involvement of the neurotransmitter release machinery in

388

anesthetic action in favor of presynaptic Na+ channels after clinical concentrations of

389

isoflurane were found to have little effect on neurotransmitter release from KCl-treated

390

chromaffin cells (Pocock and Richards 1988). Similarly, subsequent studies conducted by

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Hemmings and colleagues also suggested an insensitivity of KCl-evoked neurotransmitter

392

release to clinical concentrations of isoflurane in cerebrocortical synaptosomes

393

(Lingamaneni et al. 2001; Westphalen and Hemmings 2003). Using a different assay of

394

vesicular fusion in individual neuronal synaptic terminals this group reported a dramatic

395

inhibition of neurotransmitter release (~56%) by 1 mM isoflurane; they attributed most of

396

the effect to an inhibition of pre-synaptic Na+ channels but ~one third of the response could

397

have been due to suppression of the release machinery (Hemmings et al. 2005). These

398

results are similar to those of Wu et al. who found that clinically relevant concentrations of

399

isoflurane dose-dependently reduce action potential amplitude in the presynaptic terminal

400

of glutamatergic calyx-type synapses in rat brain stem (Wu et al. 2004). Simulations

401

suggested that the effects of isoflurane on the electrical properties of neurons can account

402

for between 62-78% of the inhibitory effects of isoflurane on excitatory post synaptic

403

currents (Wu et al. 2004). In contrast with the studies from Hemmings, Richards and Wu,

404

other groups have reported more robust dose-dependent inhibition of KCl-evoked

405

neurotransmitter release by clinically relevant concentrations of isoflurane (Larsen et al.

19

406

1994; Liachenko et al. 1999; Miao et al. 1995). Larsen et al., for example, reported ~0.33, 1

407

and 2 MAC isoflurane inhibited KCl-evoked glutamate release from rat hippocampal slices

408

by 31, 42 and 51%, respectively (Larsen et al. 1994). Furthermore, Miao et al. reported that

409

volatile anesthetics, including isoflurane, dose-dependently inhibited KCl-evoked

410

neurotransmitter release from guinea pig cerebrocortical synaptosomes by ~20-25% per

411

MAC (Miao et al. 1995). Surprisingly, a recent study reported that isoflurane facilitated

412

KCl-evoked norepinephrine release from mouse spinal cord slices at ~0.5 MAC (Rowley

413

and Flood 2008).

414

In an attempt to directly investigate anesthetic effects on the neurotransmitter

415

release machinery the present study examined evoked release from both neurosecretory

416

cells and hippocampal neurons, using experimental paradigms that elevated [Ca2+]i at the

417

release sites. These methods of stimulation were used in lieu of KCl-evoked release in

418

order to avoid potential confounding effects of anesthetics on channels and receptors. Our

419

data from individual PC12 cells and individual hippocampal neuron synaptic terminals,

420

strongly suggests clinical concentrations of isoflurane inhibit the neurotransmitter release

421

machinery, and is most consistent with reports of dose-dependent inhibition of KCl-evoked

422

release with clinical concentrations of isoflurane reviewed above. At this time it is difficult

423

to draw firm conclusions from the literature, as the effects of anesthetics remain somewhat

424

unclear. This may be due to the fact that anesthetics target a variety of different proteins.

425

For instance, anesthetics appear to target both pre-synaptic Na+ channels as well as pre-

426

synaptic TREK channels (Hemmings et al. 2005; Westphalen et al. 2007). Activation of

427

pre-synaptic K+ channels along with a concomitant decrease in Na+ channel activity may

428

produce significant suppression of neurotransmitter release, when neurons are induced to

20

429

fire action potentials. But cells may exhibit a different response when exposed to high-K+

430

solutions. Neurons typically do not respond to high-K+ solutions in a Nernst-like manner,

431

due to the activation of a variety of non-K+ conductances (Augustine et al. 2008). In this

432

case, activation of background K+ channels by anesthetics may cause cells to depolarize to

433

different potentials in response to elevated K+, making comparisons in the presence and

434

absence of anesthetics somewhat difficult. Therefore, we chose to try to elevate Ca2+

435

directly at the release sites in an effort to bypass these effects. But the suppression of

436

neurotransmitter release in intact neurons probably corresponds to a combination of all

437

these effects, as well as inhibitory effects due to the facilitation of GABAA receptors.

438

Please note that the use of amperometry in these studies precludes the determination of

439

whether isoflurane induces changes in endocytosis.

440

The clinical effects of general anesthetics are dose-dependent. If the inhibition of

441

the neurotransmitter release machinery plays a role in the production of anesthesia,

442

measurable effects on the neurotransmitter release machinery should be observed

443

throughout isoflurane’s clinically effective range (~0.3-1 mM). Therefore, we tested the

444

effects of five concentrations of isoflurane (0.3, 0.5, 1, 2 and 3 mM) on permeabilized

445

PC12 cells. Dose-dependent inhibition of the release machinery was indeed observed

446

throughout isoflurane’s clinically relevant range and beyond. The best possible fit of the

447

data suggests the effects of isoflurane on the neurotransmitter release machinery saturate at

448

concentrations > 1 mM, which maximally reduce neurotransmitter release by ~70%. The

449

calculated EC50 for inhibition of the release machinery was 0.38 mM. Reassuringly, this

450

concentration is very similar to the MAC value of isoflurane. However, caution must be

451

exercised when drawing quantitative conclusions based on dose-response curves fit to

21

452

relatively few data points. Nevertheless, robust inhibitory effects at 0.3 mM and 0.5 mM

453

isoflurane strongly suggest a biologically important effect on the neurotransmitter release

454

machinery.

455

Interestingly, isoflurane inhibition of total neurotransmitter release peaked at

456

<100%. This observation leaves open the possibility that anesthetics operate as partial

457

agonists with regards to neurotransmitter inhibition. This, coupled with the observation that

458

higher concentrations of isoflurane are able to inhibit 100% of neurotransmitter release in

459

rat synaptosomes and in cultured hippocampal neurons (Hemmings et al. 2005; Westphalen

460

and Hemmings 2003), suggests that multiple mechanisms may come together to produce

461

the complete inhibition observed. This idea is consistent with more recent studies that have

462

sought to determine the relative contributions of multiple presynaptic targets of anesthetic

463

action (Hemmings et al. 2005; Westphalen et al. 2007; Wu et al. 2004).

464

In this study we have also demonstrated the importance of the SNARE protein,

465

syntaxin 1A, in isoflurane’s ability to inhibit the neurotransmitter release machinery. The

466

expression of a syntaxin 1A truncation mutant, md130A, completely blocked isoflurane-

467

mediated inhibition of neurotransmitter release in permeabilized PC12 cells. This data is in

468

agreement with an observed reduction in behavioral sensitivity of C. elegans md130A

469

heterozygotes to isoflurane (van Swinderen et al. 1999), and suggests that isoflurane-

470

mediated inhibition of the release machinery in mammalian cells is dependent on an

471

interaction between isoflurane and syntaxin 1A. Surprisingly, cells expressing md130A

472

produced more amperometric events when they were stimulated in the presence of

473

isoflurane than in the absence of isoflurane (see Fig. 5B). An increase in amperometric

474

event number was also observed in permeabilized PC12 cells exposed to the non-

22

475

immobilizer, F6 (see fig. 3). While in both cases increases in neurotransmitter release failed

476

to reach significance, increasing the number of recordings in these experiments may

477

ultimately lead to significant results. Thus, in blocking isoflurane’s ability to inhibit

478

neurotransmitter release with md130A, we may have unmasked a weaker stimulatory

479

effect. It is also interesting that overexpression of md130A was found to completely block

480

the effects of isoflurane on the neurotransmitter release machinery despite the presence of

481

endogenous syntaxin 1A. One possible explanation is md130A operates in a dominant

482

fashion with regards to endogenous syntaxin 1A. Additional study will be necessary to test

483

these hypotheses.

484

The details of syntaxin’s involvement in the response to isoflurane remain unclear.

485

While anesthetic interactions with other SNARE and/or SNARE-related proteins are

486

possible, our data raises the possibility that syntaxin 1A is involved in the response to

487

isoflurane. This hypothesis is supported by NMR binding studies demonstrating the ability

488

of isoflurane to bind to syntaxin monomers (Nagele et al. 2005). It is currently unknown

489

whether the md130A mutant is capable of supporting exocytosis, although replacing wild-

490

type syntaxin with the md130A mutant produced C. elegans that were not viable; animals

491

that had both md130A and wild-type syntaxin were viable and were resistant to isoflurane

492

(van Swinderen et al. 1999). Our own studies found that overexpressing the md130A

493

mutant in cells containing endogenous syntaxin resulted in anesthetic insensitive PC12

494

cells. It is possible that the truncation of the C-terminal portion present in the md130A

495

mutant may produce a functional form of syntaxin lacking the isoflurane binding pocket. It

496

is also possible that md130A, lacking its transmembrane domain, may be more mobile than

497

endogenous syntaxin allowing for preferential access to conjugate SNARE proteins. This

23

498

may explain the potential dominant characteristics of md130A. An alternate hypothesis

499

concerning isoflurane’s actions on the release machinery has recently been put forth by

500

Crowder and colleagues whereby isoflurane inhibits the recruitment of the syntaxin

501

activator, UNC-13, to the plasma membrane, thereby reducing syntaxin 1A activation

502

(Metz et al. 2007). This group goes on to speculate that the md130A mutant may bind to

503

UNC-13, preventing the association of isoflurane with the syntaxin activator. Our data is

504

consistent with this model as well. In this scenario the potential of md130A to act

505

dominantly may also be explained as a consequence of increased motility. A soluble

506

md130A might preferentially bind to soluble (unactivated) UNC-13 and prevent the

507

binding of anesthetic molecules to this syntaxin 1A activator. An md130A/UNC-13

508

association prior to the recruitment of UNC-13 to the plasma membrane (activation) may

509

“protect” this syntaxin 1A activator from anesthetic molecules until md130A can be

510

replaced with endogenous syntaxin. Additional study will undoubtedly be necessary to test

511

these hypotheses or to determine whether other as yet unidentified release machinery

512

proteins play a role in the response to isoflurane.

513

Regardless of how isoflurane interacts with the release machinery, this mechanism

514

is likely to operate in humans due to the highly conserved nature of the neurotransmitter

515

release machinery among a variety of species that span invertebrates to mammals. While

516

our data seems to suggest biologically relevant inhibition of the release machinery by

517

isoflurane, it is unclear at the present time as to whether this mechanism participates in the

518

production of the anesthetic state. In the future it will be necessary to generate knockout or

519

transgenic animals in which the effects of anesthetics on the release machinery are blocked

24

520

in order to determine the relative contribution of this mechanism to the production of

521

anesthesia.

522 523

Acknowledgements: We thank Janice Wang for preparation of hippocampal neuron

524

cultures and Anne Cahill for her assistance in the construction of the syntaxin 1A

525

expression plasmids. We also thank Vytautas Bindokas, Xue Qing Wang and Mitch

526

Villereal for their assistance with the RH414 and Fura-2 imaging experiments. This study

527

was supported by a NIGMS grant to APF, a NINDS grant to BEH and by Foundation for

528

Anesthesia Education and Research and Brain research Foundation grants to ZX.

529 530

25

531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

References Augustine GJ, Fitzpatrick D, Hall WC, LaMantia AS, McNamara JO, Mooney RD, Platt ML, Purves D, Simon SA, White LE, and Willams SM. Electrical Signals of Nerve cells. In: Neuroscience, edited by Augustine GJ, Fitzpatrick D, Hall WC, White WE, LaMantia AS and Purves D. Sunderland, MA: Sinauer Associates, Inc., 2008, p. 25-39. Campagna JA, Miller KW, and Forman SA. Mechanisms of actions of inhaled anesthetics. N Engl J Med 348: 2110-2124, 2003. Chen Y, Dai TJ, and Zeng YM. Strychnine-sensitive glycine receptors mediate the analgesic but not hypnotic effects of emulsified volatile anesthetics. Pharmacology 80: 151-157, 2007. Eger EI, 2nd, Saidman LJ, and Brandstater B. Minimum alveolar anesthetic concentration: a standard of anesthetic potency. Anesthesiology 26: 756-763, 1965. Franks NP and Lieb WR. Temperature dependence of the potency of volatile general anesthetics: implications for in vitro experiments. Anesthesiology 84: 716-720, 1996. Grabner CP, Price SD, Lysakowski A, and Fox AP. Mouse Chromaffin Cells Have Two Populations of Dense Core Vesicles. Journal of Neurophysiology 94: 2093-2104, 2005. Graham ME, O'Callaghan DW, McMahon HT, and Burgoyne RD. Dynamindependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc Natl Acad Sci U S A 99: 7124-7129, 2002. Hemmings HCJ, Yan W, Westphalen RI, and Ryan TA. The general anesthetic isoflurane depresses synaptic vesicle exocytosis. Mol Pharmacol 67: 1591-1599, 2005. Jankowski JA, Schroeder TJ, Holz RW, and Wightman RM. Quantal secretion of catecholamines measured from individual bovine adrenal medullary cells permeabilized with digitonin. J Bio Chem 267: 18329-18335, 1992. Jones MV, Brooks PA, and Harrison NL. Enhancement of gamma-aminobutyric acidactivated Cl- currents in cultured rat hippocampal neurones by three volatile anaesthetics. J Physiol 449: 279-293, 1992. Jones MV and Harrison NL. Effects of volatile anesthetics on the kinetics of inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurophysiol 70: 1339-1349, 1993. Krasowski MD and Harrison NL. General anaesthetic actions on ligand-gated ion channels. Cell Mol Life 55: 1278-1303, 1999. Larsen M, Grondahl TO, Haugstad TS, and Langmoen IA. The effect of the volatile anesthetic isoflurane on Ca(2+)-dependent glutamate release from rat cerebral cortex. Brain Res 663: 335-337, 1994. Liachenko S, Tang P, Somogyi GT, and Xu Y. Concentration-dependent isoflurane effects on depolarization-evoked glutamate and GABA outflows from mouse brain slices. Br J Pharmacol 127: 131-138, 1999. Lingamaneni R, Birch ML, and Hemmings HC, Jr. Widespread inhibition of sodium channel-dependent glutamate release from isolated nerve terminals by isoflurane and propofol. Anesthesiology 95: 1460-1466, 2001. Llinas R, Sugimori M, and Silver RB. Presynaptic calcium concentration microdomains and transmitter release. J Physiol Paris 86: 135-138, 1992.

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575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619

Maclver MB, Mikulec AA, Amagasu SM, and Monroe FA. Volatile anesthetics depress glutamate transmission via presynaptic actions. Anesthesiology 85: 823-834, 1996. Metz LB, Dasgupta N, Liu C, Hunt SJ, and Crowder CM. An evolutionarily conserved presynaptic protein is required for isoflurane sensitivity in Caenorhabditis elegans. Anesthesiology 107: 971-982, 2007. Miao N, Frazer MJ, and Lynch C, 3rd. Volatile anesthetics depress Ca2+ transients and glutamate release in isolated cerebral synaptosomes. Anesthesiology 83: 593-603, 1995. Mihic SJ, McQuilkin SJ, Eger EI, 2nd, Ionescu P, and Harris RA. Potentiation of gamma-aminobutyric acid type A receptor-mediated chloride currents by novel halogenated compounds correlates with their abilities to induce general anesthesia. Mol Pharmacol 46: 851-857, 1994. Nagele P, Mendel JB, Placzek WJ, Scott BA, D'Avignon DA, and Crowder CM. Volatile anesthetics bind rat synaptic snare proteins. Anesthesiology 103: 768-778, 2005. Perouansky M, Baranov D, Salman M, and Yaari Y. Effects of halothane on glutamate receptor-mediated excitatory postsynaptic currents. A patch-clamp study in adult mouse hippocampal slices. Anesthesiology 83: 109-119, 1995. Petrenko AB, Tsujita M, Kohno T, Sakimura K, and Baba H. Mutation of alpha1G Ttype calcium channels in mice does not change anesthetic requirements for loss of the righting reflex and minimum alveolar concentration but delays the onset of anesthetic induction. Anesthesiology 106: 1177-1185, 2007. Pocock G and Richards CD. The action of volatile anaesthetics on stimulus-secretion coupling in bovine adrenal chromaffin cells. Br J Pharmacol 95: 209-217, 1988. Rettig J and Neher E. Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science 298: 781-785, 2002. Richards CD. The depression of evoked cortical EPSPs by halothane. J Physiol 222: 152P153P, 1972. Richards CD and White AE. The actions of volatile anaesthetics on synaptic transmission in the dentate gyrus. J Physiol 252: 241-257, 1975. Rowley TJ and Flood P. Isoflurane prevents nicotine-evoked norepinephrine release from the mouse spinal cord at low clinical concentrations. Anesth Analg 107: 885-889, 2008. Schlame M and Hemmings HC, Jr. Inhibition by volatile anesthetics of endogenous glutamate release from synaptosomes by a presynaptic mechanism. Anesthesiology 82: 1406-1416, 1995. van Swinderen B, Saifee O, Shebester L, Roberson R, Nonet ML, and Crowder CM. A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans. Proc Natl Acad Sci U S A 96: 2479-2484, 1999. Wang XQ, Deriy LV, Foss S, Huang P, Lamb FS, Kaetzel MA, Bindokas V, Marks JD, and Nelson DJ. CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons. Neuron 52: 321-333, 2006. Westphalen RI and Hemmings HC, Jr. Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals. J Pharmacol Exp Ther 304: 1188-1196, 2003. Westphalen RI, Krivitski M, Amarosa A, Guy N, and Hemmings HC, Jr. Reduced inhibition of cortical glutamate and GABA release by halothane in mice lacking the K+ channel, TREK-1. Br J Pharmacol 152: 939-945, 2007.

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Wu XS, Sun JY, Evers AS, Crowder M, and Wu LG. Isoflurane inhibits transmitter release and the presynaptic action potential. Anesthesiology 100: 663-670, 2004. Xie Z, Herring BE, and Fox AP. Excitatory and inhibitory actions of isoflurane in bovine chromaffin cells. J Neurophysiol 96: 3042-3050, 2006.

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641

Figure Legends

642

Fig. 1. Isoflurane inhibits neurotransmitter release in permeabilized PC12 cells. Digitonin-

643

permeabilized cells were exposed to Ca2+ (100 µM), indicated by the bars below the traces,

644

to elicit neurotransmitter release. (A-B) Representative amperometric recording in the

645

absence of isoflurane and in the presence of isoflurane (0.5 mM). (C) Bar chart plots the

646

averaged number of events in the absence (“Control”) and presence of isoflurane (0.5 mM).

647

Control cells produced an average of 94 ± 13 events per stimulation (mean ± SEM, n = 25).

648

Isoflurane-treated cells produced an average of 58 ± 11 events (n = 24), a 38% reduction.

649

*P < 0.05, student’s t-test. (D) Mean inhibition of neurotransmitter release plotted as a

650

function of isoflurane concentration (log10).

651

concentration is indicated above or below each data point. Data was fit with Y = Ymax × 1 /

652

1 + (EC50 / X). Y is the percentage of release inhibited. X is the isoflurane concentration.

653

This equation assumes 1:1 binding. Inset re-plots data on a linear scale to show saturation at

654

higher isoflurane concentrations. The open data point denotes data obtained from patch

655

dialysis stimulation.

The number of cells studied at each

656 657

Fig. 2. Isoflurane inhibits neurotransmitter release in patch-clamped PC12 cells. Cells were

658

dialyzed via a patch pipette with Ca2+ (100 µM), indicated by the bars below the traces, to

659

elicit neurotransmitter release. (A-B) Representative amperometric recording in the absence

660

(“Control”) and in the presence of isoflurane (1 mM). (C) Bar chart plots averaged data

661

from control cells and isoflurane-treated cells. During a 2.5-minute stimulation control cells

662

produced an average of 126 ± 22 events (n = 7). Isoflurane (1 mM)-treated cells produced

29

663

an average of 51 ± 10 events (n = 6), a 59% reduction. *P < 0.05, student’s t-test. (D) Re-

664

plots the same data as box plots which span 25% - 75% of each data range. The line in each

665

box represents the median data point.

666 667

Fig. 3. The non-immobilizing agent, 1, 2-dichlorohexaflurocyclobutane (F6), does not

668

inhibit neurotransmitter release in permeabilized PC12 cells. Digitonin-permeabilized cells

669

were exposed to Ca2+ (100 µM), indicated by the bars below the traces, to elicit

670

neurotransmitter release. (A-B) Representative amperometric recording in the absence of

671

F6 (“Control”) and in the presence of F6 (36 μM). (C) Bar chart plots averaged data from

672

control cells and F6-treated cells. During a 2.5-minute stimulation control cells produced an

673

average of 103 ± 22 events (n = 14). F6-treated cells produced an average of 137 ± 24

674

events (n = 15). This difference was not significant, P = 0.29 (student’s t-test). (D) Re-plots

675

the same data as box plots which span 25% - 75% of each data range. The line in each box

676

represents the median data point.

677 678

Fig. 4. Isoflurane inhibits the neurotransmitter release machinery in rat hippocampal

679

neurons. (A) Fluorescence micrograph illustrating nerve terminals containing RH414-

680

loaded synaptic vesicles (arrows). “S”=soma. (B) Fluorescence intensity of terminals in the

681

presence and absence of isoflurane (0.5 mM) plotted as a function of time. (C) Average

682

fluorescence intensity of control and isoflurane-treated terminals 2 min following

683

ionomycin exposure. Stimulation with ionomycin caused a de-staining of 51 ± 3% (mean ±

684

SEM, n = 58) but isoflurane (0.5 mM) treated cells de-stained by only 37 ± 2% (n = 98), a

30

685

27.5% reduction. **P < 0.0001, student’s t-test. (D) Plots normalized average increases in

686

peak [Ca2+]i, in the presence and absence of isoflurane, in response to 5 µM ionomycin.

687 688

Fig. 5. Overexpression of the syntaxin 1A mutant, md130A, eliminates isoflurane’s effect

689

on the neurotransmitter release machinery. (A) Cells were transfected with the md130A

690

expression plasmid. This immunoblot illustrates the levels of md130A and endogenous

691

syntaxin expressed in transfected and untransfected PC12 cells. The relative faintness of the

692

26 kDa md130A band relative to the 33 kDa endogenous syntaxin band is due to the fact

693

that only ~13% of cells were transfected in these experiments. (B) Normalized average

694

number of amperometric events produced by PC12 cells overexpressing md130A in the

695

absence (“Control”) and presence of isoflurane (1 mM). Isoflurane treated cells exhibited

696

~40% more events than control (n = 15, P > 0.05), student’s t-test. (C) Normalized average

697

number of amperometric events produced by PC12 cells overexpressing wild-type (wt)

698

syntaxin 1A in the absence and presence of isoflurane (1 mM). Isoflurane treatment

699

resulted in ~57% reduction in the number of events (n = 16). *P < 0.05, student’s t-test.

700

A.

C.

Control

120

20 pA

100

B.

# of events/stim

20 s

0.5 mM isoflurane

80

*

60

40 20 0

control

0.5 mM Isoflurane

D. 0.8

(n=6)

0.6

(n=6) (n=13)

0.4

EC50=0.38 mM

(n=24) (n=10)

0.2

0.6

Percent Inhibition

Percent Inhibition

(n=10)

0.4

0.2

0.0

0.0

0.01

0.1

1

Isoflurane Concentration

10

0

1

2

3

Isoflurane Concentration

4

A.

B.

Control

1 mM Isoflurane

20 pA 20 s

D. 250

140 120 100 80 *

60 40 20 0

control

1 mM Isoflurane

# of events/stim

# of events/stim

C.

200 150 100

50 0

control

1 mM Isoflurane

A.

B.

Control

F6

25 pA 25 s

D.

C.

300

160

# of events/stim

# of events/stim

200

120 80 40 0

control

F6

200

100

0

control

F6

A.

S

C.

1.1

0.9 0.8

0.7 0.6 0.5 0.4

**

0.4

0.3 0.2

0.1

control

0.5 mM Isoflurane

Normalized Ca2+ Increase

0.5

Ionomycin (5 mM)

1.0

D. 0.6

Percent De-staining

Fluorescence (normalized)

B.

control isoflurane (0.5 mM)

-20 0 20 40 60 80 100 120 140

Time (seconds)

1.2 1.0 0.8 0.6 0.4 0.2 0.0

control

0.5 mM Isoflurane

tra ns fe nt ct ra ed ns ce fe ct lls ed ce lls U

33

Syntaxin 1A

26

md130A mutant

45

b-Actin

md130A transfected cells

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

control

1 mM Isoflurane

C. Normalized # of events/stim

Normalized # of events/stim

B.

m

kD

d1 30 A

A.

wt syntaxin 1A transfected cells

1.2 1.0 0.8 0.6

*

0.4 0.2 0

control

1 mM Isoflurane