Isoflurane Inhibits Transmitter Release and the Presynaptic Action Potential
Wu, Xin-Sheng M.D., Ph.D.*; Sun, Jian-Yuan Ph.D.†; Evers, Alex S. M.D.‡; Crowder, Michael M.D., Ph.D.§; Wu, Ling-Gang M.D., Ph.D.∥
Background: Isoflurane inhibits the excitatory postsynaptic current (EPSC) at many synapses. Accumulated evidence suggests the involvement of a presynaptic mechanism. However, the extent of the presynaptic contribution has not been quantitatively studied. Furthermore, the mechanism underlying the presynaptic contribution remains unclear.
: To estimate the presynaptic contribution, the authors compared the effects of isoflurane on the presynaptic capacitance jump, which is proportional to vesicle release, and the postsynaptic glutamate receptor–mediated EPSC at a calyx-type synapse in rat brainstem. The authors determined whether isoflurane affects the waveform of the action potential recorded from nerve terminals. By studying the relation between the EPSC and the presynaptic action potential at the same synapse, the authors determined whether isoflurane inhibits the EPSC by decreasing the presynaptic action potential.
: Isoflurane at 0.35–1.05 mm reduced the EPSC and the presynaptic capacitance jump to a similar degree without affecting the miniature EPSC (an indicator of quantal size), suggesting that isoflurane inhibits the EPSC predominantly by reducing glutamate release. Isoflurane reduced the presynaptic action potential by approximately 3–8%. The EPSC was proportional to the presynaptic action potential amplitude raised to a power of 10.2. Based on this relation, inhibition of the presynaptic action potential contributed to 62–78% of isoflurane-induced inhibition of the EPSC.
Conclusions: Isoflurane inhibits the EPSC predominantly by inhibition of transmitter release. Isoflurane reduces the presynaptic action potential amplitude, which may contribute significantly to its inhibitory effect on the EPSC.
VOLATILE anesthetics inhibit the excitatory postsynaptic current (EPSC) or potential at many synapses. 1,2
This inhibitory effect, together with an enhancement of inhibitory synaptic transmission, may contribute to the generation of general anesthesia. 1,2
The mechanism underlying volatile anesthetic–induced inhibition of the EPSC is not completely understood. Multiple lines of evidence indirectly suggest a presynaptic contribution. 3–12
However, direct evidence indicating the extent of a presynaptic contribution is missing.
The mechanism mediating inhibition of transmitter release by volatile anesthetics remains not well understood. Transmitter release is triggered by Ca2+
influx through Ca2+
channels, which are activated by action potentials. Each of these steps could be the target for volatile anesthetics, as proposed by studies on somatic or expressed channels. 9,13–18
The relative contribution of each of these presynaptic mechanisms remains unclear.
Here, we studied how isoflurane, a clinically used volatile anesthetic, inhibits the EPSC at a large, glutamatergic calyx-type synapse in the medial nucleus of the trapezoid body (MNTB) in rat brainstem. We chose this synapse because both the nerve terminal and the postsynaptic neuron can be patch clamped, 19
which allows for a direct and quantitative study of presynaptic mechanisms underlying isoflurane-induced inhibition of the EPSC. By monitoring vesicle release with presynaptic capacitance measurements, combined with measurements of EPSCs, we found that isoflurane inhibited the EPSC by depression of vesicle release. By quantitatively examining the relation between the ESPC and the presynaptic action potential, we found that isoflurane reduced the presynaptic action potential amplitude, which may contribute significantly to its inhibition of the EPSC.
Materials and Methods
Slice Preparation and Electrophysiology
The experiments were performed with permission from the Animal Studies Committee of Washington University (St. Louis, Missouri). All methods were described previously. 20,21
Briefly, Wistar rats (7–10 days old) were decapitated. Transverse or parasagittal slices, 200 μm thick, were cut from the auditory brainstem with a vibratome. Recordings were made at room temperature (23–25°C). The holding potential was −80 mV, and the potential was corrected for a liquid junction potential of −11 mV between the extracellular and the pipette solution. Currents were low-pass filtered at 5 KHz and digitized at 20 KHz with a 16-bit analog-to-digital converter (Instrutech, Greatneck, NY). Data were expressed as mean ± SEM. The statistical test for calculation of P
values was a t
For postsynaptic whole cell voltage clamp recordings of EPSCs and miniature EPSCs (mEPSCs;figs. 1–3
) in transverse slices, an Axopatch 200B amplifier (Axon Instruments Inc., Foster City, CA) was used. The postsynaptic pipette (2–3 MΩ) solution contained 125 mm K-gluconate, 20 mm KCl, 4 mm MgATP, 10 mm Na2
-phosphocreatine, 0.3 mm GTP, 10 mm HEPES, and 0.5 mm EGTA, at a pH of 7.2, adjusted with KOH. The series resistance (< 15 MΩ) was compensated by 95% (lag, 10 μs). The mEPSCs were analyzed with use of a software program (Jaejin Software, Leonia, NJ). The bath solution contained 125 mm NaCl, 2.5 mm KCl, 1 mm MgCl2
, 2 mm CaCl2
, 25 mm NaHCO3
, 1.25 mm NaH2
, 25 mm dextrose, 0.4 mm ascorbic acid, 3 mm myo
- inositol, 2 mm sodium pyruvate, 0.05 mm D-APV, 0.01 mm bicuculline, and 0.01 strychnine, at a pH of 7.4 when bubbled with 95% O2
and 5% CO2
WaveMetrics, Inc., Lake Oswego, OR) using the principle of the least squares optimization, the data were fit with a Hill function (curve) with an EC50 of 0.49 mm, a Hill coefficient of 1.7, and a maximal inhibition of 80%.
For capacitance recordings at calyces (fig. 4
), an EPC-9 amplifier, together with the software lock-in amplifier (HEKA, Lambrecht, Germany), was used at parasagittal slices (see Sun and Wu 21
for selection of calyces). The bath solution was the same as described in the previous paragraph (starting with “The bath solution contained …”). The series resistance (< 20 MΩ) was compensated by 60%. A sinusoidal stimulus (1,000 Hz) was applied in addition to the DC holding potential (−80 mV). The peak-to-peak voltage of the sine wave was less than 60 mV. The presynaptic pipette (3.5–5 MΩ) solution contained 125 mm K-gluconate, 20 mm KCl, 4 mm MgATP, 10 mm Na2
-phosphocreatine, 0.3 mm GTP, 10 mm HEPES, and 0.05 mm BAPTA, at a pH of 7.2, adjusted with KOH.
For voltage clamp recordings of presynaptic Ca2+
currents (figs. 5 and 6
), the presynaptic pipette (3.5–5 MΩ) solution contained 125 mm Cs-gluconate, 20 mm CsCl, 4 mm MgATP, 10 mm Na2
-phosphocreatine, 0.3 mm GTP, 10 mm HEPES, and 0.05 mm BAPTA, at a pH of 7.2, adjusted with CsOH. The bath solution contained 105 mm NaCl, 20 mm TEA-Cl, 2.5 mm KCl, 1 mm MgCl2
, 2 mm CaCl2
, 25 mm NaHCO3
, 1.25 mm NaH2
, 25 mm dextrose, 0.4 mm ascorbic acid, 3 mm myo
-inositol, 2 mm sodium pyruvate, 0.001 mm tetrodotoxin, 0.1 mm 3,4-diaminopyridine, and 0.05 mm D-APV, at a pH of 7.4 when bubbled with 95% O2
and 5% CO2
Delivery of Isoflurane and Measurements of Isoflurane Concentration
Delivery of isoflurane and measurements of isoflurane concentration at the room temperature were similar to those described previously. 22,23
Briefly, the 95% O2
mixture was directed via
a flowmeter through a calibrated commercial vaporizer (Fortec; Fraser Harlake, Orchard Park, NY) containing isoflurane. The gas mixture of O2
–isoflurane was then used to bubble the experimental saline in a reservoir bottle for at least 20 min before it was applied to the slice chamber with a pump (approximately 4 ml/min, Ismatec pump; Cole-Parmer, Vernon Hills, IL). The aqueous isoflurane concentration, measured with the standard gas chromatography methods, 24
dropped from the saline reservoir to the slice chamber by 28 ± 2% (n = 3). The aqueous isoflurane concentration was linearly proportional to the gaseous partial pressure of isoflurane, detected by a Raman light-scattering gas analyzer (Rascal II; Ohmeda, Salt Lake City, UT; data not shown). Based on these data, the continuously measured gaseous partial pressure was used to estimate the aqueous isoflurane concentration in the slice chamber.
Isoflurane Inhibits the EPSC Dose Dependently at Calyx-type Synapses
The postsynaptic neurons of the MNTB were whole cell voltage clamped at a holding potential of −80 mV, a potential at which the EPSC is mediated by AMPA receptors. 25
A bipolar stimulating electrode was positioned at the midline of the trapezoid body, through which the presynaptic axons of MNTB synapses pass. A stimulus (5–20 V, 0.1 ms) was delivered every 20 s to induce a presynaptic action potential, 20
and the resulting EPSC was recorded at the postsynaptic cell. The EPSC amplitude in control was 5.6 ± 0.7 nA (n = 8). Application of 0.35 mm isoflurane (see Materials and Methods) reduced the EPSC amplitude by 34 ± 3% (n = 5 synapses) without significantly affecting the 20–80% rise and decay time (fig. 1
). The blocking effect reached steady state in less than 10 min after applying isoflurane and reversed in 5 min after washout (fig. 1A
). This effect was dose dependent (e.g.
, fig. 1, A and B
). The percent inhibition as a function of the isoflurane concentration (eight synapses) was fit with a Hill function in which the EC50
, the Hill coefficient, and the maximal inhibition were free parameters. The fitting results were an EC50
of 0.49 mm, a Hill coefficient of 1.7, and a maximal inhibition of 80% (fig. 1C
). Here, the EC50
referred to the concentration at which isoflurane achieved 50% of its maximal inhibition, i.e.
, 40% (= 50% × 80%) inhibition of the EPSC. These results are quantitatively similar to those observed at hippocampal synapses. 5,23
The minimum alveolar concentration (MAC) of isoflurane, at which general anesthesia occurs in 50% of animals, is 0.35 mm in rats. 26
Therefore, clinically relevant concentrations (≤ 2 MAC) of isoflurane may significantly inhibit the EPSC at MNTB synapses.
Isoflurane Reduces Paired-pulse Depression
When a pair of electrical stimuli with a short interval (e.g.
, < 50 ms) is applied to axons, it generates a pair of action potentials, which in turn causes a pair of EPSCs. The ratio between the amplitudes of the second and the first EPSC is less than 1 at synapses, including MNTB synapses, 27
which release a large number of vesicles during the first stimulus. Such a paired-pulse depression is conventionally interpreted as a result of depletion of the releasable pool by the first pulse. 28
Consequently, if isoflurane reduces transmitter release, depletion is relieved, and thus, paired-pulse depression is reduced. We tested this prediction as follows. A pair of electrical stimuli (5–20 V, 0.1 ms) with an interval of 20 ms was applied to generate a pair of EPSCs (e.g.
, fig. 2, A and B
). The ratio between the second and the first EPSC amplitudes was 0.61 ± 0.07 (n = 16;fig. 2
). This ratio was significantly increased to 0.97 ± 0.18 (n = 5; P
= 0.02) and 1.42 ± 0.31 (n = 3; P
= 0.0002) in the presence of 0.35 and 1.05 mm isoflurane, respectively (fig. 2
). These results, similar to those observed at hippocampal synapses, 5–7
are consistent with the hypothesis that isoflurane inhibits the EPSC by a presynaptic mechanism.
Isoflurane Does Not Affect the Miniature EPSC
If isoflurane inhibits the EPSC by decreasing the sensitivity of postsynaptic glutamate receptors, it should decrease the mEPSC, the postsynaptic response to release of a single vesicle. We tested this prediction by collecting mEPSCs for 3–5 min before and after a 10-min application of 0.7 mm isoflurane (fig. 3A
). Before application of isoflurane, the peak amplitude of mEPSCs was 24.1 ± 3.1 pA (n = 5 synapses). Application of isoflurane did not significantly reduce the amplitude of the mean mEPSC, 20–80% rise time and half decay time (e.g.
, fig. 3, B and C
; n = 5 synapses; P
> 0.4). The frequency of mEPSCs ranged from approximately 1 to 5 Hz in control. Isoflurane reduced the frequency to 91 ± 4% of control, which was not statistically significant (P
= 0.18; n = 5). Because AMPA receptors were not saturated by release of a single quantum at MNTB synapses, 29
these results suggest that isoflurane does not inhibit the ESPC by decreasing the sensitivity of postsynaptic AMPA receptors.
Isoflurane Reduces the Presynaptic Capacitance Jump
To determine whether isoflurane inhibits the EPSC by reducing the number of released vesicles, we measured the membrane capacitance increase at the nerve terminal (the calyx of Held), which is proportional to the number of released vesicles. 21,30
Capacitance measurements were made in the voltage clamp mode (fig. 4A
). The voltage clamp mode was switched to a current clamp mode for 500 ms, during which 10 pulses of 3-ms current of 300–600 pA at 200 Hz were injected into the calyx to induce 10 action potentials (fig. 4B
). We induced 10 action potentials instead of 1 to increase the signal-to-noise ratio. The capacitance jump, calculated as the difference between the capacitance values before and 500 ms after stimulation, was 293 ± 31 fF (n = 6;fig. 4A
). The jump returned to the baseline in 10s of seconds (not shown, but see Sun and Wu, 21
which reflects the time course of endocytosis). Therefore, slow endocytosis does not significantly affect measurements of capacitance jumps after stimulation. Consistent with our previous study, 21
the capacitance change did not parallel the change of the membrane resistance or the series resistance (fig. 4A
). Application of isoflurane (0.7 mm) significantly reduced the capacitance jump by 43 ± 6% (n = 6) of control. This decrease was similar to the decrease in the EPSC (approximately 50% at 0.7 mm as calculated from the fit curve in fig. 1C
), suggesting that isoflurane inhibits the EPSC largely by reducing vesicle release.
Isoflurane Decreases the Amplitude of the Presynaptic Action Potential
The effects of isoflurane on the capacitance jump were accompanied by a decrease in the amplitude of the presynaptic action potential. For example, isoflurane at 0.7 mm reduced the presynaptic action potential amplitude from 106.3 ± 1.3 mV (n = 6) to 100.5 ± 1.7 mV (n = 6; P
< 0.01;fig. 4B
). That is, isoflurane reduced the action potential amplitude by 5.8 ± 1.3 mV or 5.5 ± 1.3% (n = 6). Isoflurane (0.7 mm) also slightly hyperpolarized the resting membrane potential (ranged from −56 to −68 mV) by 1.2 ± 0.3 mV (n = 6; P
< 0.01). These effects were reversible on drug washout (fig. 4B
). Isoflurane (0.7 mm) did not significantly affect the half-width of the action potential (0.36 ± 0.02 ms in control; 0.39 ± 0.03 ms in isoflurane; n = 6; P
> 0.2). The effect of isoflurane on the action potential amplitude was dose dependent. At 0.35 mm, isoflurane reduced the amplitude by 3.3 ± 1.5% (n = 4). At 1.05 mm, isoflurane reduced the amplitude by 7.6 ± 0.8% (n = 4).
The above analysis of the action potential waveform (APW) was made at the first action potential during a train of 10 action potentials (fig. 4B
). Similar results were obtained when the last action potential during the 10-action-potential train was analyzed. For example, isoflurane at 0.7 mm reduced the amplitude of the last action potential by 4.1 ± 1.1% (n = 6), without significantly affecting its half-width (P
> 0.4). Therefore, the inhibition of the action potential during the 10-action-potential train was not activity dependent.
Relation between the EPSC and the Presynaptic Action Potential Amplitude
To determine the extent to which a decrease in the action potential accounts for inhibition of release, we examined the relation among the presynaptic action potential, the action potential–induced presynaptic Ca2+
current (ICa), and the EPSC. All of these measurements were made in the absence of isoflurane. Both the nerve terminal and the postsynaptic neuron were whole cell voltage clamped at the same synapse. ICa was recorded by pharmacologically blocking Na+
channels (see Materials and Methods). An experimentally recorded action potential of 110 mV evoked by nerve stimulation (stimulation electrode positioned at the midline of the trapezoid body) 20
was used as the voltage command to mimic an action potential. Application of this command induced a presynaptic ICa and a postsynaptic EPSC at the same synapse (fig. 5A
). By scaling the typical APW to 95, 90, 85, 80, or 75%, the APW amplitude was reduced by 5.5, 11, 16.5, 22, or 27.5 mV, respectively. Consequently, the presynaptic ICa and the postsynaptic EPSC were reduced (e.g.
, fig. 5A
). The amplitude of the resulting EPSC was highly nonlinearly related to the presynaptic APW amplitude (fig. 5B
). For example, the EPSC amplitude was decreased to 72 ± 5% (n = 7) or 43 ± 3% (n = 7) of control when the APW amplitude was decreased from 110 mV to 104.5 or 99 mV, respectively (fig. 5B
). The normalized EPSC amplitude and the normalized APW amplitude obtained from seven synapses was fit with the following equation:EQUATION
in which c1
= 1.15 and n = 10.2 (fig. 5B
To study why there was such an extremely nonlinear relation, we plotted the ICa charge versus
the APW amplitude (fig. 5C
) and the EPSC amplitude versus
the ICa charge (fig. 5D
). The relation between the normalized ICa charge (QICa
) and the normalized APW amplitude was fit with the following equation:EQUATION
in which c2
= 1.04 and k = 2.8 (fig. 5C
, right). Therefore, a small decrease in the APW amplitude may cause a larger decrease in the QICa
. The relation between the normalized EPSC amplitude and the normalized QICa
was fit with the following equation:EQUATION
in which c3
= 1.01 and m = 3.7 (fig. 5D
, right). This nonlinear relation was consistent with previous studies at MNTB synapses and other synapses. 19
Therefore, equations 2 and 3 explain why the EPSC amplitude was extremely nonlinearly related to the APW amplitude.
The EPSC induced by a typical APW command was 1.3 ± 0.3 nA (n = 7), which was smaller than the EPSC induced by a real action potential. 20,31
This is likely caused by imperfect voltage clamp. To determine whether the results shown in figure 5
still hold when the EPSC is similar to that induced by a real action potential, we induced EPSC by a 1-ms step voltage command from −80 to +7 mV (fig. 6
, solid traces). We have shown such a step depolarization to induce release similar to that induced by a real action potential. 32
Here, this depolarization induced an EPSC of 5.2 ± 1.4 nA (n = 4; e.g.
, fig. 6
). By decreasing the 1-ms step voltage command by 5 mV (the step was from −80 to +2 mV;fig. 6
, dash trace), i.e.
, by approximately 6% (5/87 ≈ 6%), the charge of the presynaptic calcium current and the postsynaptic EPSC decreased to 88 ± 2 and 67 ± 8% (n = 4, e.g.
, fig. 6
) of control, respectively. These results were similar to those shown in figure 5
, confirming that a small decrease in a brief presynaptic depolarization results in a large decrease in the EPSC.
Based on the relation between the EPSC and the isoflurane concentration (fit curve in fig. 1C
), 0.35, 0.7, and 1.05 mm isoflurane inhibited the EPSC by 29, 52, and 63%, respectively. Isoflurane at these concentrations reduced the action potential amplitude by means of 3.3, 5.5, and 7.6%, respectively. These results (fig. 5B
, right, triangles) were plotted together with those obtained with voltage clamp experiments in control (fig. 5B
, right, circles). It was clear from the plot that the decrease in the action potential amplitude may largely, but not completely account for the decrease in the EPSC induced by isoflurane. According to equation 1, 3.3, 5.5, and 7.6% decreases in the action potential amplitude would decrease the EPSC by 18, 35, and 49%, respectively. Therefore, on average, the decrease in the ESPC induced by the decrease in the action potential amplitude accounted for approximately 62% (= 18%/29%), 70% (= 35%/50%), or 78% (= 49%/63%) of inhibition of the EPSC by isoflurane at 0.35, 0.7, and 1.05 mm, respectively.
The amplitude of the APW we used was 110 mV rather than 106 mV as measured here. This was to be consistent with several previous studies that used a 110-mV APW as the voltage command. 31,33,34
However, such a small difference should not significantly affect our conclusion because a similar extent of decrease in the EPSC was observed when the APW amplitude was decreased by 5.5 mV from 110 or 104.5 mV (fig. 5B
Isoflurane Depresses the EPSC by Inhibition of Vesicle Release
We found that isoflurane dose-dependently inhibited the EPSC at MNTB synapses. By the use of capacitance measurements to monitor vesicle release at nerve terminals, we showed that isoflurane inhibits vesicle release induced by a train of 10 action potentials at 200 Hz. The stimulation train lasted for only 50 ms, during which mobilization of synaptic vesicles from the reserve pool to the releasable pool is negligible. 34,35
Therefore, the capacitance jump induced by the stimulation train was proportional to the averaged release evoked by an action potential during the train. Because isoflurane inhibited the capacitance jump and the EPSC to a similar degree and reduced paired-pulse depression but did not affect the mEPSC amplitude, we concluded that isoflurane inhibited the EPSC predominantly by a presynaptic mechanism. These results provide strong evidence confirming many previous studies suggesting a presynaptic contribution to volatile anesthetic–induced inhibition of synaptic transmission. 3–12
Isoflurane inhibited the presynaptic capacitance jump (43 ± 6%) and the EPSC (50%) to slightly different extents. Therefore, the possibility that isoflurane reduced the EPSC to a minor extent by a mechanism other than presynaptic mechanisms cannot be ruled out. These minor mechanisms might include effects on the number of glutamate receptors, 12
glutamate receptor desensitization, and clearance of glutamate from the synaptic cleft. However, the slight difference in the inhibition of the capacitance jump and the EPSC might also be caused by the difference in the stimuli used to evoke capacitance jump and the EPSC. The EPSC was evoked by a single action potential. Such an EPSC was linearly proportional to the capacitance jump, suggesting that postsynaptic receptor saturation does not occur at such a minimal stimulus. 21
In contrast, the capacitance jump was evoked by 10 action potentials at 200 Hz, which may cause a larger extent of depletion of a limited pool of vesicles immediately available for release 21,34,35
and thus might reduce the percent inhibition of the capacitance jump by isoflurane.
Isoflurane Inhibits the EPSC Largely by Depressing the Presynaptic Action Potential
We found that 0.35–1.05 mm isoflurane reduced the presynaptic action potential amplitude by approximately 3.3–7.6%. This is consistent with many previous studies showing little effect or minor effects of general anesthetics on the action potential. 1,2
However, the EPSC was proportional to the presynaptic APW amplitude raised to a power of 10.2 (equation 1). Such an extremely nonlinear relation was caused by two highly nonlinear relations, one between the charge of the ICa induced by the APW and the APW amplitude (equation 2), and the other between the EPSC amplitude and the ICa charge (equation 3). Based on these quantitative relations, we concluded that inhibition of the presynaptic action potential accounts for approximately 62–78% of the inhibitory effects of isoflurane on the EPSC, whereas the remaining fraction might be mediated by mechanisms downstream of the action potential.
The decrease in the action potential amplitude could be due to inhibition of Na+
or activation of K+
The mechanism downstream of the action potential may be mediated by inhibition of Ca2+
or the release machinery downstream of the calcium influx. 16
It would be of great interest to dissect each of these potential mechanisms. We attempted to perform voltage clamp recordings of Na+
currents at the calyx and found that the current was too large (> 5–10 nA) and too fast to reliably voltage clamp. We also attempted to study whether isoflurane (0.7 mm) inhibits the presynaptic Ca2+
currents. We did not observed consistent inhibition of Ca2+
current by more than 15% (unpublished results, Xin-Sheng Wu, M.D., Ph.D., and Ling-Gang Wu, M.D., Ph.D., National Institute of Neurologic Disorders and Stroke, Bethesda, Maryland, May–September 2002). However, the Ca2+
current evoked by an APW was extremely sensitive to a very small change (e.g.
, 1- to 2-MΩ change) in the series resistance. As a result, stable recordings of the Ca2+
current for as long as 10 min, which is needed for the effects of isoflurane to reach a steady state, was extremely difficult (note that experiments shown in figures 5A and 6
may take < 2 min). We could not consistently resolve an inhibition of less than 10–15% in a 10 min, which may well be in the range of inhibition induced by isoflurane. In summary, resolving small changes (perhaps < 5–10%) in presynaptic Na+
currents is a difficult and substantial undertaking that is beyond the scope of the current study.
A single calyx in the MNTB contains approximately 600 conventional active zones 40
and thus is viewed as equivalent to approximately 600 boutons in parallel. 19,41
Except for this difference, the calyx-type MNTB synapse shares many similar properties with central synapses, such as paired-pulse depression or facilitation, a nonlinear relation between release and Ca2+
channel types and their modulation by transmitters, AMPA and N
-methyl-d-aspartate receptors, and the ultrastructure of active zones. 19,41
More importantly, isoflurane dose-dependently inhibited the EPSC and relieved paired-pulse depression while producing a minor reduction of the presynaptic action potential at MNTB synapses. These effects are qualitatively and quantitatively similar to those observed with volatile anesthetics at other synapses. 5–8,10,11,23,42
Isoflurane inhibited the capacitance jump evoked by action potentials at MNTB synapses, which is consistent with the result that volatile anesthetics inhibit glutamate release induced by prolonged depolarization at synaptosomes. 3,4
Therefore, the principles learned in the current study are likely to apply to other glutamatergic synapses.
We performed experiments in brain slices of 7- to 10-day-old rats at room temperature (23–25°C). Whether our findings can be applied to adult rats at body temperature remains unclear. We think this possibility is likely for the following reasons. The electrical properties of MNTB synapses of 9-day-old rats, such as the action potential width, the EPSC size, the quantal size, the calcium channel types, and paired-pulse depression and facilitation, are qualitatively similar but quantitatively slightly different from those of adult rats. 19
Therefore, the results observed here might be quantitatively slightly different in adult rats. However, the major conclusions are likely to remain the same. Volatile anesthetics at similar aqueous concentrations inhibit excitatory synaptic transmission to a similar degree at other synapses at both room temperature and a temperature close to body temperature. 9,22,23,43–45
Therefore, it is likely that the principles learned from brain slices at room temperature apply to those at body temperature. In conclusion, isoflurane-induced inhibition of the presynaptic action potential amplitude may contribute to inhibition of glutamate release, which may be relevant to certain end points of anesthesia. 1,2
The authors thank Steve Mennerick, Ph.D. (Assistant Professor, Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri), and Joe Henry Steinbach, Ph.D. (Professor, Department of Anesthesiology, Washington University School of Medicine), for suggestions during the course of this work and comments on the manuscript.
1. Pocock G, Richards CD: Excitatory and inhibitory synaptic mechanisms in anesthesia. Br J Anaesth 1993; 71: 134–47
2. MacIver MB: General anesthetic actions on transmission at glutamate and GABA synapses, Anesthesia: Biologic Foundations. Edited by Yaksh TL, Lynch C, Zapol WM, Maze M, Biebuyck JF, Saidman LJ. Philadelphia, Lippincott–Raven, 1997, pp 277–86
3. Miao N, Frazer MJ, Lynch C: Volatile anesthetics depress Ca2+ transients and glutamate release in isolated cerebral synaptosomes. A nesthesiology 1995; 83: 593–603
4. Schlame M, Hemmings HC: Inhibition by volatile anesthetics of endogenous glutamate release from synaptosomes by a presynaptic mechanism. A nesthesiology 1995; 82: 1406–16
5. Maclver MB, Mikulec AA, Amagasu SM, Monroe FA: Volatile anesthetics depress glutamate transmission via presynaptic actions. A nesthesiology 1996; 85: 823–34
6. Hirota K, Roth SH: The effects of sevoflurane on population spikes in CA1 and dentate gyrus of the rat hippocampus in vitro. Anesth Analg 1997; 85: 426–30
7. Hirota K, Roth SH, Fujimura J, Masuda A, Ito Y: GABAergic mechanisms in the action of general anesthetics. Toxicol Lett 1998; 100–101: 203–7
8. Galindo A: Effects of procaine, pentobarbital and halothane on synaptic transmission in the central nervous system. J Pharmacol Exp Ther 1969; 169: 185–95
9. Richards CD, White AE: The actions of volatile anaesthetics on synaptic transmission in the dentate gyrus. J Physiol 1975; 252: 241–57
10. Sugiyama K, Muteki T, Shimoji K: Halothane-induced hyperpolarization and depression of postsynaptic potentials of guinea pig thalamic neurons in vitro. Brain Res 1992; 576: 97–103
11. Kirson ED, Yaari Y, Perouansky M: Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus. Br J Pharmacol 1998; 124: 1607–14
12. Dildy-Mayfield JE, Eger EI, Harris RA: Anesthetics produce subunit-selective actions on glutamate receptors. J Pharmacol Exp Ther 1996; 276: 1058–65
13. Rehberg B, Xiao YH, Duch DS: Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. A nesthesiology 1996; 84: 1223–33
14. Franks NP, Lieb WR: Volatile general anaesthetics activate a novel neuronal K+ current. Nature 1988; 333: 662–4
15. Takenoshita M, Steinbach JH: Halothane blocks low-voltage-activated calcium current in rat sensory neurons. J Neurosci 1991; 11: 1404–12
16. van Swinderen B, Saifee O, Shebester L, Roberson R, Nonet ML, Crowder CM: A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans. Proc Natl Acad Sci U S A 1999; 96: 2479–84
17. Hall AC, Lieb WR, Franks NP: Insensitivity of P-type calcium channels to inhalational and intravenous general anesthetics. A nesthesiology 1994; 81: 117–23
18. Study RE: Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons. A nesthesiology 1994; 81: 104–16
19. Von Gersdorff H, Borst JGG: Short-term plasticity at the calyx of Held. Nature Rev Neurosci 2002; 3: 53–64
20. Borst JGG, Helmchen F, Sakmann B: Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J. Physiol 1995; 489: 825–40
21. Sun JY, Wu LG: Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse. Neuron 2001; 30: 171–82
22. MacIver MB, Roth SH: Inhalation anaesthetics exhibit pathway-specific and differential actions on hippocampal synaptic responses in vitro. Br J Anaesth 1988; 60: 680–91
23. Perouansky M, Baranov D, Salman M, Yaari Y: Effects of halothane on glutamate receptor–mediated excitatory postsynaptic currents: A patch-clamp study in adult mouse hippocampal slices. A nesthesiology 1995; 83: 109–19
24. Stern RC, Weiss CI, Steinbach JH, Evers AS: Isoflurane uptake and elimination are delayed by absorption of anesthetic by the Scimed membrane oxygenator. Anesth Analg 1989; 69: 657–62
25. Forsythe ID, Barnes-Davies M: The binaural auditory pathway: Excitatory amino acid receptors mediate dual time course excitatory postsynaptic currents in the rat medial nucleus of the trapezoid body. Proc R Soc Lond B 1993; 251: 151–7
26. Mazze RI, Rice SA, Baden JM: Halothane, isoflurane, and enflurane MAC in pregnant and nonpregnant female and male mice and rats. A nesthesiology 1985; 62: 339–41
27. Barnes-Davies M, Forsythe ID: Pre- and postsynaptic glutamate receptors at a giant excitatory synapse in rat auditory brainstem slices. J. Physiol 1995; 488: 387–406
28. Zucker RS: short-term synaptic plasticity. Annu Rev Neurosci 1989; 12: 13–31
29. Ishikawa T, Sahara Y, Takahashi T: A single packet of transmitter does not saturate postsynaptic glutamate receptors. Neuron 2002; 34: 613–21
30. Von Gersdorff H, Sakaba T, Berglund K, Tachibana M: Submillisecond kinetics of glutamate release from a sensory synapse. Neuron 1998; 21: 1177–88
31. Borst JGG, Sakmann B: Calcium influx and transmitter release in a fast CNS synapse. Nature 1996; 383: 431–4
32. Sun JY, Wu XS, Wu LG: Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse. Nature 2002; 417: 555–9
33. Borst JG, Sakmann B: Effect of changes in action potential shape on calcium currents and transmitter release in a calyx-type synapse of the rat auditory brainstem. Philos Trans R Soc Lond B Biol Sci 1999; 354: 347–55
34. Wu LG, Borst JGG: The reduced release probability of releasable vesicles during recovery from short-term synaptic depression. Neuron 1999; 23: 821–32
35. Schneggenburger R, Meyer AC, Neher E: Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse. Neuron 1999; 23: 399–409
36. Haydon DA, Urban BW: The effects of some inhalation anaesthetics on the sodium current of the squid giant axon. J Physiol 1983; 341: 429–39
37. Patel AJ, Honore E, Lesage F, Fink M, Romey G, Lazdunski M: Inhalational anesthetics activate two-pore-domain background K+ channels. Nature Neuroscience 1999; 2: 422–6
38. Ries CR, Puil E: Mechanism of anesthesia revealed by shunting ac-tions of isoflurane on thalamocortical neurons. J Neurophysiol 1999; 81: 1795–801
39. Ries CR, Puil E: Ionic mechanism of isoflurane’s actions on thalamocortical neurons. J Neurophysiol 1999; 81: 1802–9
40. Sätzler K, Sohl L, Bollmann JH, Borst JGG, Frotscher M, Sakmann B, Lubke JH: Three-dimensional reconstruction of a calyx of Held and its postsynapticprincipal neuron in the medial nucleus of the trapezoid body. J Neurosci 2002; 22: 10567–79
41. Schneggenburger R, Sakaba T, Neher E: Vesicle pools and short-term synaptic depression: Lessons from a large synapse. Trends Neurosci 2002; 25: 206–12
42. Richards CD, White AE: The actions of volatile anaesthetics on synaptic transmission in the dentate gyrus. J Physiol 1975; 252: 241–57
43. Richards CD: On the mechanism of halothane anaesthesia. J Physiol 1973; 233: 439–56
44. Kullmann DM, Martin RL, Redman RJ: Reduction by general anesthetics of group 1a excitatory postsynaptic potentials and currents in the cat spinal cord. J Physiol 1989; 412: 227–96
45. Nishikawa K, MacIver MB: Membrane and synaptic actions of halothane on rat hippocampal pyramidal neurons and inhibitory interneurons. J Neurosci 2000; 20: 5915–23
This article has been cited 57 time(s).
Proceedings of the 8th Biennial Congress of the Asian & Oceanic Society of Regional Anesthesia and Pain Medicine
Anesthetics differentially affect nerve regeneration synapse and synaptic plasticity
Proceedings of the 8th Biennial Congress of the Asian & Oceanic Society of Regional Anesthesia and Pain Medicine, ():
British Journal of Pharmacology
Molecular targets underlying general anaesthesia
British Journal of Pharmacology, 147():
Basic & Clinical Pharmacology & Toxicologyalpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors participate in the analgesic but not hypnotic effects of emulsified halogenated anaestheticsBasic & Clinical Pharmacology & Toxicology
British Journal of AnaesthesiaDifferential effects of halothane and isoflurane on lumbar dorsal horn neuronal windup and excitabilityBritish Journal of Anaesthesia
PharmacologySpinal N-methyl-D-aspartate receptors may mediate the analgesic effects of emulsified halogenated anestheticsPharmacology
Pharmacology & TherapeuticsMechanisms of neurosteroid interactions with GABA(A) receptorsPharmacology & Therapeutics
British Journal of AnaesthesiaVolatile anaesthetic effects on phospholipid binding to synaptotagmin 1, a presynaptic Ca2+ sensorBritish Journal of Anaesthesia
Clinical and Experimental Pharmacology and PhysiologySpinal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors may mediate the analgesic effects of emulsified halogenated anaestheticsClinical and Experimental Pharmacology and Physiology
Journal of Pharmacology and Experimental TherapeuticsDepression by isoflurane of the action potential and underlying voltage-gated ion currents in isolated rat neurohypophysial nerve terminalsJournal of Pharmacology and Experimental Therapeutics
Bmc NeuroscienceMultiple synaptic and membrane sites of anesthetic action in the CA1 region of rat hippocampal slicesBmc Neuroscience
Physical Review EModeling the effects of anesthesia on the electroencephalogramPhysical Review E
EndocrinologyLarge Neurohypophysial Varicosities Amplify Action Potentials: Results from Numerical SimulationsEndocrinology
Trends in Pharmacological SciencesEmerging molecular mechanisms of general anesthetic actionTrends in Pharmacological Sciences
Experimental Brain ResearchContributions of GABAergic and glutamatergic mechanisms to isoflurane-induced suppression of thalamic somatosensory information transferExperimental Brain Research
Journal of NeurophysiologySubunit-Specific Effects of Isoflurane on Neuronal I-h in HCN1 Knockout MiceJournal of Neurophysiology
Trends in NeurosciencesThe low synaptic release probability in vivoTrends in Neurosciences
Journal of NeuroscienceAmplitude and kinetics of action potential-evoked Ca2+ current and its efficacy in triggering transmitter release at the developing calyx of held synapseJournal of Neuroscience
Basic & Clinical Pharmacology & ToxicologyStrychnine-sensitive glycine receptors mediate analgesia induced by emulsified inhalation anaesthetics in thermal nociception but not in chemical nociceptionBasic & Clinical Pharmacology & Toxicology
Journal of NeuroscienceCortical Inhibition during Burst Suppression Induced with Isoflurane AnesthesiaJournal of Neuroscience
Proceedings of the National Academy of Sciences of the United States of America
Nitrous oxide (N2O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans
Proceedings of the National Academy of Sciences of the United States of America, 101():
British Journal of AnaesthesiaSodium channels and the synaptic mechanisms of inhaled anaestheticsBritish Journal of Anaesthesia
Journal of Pharmacology and Experimental TherapeuticsIsoflurane inhibits NaChBac, a prokaryotic voltage-gated sodium channelJournal of Pharmacology and Experimental Therapeutics
Anesthesia and AnalgesiaIsoflurane bidirectionally modulates the paired-pulse responses in the rat hippocampal CA1 field in vivoAnesthesia and Analgesia
British Journal of PharmacologyBidirectional modulation of isoflurane potency by intrathecal tetrodotoxin and veratridine in ratsBritish Journal of Pharmacology
Bmc NeuroscienceIsoflurane depresses hippocampal CA1 glutamate nerve terminals without inhibiting fiber volleysBmc Neuroscience
Experimental NeurologyThe impact of anesthetics and hyperoxia on cortical spreading depressionExperimental Neurology
Journal of NeurophysiologyIsoflurane Inhibits the Neurotransmitter Release MachineryJournal of Neurophysiology
Cerebral CortexRelationship between neural, vascular, and BOLD signals in isoflurane-anesthetized rat somatosensory cortexCerebral Cortex
NeuroscienceResponse to neonatal anesthesia: Effect of sex on anatomical and behavioral outcomeNeuroscience
Molecular PharmacologyThe general anesthetic Isoflurane depresses synaptic vesicle exocytosisMolecular Pharmacology
Magnetic Resonance in MedicineAssessing transneuronal dysfunction utilizing manganese-enhanced MRI (MEMRI)Magnetic Resonance in Medicine
Journal of Physiology-LondonIsoflurane depresses glutamate release by reducing neuronal excitability at the Drosophila neuromuscular junctionJournal of Physiology-London
NeuroscienceHeterogeneous Ca2+ influx along the adult calyx of held: A structural and computational studyNeuroscience
European Journal of NeuroscienceActions of propofol on substantia gelatinosa neurones in rat spinal cord revealed by in vitro and in vivo patch-clamp recordingsEuropean Journal of Neuroscience
Plos OnePaired Assessment of Volatile Anesthetic Concentrations with Synaptic Actions Recorded In VitroPlos One
European Journal of NeuroscienceDose-dependent effect of isoflurane on neurovascular coupling in rat cerebral cortexEuropean Journal of Neuroscience
Anesthesia and AnalgesiaThe Effects of Volatile Aromatic Anesthetics on Voltage-Gated Na+ Channels Expressed in Xenopus OocytesAnesthesia and Analgesia
Suppressing the Mind: Anesthetic Modulation of Memory and ConsciousnessMolecular Targets of General Anesthetics in the Nervous SystemSuppressing the Mind: Anesthetic Modulation of Memory and Consciousness
Journal of NeurosciencePresynaptic Na+ channels: Locus, development, and recovery from inactivation at a high-fidelity synapseJournal of Neuroscience
British Journal of AnaesthesiaEffects of isoflurane and xenon on Ba2+-currents mediated by N-type calcium channelsBritish Journal of Anaesthesia
NeuronThe decrease in the presynaptic calcium current is a major cause of short-term depression at a calyx-type synapseNeuron
PharmacologyStrychnine-sensitive glycine receptors mediate the analgesic but not hypnotic effects of emulsified volatile anestheticsPharmacology
Journal of NeurophysiologyNeural origin of evoked potentials during thalamic deep brain stimulationJournal of Neurophysiology
Environmental Toxicology and PharmacologyEffects of isoflurane inhalation on the male reproductive system in ratsEnvironmental Toxicology and Pharmacology
Journal of NeurophysiologyInteraction of anesthetics with neurotransmitter release machinery proteinsJournal of Neurophysiology
Plos OneMembrane Properties of Striatal Direct and Indirect Pathway Neurons in Mouse and Rat Slices and Their Modulation by DopaminePlos One
British Journal of AnaesthesiaPresynaptic inhibition of the release of multiple major central nervous system neurotransmitter types by the inhaled anaesthetic isofluraneBritish Journal of Anaesthesia
© 2004 American Society of Anesthesiologists, Inc.
Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.