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Anesth Analg 2007; 105:1006-1011 © 2007 International Anesthesia Research Society doi: 10.1213/01.ane.0000281433.73260.8d
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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BACKGROUND: We studied the effects of isoflurane on hippocampal synaptic transmission and paired-pulse plasticity, under in vivo intact interneuron circuitry.
METHODS: Using rats chronically implanted with electrodes, excitatory postsynaptic potential (EPSP) and population spike amplitude (PSA) were measured in the hippocampal CA1 field by stimulating Schaffer collaterals. The lungs of the rats were mechanically ventilated with 0.25–1.5 minimum alveolar anesthetic concentration (MAC) isoflurane. A control value was obtained in the absence of isoflurane.
RESULTS: Isoflurane depressed EPSP responses and enhanced synaptic efficacy. PSA was not depressed except under high concentrations, presumably reflecting a well-balanced combination with the decreased EPSP and enhanced synaptic efficacy. Low concentrations of isoflurane (0.25 and 0.5 MAC) increased paired-pulse facilitation (PPF), whereas a high concentration of isoflurane (1.5 MAC) prolonged the paired-pulse depression.
CONCLUSIONS: Isoflurane appeared to affect multiple sites of CA1 synapses: 1) the depression of presynaptic glutamatergic transmission as shown by depressed EPSP and increased PPF; 2) the depression of pyramidal neurons as shown by prolonged PPF and depressed PSA under high concentration; and 3) the depression of interneurons as shown by the greater synaptic efficacy. The degree of each of these inhibitory effects seemed to vary at different concentrations, and the overall direction of the synaptic properties may depend on the balances between these inhibitory effects in vivo.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Depression of neuronal responses by volatile anesthetics involves reduced excitatory glutamatergic neurotransmission and enhanced inhibitory
-aminobutyric-acid (GABA)ergic neurotransmission (1). The hippocampal CA1 region is a laminated structure and its comparatively simple synaptic organization consists of glutamate-mediated excitatory pathways and GABA-mediated inhibitory interneurons. Many electrophysiological studies have demonstrated the inhibitory effects of general anesthetics on glutamate-mediated excitatory synaptic transmission in the CA1 region (2–7) via pre- and postsynaptic mechanisms. Volatile anesthetics can also inhibit the hippocampal interneurons (8–10). Since the excitation of pyramidal cells depends critically on the inhibitory drive contributed by interneurons, the inhibitory action on the interneurons could counteract the depressant effects on pyramidal neurons, i.e., cause the disinhibition in pyramidal neurons (8,10). In other words, anesthetic-induced depressant action on interneurons and those on pyramidal neurons may be competing. Thus, it is reasonable to suppose that the balances between these competing actions may play key roles in the direction of the anesthetic-induced synaptic responses.
Most studies of anesthetic-induced synaptic properties have been conducted under slice preparations. Under physiological conditions with intact interneuron circuitry, anesthetic-induced synaptic responses may differ from those under slice preparations. Therefore, in vivo experiments may greatly help us to elucidate the overall effects of anesthetics on synaptic properties. However, less information is available on the in vivo effects of volatile anesthetics on hippocampal synaptic properties (2). The purpose of our study was to elucidate the effects of isoflurane on hippocampal synaptic transmission and the response to paired-pulse stimuli in vivo.
Elucidating the effects of general anesthetics on synaptic responses in vivo has proven technically difficult, in part because reliable electrophysiological recording in animals often requires sedating them with other anesthetics (2,11). In this study, we used chronically electrode-implanted rats to assess synaptic properties. This allows measurement of synaptic responses regardless of the movement of the rats, thus enabling us to assess synaptic plasticity, even under a low concentration of anesthesia or in the absence of anesthetics. To our knowledge, this is the first study that examines the changes in synaptic properties of volatile anesthetics in vivo, compared with conscious controls.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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General
This study was approved by the Hokkaido University School of Medicine Animal Care and Use Committee. Male Wistar rats (10–12-wk old) were used. Before the electrophysiological study, electrodes were implanted under 1% halothane anesthesia. A monopolar stainless steel recording electrode was placed in the pyramidal cell layer of CA1 (5.0 mm posterior, 3.0 mm lateral to the bregma, approximately 2.3 mm ventral to the dura), and a bipolar electrode was used to stimulate the ipsilateral Schaffer collaterals (3.0 mm posterior, 1.5 mm lateral to the bregma, 2.8 mm ventral to the dura). The electrodes were fixed to screws on the skull with dental cement. The rats were allowed at least 5 days to recover from surgery. Nine rats were implanted with electrodes, and those that showed typical responses to the Schaffer collaterals (Fig. 1) were used for the following measurements. Because the measurements were occasionally affected by noise, the data with apparent noise were eliminated.
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Electrophysiological experiments were performed under 0.25, 0.5, 1.0, and 1.5 minimum alveolar anesthetic concentrations (MAC) of isoflurane anesthesia, where 0.25 and 0.5 MAC were defined as low concentrations. The control value was evaluated in the home cage before anesthesia (in room air: fraction of inspired O2 concentration (Fio2) was approximately 0.21). Anesthesia was induced by placing the rats in a chamber flushed with 5% isoflurane and 95% oxygen. After intubating the trachea with a 14-gauge catheter, anesthesia was maintained under the various concentrations of isoflurane in a 21% O2 and 79% N2 mixture to simulate the Fio2 of the control preparation. The concentrations of isoflurane and expired CO2 were continuously monitored through the tracheal catheter (anesthetic gas monitor, Brüel & Kjær, Denmark). The MAC for isoflurane was taken as 1.4%. Under isoflurane anesthesia, the rats were mechanically ventilated, if appropriate, to keep the expired CO2 concentration between 35 and 45 mm Hg. Rectal temperature was controlled at 37°C ± 0.5°C with a heating pad.
Electrophysiological Study
Basal synaptic transmission was measured by measuring EPSP transmission slope and PSA caused by a single stimulation of Schaffer collaterals. Activation of CA1 pyramidal cells by stimulation of the Schaffer collaterals input results in the recording of a triphasic extracellular field potential. Four component points were identified (Fig. 1), including (a) EPSP onset, (b) population spike onset, (c) population spike minimum, and (d) population spike offset. EPSP slope was calculated from EPSP onset and population spike onset. The PSA was defined as the absolute voltage of a vertical line running from the population spike minimum to its intersection with a line tangential to the population spike onset and population spike offset. Test stimulation (frequency 0.1 Hz, pulse duration 250 µs) was delivered following our previous methods (12). Changes in EPSP and PSA by test stimulation (frequency 0.1 Hz, pulse duration 250 µs) were monitored and recorded by averaging five responses at each stimulus intensity following our previous methods (12).
Two input–output curves were constructed. To determine the effects of isoflurane on the synaptic activation, the input–output curve relating the EPSP slope to the stimulus intensity was plotted in the presence and absence of isoflurane. To test the basal synaptic efficacy, the input–output curve relating the PSA to the EPSP slope was plotted. The changes in the latter input–output curve were quantified as the percentage change of the EPSP slope corresponding to 50% spike probability (E50) (13). To test the isoflurane-mediated changes in PSA, a fixed stimulus intensity was delivered that was determined to elicit a PSA of approximately 50% maximum amplitude under 1.0 MAC.
Paired-pulses were delivered at a frequency of 0.1 Hz whereas the interpulse interval was varied from 50 to 300 ms. The intensity of stimulation was adjusted to elicit a PSA of approximately 50% maximum amplitude under each concentration. Three successive stimuli were recorded every 1 min during anesthesia. The ratio of second/first response was calculated.
To conduct these electrophysiological tests on moving rats (control group and some rats under 0.25 MAC of isoflurane), stimulation was only delivered selectively during immobility to reduce the behavioral variation of the hippocampal synaptic responses (14). Immobility was operationally defined to be the behavior when the rat made no body movements (other than respiratory and small vibrating movements). After the experiments, small electrolytic lesions were made and the positions of electrodes were histologically examined.
Measurement of Arterial Blood Pressures
A pilot study was undertaken to elucidate the effect of isoflurane on hemodynamic and blood gas changes. Five rats were used in this pilot study. A left femoral arterial catheter was inserted to continuously monitor systolic arterial blood pressure and to draw samples for blood gas analysis. Systemic blood pressure and mean arterial blood pressure were measured at each concentration of isoflurane (0.25, 0.5, 1.0, and 1.5 MAC) in an increasing order of concentration from low to high. At least 15 min elapsed between adjusting the vaporizer and making subsequent measurements. The blood gas analysis under the isoflurane anesthesia was conducted at the end of the blood pressure measurement, i.e., at 1.5 MAC under the mechanical ventilation. Control blood-gas values were determined in room air before initiating isoflurane anesthesia.
Statistics
All experimental values are presented as the mean ± se of the mean (sem). Statistics were analyzed using Student's t-test or one-factor analysis of variance (ANOVA) followed by a post hoc Scheffé test to calculate the differences compared with control groups, except for the analysis for paired-pulse plasticity. Changes in paired-pulse plasticity were analyzed using repeated-measures ANOVA. Probability values of <5% were considered statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Isoflurane on Synaptic Efficacy in the CA1 Field
Figure 2A shows isoflurane depressed synaptic activation (EPSP) in a concentration-dependent manner. Maximum responses to control value were 77.4% ± 11.7% (n = 7), 63.5% ± 10.8% (P < 0.05, n = 8), 59.3% ± 11.7% (P < 0.05, n = 7), and 43.0% ± 10.8% (P < 0.01, n = 6) at 0.25, 0.5, 1.0, and 1.5 MAC, respectively.
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As Fig. 2B shows, isoflurane-anesthetized rats exhibited higher PSA values along with EPSP slope measures equivalent to those of the conscious controls. The input–output curve was shifted to the left, as reflected by E50 (0.25 MAC to 78.5% ± 5.1% [P < 0.05], 0.5 MAC to 61.6% ± 8.5% [P < 0.05], 1.0 MAC to 59.4% ± 7.5% [P < 0.05], and 1.5 MAC to 50.8% ± 10.4% [P < 0.01]). These data indicate the ability to convert synaptic activation (EPSP) to cellular discharge (PSA) under isoflurane anesthesia, i.e., greater synaptic efficacy.
Impairment of PSA was not observed under the low and moderate concentrations of isoflurane (0.25, 0.5, and 1.0 MAC) (Fig. 3). Post hoc Scheffé test showed that PSA under 1.5 MAC isoflurane significantly decreased compared with that in the control rats (P < 0.05). These findings indicate that overall cellular discharge is well maintained under low concentrations of isoflurane, presumably because of the well-balanced interaction between the decreased activation (EPSP) and the enhanced synaptic efficacy. There was no significant difference between the maximum stimulation intensity of the groups. In addition, the test stimulation intensity, i.e., the PSA of approximately 50% maximum amplitude did not differ between the groups.
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Effects of Isoflurane on Paired-Pulse Plasticity in the CA1 Field
When the Schaffer collaterals are stimulated by two closely paired stimuli, a series of temporal changes occurs in pyramidal cell excitation to the second pulse. Usually, pyramidal cell response to the second stimulus is more than that to the first evoked response at interpulse interval 40–200 ms (15) because of facilitation of presynaptic Ca2+ influx. The low concentrations of isoflurane (0.25 and 0.5 MAC) significantly increased the PSA evoked by the second pulse, i.e., induced the paired-pulse facilitation (PPF) (P = 0.025 at 0.25 MAC and P = 0.026 at 0.5 MAC versus control, Figs. 4A and B). In contrast, under 1.5 MAC isoflurane, the rate of the paired-pulse response was significantly reduced, i.e., paired-pulse depression (PPD) was prolonged (P = 0.001 at 1.5 MAC versus control, Figs. 4A and C).
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Changes in Hemodynamic Variables Under Isoflurane Anesthesia
Both hypoxia and severe hypotension can decrease hippocampal blood flow, decrease oxygen delivery, and depress the evoked EPSP to the hippocampus (16). To eliminate the effect of these physiological inhibitory factors on synaptic transmission, arterial blood pressure and arterial blood gas were analyzed in the pilot study. Systolic blood pressure was similar in the low concentration groups (0.25 and 0.5 MAC) and the conscious controls, whereas it was significantly decreased under the higher concentrations of isoflurane (P = 0.041 at 1.0 MAC and P < 0.001 at 1.5 MAC; n = 4, Fig. 5). It has been shown that cerebral blood flow is well maintained even under 70 mm Hg of systemic blood pressure during isoflurane anesthesia (17), leading to the conclusion that the neurophysiological changes cannot be based on the changes in systemic blood pressure. There were no signs of hypoxia (pH and Po2 were stable) or significant hypercarbia (pH and Pco2 were stable) during isoflurane anesthesia compared with the controls (Table 1). These data eliminated the modulation of synaptic responses by hypotension, hypoxemia, or respiratory acidosis in this study.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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In this study, isoflurane depressed the single-evoked EPSP responses in a concentration-dependent manner. Since EPSP of CA1 pyramidal cells exclusively reflects glutamatergic excitatory synaptic transmission (7,18), the depressed EPSP in this study confirms previous findings that isoflurane depresses glutamate-mediated transmission (4,6,7). However, isoflurane did not affect the single-evoked PSA except at high concentration, as evident in a previous in vivo study (2). Volatile anesthetics usually depress PSA under slice preparation, reflecting the decrease in EPSP (4,5). The reason for this difference between preparations in not clear, but could be due to loss of afferent modulations, such as muscarinic (19,20), adrenergic (21), and glutamatergic (19) inputs, which may cause the altered balance between excitatory and inhibitory mechanisms (3). In this study, the probability of postsynaptic discharge in response to a given EPSP is enhanced by isoflurane, as shown in higher PSA values along with EPSP slope. This isoflurane-induced enhancement of synaptic efficacy has not been demonstrated under slice preparations. Thus, the enhancement of synaptic efficacy may affect the unchanged PSA in vivo, through the well-balanced combination with the decreased EPSP.
Generally, enhancement of synaptic efficacy could be the result of decreased inhibition within the hippocampal inhibitory circuitry (22). When the local inhibition is reduced, excitatory synapses on pyramidal cells will be more effective at driving CA1 pyramidal cells to threshold, powerfully enhancing excitation: i.e., cause the enhancement of synaptic efficacy (23). Several studies have demonstrated that volatile anesthetics can enhance the inhibition in hippocampal interneurons as well as in pyramidal neurons (10). The depression of interneurons would counteract the anesthetic-induced depressant effects on pyramidal neurons by decreasing polysynaptic tonic feedforward synaptic inhibition (8). The relative loss of inhibition in pyramidal cells compared with interneurons may, therefore, cause hyperactivity of CA1 pyramidal neurons. These speculations led us to suppose that combined modulation on interneurons and pyramidal neurons may play a key role in isoflurane-mediated postsynaptic properties in vivo. Under a high concentration of isoflurane, the depressant effects on pyramidal neurons may presumably overcome the disinhibition by inhibited interneurons; thus, PSA might be depressed.
In this study, isoflurane bidirectionally altered the paired-pulse responses; that is, an increase in PPF under the low concentrations (0.25 and 0.5 MAC) and a prolongation of PPD under the high concentration of isoflurane (1.5 MAC) were shown. Generally, the mechanisms that underlie increased PPF are decreased presynaptic transmitter release (24). Presynaptically, activation of GABAA receptors by volatile anesthetics inhibits Schaffer collateral axons, reducing the release of glutamate through the inhibition of voltage-gated Ca2+ channels at synaptic terminals (7,25). The depression of EPSP and the increase in PPF, therefore, suggest the presynaptic sites of action contribute to isoflurane-induced depression of excitatory transmission, as evident in biochemical measures of glutamate release from synaptosomes and previous in vitro electrophysiological studies (6,7,25,26).
Prolongation of PPD is attributed not only to the presynaptic alterations but also to the postsynaptic processes (24). Usually, the activation of GABAergic interneurons feeding back to the pyramidal cells induces the smaller response to second stimuli, i.e., the PPD. Indeed, early studies have demonstrated that the enhancement of PPD in vivo and in vitro by a variety of anesthetics has been attributed to effects on recurrent GABAA-mediated inhibition (27). In this study, the enhancement of synaptic efficacy was present even under the high concentration of isoflurane, suggesting depressed inhibitory circuitry under that concentration. It was likely, therefore, that PPD under the high concentration of isoflurane could be independent of the activation of recurrent inhibitory circuitry.
Pearce et al. (3) demonstrated that the volatile anesthetic- induced prolongation of PPD is a result of the prolonged decay rate of the slow dendritic GABAA response on the CA1 pyramidal cells. This evidence suggests the dominant effect of depressed pyramidal neuron activity in the isoflurane-induced prolongation of PPD, which appeared to be independent of the interneuron activity (3). Indeed, single-evoked PSA was depressed under the high concentration of isoflurane in the current study, suggesting the strong depressant effects on pyramidal neurons. This evidence led us to suppose that the depressant effect on pyramidal neurons may overcome the disinhibition by the suppressed interneuron activity under the high concentration of isoflurane. Moreover, this effect may mask the PPF that should appear by depressed glutamate release, causing PPD under the high concentration of isoflurane in vivo.
In conclusion, isoflurane appeared to act on multiple sites of CA1 synapses: the depression of transmitter release, the depression of pyramidal neurons, and the inhibition of the interneurons. These effects were individually evident in previous in vitro studies (6,7,10,25,26). However, the degree of each inhibitory effect seemed to vary at different concentrations in vivo. The complex balances between these inhibitory effects may be important in understanding hippocampal synaptic properties under isoflurane anesthesia.
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Footnotes |
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Accepted for publication June 18, 2007.
Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.
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