Halothane and Isoflurane Increase Spontaneous but Reduce the N-methyl-D-aspartate–evoked Dopamine Release in Rat Striatal Slices : Evidence for Direct Presynaptic Effects

Handlings procedures according to the Guide for the Care and Use of Laboratory Animals  24were followed throughout. Experiments were performed on male Sprague-Dawley rats (Iffa-Credo, L'Arbresle, France) weighing 200–225 g and housed on a 12:12 light/dark cycle with food and water ad libitum . Approval was obtained from the Institutional Animal Care and Use Committee at the University of Paris 7.

Animals were killed by stunning (thoracic shock followed by reflex cardiac arrest) and decapitation. After longitudinal hemisection of the brain, striata were quickly removed and processed to a purified synaptosomal preparation as previously reported in detail. 20,21Synaptosomes were diluted up to 0.16 mg/ml in an ice-cold Mg²⁺-free artificial cerebrospinal fluid containing 126.5 mM NaCl, 27.5 mM NaHCO³, 1 mM KCl, 0.5 mM KH²PO⁴, 1.1 mM CaCl², 0.5 mM Na²SO⁴, 5.9 mM glucose, 10⁻²mM pargyline, and 1 mM ascorbic acid, adjusted at p  H 7.3 with 95%/5%[vol/vol] oxygen/carbon dioxide mixture. In Ca²⁺-free experiments, CaCl²was omitted from the cerebrospinal fluid.

Microdiscs of striatal tissue were prepared according to a slightly modified version of the procedure described by Galli et al.  22Brains were quickly removed from the skull and placed for 10 min in the same cooled cerebrospinal fluid. Microdiscs (0.9-mm diameter, 0.35 mg protein per disc) of tissue were punched out with a stainless steel tube from 300-μm sagittal rat brain slices prepared with a vibratome (Campden Instruments, London, United Kingdom). Microdiscs of tissue from the dorsolateral striatal matrix-enriched area were obtained from three consecutive slices in each hemisphere (laterality 4.80–3.75 mm according to the atlas of Paxinos and Watson 23), with one microdisc punched out in each slice.

Uptake of DA by synaptosomes and tissue microdiscs was performed by incubating (15 min at 37°C) the preparations (final volume, 12 ml for synaptosomes and 5 ml for microdiscs) with [2,5,6]³H-DA (Amersham, Little Chalfont, United Kingdom; 25 μl, 44 nM, 24 Ci/mM, 1 mCi/ml). Aliquots of the synaptosomal preparation (1 ml) were pipetted into each of the superfusion chambers (15-mm diameter, 2-mm height) and embedded in Whatman (Whatman International Ltd., Maidstone, England) GF/F glass filters (0.70-μm retention capacity) by light suction. Each microdisc of the striatum was introduced individually in a microsuperfusion chamber (6.35-mm diameter, 9.5-mm height). Synaptosomes or microdiscs were then superfused at a 1-ml/min flow rate using a superfusion device equipped with an automatic fraction collector (Brandel, Gaithersburg, MD). The 1-ml/min flow rate was selected because of its ability to prevent reuptake of ³H-DA by the preparations. 11,25After a 30-min washing step (37°C), serial fractions were collected every minute during three consecutive periods; the mean value of the radioactivity (counts/min) measured over the first 5 min by liquid scintillation spectrometry using Aquasol-2 (New England Nuclear, Boston, MA) was considered the basal (spontaneous)³H-DA release. During the next 5 min, either no treatment (time-dependent control) or various pharmacologic or anesthetic agents were delivered to the preparations, and radioactivity was again estimated in each fraction. Because of a 3-ml dead volume occupied by the inside of the connect tubes, pharmacologic agents delivered at a 1-ml/min flow rate reached striatal tissue or synaptosomes only after a delay averaging 180 s. For the final 9 min, radioactivity was determined from 1-min fractions in the absence of any treatment.

The effects of the following pharmacologic agents were studied: nomifensine (a blocker of the high affinity DA uptake, 10⁻⁵M; Sigma, L'Isle d'Abeau, France), NMDA (5 × 10⁻⁷–10⁻³M; Sigma), MK 801 (an NMDA receptor antagonist, 10⁻⁸–10⁻⁵M; Merck-Sharp and Dohme, Darmstadt, Germany), glycine (10⁻⁶M; Sigma), 7-chloro-kynurenate (an antagonist of the glycine site at the NMDA receptor, 10⁻⁴M; Research Biochemical Incorporated, Illkirch, France), TTX (a blocker of the voltage-operated sodium channels that suppresses action potentials, 10⁻¹⁰–10⁻⁷M; Sigma), and bicuculline (an antagonist of the GABA^Areceptors, 10⁻⁵M; Sigma). Halothane (Fluothane; Zeneca, Cergy, France) and isoflurane (Forane; Abbott, Rungis, France) were delivered through a calibrated vaporizer in an oxygen/carbon dioxide mixture (95%/5% vol/vol, 3 l/min) at various concentrations ranging from 0.25% to 5%. The minimum alveolar concentration value was considered to be 1% for halothane and 1.5% for isoflurane, as previously reported for male rats. 26Volatile anesthetics were equilibrated with the reservoir of artificial cerebrospinal fluid for 60 min at 37°C, and aqueous concentrations in the superfusion chambers were determined by gas phase chromatography according to a slightly modified version of the method of Brachet-Liermain et al.  27 

Results were considered reliable only if they had been reproduced in four independent experiments (each of them performed in triplicate). The influence of a drug on ³H-DA release was assessed by calculating the difference between the release observed in the presence and absence (time-dependent control) of the pharmacologic agent applied over the 5-min period. This was expressed as a fractional increase from spontaneous release. Only minor, nonsignificant differences in ³H-DA release evoked by NMDA were observed from one microdisc of tissue to the other. Therefore, results obtained with microdiscs of tissues punched out on three successive sagittal slices on each side of the brain were combined for statistical analysis.

Concentration–response curves, IC⁵⁰values, and Hill coefficients were computer-generated using GraphPAD software (Intuitive Software for Science, San Diego, CA). The following four-parameter logistic equation was used to fit the curves to the data: where X  represents the logarithm of drug concentration, and A  and B  represent the minimum (bottom) and maximum (top) of X values, respectively. C  is the logarithm of the IC⁵⁰value, and D  is the Hill coefficient or slope factor that is positive for curves in which the Y  value increases with increasing X  and is negative for curves in which the Y  value decreases with increasing X .

Normality of distributions was first assessed by the Fisher test for equality of variances. Statistical analysis was then performed by analysis of variance with Scheffé’s post hoc  correction for multiple comparisons. A P  value < 0.05 was considered the threshold for significance. Data are expressed as mean ± SD.

The ³H-DA concentration estimated in the incubation medium during the uptake phase was 250 ± 29 pmol/mg protein, and the initial basal release rate measured over 5 min averaged 1.08 ± 0.10 and 2.9 ± 0.32 pmol · mg protein⁻¹· min⁻¹for synaptosomes and microdiscs, respectively. Omitting Ca²⁺from the superfusion medium did not affect basal release rate. The aqueous concentrations of volatile anesthetics measured in the chambers after 1 h of equilibration with the superfusion medium at 37°C are given in table 1.

In striatal slices, no significant variation in basal (spontaneous) release was observed with time. NMDA application induced a concentration-related increase in ³H-DA release (fig. 1A). The 10⁻⁴-M concentration of NMDA produced a 1.62 (in fractional units, P < 0.05) increase from spontaneous release and was used as the reference depolarizing stimulus to elicit ³H-DA release in microdiscs of striatal tissue. This release was markedly decreased by MK 801 in a concentration-related fashion (fig. 1B). It was significantly enhanced by a 10⁻⁶-M glycine concentration (in fractional units: 1.84 ± 0.13;P < 0.01), but coapplication of 7 chloro-kynurenate (10⁻⁴M) with glycine suppressed the glycine-induced potentiation of NMDA on ³H-DA release (fig. 1C). 28MK 801, glycine, and kynurenate had no effect per se  on spontaneous release. As shown in figure 1D, TTX induced a significant, concentration-related decrease in the NMDA-evoked ³H-DA release from striatal slices. A ceiling effect was noted in TTX action. The TTX concentration of 5 × 10⁻⁸M was used for testing the effects of anesthetics, because this concentration attenuated ≈90% of the TTX-sensitive NMDA-evoked release in the slices. Consistent with previous reports, 28we found that this TTX concentration decreased basal DA release by 40% in striatal slices. Nomifensine (10⁻⁵M) failed to affect basal or NMDA-evoked release under these experimental conditions.

Halothane and isoflurane produced a significant increase in spontaneous ³H-DA release in TTX-treated slices (5 × 10⁻⁸M; EC ⁵⁰= 0.33 mM and 0.41 mM for halothane and isoflurane, respectively;fig. 2). Both agents induced a significant, concentration-related decrease of ³H-DA release evoked by NMDA in slices (fig. 3). The IC⁵⁰values obtained for these inhibitory effects of halothane and isoflurane were 0.15 mM and 0.14 mM, respectively. The best fit of the curves to the data was always obtained when the Hill coefficient was forced to 1. The attenuation of the NMDA-evoked release by volatile anesthetics yielded a ceiling effect. The IC⁵⁰values for the inhibitory effects of halothane and isoflurane in microdiscs of tissue were not significantly different in either the presence or absence of TTX (5 × 10⁻⁸M) in the medium (IC⁵⁰values: 0.15 mM and 0.15 mM for halothane and isoflurane, respectively;fig. 3).

Muscimol (10⁻⁵M) did not affect basal release. In contrast, it induced a mild but significant decrease in NMDA-evoked 3H-DA release in slices (fig. 4). In contrast, bicuculline application (10⁻⁵M) produced a marked increase in NMDA-evoked 3H-DA release. In the presence of bicuculline, the inhibitory effect of halothane and isoflurane was significantly attenuated (fig. 4).

In the synaptosomal preparation, the ³H-DA release elicited by NMDA (10⁻⁵-M, 10⁻⁴-M, 5 × 10⁻⁴-M, and 10⁻³-M concentrations) was (in fractional units): 1.10 ± 0.12 (not significant), 1.24 ± 0.13 (P < 0.05), 1.30 ± 0.15 (P < 0.05), and 1.37 ± 0.20 (P < 0.01), respectively. For comparative purposes with previous studies, the 10⁻³-M NMDA concentration was selected. 11The effect of NMDA (10⁻³M) was completely blocked by MK 801 (10⁻⁵M; 97%± 13%) but was not significantly affected by TTX application (10⁻⁷M). Halothane and isoflurane produced a concentration-related decrease in the NMDA-evoked ³H-DA release (fig. 5). The IC⁵⁰values for these inhibitory effects were 0.19 mM for both anesthetics.

The present study indicates that halothane and isoflurane exert complex effects on DA release in striatal slices: they enhance the spontaneous release of striatal DA but decrease the NMDA-evoked in a concentration-related fashion. The latter effects are observed at particularly low concentrations of anesthetics whether the activity of the local interneurons and afferent fibers involved in DA release is depressed or not. These findings show that volatile agents modulate transmitter release mainly via  direct presynaptic effects at DA terminals in the striatum and the involvement of presynaptic GABA^Areceptors in at least part of their inhibitory effects on NMDA-evoked release.

The synaptosomal preparation provides a useful tool to examine direct presynaptic effects of anesthetics on transmitter release. 10,11,14,16However, it precludes examination of indirect, TTX-sensitive mechanisms or the role of diffusible mediators, such as nitric oxide, involved in NMDA-stimulated DA release. 29The use of striatal slices is particularly interesting because it allows separate examination of the effects of anesthetics on the direct, TTX-insensitive and indirect, TTX-sensitive DA release elicited by NMDA application. However, the fact that drug-induced neurotransmitter release is sensitive to TTX application does not rule out the possibility of direct presynaptic action at DA terminals. Indeed, several nonexclusive mechanisms could contribute to the NMDA-evoked GABAergic inhibitory control of ³H-DA release resistant to TTX. 21These include local modifications of the GABA neuronal uptake process, 30release of GABA either from collaterals of medium-sized spiny neurons or GABAergic interneurons through processes involving TTX-insensitive sodium channels, 31or dendritic release of GABA as already reported in the rat olfactory bulb. 28In the present study, the NMDA-evoked ³H-DA release was insensitive to TTX in the synaptosomal preparation.

We used ³H-DA to preload DA nerve endings in the present study. This technique allows a specific prelabeling of DA terminals in the striatum, because only DA nerve endings possess the high affinity carrier for this neurotransmitter among the various neuronal subpopulations present in this brain area. Release of the newly synthesized DA from ³H-tyrosine could also have been used. 15The newly synthesized DA is preferentially released from nerve endings by depolarization, and this phenomenon is also calcium-dependent. 32However, this technique does not separate effects of anesthetics on release and synthesis of DA.

Halothane and isoflurane were found to significantly enhance spontaneous ³H-DA release in a concentration-related fashion in TTX-treated slices. These findings are consistent with the effects of a restricted number of concentrations of volatile anesthetics on ³H-DA release reported in striatal synaptosomes. 11This effect may have been caused by the blockade of the DA carrier, because halothane and isoflurane show significant effects on this transporter in vitro  10; however, this was likely not to be the case for two reasons:(1) the 1-ml/min flow prevents from DA reuptake subjected to both KCl- and NMDA-induced release from synaptosomes 11,25; and (2) nomifensine, an inhibitor of DA uptake, failed to affect either spontaneous or NMDA-elicited release in the present study.

According to the dose–response curve shown in figure 1, the TTX concentration used was expected to block action potential propagation along nerve membranes ≈90% of the striatal interneurons or fibers. Consistent with previous reports, we found that TTX alone reduced spontaneous ³H-DA release by 40% from slices. 19This might be a result of the inhibition of striatal interneurons such those containing acetylcholine, substance P, or nitric oxide, all of which exert presynaptic facilitative control on DA release. 17–19Indeed, recent electrophysiologic and biochemical experiments indicate that presynaptic voltage-operated sodium channels show some sensitivity to volatile agents. 33,34In addition, halothane blocks synaptic excitation of local interneurons in some brain areas, such as the hippocampus. 35However, this hypothesis is not consistent with the lack of effect of the anesthetics on the TTX-sensitive portion of ³H-DA release observed in the present study. Therefore, the concentration-related enhancement by halothane and isoflurane of the spontaneous ³H-DA release in TTX-treated slices together with our previous data 11support that this effect is mainly caused by direct stimulatory action of volatile anesthetics at DA terminals. Membrane fluidization—which correlates with the ability of volatile agents to increase resting calcium levels, 36increase by volatile anesthetics of Ca²⁺permeability of synaptosomal membranes, 37or release of intraneuronally stored neurotransmitter, as was demonstrated for GABA 38,39—may contribute to the stimulatory effect of inhalational anesthetics on spontaneous ³H-DA release.

A major finding made in the present study is that halothane and isoflurane used at particularly low concentrations markedly decreased the NMDA-evoked DA release in striatal slices in a concentration-related fashion. With respect to the IC⁵⁰values reported for each anesthetic, this observed effect was with the same potency in the presence or absence of activity of the striatal interneurons. In addition, it was present in synaptosomes in a dose-related fashion. Taken together, these data confirm and extend our previous observations 11and show that volatile anesthetics decrease the NMDA receptor-mediated DA release from striatal slices by acting directly at presynaptic DA terminals.

NMDA application evoked a potent, concentration-related stimulation of ³H-DA release in striatal slices, which is consistent with previous observations. 21The stimulatory effect of NMDA on DA release was enhanced by glycine, a positive allosteric effector at the NMDA receptor, and blocked by MK 801, an antagonist of the NMDA receptor. The potentiation by glycine of NMDA-evoked DA release was no more observed in the presence of 7-chloro-kynurenate, an antagonist of the glycine site at the NMDA receptor. These findings show that DA release observed in the present study was actually caused by stimulation of striatal NMDA receptors. The release of ³H-DA evoked by NMDA in the presence of TTX was markedly reduced in comparison with that elicited without TTX in the superfusion medium. In contrast, NMDA-evoked ³H-DA release was insensitive to TTX in synaptosomes. This further shows that, in slices, NMDA stimulates DA release both directly and indirectly. 14The higher NMDA concentrations (10⁻³M vs.  10⁻⁴M) required to significantly stimulate ³H-DA release in synaptosomes compared with slices could be a result of a lower extracellular concentration of endogenous glycine in the synaptosomal preparation.

The fractional equivalent reduction by volatile agents of TTX-sensitive and -insensitive responses to NMDA stimulation in slices suggests direct presynaptic actions of volatile agents at DA terminals. Interestingly, the inhibitory effect of an IC⁵⁰concentration of halothane and isoflurane was significantly, but not totally, reversed by bicuculline used at a concentration that blocked the tonic GABAergic inhibitory control of NMDA-evoked DA release. 20,21Consistent with this finding, we observed a slight but significant decrease in NMDA-induced ³H-DA release by muscimol, an agonist of GABA^Areceptors. The presence of these receptors on striatal DA terminals is strongly supported by previous pharmacologic experiments. 21,40On the other hand, volatile anesthetics have been shown to potently enhance the activity of the GABA^Areceptor-coupled-chloride current. 41Whether muscimol and volatile anesthetics share common target sites at GABA^Areceptors cannot be determined from our data. It can be proposed that that the inhibitory effects of inhalational anesthetics on NMDA-evoked ³H-DA release are mediated in part via  enhancement of the action of GABA released from recurrent collaterals or dendrites of striatal output neurons on the GABA^Areceptors present on DA terminals. The potency of volatile anesthetics to block NMDA-induced ³H-DA release was greater in slices than in synaptosomes. The potentiation of GABA action on its receptors in slices may account for this difference, because no NMDA-evoked GABA release occurs in the synaptosomal preparation. 22 

Although full dose–response curves have not been generated with bicuculline, we observed that the reversal of the inhibitory effect of inhalational agents by bicuculline was only partial. This may suggest that volatile agents also act directly at either NMDA receptors or their environment to reduce ³H-DA release. Our findings may suggest that downstream sites common to both KCl- and NMDA-elicited challenges are involved in halothane and isoflurane effects. However, some data show that the NMDA receptor-mediated response is particularly sensitive to isoflurane. Volatile anesthetics have been shown to depress NMDA-induced Ca²⁺entry in cultured hippocampal neurons. 42In addition, isoflurane is particularly effective at blocking glutamate transmission in numerous models. 43,44This is in agreement with the greater sensitivity to isoflurane of the NMDA-evoked ³H-DA release reported in our study. In addition, recent patch-clamp experiments indicate that isoflurane action at the NMDA receptor may show a high degree of specificity in some models. 45Conversely, the KCl-evoked release of ³H-GABA was not affected by these inhalational anesthetics in similar experimental conditions, 46whereas synaptosomal glutamate release, which is also closely coupled to specific Ca²⁺channel activation, is not highly sensitive to volatile anesthetics. 47However, the N- type channels, which play a key role in presynaptic stimulus-secretion coupling in the brain, 48are depressed by clinical concentrations of halothane or isoflurane. 37Therefore, it cannot be excluded that a decrease in the activity of these voltage-gated Ca²⁺channels may contribute to the anesthetic-induced decrease in depolarization-evoked DA release in striatal slices.

A potent inhibitory action of volatile anesthetics was observed on the NMDA-induced ³H-DA release from striatal terminals. In contrast, a stimulatory effect of these agents on spontaneous release was observed in either the slice preparation and synaptosomes in vitro , although at higher concentrations than the effect on NMDA-evoked release, or in in vivo  studies. Several explanations can be proposed to account for this apparent discrepancy. Isoflurane may both stimulate spontaneous DA release from striatal terminals and reduce glutamate release from striatal nerve endings, as was reported in some brain areas. 44,47Alternatively, isoflurane depresses neuronal excitability by hyperpolarizing the cell membrane of all neocortical neurons in humans. 49This results in reduction of the activity of the corticostriatal glutamate pathway and subsequent decrease in glutamate-stimulated DA release. Thus, the stimulatory effect of inhalational anesthetics on spontaneous DA release would be more perceptible than the inhibitory one on depolarization-evoked DA release, resulting in an increase in DA extracellular concentrations in the striatum. The lack of effect of anesthetics on the local circuits involved in the regulation of DA release is consistent with this hypothesis.

In conclusion, we provide evidence that clinical concentrations of halothane and isoflurane enhance basal but decrease NMDA-evoked DA release in the rat striatum via , at least in part, actions at GABA^Areceptors. It can be speculated that the inhibitory effects of volatile agents on glutamate transmission might contribute to some of their neuroprotective effects against striatal ischemic injury. 50