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ANESTHETIC PHARMACOLOGY

Ketamine Stereoselectively Inhibits Spontaneous Ca²⁺-Oscillations in Cultured Hippocampal Neurons

Barbara Sinner, MD^*, Oliver Friedrich, MD, PhD, Wolfgang Zink, MD^*, Eike Martin, MD^*, Rainer H. A. Fink, PhD, and Bernhard M. Graf, MD, PhD^*

*Department of Anesthesiology, and Institute for Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany

Address correspondence and reprint requests to Bernhard M. Graf, MD, PhD, Department of Anesthesiology, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany. Address e-mail to Bernhard_graf{at}med.uni-heidelberg.de.

	Abstract

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Spontaneous Ca²⁺-oscillations are a result of periodic increases and decreases of cytosolic Ca²⁺. In neurons, they are thought to possess integrative properties because amplitude and frequency influence axon outgrowth, neuronal growth cone migration, and long distant wiring within the developing cortex. Ketamine stereoisomers differ in their affinities for the N-methyl-d-aspartic acid receptor and analgesic and anesthetic effects. Using a dual-excitation Ca²⁺ ratiometric fluorescence technique with the Ca²⁺-sensitive dye fura-2 AM, we detected spontaneous Ca²⁺-oscillations in neurons of hippocampal cell cultures. Spontaneous Ca²⁺-oscillations development is dependent on external Ca²⁺, and their amplitude and frequency increased in Mg²⁺-free solution. Ca²⁺-oscillations are glutamate dependent because blocking of the N-methyl-d-aspartic acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic, or kainate receptor resulted in a complete disruption of the oscillations. The ketamine stereoisomers dose-dependently and reversibly suppressed the amplitude and frequency of the spontaneous Ca²⁺-oscillations. This effect was highly stereoselective with the S(+) isomer being nearly four times more potent than the R(–) enantiomer. These results correlate well with the clinical anesthetic and analgesic potency of the stereoisomers and therefore our experimental approach might represent a model system to study mechanisms of anesthetic action on Ca²⁺-dependent integration of neuronal information.

	Introduction

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The hippocampus has an important role in learning and memory (1). Hippocampal neuronal circuits express strong plasticity which is partly mediated through changes in synaptic strength, as revealed by long-term enhancement and depression (1). In these mechanisms, the glutamate receptors are mainly involved, of which the N-methyl-d-aspartic acid (NMDA) receptor, a calcium (Ca²⁺) permeable subtype of glutamate receptors, seems to be very important (1). Blocking this receptor impairs neuronal plasticity and learning in memory processes requiring the hippocampus, and may result in amnesia (1).

In addition to their involvement in the generation of action potentials, regulation of neuronal excitability and neurotransmitter release, Ca²⁺ ions are responsible for regulation of neuronal cell growth, differentiation, and neuronal cell death (2–4). In neurons under physiological conditions, resting intracellular Ca²⁺ ([Ca²⁺]_i) concentrations are usually very small (approximately 100 nM). Small changes or local fluctuations of [Ca²⁺]_i, as induced by spontaneous Ca²⁺-oscillations, might result in relatively large alterations of the intracellular concentration. Usually, spontaneous Ca²⁺-oscillations are triggered by extracellular Ca²⁺ which enters the neuron via receptor-operated or voltage-gated Ca²⁺ channels (4,5) such as the NMDA receptor (6,7). These Ca²⁺ ions then induce Ca²⁺ release from the endoplasmic reticulum. Increased [Ca²⁺]_i are rapidly restored to baseline levels by Ca²⁺-reuptake, mainly into the endoplasmic reticulum. Spontaneous Ca²⁺-oscillations are caused by periodical increase and decrease of the free [Ca²⁺]_i (5,6,8). They are thought to possess integrative properties which is reflected by the influence of the amplitude and frequency of spontaneous Ca²⁺-oscillations on neuronal growth cone migration, axon outgrowth, and long distant wiring within the developing cortex (2–4).

Although the exact mechanism of its anesthetic action is unknown, the IV anesthetic ketamine is considered to act primarily by a noncompetitive block of the NMDA receptor in a use-dependent manner (9). Thus, ketamine mainly affects network activity involving NMDA receptor-mediated excitation. Ketamine is a racemic mixture of S(+) and R(–) isomers. Clinically, ketamine isomers differ in their anesthetic and analgesic potency, with the S(+) isomer being about three to four times more potent than the R(–) enantiomer (10–12). Patch clamp examinations of the NMDA receptor revealed a potency ratio between S(+) and the R(–) isomer of 1:1.9 (13).

Because spontaneous Ca²⁺-oscillations in hippocampal neurons are involved in encoding of neuronal information, they could be an interesting system to test the effects of anesthetics on neuronal information processing. To address this question, in the present study, we characterized spontaneous Ca²⁺-oscillations and evaluated the effects of the pure optical isomers of ketamine on spontaneous Ca²⁺-oscillations in hippocampal neuronal cell cultures of embryonic Wistar rats.

	Methods

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Preparation and Cell Culture
According to the guidelines of the local Animal Care Committee, hippocampal neuronal cell cultures were cultured based on a modification of the protocol by Banker and Cowan (14). The Hippocampi of 19-day-old embryonic Wistar rats were prepared in phosphate buffered saline containing 0.05% gentamicin (both Invitrogen Life Technologies, Karlsruhe, Germany) and incubated for 15 min at 37°C in saline containing 0.02% trypsin (Sigma Chemicals, Steinheim, Germany). Hippocampi were then rinsed 3 times in minimum essential medium (MEM) (Invitrogen Life Technologies, Karlsruhe, Germany) dissociated by repeated passings through a fire-polished pipette and plated on 0.01% poly l-ornithine (Sigma Chemicals) coated coverslips. Cultures were kept sterile in MEM supplemented with 10% horse serum (Invitrogen Life Technologies) at 37°C and a 5% CO₂ atmosphere. Cells were used for experiments on days 17 and 18.

Microfluorimetry
For experiments, coverslips were mounted in a perfusion chamber and exposed to standard extracellular solution containing (in mM) 116 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.9 MgCl₂, 0.9 NaH₂PO₄, 10 glucose, 20 HEPES (N-[2-Hydroxyethyl]piperazine-n-[2-ethanesulfonic acid]; Sigma Chemicals) pH 7.4 adjusted with NaOH. Cells were loaded with fura-2 by incubation with 20 µM fura-2 AM in standard extracellular solution containing 0.08% pluronic-127 (both Molecular Probes, Göttingen, Germany) for 1 h at room temperature. Loading solution was replaced by MEM and cells were stored at 37°C and 5% CO₂ for 30 min to allow de-esterification of the dye.

Measurements
Dual wavelength excitation Ca²⁺-fluorescence experiments were conducted using an Olympus OSP-3-photometry system with a Xenon UV light source added to an IMT-2 inverted microscope (Olympus, Hamburg, Germany). A fast filter wheel allows excitation at 340 and 380 nm. Emission was detected at 510 nm by a photomultiplier unit (at a fixed voltage of 650 V) and the ratio of the dual wavelength excitation F [340/380] (fluorescence ratio of 340–380 nm) was calculated.

In Situ Calibration
To quantify free [Ca²⁺]_i concentrations, an "in situ" calibration was performed by exposing the neurons to 10 µM of the Ca²⁺-sensitive ionophore Br-A 23187 (Molecular Probes) dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany) in EGTA (ethylene glycol-bis[ß-aminoethylether]-N,N,N',N',-tetraacetic acid) buffered intracellular solution containing (in mM) HEPES 60, Mg(OH₂) 5.31, ATP (adenosine triphosphate) 8.0, creatine phosphate 10.0, EGTA 50.0 (Sigma Chemicals) at varying levels of Ca²⁺ ranging from 1 nM to 1 µM. By least-square Hill fits to the data, the in situ dissociation constant (K_d) of fura-2 could be determined and the free [Ca²⁺]_i concentration was calculated from fura-2 fluorescence ratios (15,16)

Experimental Protocol
To investigate the effect of ketamine stereoisomers on spontaneous Ca²⁺-oscillations, all drugs were dissolved in standard extracellular solution according to their solubility. Each single ketamine isomer was applied in concentrations of 3, 25, 50, 100, and 250 µM, respectively.

Before drug application, [Ca²⁺]_i-oscillations of a single pyramidal neuron were recorded over a period of 3 min. Immediately after complete washin of the drug, Ca²⁺-oscillations were monitored over a period of 6 min and the reversibility of drug effects was verified by recording the same cell for 3 min after washout. Each preparation was used only for one type of stereoisomer. All recordings were made from individual isolated neurons and were performed at room temperature (21°C). In in vitro control experiments, ketamine stereoisomers had no effect on the fluorescence of fura-2.

For data analysis, oscillation frequency was evaluated and amplitudes were calculated by converting the ratiometric signal into free [Ca²⁺]_i. The relative amplitude of each oscillation relative to baseline [Ca²⁺]_i was then calculated. Paired t-tests were performed and significance was considered to be at the P < 0.05 level. Data were presented as mean ± se with number of n observations where indicated.

Drugs and Chemicals
CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) was obtained from Sigma Chemicals. MK801 was from Calbiochem (Beeston, UK). ATP and creatine phosphate were from Boehringer (Ingelheim, Germany). S(+) and R(–) ketamine were a gift from Pfizer (Karlsruhe, Germany).

	Results

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Basic Properties of Spontaneous Ca²⁺-Oscillations
Spontaneous Ca²⁺-oscillations were detected in almost every hippocampal neuron and lasted for at least 4 h. Calibration of the [Ca²⁺]_i revealed an in situ K_d of 659 ± 104 nM for fura-2. Spontaneous Ca²⁺-oscillations were recorded in 76 hippocampal neurons. The mean resting [Ca²⁺]_i concentration was 82.7 ± 8.3 nM. During spontaneous Ca²⁺-oscillations, the mean amplitude of Ca²⁺ transients was 239.6 ± 23.5 nM and mean frequency was 1.53 ± 0.02/min (0.025 ± 0.0003 Hz). There was no significant difference in amplitude and frequency between day 17 and 18 in culture (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. The right panel shows hippocampal neurons (day 18) in culture. The arrow indicates the soma of a pyramidal neuron used for experiments. The circle marks the area that was covered by the pinhole during the fluorescence experiments. Within this area, spontaneous Ca2+-oscillations could be detected. The Ca2+ fluorescence was displayed as the ratio of the Ca2+ bound and free dye. Representative transients are shown on the right panel of the figure. The dimension of the fluorescence ratio F340/380 and the time scale are shown in the index.

Spontaneous Ca²⁺-Oscillations Are Dependent on Extracellular Ca²⁺
To investigate the influence of extracellular Ca²⁺ on Ca²⁺-oscillations, extracellular solution was replaced by Ca²⁺-free EGTA buffered (20 mM) standard extracellular solution. Spontaneous Ca²⁺-oscillations were abolished and almost completely restored after return to standard extracellular solution (n = 4) (Fig. 2a).

View larger version (12K): [in this window] [in a new window]   Figure 2. Pharmacological characterization of the spontaneous Ca2+-oscillations. The panels show spontaneous Ca2+-oscillations before (left), after application of the drug indicated (middle), and after washout of the drug and return to the standard extracellular solution. A, Spontaneous Ca2+-oscillations are dependent on extracellular Ca2+. Substitution of the extracellular solution with a Ca2+-free EGTA (ethylene glycol-bis[ß-aminoethylether]-N,N,N',N',-tetraacetic acid)-buffered (20 mM) solution results in a complete and reversible block of the oscillations. B, Removal of Mg2+ results in a marked and reversible increase in amplitude and frequency of the spontaneous Ca2+-oscillations. C and D, Spontaneous Ca2+-oscillations are glutamate receptor-dependent. C, Blocking the oscillations with the N-methyl-d-aspartic acid (NMDA) receptor antagonist MK 801 (40 µM) resulted in a complete and reversible block of the oscillations. D, Application of the non-NMDA receptor antagonist (CNQX) 6-cyano-7-nitroquinoxaline-2,3-dione which blocks -amino-3-hydroxy-5-methyl-4-isoxazolepropionic and kainate receptors results in a complete block of the spontaneous Ca2+-oscillations.

Spontaneous Ca²⁺-Oscillations Are Dependent on Extracellular Mg²⁺
The removal of extracellular Mg²⁺ (n = 4) led to a 3- to 4-fold increase in the amplitude of the spontaneous Ca²⁺-oscillations from control values of 144.8 ± 67.6 to 492.2 ± 157.8 nM Ca²⁺ in the neurons. This was accompanied by a 2-fold increase in the frequency of the spontaneous Ca²⁺-oscillations (P < 0.05). Adding Mg²⁺ to the solution rapidly reduced amplitude and frequency to baseline levels (Fig. 2b).

Spontaneous Ca²⁺-Oscillations Are Glutamate-Receptor Mediated
As illustrated in Figure 2c, the washin of the noncompetitive NMDA receptor antagonist dizocilpine (MK 801, 40 µM) resulted in complete inhibition (>98%) of the amplitude and frequency of the spontaneous oscillations in the studied neurons (n = 6). The washout of the antagonist restored spontaneous Ca²⁺-oscillations.

To determine whether non-NMDA ionotropic glutamate receptors such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) or kainate are required for Ca²⁺-oscillations, we applied 100 µm CNQX, a competitive antagonist of AMPA and kainate receptors. As shown in a representative recording (Fig. 2d), Ca²⁺-oscillations were reversibly abolished (n = 4), suggesting that the activation of all ionotropic glutamate receptors is essential for the initiation and propagation of the spontaneous Ca²⁺-oscillations.

-Aminobutyric Acid (GABA)_A Receptor
GABA is the major inhibitory neurotransmitter in the mammalian brain. To examine the role of the synaptic inhibitory mechanism on spontaneous oscillations, we exposed the oscillating neurons to 50 µM of the GABA_A receptor antagonist bicuculline (n = 4). Blocking the GABA_A receptor significantly increased the amplitude of the spontaneous Ca²⁺-oscillations reversibly from 121.4 ± 17.8 to 351.8 ± 69.9 nM. In contrast, there was no significant change of the frequency of the spontaneous Ca²⁺-oscillations (from 1.14 ± 0.36/min [0.019 ± 0.006 Hz] to 0.78 ± 0.12/min [0.013 ± 0.002 Hz]) (Fig. 3).

View larger version (5K): [in this window] [in a new window]   Figure 3. Spontaneous Ca2+-oscillations after blocking the -aminobutyric acid (GABA)A receptor with the specific antagonist bicuculline. Representative example of Ca2+-oscillations in a hippocampal neuron before (left), after the application of bicuculline (middle), and after washout of the drug (right). The suppression of the GABAergic input leads to a reversible increase in the amplitude and a mild reduction of the frequency.

Ketamine Stereoisomers
Ketamine stereoisomers suppressed the amplitude and frequency of the spontaneous Ca²⁺-oscillations significantly in a dose-dependent manner (n = 60) (Fig. 4, a and b).

View larger version (16K): [in this window] [in a new window]   Figure 4. The amplitude and frequency of spontaneous Ca2+-oscillations are stereospecifically and dose-dependently suppressed by ketamine. A, Relative amplitude of the spontaneous Ca2+-oscillations with a concentration of the applied S(+) and R(–) ketamine stereoisomer in relation to the control value before drug application. Both ketamine stereoisomers suppress the amplitude in a dose-dependent manner. The S(+) is more potent than the R(–) ketamine resulting in an approximated potency ratio of 3:1. At concentrations of 250 µM, the amplitude was completely suppressed by both stereoisomers (bars indicate standard error, *P < 0.05 versus control #P < 0.05 S(+) versus R(–) ketamine). B, Reduction of the frequency with the applied ketamine stereoisomer concentration. The ordinate indicates the resulting relative frequency after addition of the drug. Both stereoisomers reduce the frequency of the spontaneous Ca2+-oscillations in a dose-dependent manner (*P < 0.05 versus control). At concentrations of 3 µM, the R(–) isomer slightly increased the oscillation frequency which did not reach statistical significance. The frequency of the spontaneous Ca2+-oscillations was significantly more reduced by the S(+) ketamine. The approximated 50% effective concentration for S(+) and R(–) were 25 and 150 µM, respectively (bars indicate standard error).

The amplitude of the spontaneous Ca²⁺-oscillations was more potently attenuated by the S(+) isomer than the R(–) enantiomer for concentrations up to 100 µM (Fig. 4a) (P < 0.05). For concentrations 250 µM, spontaneous Ca²⁺-oscillations were completely suppressed by both isomers. The 50% effective concentration was empirically derived from the dose-response curve. The S(+) ketamine-induced reduction of Ca²⁺ amplitude is approximately 20 µM and for the R(–) ketamine 60 µM, resulting in an approximated potency ratio for amplitude attenuation of S(+)/R(–) = 3:1.

Ketamine also reduced the frequency of the spontaneous Ca²⁺-oscillations in a dose-dependent manner (Fig. 4b). The S(+) ketamine reduced the frequency more potently than the R(–) enantiomer. From concentrations of 250 µM, the frequency was completely and reversibly abolished. The 50% effective concentration for ketamine-induced reduction of Ca²⁺-oscillation frequency is approximately 25 and 150 µM for the R(–) ketamine, respectively, resulting in a potency ratio for frequency attenuation of approximately 1:6. At a concentration of 3 µM, the application of S(+) ketamine did not decrease the amplitude and frequency. Interestingly, the application of R(–) ketamine at this concentration resulted in a slight increase in oscillation frequency.

	Discussion

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Spontaneous Ca²⁺-oscillations of [Ca²⁺]_i represent an interesting form of neuronal signaling. The results of our experiments provide insights into pharmacological profiles of spontaneous Ca²⁺-oscillations in hippocampal neuronal cell cultures and demonstrate a major contribution of glutamate receptors. The stereoisomers of the IV anesthetic ketamine led to a dose-dependent, stereospecific, and reversible reduction of the amplitude and frequency of the spontaneous Ca²⁺-oscillations. The reduction of the oscillation amplitude resulted in a potency ratio of about 1:3 and for the frequency of 1:6, respectively, between R(–) and S(+) ketamine. These results correlate well with the amnesic, analgesic, and anesthetic potency ratio of both isomers (10–13). Hippocampal neurons have a decisive role in memory formation and therefore the influence of ketamine on NMDA receptor-mediated spontaneous Ca²⁺-oscillations might constitute its amnesic, anesthetic, and analgesic mechanism.

The development of spontaneous Ca²⁺-oscillations in various neuronal cell types is dependent on extracellular Ca²⁺ (6,17). Moreover, extracellular Ca²⁺ is necessary for oscillations induced by the presence of caffeine or K⁺, respectively, or in the absence of extracellular Mg²⁺ (18). In our experiments, the removal of extracellular Mg²⁺ resulted in a significant increase in the amplitude and the frequency of the spontaneous oscillations. The NMDA receptor is most sensitive to changes in extracellular Mg²⁺. In the inactivated state of the channel, this divalent cation binds to a site located within the NMDA receptor ion channel and blocks the NMDA receptor voltage-dependently (18,19). Besides the increase of the amplitude and frequency of spontaneous Ca²⁺-oscillations, the removal of extracellular Mg²⁺ initiates Ca²⁺-oscillations in cortical neurons (18). The strong influence of Mg²⁺ on amplitude and frequency of the spontaneous Ca²⁺-oscillations suggests the involvement of the glutamatergic transmission via the NMDA receptor (6–8,18). This was confirmed by the application of the noncompetitive NMDA receptor antagonist MK 801 which led to a complete disruption of the spontaneous neuronal Ca²⁺-oscillations. The role of the ionotropic glutamate receptors AMPA and kainate in Ca²⁺-oscillation development seems to be different and may depend on the neuronal cell type. Similar to our experiments, a reversible block of spontaneous Ca²⁺-oscillations in hippocampal neuronal cell cultures has been found (6). In contrast, in brain slices the involvement of the AMPA and kainate receptors seem to be dependent on neuronal age and cell type (2,8).

The physiological relevance of spontaneous Ca²⁺-oscillations is not yet fully understood. In the immature central nervous system, transient increases of [Ca²⁺]_i are important for many aspects of cell growth and differentiation. These include cell proliferation, neuronal, axonal, and dendritic migration and activity-dependent maturation of glutamatergic synapses. In the spinal cord, the speed of growth cone migration is controlled by the frequency of spontaneous Ca²⁺ transients (3). Spontaneous Ca²⁺-oscillations exhibit a characteristic developmental profile that seems to be dependent on the development of the GABA_A receptor (2). In contrast to the adult brain where the GABA_A receptor is the major inhibitory receptor, in the developing hippocampus, the GABA_A receptor seems to be excitatory and GABA application depolarizes developing hippocampal pyramidal cells (20). At this stage, the application of GABA_A receptor antagonists led to a suppression of spontaneous Ca²⁺-oscillations (2). In contrast, in the present study, the application of the GABA_A receptor antagonist bicuculline resulted in an increase in the amplitude and a decrease in frequency which might indicate an inhibitory state of the GABA_A receptor. This is supported by similar results found in cortical neurons, where Ca²⁺-oscillations were induced in the absence of Mg²⁺. Bicuculline reduced the frequency but increased the amplitude of the Ca²⁺-oscillations. This increase was described as being consistent with a proposed role for inhibitory postsynaptic potentials in the termination of each individual calcium spike (18).

Ketamine, a phencyclidine derivate, is an IV anesthetic with a well defined effect on the NMDA receptor in clinical concentrations (9). It blocks the open channel by reducing channel mean open time and decreasing the frequency of channel opening by an allosteric mechanism in the open state (21). In the present study, ketamine reversibly suppressed the amplitude and frequency of the spontaneous Ca²⁺-oscillations. This effect was stereoselective, resulting in a potency ratio of R(–)/S(+) ketamine correlating especially with the clinical observations of a 3.4-fold larger potency of the S(+) compared with the R(–) ketamine regarding its anesthetic, a 4-fold larger potency regarding its analgesic, and a 4.8-fold larger potency regarding its amnesic effect (10,11). In examinations using electroencephalograph, the S(+) isomer suppressed electroencephalograph activity more potently than the R(–) ketamine (11,12). For ketamine concentrations up to 10 µM, using the whole cell patch clamp technique, a potency ratio on NMDA receptor currents between R(–) and S(+) ketamine of 1:1.9 was found in hippocampal neurons (13). There is no significant difference for both isomers for concentrations of 3 µM which parallels the plasma concentration for regaining consciousness (11). Tissue brain concentrations for ketamine are considered to be 6.5 times of the plasma concentration and therefore the significant differences for concentrations of 25 µM represent clinical relationships (22).

Besides the NMDA receptor, ketamine also affects other receptors including the glutamate receptors AMPA, kainate, the nicotinic or muscarinic acetylcholine, or the sigma opioid receptor at concentrations necessary for anesthesia (23,24). Except for the sigma opioid receptor, on which the R(–) ketamine acts more potently than the S(+) ketamine, there seems to be a lack of stereoselectivity of ketamine on the above-mentioned receptors in hippocampal neurons (25,26). Therefore, non-NMDA receptor suppression seems to be an unlikely explanation for the stereoselective effects. For concentrations exceeding the clinical range of 50–100 µM, additional receptors or channels might be involved in ketamine-induced depression of spontaneous Ca²⁺-oscillations. For example, ketamine might be an antagonist on the GABA_A receptor in supraclinical concentrations (27).

In conclusion, spontaneous Ca²⁺-oscillations in hippocampal neurons strongly depend on the main excitatory and inhibitory transmitter systems represented in neuronal networks. For the first time, we demonstrated that spontaneous Ca²⁺-oscillations were reversibly and stereospecifically attenuated by ketamine with potency ratios that were similar to the clinical ratios. Therefore, our experimental approach might represent a suitable model system for studying mechanisms of anesthetic action on neuronal receptor channels.

The authors thank Prof. Dr. A. Draguhn for helpful discussion of the manuscript.

	Footnotes

 
This study was supported by a grant of the University of Heidelberg: Juniorantrag 127/2000.

Accepted for publication November 1, 2004.

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