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.
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.
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.
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[β-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 (Kd) of fura-2 could be determined and the free [Ca2+]i concentration was calculated from fura-2 fluorescence ratios (15,16)
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).
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 Kd 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).
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).
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 α-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.
γ-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 GABAA receptor antagonist bicuculline (n = 4). Blocking the GABAA 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).
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).
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.
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 GABAA receptor (2). In contrast to the adult brain where the GABAA receptor is the major inhibitory receptor, in the developing hippocampus, the GABAA receptor seems to be excitatory and GABA application depolarizes developing hippocampal pyramidal cells (20). At this stage, the application of GABAA receptor antagonists led to a suppression of spontaneous Ca2+-oscillations (2). In contrast, in the present study, the application of the GABAA receptor antagonist bicuculline resulted in an increase in the amplitude and a decrease in frequency which might indicate an inhibitory state of the GABAA 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 GABAA 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.
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