N-Methyl-D-Aspartate-Induced Oscillations in Whole Cell Clamped Neurons From the Isolated Spinal Cord of Xenopus laevis Embryos

L. Prime,1,2 Y. Pichon,2 and L. E. Moore1

 1Laboratoire de Neurobiologie des Réseaux Sensorimoteurs, Unité Propre de Recherche de l'Enseignement Supérieur Associée 7060 au Centre National de la Recherche Scientifique, Université de Paris V, VII, 75270 Paris Cedex; and  2Equipe Canaux et Récepteurs Membranaires, Unité Propre de Recherche de l'Enseignement Supérieur Associée 6026 au Centre National de la Recherche Scientifique, Université de Rennes 1, F-35042 Rennes Cedex, France


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Prime, L., Y. Pichon, and L. E. Moore. N-Methyl-D-Aspartate-Induced Oscillations in Whole Cell Clamped Neurons From the Isolated Spinal Cord of Xenopus laevis Embryos. J. Neurophysiol. 82: 1069-1073, 1999. The patch-clamp technique was used to measure the effect of N-methyl-D-aspartate (NMDA) on Xenopus embryonic neurons in an isolated, but intact spinal cord. Whole cell recordings were done at external calcium concentrations of 1 mM. NMDA alone (50-200 µM) or in association with 10 µM serotonin or glycine induced oscillatory activity in most presumed motoneurons, which were therefore considered part of rhythm generating networks. In the presence of TTX, one-half of these neurons maintained this activity. The oscillations fell into two main categories: voltage-dependent, low-frequency (0.3-0.5 Hz) and voltage-independent, high-frequency (3-8 Hz) oscillations. NMDA alone induced TTX-insensitive oscillations in one-third of the neurons; however, the percentage of neurons showing oscillations was greater in the presence of exogenous 5-hydroxytryptamine (5-HT) or glycine. Because these observations were made at embryonic stages where little or no serotonergic innervation exists, it is likely that NMDA-induced intrinsic oscillatory activity in Xenopus embryonic neurons does not require 5-HT.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Rhythmic activity in neural systems is associated with the activity of both conditional pacemaker neurons and oscillatory networks. In many cases, part of this oscillatory behavior is linked to the activation of one category of glutamate receptors: N-methyl-D-aspartate (NMDA) receptors. This is true for lower vertebrates such as the lamprey in which TTX-resistant membrane potential oscillations are induced in the spinal cord by NMDA (Wallen and Grillner 1987), as well as amphibian embryos and larvae in which the locomotor activity is associated with the activation of NMDA receptors of the spinal motoneurons (Dale and Roberts 1984; Reith and Sillar 1998; Roberts et al. 1995; Scrymgeour-Wedderburn et al. 1997; Sillar and Simmers 1994a,b; Soffe 1993, 1996) and for the CNS of mammals (Durand 1993; Hochman et al. 1994a,b; Kiehn et al. 1996; MacLean et al. 1997, 1998; Tell and Jean 1993; Vincent et al. 1996).


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Prehatching and hatching (32-37/38 stages of Nieuwkoop and Faber 1956) Xenopus laevis embryos were obtained from the local laboratory culture. The eggs, which were taken from adult females injected with human chorionic gonadotrophin (HCG), were fertilized with sperm and kept at room temperature (15-24°C). The embryos were anesthetized using 0.5 mg/ml tricaïne methane sulfate (MS222, Sigma). The CNS surrounded by myotomes was cut away using sharp needles and transferred to a solution of 0.5 mg/ml dispase (Sigma) in the external Ringer solution. The preparation was gently agitated for ~20 min and then thoroughly rinsed with enzyme-free saline. The spinal cord was dissected free and transected at the level of the otic capsule. This procedure removed the rostral part of the hindbrain where the raphe nuclei containing the serotonergic neurons are located (Reith and Sillar 1998; Sillar et al. 1992, 1995; Van Mier et al. 1986). The spinal cord preparation was then placed ventral side up in a continuously perfused 200-µl chamber.

The two ends of the preparations were fixed on the bottom of the dish using two glass suction electrodes that were used for extracellular recording of spontaneous activity. Neurons located in ventral quarters of the cord, presumed to be motoneurons, were selected for the experiments. Whole cell patch-clamp measurements (Hamill et al. 1981) were made on single neurons emerging from the main body of the cord (Desarmenien et al. 1993). The electrode resistance was 10-12 MOmega before the seal was made. White noise measurements (Moore et al. 1993) were used to estimate an electrode series resistance of 20-30 MOmega in the whole cell current clamp. All experiments were done with an Axoclamp-2A amplifier (Axon Instruments) in a constant current (bridge) mode. The signals were filtered at 2 kHz using a 24-dB/octave Flat delay (Linear Phase) filter (Rockland Systems) and digitized with a 12-bit A/D converter (Labmaster, Scientific Solutions). Data acquisition and some analysis was done with ACQUIS1 (Bio-Logic/CNRS License) software. In addition, Sigmaplot (Jandel Scientific) was used for statistical analysis and figure preparation.

The external Ringer solution contained (in mM) 110 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 2.4 NaHCO3, and 10 HEPES buffered at pH 7.4. The internal (pipette) solution contained (in mM) 90 K-gluconate, 10 KCl, 10 EGTA, 10 HEPES, 2 MgCl2, 3 ATP, and 0.5 GTP buffered at pH 7.3. NMDA (50-200 µM) and 1 µM TTX were added to the perfusing solution. In 50% of the experiments, 10 µM 5-hydroxytryptamine (5-HT) or 10 µM glycine was also added to the perfusing solution to stabilize the NMDA oscillations (Hochman et al. 1994b). It should be noted that the whole cell patch recording was done in Ringer solution containing a relatively normal calcium concentration of 1 mM, whereas most previous microelectrode measurements required high calcium levels of 4 mM.


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NMDA induced rhythmic activity in whole cell clamped neurons

The neurons had a mean resting potential of -53 ± 13 mV (mean ± SD, N = 38), and in most cases, clear excitatory synaptic potentials up to 10 mV were observed. Bath application of 50-200 µM NMDA, with or without exogenous glycine, caused a 10 to 30 mV depolarization of the membrane potential generally associated with an increase in synaptic noise and oscillatory spike activity. A hyperpolarizing current enhanced the membrane potential oscillatory response underlying the action potentials (Figs. 1A and 2A) in 30 of the 38 tested neurons. The means ± SD of the frequencies (Hz) and amplitudes (mV) of these NMDA-induced oscillations fell into two categories: slow oscillations (Fig. 1A), 0.5 ± 0.13 Hz (20.9 ± 10.1 mV, N = 3), 0.6 ± 0.3 Hz (26.7 ± 18.6 mV, N = 5) with glycine and 0.6 Hz (33.1 mV, N = 1) Hz with 5-HT; and fast oscillations (Fig. 2A), 3.9 ± 2.6 Hz (15.6 ± 6.0 mV, N = 9), 12.1 Hz (36.6 mV, N = 1) with glycine and 5.3 ± 3.9 Hz (20.8 ± 9.6 mV, N = 6) Hz with 5-HT, where N is the number of neurons in each category. As can be seen from this data, the rhythmic activity was present with (13 neurons) or without serotonin (5-HT) or glycine (12 neurons).



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Fig. 1. Slow oscillatory activity induced by N-methyl-D-aspartate (NMDA) in presumed motoneurons from an isolated spinal cord of a Xenopus embryo. A: slow (0.55 ± 0.05 Hz, n = 11) sinusoidal activity induced by 200 µM NMDA alone. A -80-pA current (as indicated) was used to hold the membrane potential to about -70 mV. Note the existence of a few truncated spikes on top of the (48.7 ± 2.9 mV, n = 11 peak-to-peak) depolarizations. B1 and B2: oscillatory activity in the presence of NMDA and tetrodotoxin (TTX); 1: beginning of NMDA application; 2: relatively stable activity after 22 s in NMDA (0.72 ± 0.4 Hz, 33.4 ± 4.2 mV, n = 15). A -5-pA current was continuously injected in this neuron. C: effects of NMDA, TTX, and glycine on another neuron. C1: the 1st part of the recording shows the electrical activity induced by 50 µM NMDA. At the arrow, the holding current was changed from -20 pA to -30 pA, and 1 µM TTX + 10 µM glycine were added to the bath. The activity was transformed into large-amplitude (50 ± 7.5 mV, n = 5) low-frequency (0.5 ± 0.05 Hz, n = 5) irregular oscillations that are illustrated with an expanded scale in C2. D1-D4: effects of current on the amplitude and frequency of the slow oscillatory activity (same neuron as in C1 and C2). D1: reduction of the injected current from -30 pA (as indicated) to -20 pA, reversibly inhibited the oscillations (interval between the 2 arrows) resulting in a constant membrane potential at its plateau value of -20 mV. D2: increase of the current to -40 pA hyperpolarized the cell by ~15 mV to -75 mV, increased the amplitude of the oscillations to 55 mV, and decreased their frequency. D3: a transient increase of the injected current from -40 pA (as indicated) to -55 pA reversibly inhibited the activity (interval between the 2 arrows). D4: plot of the relationship between the intensity of the injected current and the frequencies and amplitudes of the oscillations (mean values) for the same neuron. Means ± SD for the frequencies and amplitudes are given for the number, n, of oscillations used.



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Fig. 2. Fast oscillatory activity induced by NMDA in a (presumed) motoneuron of an isolated spinal cord of a Xenopus embryo. A: effects of TTX (1 µM) and 5-hydroxytryptamine (5-HT; 10 µM) on the electrical activity of the neuron previously perfused with 50 µM NMDA with a holding current of -100 pA. The action potentials were abolished, and the fast oscillatory activity decreased from 13.1 ± 2.6 (n = 66) to 7.8 ± 1.7 Hz (n = 38). B: changes of the injected current (as indicated on the bottom trace) failed to modify the frequency of the oscillations, whereas their amplitude and the time course were altered. The amplitude increased with membrane depolarization, which was associated with the appearance of spikelike events (see C and D). E: the oscillations progressively disappeared, and the membrane repolarized following return to a normal drug-free bathing solution. F: plot of the relationship between the intensity of the injected current and the frequencies and amplitudes of the oscillations (mean values) for the same neuron.

NMDA-induced oscillations in the presence of TTX

In the presence of TTX, NMDA-induced oscillations were observed in 5 of 14 motoneurons, three with slow (Fig. 1, B1 and B2) 0.6 ± 0.2 Hz (26.9 ± 5.8 mV, N = 3) and two with fast 2.3 ± 0.7 Hz (14.8 ± 1.9 mV, N = 2) oscillations. The mean holding potential in these experiments with or without TTX was -60 to -50 mV. The observation of oscillations in TTX required a bias current of -5 to -50 pA. Serotonin in combination with NMDA led to four of seven neurons showing TTX-resistant oscillations (Fig. 2), namely, 0.6 Hz (31.5 mV, N = 1) and 4.6 ± 3.1 Hz (20.6 ± 5.4 mV, N = 3). Similarly, in glycine with NMDA (Fig. 1, C1, C2, and D1-D3), four of six cells had oscillatory activity in TTX: 0.4 ± 0.1 Hz (23.2 ± 10.1 mV, N = 3) and 7.5 Hz (10.2 mV, N = 1). The finding that higher ratios of neurons showed NMDA-induced oscillations with the addition of serotonin (4/7) or glycine (4/6) compared with NMDA alone (5/14) is consistent with the hypothesis that these two amino acids enhance NMDA-induced intrinsic oscillatory behavior. The maintenance of the fast oscillations in Fig. 2, A and B, after TTX was added, suggests that although the synaptic input contributes to the oscillatory activity, the rapid sinusoidal form is due to the activation of intrinsic TTX-insensitive receptors.

Voltage dependency of the oscillations in TTX

The effects of membrane potential on the properties of the two categories of oscillations were studied by injecting current through the patch electrode. The voltage dependency of the two categories of oscillations was clearly different. The frequency of the slower patterned activity was sensitive to changes in the membrane voltage and was similar to that observed in other preparations (Chub et al. 1997; Wallen and Grillner 1987). The oscillations, illustrated in Fig. 1, D1-D3, were characterized by plateau potentials around -20 mV that had variable durations and abrupt terminations. The frequency and plateau duration of these oscillations decreased with membrane hyperpolarization, whereas their amplitude increased (Fig. 1D4).

By contrast, the frequency of the fast oscillations was not modified by changes in membrane potential, but their amplitude was found to decrease with membrane hyperpolarization (Fig. 2F). This change was associated with a modification of the time course of the oscillations as shown on Fig. 2, C and D. The more hyperpolarized responses (Fig. 2C) were nearly sinusoidal; however, at depolarized potentials, a 20 to 30 mV abortive spike appeared on the rising phase of the oscillation (Fig. 2D). These findings suggest that increased amplitude is related to the turning on of voltage-dependent conductances that are activated in concert with the direct NMDA effects.


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These experiments clearly show that in the presence of TTX, NMDA (50-200 µM) together with a negative current bias can induce oscillatory behavior in whole cell clamped (presumed) motoneurons of the spinal cords of Xenopus embryos at stages 32-37/38. This is in contrast to the slow (sinusoidal) pattern of activity observed by Scrymgeour-Wedderburn et al. (1997) in postembryonic and larval neurons of the same preparation, which required exogenous 5-HT and NMDA. At least two differences in the experimental procedures could be used to explain why 5-HT was needed in their experiments and not in the present ones: 1) the internal milieu of the cell was not the same since, in patch-clamped neurons, the interior of the cell was perfused with the pipette solution, and 2) the external calcium concentration was 1 mM in the patch-clamp experiments compared with 4 mM in the intact preparation. Because oscillations depend on a complex balance between multiple conductances, it is not surprising that changes in both external and interior environments would alter this behavior.

We cannot completely exclude the presence of endogenous serotonin playing some role in NMDA-induced oscillations, nevertheless the levels must be relatively low because our isolated spinal cord was continuously perfused, and the serotonergic neurons of the brain stem were not present. It has been suggested that 5-HT may have a direct agonist effect on the NMDA receptor (Scrymgeour-Wedderburn et al. 1997), however, its influence on fictive swimming at stage 37/38 (Sillar et al. 1992) could be caused by other mechanisms, such as an alteration in a potassium conductance. Although 5-HT may not be obligatory for the activation of the NMDA receptor, both the microelectrode and whole cell clamp measurements are consistent with a modulatory effect of 5HT on NMDA-induced oscillations.

Soffe (1996) reported that rapid rhythmic behavior, similar to that induced by NMDA in whole cell clamped neurons, was induced by non-NMDA agonists (alpha -amino-3-hydroxy-5methyl-4-isoxazolepropionic acid and kainate), but not NMDA. Some of the discrepancies between our observations and those of Soffe (1993, 1996), as well as Scrymgeour-Wedderburn et al. (1997), could also be reasonably well accounted for by differences in the techniques used to record the electrical activity of the neurons. Thus the significantly (~5 times) higher impedance of the patch-clamped neurons (601 ± 98.7 MOmega , n = 27) as compared with the microelectrode-impaled neurons (118 ± 17 MOmega ) (Soffe 1990) is indicative of a smaller leak that could theoretically facilitate oscillatory activity.

Our slow voltage-dependent oscillations resemble the NMDA-induced inherent oscillatory activity associated with fictive locomotion in the lamprey (Wallen and Grillner 1985, 1987). NMDA-induced plateau potentials similar to those recorded in the present experiments have been seen in a variety of preparations such as the interneurons of the spinal cord of neonatal rats (Kiehn et al. 1996), cat neocortical neurons (Flatman et al. 1983, 1986), or isolated turtle spinal cords (Guertin and Hounsgaard 1998).

Besides the fact that 5-HT was needed in their experiments to obtain the activity, the frequency of the oscillations recorded in larval neurons by Scrymgeour-Wedderburn et al. (1997) and Reith and Sillar (1998) was voltage independent, whereas it is strongly voltage dependent in one of our two types of oscillations. Electrical coupling between the motoneurons in intact Xenopus embryos (Perrins and Roberts 1995) has been proposed by Scrymgeour-Wedderburn et al. (1997) as an explanation for the voltage independence of the NMDA-induced oscillations in TTX of intact Xenopus larvae. It is plausible that the experimental procedure used to isolate the spinal cord in our experiments, which requires enzymatic treatment, partially disrupts this coupling between the neurons, as well as synaptic connections. This hypothesis is also consistent with the observation of slow rhythmic oscillations with action potentials in Fig. 1A1 that resemble those observed by Reith and Sillar (1998) on Xenopus larvae when the inhibitory synapses had been blocked by strychnine and bicuculline.

The fast oscillatory activity seen with or without TTX resembles in some respects the "struggling" activity induced by 1 mM kynurenic acid (Soffe 1993) in stage 37-38 Xenopus embryos. Although some of the rapid behavior is a consequence of network connections, our experiments clearly indicate that the fast oscillations are also part of the intrinsic oscillatory properties of some neurons. TTX-resistant voltage-independent fast oscillatory activity, very similar to that observed in our experiments, has been recorded with the patch-clamp technique in neonatal rat lumbar motoneurons in vitro by MacLean et al. (1997, 1998). The mean frequency of these oscillations (~ 4.5 Hz) falls precisely in the range of frequencies observed in our experiments (3-8 Hz).

The ionic basis of the slow oscillatory activity is most probably the same as in the other preparations exhibiting a similar activity, i.e., the alternation of an inward (depolarizing) current corresponding to the opening of nonselective NMDA-activated channels followed by an outward potassium current corresponding to the opening of calcium-activated potassium channels as described by Wallen and Grillner (1987) (see also Moore and Buchanan 1993; Moore et al. 1993). The ionic basis of the fast oscillations may simply be a continuum that includes the voltage-dependent ionic channels, especially calcium (Gu and Spitzer 1993) and those of the delayed rectifier potassium currents (Dale 1995). The increase in amplitude of these oscillations with membrane depolarization might correspond to the activation of high-threshold channels, which have been observed in isolated Xenopus neurons (Dale 1995).

The physiological relevance of these intrinsic oscillatory properties remains speculative. It is reasonable, however, to suggest that the low-frequency oscillations might control the frequency of the swimming episodes observed in Xenopus larvae as proposed by Reith and Sillar (1998) and to assume that the high-frequency oscillations are related to the struggling behavior of the embryo.


    FOOTNOTES

Address for reprint requests: L. E. Moore, UPRESA-7060, Centre National de la Recherche Scientifique, Université Paris 5-7, 45 rue des Saint Pères, F-75270 Paris Cedex 06, France.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 December 1998; accepted in final form 5 April 1999.


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