Local facilitation of plateau potentials in dendrites of turtle motoneurones by synaptic activation of metabotropic receptors

Abstract

1. The spatial distribution of synaptic facilitation of plateau potentials in dendrites of motoneurones was investigated in transverse sections of the spinal cord of the turtle using differential polarization by applied electric fields.

2. The excitability of motoneurones in response to depolarizing current pulses was increased following brief activation of either the dorsolateral funiculus (DLF) or the medial funiculus (MF) even when synaptic potentials were eliminated by antagonists of ionotropic receptors.

3. The medial and lateral compartments of motoneurones were differentially polarized by the electric field generated by passing current between two electrodes on either side of the preparation. In one direction of the field lateral dendrites were depolarized while the cell body and medial dendrites were hyperpolarized (S_- configuration). With current in the opposite direction the cell body and medial dendrites were depolarized while lateral dendrites were hyperpolarized (S₊ configuration).

4. Following brief activation of the DLF the excitability and the generation of plateau potentials were facilitated during differential depolarization of the lateral dendrites but not during differential depolarization of the cell body and medial dendrites. Following brief activation of the MF the excitability and generation of plateau potentials were facilitated during differential depolarization of the cell body and medial dendrites but not during differential depolarization of the lateral dendrites.

5. It is concluded that the synaptic facilitation of the dihydropyridine-sensitive response to depolarization is compartmentalized in turtle motoneurones.

Several types of neurones in the central nervous system of vertebrates have a non-homogeneous distribution of voltage-sensitive ion channels in the soma-dendritic membrane (Llinas & Hess, 1976; Chan et al. 1988; Schiller et al. 1997; Wolff et al. 1998). This gives rise to highly compartmentalized changes in concentration of permeant ions during electrical activity. For biologically active ions such as Ca²⁺ the spatial distribution of concentration changes may be of functional significance. In turtle motoneurones it was previously shown that plateau potentials mediated by L-type Ca²⁺ channels can be activated in distal dendrites by differential polarization (Hounsgaard & Kiehn, 1993). In these neurones the responses mediated by L-type Ca²⁺ channels are regulated by activation of certain metabotropic receptors (Svirskis & Hounsgaard, 1998). These receptors can be activated synaptically (Delgado-Lezama et al. 1997). This shows that the magnitude of Ca²⁺ influx in turtle motoneurones is regulated by presynaptic activity. In the present study we use differential polarization to show that activation of afferent tracts in the lateral spinal cord preferentially facilitates generation of plateau potentials in lateral dendrites while activation of medial tracts preferentially facilitates the generation of plateau potentials in medial dendrites. It is concluded that the spatial distribution of Ca²⁺ influx through L-type Ca²⁺ channels or the response that this influx gives rise to is regulated by the regional level of activation of metabotropic receptors.

METHODS

Transverse slices (2-3 mm thick) were obtained from the lumbar enlargement of adult turtles (Pseudemys scripta elegans) deeply anaesthetized with pentobarbitone (100 mg kg⁻¹) and subsequently killed by decapitation. The surgical procedures comply with Danish legislation and are approved by the controlling body under The Ministry of Justice. Experiments were performed at room temperature (20-22°C) in a solution containing (mM): 120 NaCl, 5 KCl, 15 NaHCO₃, 2 MgCl₂, 3 CaCl₂ and 20 glucose, saturated with 98 % O₂ and 2 % CO₂ to obtain pH 7.5.

Intracellular recordings in current clamp mode were performed with an Axoclamp-2A amplifier. Pipettes (50-60 MΩ) were filled with a solution containing 1 M potassium acetate. Recordings from 14 motoneurones with a stable membrane potential of more than -60 mV were included in this study. Trains of 40 constant current pulses (0.2 ms duration) at 20 Hz were applied on the dorsolateral funiculus (DLF) and the medial funiculus (MF) by bipolar wire electrodes.

Field stimulation

A slice was placed in the recording chamber between silver electrodes as described before (Hounsgaard & Kiehn, 1993; Svirskis & Hounsgaard, 1997). The electric field was applied in the medio-lateral direction of motoneuronal dendrites (Fig. 1A, scheme). With this orientation of the electric fields maximal polarization is obtained in the distal compartments of lateral and medial dendrites while an indifference point with no change in membrane potential is located proximally in lateral dendrites (Baginskas et al. 1993; Hounsgaard & Kiehn 1993; Svirskis et al. 1997a,b; Svirskis & Hounsgaard, 1998). With the cathode lateral to the ventral horn recorded from, the lateral dendrites are depolarized while the medial dendrites and the soma are hyperpolarized. This configuration is termed a soma-hyperpolarizing field (S_-) (Fig. 1A, scheme). The field induced by current in the opposite direction hyperpolarizes lateral dendrites while depolarizing medial dendrites and the soma. This is termed a soma-depolarizing field (S ₊) (Fig. 2A, scheme). When the recording electrode was withdrawn from a motoneurone the extracellular potentials induced by the fields applied during intracellular recording were measured. Off-line the transmembrane potential was obtained as the difference between intra- and extracellular potentials for each intensity and polarity of the electric field applied.

Figure 1. Selective facilitation of plateau potentials in lateral dendrites.

A, left: scheme showing the spinal cord slice between the two silver electrodes used to apply electric fields. The slice was oriented so that lateral dendrites were polarized with opposite polarity of soma and medial dendrites. The dark-grey area was stimulated with a bipolar electrode. Right, with the field electrode lateral to the horn recorded from as cathode, the lateral dendrites were depolarized while the medial dendrites and the soma were hyperpolarized (S_-) by an applied field. B, DLF stimulation (horizontal line) facilitates subthreshold depolarizing response to intracellular current pulse in motoneurone. C, DLF stimulation facilitates response to differential depolarization of lateral dendrites with a soma-hyperpolarizing field (S_-). D, DLF stimulation without effect on the response to differential hyperpolarization of lateral dendrites by a soma-depolarizing field (S ₊). E, facilitation of response to S_- field induced by DLF stimulation was blocked when nifedipine (10 μM) was added to the bath solution. All the records in Figs 1 and 2 were obtained from the same motoneurone. The medium contained CNQX, APV and strychnine. Mn, motoneurone; IC, intracellular current.

Figure 2. Selective facilitation of plateau potentials in medial dendrites.

A, left: scheme showing a spinal cord slice between the two silver electrodes used to apply electric fields. The slice was oriented so that lateral dendrites were polarized with opposite polarity of soma and medial dendrites. The dark-grey area was stimulated with a bipolar electrode. Right, with the field electrode lateral to the horn recorded from as anode, the medial dendrites and the soma were depolarized while the lateral dendrites were hyperpolarized (S_-) by an applied field. B, MF stimulation (horizontal line) facilitates subthreshold depolarizing response to intracellular current pulse in motoneurone. C, MF stimulation facilitates response to differential depolarization of the soma and medial dendrites with a soma-depolarizing field (S ₊). Arrows indicate the subthreshold activation of a plateau potential during and after the facilitated response to the field stimulus. D, MF stimulation without effect on the response to differential hyperpolarization of soma and medial dendrites by a soma-hyperpolarizing field (S_-). E, facilitation of response to S ₊ field induced by MF stimulation was blocked when nifedipine (10 μM) was added to the bath solution. The medium contained CNQX, APV and strychnine.

Experimental procedure

Intracellular recordings were obtained from motoneurones deeper than 100 μm from the surface of the slice. Cells were selected for experiments if the monosynaptic EPSPs evoked by a single stimulus at supramaximal intensity in the DLF and in the MF had amplitudes of more than 5 mV. In such cells experiments proceeded in medium containing antagonists of ionotropic receptors for glutamate and glycine (20 μM CNQX, 50 μM AP-5 and 1-10 μM strychnine). Under these conditions spontaneous and evoked synaptic potentials were not observed after 15 min.

The response to a depolarization evoked by current pulse applied through the recording electrode or an applied field served to monitor the excitability before and after a stimulus train in either the DLF or the MF. The stimulus intensity in the DLF and MF was adjusted to give a maximal synaptic response in normal medium.

The data were sampled at 16.6 kHz (current clamp recordings) with a 12-bit analog-to-digital converter (DIGIDATA 1200, Axon Instuments) and displayed by means of Axoscope software and stored on a hard disk for later analysis.

Drugs used

Synaptic potentials were eliminated by adding the following drugs to normal medium: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 μM; RBI), (±)-2-amino-5-phosphonopentanoic acid (AP-5; 50 μM; RBI), strychnine (1-10 μM; Sigma). In five experiments nifedipine (10 μM) was added to the bath. In 10 additional experiments one of the following antagonists of metabotropic receptors was added to the medium: pindobind-5HT_1A (5HT_1A receptor antagonist; 10 μM; RBI), (S)-α-methyl-4-carboxyphenylglycine (MCPG; metabotropic glutamate receptor antagonist; 1 mM; Tocris) and atropine (muscarinic receptor antagonist; 0.1 μM; Sigma).

RESULTS

DLF or MF stimulation facilitate excitability of motoneurones

As in a previous study (Delgado-Lezama et al. 1997) a brief train of stimuli in the DLF was followed by a period of increased excitability in motoneurones even in the absence of ionotropic synaptic transmission. In Fig. 1B the subthreshold response to a depolarizing current pulse applied through the recording electrode was converted to a suprathreshold response when the same pulse was applied after the stimulus train in the DLF. In the same cell brief activation of the MF also induced facilitation (Fig. 2B). In all motoneurones tested the excitability was increased after brief activation of both DLF and MF (n = 8). These results show that the excitability of motoneurones is facilitated by activation of afferents in the medial and lateral funiculus. The previous study found that the increased excitability following DLF stimulation was not associated with changes in input resistance but was abolished by antagonists of metabotropic receptors for acetylcholine, serotonin and glutamate (Delgado-Lezama et al. 1997). The present study confirmed that facilitation induced by MF stimulation also occurred without changes in input resistance (n = 6) and was reduced by antagonizing muscarine receptors (0.1 μM atropine, in 3 out of 3 experiments) and metabotropic glutamate receptors (0.5 mM MCPG, in 3 out of 3 experiments). However, blocking 5HT_1A receptors (10 μM pindobind-5HT_1A) reduced facilitation in only 1 out of 4 experiments. This is in agreement with a predominant lateral location of serotonergic tracts in the spinal cord of the turtle (Kiehn et al. 1992).

The findings suggest that facilitation by stimulation of MF and DLF is mediated by the same classes of transmitters and receptors with serotonergic effects mainly in lateral dendrites.

DLF and MF stimulation facilitate different dendritic compartments

The compartmental distribution of the facilitation induced by DLF and MF activation was investigated using differential polarization by an applied electric field as the test stimulus. Lateral dendrites were differentially depolarized by a field in the soma-hyperpolarizing direction. The cell body and medial dendrites were differentially depolarized by a field with the opposite polarity. We found that DLF activation and MF activation facilitated the response in motoneurones to applied fields of opposite polarity.

As illustrated in Fig. 1C brief activation of the DLF converted a subthreshold response to a soma-hyperpolarizing electric field into a suprathreshold response. In contrast the response to a soma-depolarizing field was unaffected by DLF stimulation (Fig. 1D). Brief activation of the MF, however, converted the subthreshold response to a soma-depolarizing field into a suprathreshold response (Fig. 2C). MF activation had no effect on the response to a soma-hyperpolarizing electric field (Fig. 2D). Similar results were obtained in all cells tested with differential polarization. This shows that DLF activation facilitates the response to depolarization of lateral dendrites (n = 14) while MF activation facilitates the response to depolarization of cell body and medial dendrites (n = 8).

Facilitation mediated by L-type Ca²⁺ channels

Previous studies have shown that the facilitated response in motoneurones following DLF stimulation is mediated by L-type Ca²⁺ channels (Delgado-Lezama et al. 1997). In the present experiments the facilitated response to differential depolarization of lateral dendrites following DLF activation and the facilitated response to differential depolarization of the cell body and medial dendrites following MF activation were blocked by 10 μM nifedipine (Figs 1E and 2E). Facilitation of the response to differential depolarization induced by DLF and MF stimulation was blocked in all six cells tested. These results show that the synaptically facilitated response in confined regions of dendrites in motoneurones depends on activation of dihydropyridine-sensitive Ca²⁺ channels.

DISCUSSION

Metabotropic synaptic regulation of L-type Ca²⁺ channels has been suggested to provide a mechanism for changing the intrinsic response properties in individual motoneurones and the order of recruitment in a population of motoneurones (Delgado-Lezama et al. 1997; Svirskis & Hounsgaard, 1998). The present findings add the possibility that metabotropic transmission also regulates the spatial distribution of Ca²⁺ influx through L-type Ca²⁺ channels in motoneurones.

In the turtle spinal cord, block of L-type Ca²⁺ channels does not affect synaptic transmission (Russo & Hounsgaard, 1994; Delgado-Lezama et al. 1997). The present experiments support the conclusion that the L-type Ca²⁺ channels responsible for plateau potentials are present in dendrites of turtle motoneurones (Hounsgaard & Kiehn, 1993; Svirskis & Hounsgaard, 1997). Plateau potentials evoked in cat motoneurones in vivo were also suggested to be of dendritic origin (Lee & Heckman, 1996). This is at odds with recent immunohistochemical evidence that the α₁ subunit of L-channels is predominantly located over the cell body and proximal dendrites in adult rats (Westenbroek et al. 1998). It is not known if the discrepancy is due to a difference in distribution of functional channels and channel subunits or is a species difference. Although metabotropic regulation of L-channels is the simplest explanation for our findings we have not excluded the possibility that hypothetical Ca²⁺-dependent inward currents are the targets for facilitation (Svirskis & Hounsgaard, 1997, 1998).

Since turtle motoneurones are electrotonically compact (Svirskis et al. 1997a,b; Svirskis & Hounsgaard, 1997) the channel distribution may not be a major determinant for the intrinsic electrophysiological properties. On the other hand the distribution of the changes in intracellular Ca²⁺ concentration as a result of electrophysiological activity may be functionally essential. Many subcellular neuronal processes may require highly localized Ca²⁺ signalling (Llinas et al. 1992). In motoneurones such signals may mediate synaptically controlled focal regulation of synaptic strength and local composition of the membrane.