barium-induced bistability in rat ocular motoneurones in vitro

E L S E V I E R Neuroscience Letters 170 (1994) 158-162

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Barium-induced bistability in rat ocular motoneurones in vitro

Jean-Patrick Gueritaud*

Unitd de NeuroeybernOtique Cellulaire, CNRS UPR-418, 280 Boulevard Sainte-Marguerite, 13009 Marseille, France

Received 23 December 1993; Revised version received 2 February 1994; Accepted 2 February 1994

Abstract IntraceUular recordings were obtained from adult rat oculomotor neurones in an in vitro brainstem slice preparation. In

motoneurones hyperpolarized to levels below -90 mV, depolarizing current injections induced voltage-dependent low-threshold plateau potentials. These potentials were triggered at rest when charge transfer through Ca 2÷ channels was increased by Ba 2+ substitution. In such conditions, the membrane displayed voltage-dependent bistable properties similar to those described in other preparations and shown in vivo to be under neurotransmitter control. These results show that increasing inward currents in adult oculomotor neurones of the rat induce functionnally different responses to slight shifts of membrane potential.

Key words: Adult motoneurone; Ocular motoneurone; Plateau potential; Low-voltage-activated potential; Hyperpolarization; Bista- ble firing; Slow inward current; Barium

Motor activity is not only shaped at premotor levels but depends also on intrinsic properties of the motoneurone membrane resulting in non-linear integration of synaptic inputs. The description in vivo of plateau potentials [19] and bistable properties in cat spinal [10] and rat brainstem [5] motoneurones induced by serotonin (5- HT) [11] or N-methyl-D-aspartate (NMDA) [5,6] is of particular relevance since such properties imply that the motoneurone may switch under synaptic control between two functional states and thus reacts in very different ways to the same synaptic input. This may be partic- ularly important in the case of motoneurones involved in typical rhythmic motor patterns such as nystagmus.

In vitro techniques allowed for a detailed analysis of motoneurone membrane electroresponsiveness and pre- cise description of ionic conductances involved [7,8,17,18,22-25]. Plateau potentials and bistability were observed in vitro in spinal motoneurones of the turtle [12] and hypoglossal motoneurones of the rat [17] and shown to be intrinsic membrane properties depending on the uncovering of a persistent or slowly inactivating inward current carried by Ca 2+ or Na ÷ ions which is nor-

* With the technical collaboration of N. Seyfritz.

0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)001 14-P

mally curtailed by outward currents. C a 2÷ currents are present in mammalian spinal motoneurones [9,24] and 5-HT enhances a low-voltage-activated Ca 2+ current in rat spinal motoneurones [3].

We show in this report that increased inward current through Ca 2+ channels may also induce bistable properties in oculomotor neurones of the adult rat in vitro.

Full details of experimental procedures were previ- ously described [8]. Briefly, adult male Wistar rats (80-150 g) were deeply anaesthetized with ether and de- capitated. Brainstem parasagittal slices, 400/.tm thick. containing the oculomotor nuclei were obtained and transferred to incubation vials at 29°C in oxygenated Ringer. 30 min before a recording session, a slice was transferred to an interface recording chamber kept at 37°C and continuously perfused with Ringer solution at a rate of 1 ml/mn. The modified Krebs-Ringer solution (pH 7.35) consisted of (in raM): NaC1, 124: KC1, 5: CaC12, 2.5; KH2PO4, 1.20: MgSO4, 1.30: glucose. 10: NaHCO3, 24 continuously bubbled with a mixture of O2:95%, CO2:5%. Ba 2+ (2 mM) replaced Ca 2+ on an equimolar basis.

Intracellular recordings were obtained from ocular motoneurones (OMN) identified by their antidromic response to IIIrd nerve fibers stimulation and their pattern

J-P Gueritaud/Neuroscience Letters 170 (1994) 158 162 159

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Fig. 1. A: response of an hyperpolarized motoneurone to a 0.2-nA depolarizing current pulse. Inset shows response ofmotoneurone to same stimulus at normal resting potential (indicated by dashed line). B: subthreshold response of same hyperpolarized motoneurone. C~ C,: response of a motoneurone after substituting Ba 2+ for Ca -,+ in bathing solution, to a depolarizing current pulse applied at rest (C 0 and after shifting membrane potential 10 mV positive with 0.2-nA current injection. D~ 04: modification of response to intracellular injection of a 0.4-hA current pulse after substituting Ba2+for Ca 2+ in bathing solution. D~: control response; D2:2 min after substitution; D3:6 min after substitution; D4: spontaneous activity recorded 14 min after substitution. Voltage calibration in Dt applies to all traces. Time calibration in C2 applies also to D~ D 3.

o f response to injected currents [8]. Microelectrodes were filled with 3 M KC1 or 2 M K-citrate and had a resistance o f 50 -80 and 70 -100 Mg2, respectively. Convent iona l intracellular recording techniques were used, employ ing bridge balance and current injection facilities. Intracellu- lar potentials were recorded on a chart recorder, s tored on magnet ic tape or digitized for computer storage.

The data base for this report was obtained from 12 identified O M N , which could be recorded in stable con- dit ions after Ba 2+ subst i tut ion for up to 90 min (n = 2), and three O M N , which were polarized to levels below - 9 5 m V in standard Ringer. Al l m o t o n e u r o n e s had an initial membrane potential in the range o f - 5 0 to - 8 5 mV, with overshoot ing spikes.

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Fig. 2. Bistable behaviour of an OMN 27 rain after substituting Ba 2÷ for Ca 2÷ in bathing solution. A: at resting potential, 0.2-nA current pulses trigger short plateau potentials. B: a 7-mV depolarization of motoneurone by injection of a 0.2-nA positive current generates a bistable behaviour. Plateau potentials are triggered by 0.2-nA short depolarizing current pulses. C: when stimulations are discontinued, membrane potential jumps spontaneously between an active and an inactive level. D: a 0.2-nA hyperpolarizing current pulse resets membrane potential to inactive state (note that preceding 0.15-nA pulse is insufficient to reset membrane potential). E: a slight hyperpolarization decreases frequency of spontaneous jumps. Time calibration: 1 (A,D) and 5 s (B,C,E).

Lowering the membrane potential below - 9 0 mV drastically modified the discharge pattern. The O M N of Fig. 1A had a resting membrane potential at penetration o f - 6 7 mV and displayed typical repetitive firing with progressive frequency adaptat ion and post-burst hyperpolarization in response to intracellular current stimulation (inset). When the membrane potential was polarized to -104 mV, the normal pattern of response to depolarizing pulses was replaced by a voltage-dependent low- threshold plateau potential with an overriding burst o f action potentials (Fig. 1A). Upon cessation of current injection, the plateau potential terminated and the membrane repolarized slowly without post-burst hyperpolarization. When the stimulating current was slightly re-

duced, the plateau potential could be seen in isolation (Fig. 1B). These potentials were only triggered f rom hyperpolarized levels and were also observed as rebound potentials.

When Ca 2+ was partially replaced by Ba 2+ in the recording medium, the voltage-dependent regenerative plateau potentials were enhanced and could be triggered from rest (Fig. 1CI). Further depolarization of the membrane to 12 mV above rest increased the duration of the plateau potentials which outlasted the stimulus duration (Fig. 1C2).

ExtraceUular Ba 2+ application on motoneurones in vivo [20] was shown to depress the fast outward current carried by K + and to enhance the inward current/1 pre-

J-R Gueritaud/Neuroscience Letters 170 (1994) 158 162 161

sumably carried at least partially by Ca 2+, Ba 2+ being a better charge carrier through Ca 2+ channels. The combi- nation of these two effects would clearly unbalance the transmembrane currents. The changes in discharge pattern due to the progressive onset of this inward current are shown on Fig. I D. 2 min after switching to Ba 2+- Ringer, the long-lasting AHP was suppressed and the firing frequency increased. The post-burst AHP was modified (Fig. ID2). 6 min after Ba 2+ substitution, the regular firing was progressively replaced by spike doublets triggered on top of a large regenerative potential followed by a large AHP (Fig. 1D3). These regenerative depolarizations and associated doublets of spikes were first obtained with intracellular injection of current pulses or as rebound depolarizations, After 14 min, the neurone developed a pacemaker-like discharge pattern (Fig. 1D4).

In three cases, OMN maintained in Bae+-Ringer for > 20 min developed typical bistable properties (Fig. 2). The membrane potential displayed abrupt jumps between a polarized silent level at -70 mV and a depolar- ized active state at -55 inV. The pattern of firing was closely dependent on the resting potential. At -70 mV, plateau potentials outlasting the stimulus duration were triggered by small depolarizing current pulses (Fig. 2A). When membrane potential was shifted to -63 mV, the duration of plateau potentials was largely increased (Fig. 2B). The plateau potentials could be terminated by hyperpolarizing current pulses of sufficient intensity (Fig. 2D). At this resting potential, the membrane tended to depolarize slowly and jump to active level and then re- turn to silent level in a bistable way {Fig. 2C).

Membrane depolarization increased the duration of each burst until continuous firing was eventually observed. On the contrary, the frequency and duration of bursts were decreased by a 2-mV hyperpolarization and bursts were suppressed when the membrane was hyperpolarized to -75 mV by injection o f - 0 . 2 nA current. These results show that voltage-dependent low-threshold plateau potentials are obtained in OMN after hyperpolarization 10-15 mV below rest or after Ba 2+ substitution for Ca -'+ and are very similar to low-threshold potentials described in other mammalian neurones [15] which were shown to be dependent on a slow inward current carried by Ca -̀+ [2,4,13,25] or Na + [1,16 18,21].

With time Ba > tended to induce pacemaker properties and bistable firing. Such behaviour has been shown to be associated with a region of negative slope conduct- ance in the steady-state L Vcurve resulting from a persistent net-inward slow current carried by Ca 2+ and/or Na + [19]. In non-pacemaker cells, this current is normally curtailed by an outward current and it can only be dem- onstrated in conditions where the balance between inward and outward current is modified such as Ba 2+ substitution [20].

The triggering of low-threshold potentials in hyperpo-

larized OMN and the induction of pacemaker-like properties and bistable firing by Ba 2÷ application would, thus, tend to demonstrate the existence of a slow non-inactivating inward current in the membrane of the OMN of the adult rat.

Bistable firing was induced in vivo in hyperpolarized Abducens motoneurones by iontophoretic application of N MD A [5], suggesting that this behaviour is under synaptic control. In decerebrate cats, plateau potentials and bistable firing were shown to be dependent on serotonin- ergic and noradrenergic descending fibers (see Ref. 14 for review) and bistable firing was also induced by N M D A iontophoresis [6]. 5-HT-dependent bistable firing was described in vitro in turtle motoneurones [14] and 5-HT was shown to enhance a low-voltage-activated Ca -'+ current in rat spinal motoneurones [3]. Our results show that the ability to generate plateau potentials and bistable firing is an intrinsic property of the membrane of adult OMN depending on the enhancement o fa transmembrane current which enables them to switch between different integrative properties.

1 thank S. Tyc-Dumont and J. Durand for valuable comments on the manuscript. This work was supported by a grant from Fondation pour la Recherche Mddicale.

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barium-induced bistability in rat ocular motoneurones in vitro

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