Synchronized Oscillations in the Inferior Olive Are Controlled by the Hyperpolarization-Activated Cation Current Ih
Thierry Bal1 and
David A. McCormick2
1 Institut Alfred Fessard, Centre National de la Recherche Scientifique, Gif sur Yvette, Cedex 91198, France; and 2 Section of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06473
 |
ABSTRACT |
Bal, Thierry and David A. McCormick. Synchronized oscillations in the inferior olive are controlled by the hyperpolarization-activated cation current Ih. J. Neurophysiol. 77: 3145-3156, 1997. The participation of a hyperpolarization-activated cationic current in the generation of oscillations in single inferior olive neurons and in the generation of ensemble oscillations in the inferior olive nucleus (IO) of the guinea pig and ferret was investigated in slices maintained in vitro. Intracellular recordings in guinea pig or ferret IO neurons revealed that these cells could generate sustained endogenous oscillations (4-10 Hz) at hyperpolarized membrane potentials (
60 to
67 mV) after the intracellular injection of a brief hyperpolarizing current pulse. These oscillations appeared as the rhythmic generation of a low-threshold Ca2+ spike that typically initiated one or two fast Na+-dependent action potentials. Between low-threshold Ca2+ spikes was an afterhyperpolarization that formed a "pacemaker" potential. Local application of apamin resulted in a large reduction in the amplitude of the afterhyperpolarization, indicating that a Ca2+-activated K+ current makes a strong contribution to its generation. However, even in the presence of apamin, hyperpolarization of IO neurons results in a "depolarizing sag" of the membrane potential that was blocked by local application of Cs+ or partial replacement of extracellular Na+ with choline+ or N-methyl-D-glucamine+, suggesting that Ih also contributes to the generation of the afterhyperpolarization. Extracellular application of low concentrations of cesium resulted in hyperpolarization of the membrane potential of IO neurons and spontaneous 5- to 6-Hz oscillations in single, as well as networks, of IO neurons. Application of larger concentrations of cesium reduced the frequency of oscillation to 2-3 Hz or blocked the oscillation entirely. On the basis of these results, we propose that Ih contributes to single and ensemble oscillations in the IO in two ways: 1) Ih contributes to the determination of the resting membrane potential such that reduction of Ih results in hyperpolarization of the membrane potential and an increased propensity of oscillation through removal of inactivation of the low-threshold Ca2+ current; and 2) Ih contributes to the generation of the afterhyperpolarization and the pacemaker potential between low-threshold Ca2+ spikes.
 |
INTRODUCTION |
Inferior olivary neurons densely innervate and monosynaptically excite cerebellar Purkinje cells through characteristic axonal terminations known as climbing fibers, and thus form a major afferent pathway to the cerebellar cortex (Eccles et al. 1966
). Inferior olivary neurons fire at a frequency of 1-12 Hz and can exhibit spontaneous oscillatory activity near 10 Hz (Armstrong et al. 1968
). Likewise, the resulting complex spikes occurring in Purkinje cells are also rhythmic and display a maximum frequency of 10-12 Hz (Llinás and Sasaki 1989
). Simultaneous recordings from several Purkinje cells have revealed that complex spikes can occur synchronously, suggesting that ensembles of inferior olive nucleus (IO) neurons have the ability to fire rhythmically in unison (Bell and Kawasaki 1972
; Bower and Llinás 1983
; Fukuda et al. 1987
; Llinás and Volkind 1973
; Sasaki and Llinás 1985
; Yamamoto et al. 1986
). This synchronous oscillation has been proposed to be important in the determination of the timing and dynamic organization of motor sequences in motor coordination (Llinás 1988
; Llinás and Welsh 1993
; Vallbo and Wessberg 1993
), a hypothesis that is supported by studies in which multiple recordings from Purkinje cells in the rat cerebellum were used (Llinás and Sasaki 1989
; Welsh et al. 1995
).
Intracellular recordings from inferior olivary neurons maintained as a slice in vitro revealed that these cells have the intrinsic properties necessary not only to endogenously oscillate but also to influence one another through electrotonic gap junctions (Llinás and Yarom 1981a
,b
) and thereby generate pools of synchronously oscillating neurons (Benardo and Foster 1986
; Llinás and Yarom 1986
). These ensemble oscillations can appear as either subthreshold sine-wave-shaped oscillations of the membrane potential or as the rhythmic generation of low-threshold Ca2+ spikes, and they can occur either spontaneously or in response to the application of the alkaloid harmaline (Benardo and Foster 1986
; Llinás and Yarom 1986
). Interestingly, harmaline, when administered in vivo, results in a 10-Hz tremor, presumably through the induction of oscillations in the inferior olive (de Montigny and Lamarre 1973
; Llinás and Volkind 1973
).
Pioneering in vitro studies by Llinás and Yarom (1981a
,b
) have described many of the ionic conductances that underlie the endogenous oscillatory properties of single IO neurons, as well as these neurons' ability to generate ensemble oscillations in the 4- to 10-Hz range through electrotonic coupling (Llinás and Yarom 1981a
, 1986
; Llinás et al. 1974
). Three main conductances, in addition to those involved in action potential generation, have been proposed to account for the oscillatory properties of IO neurons: a dendritic high-threshold Ca2+ conductance, a somatic low-threshold Ca2+ conductance, and a Ca2+-activated K+ conductance (Llinás and Yarom 1981a
,b
). The interaction of the high-threshold Ca2+ current and the prominent Ca2+-activated K+ current was proposed to account for the generation of low-frequency firing (up to 6 Hz), whereas higher-frequency (up to 10 Hz) firing was proposed to occur at more hyperpolarized membrane potentials with less involvement of the high-threshold Ca2+ current.
In similarity with inferior olivary neurons, thalamocortical relay neurons also possess the endogenous ability to generate rhythmic oscillations (Jahnsen and Llinás 1984a
,b
; McCormick and Pape 1990a
; Soltesz et al. 1991
). Detailed examination of the mechanisms of oscillation in single thalamic relay neurons has revealed a critical role for the hyperpolarization-activated cation current known as Ih (McCormick and Huguenard 1992
; McCormick and Pape 1990a
; Soltesz et al. 1991
). Low-frequency (0.5-4 Hz) rhythmic burst firing can be generated in thalamocortical neurons through the interaction between the low-threshold Ca2+ current IT and Ih. In this manner, the activation/inactivation of IT results in the generation of low-threshold Ca2+ spikes and high-frequency bursts of Na+-dependent action potentials and the activation/deactivation of Ih provides a "pacemaker-potential" that times the generation of these low-threshold Ca2+ spikes (McCormick and Huguenard 1992
; McCormick and Pape 1990a
). Interestingly, alterations in the amplitude or voltage dependence of activation/deactivation of Ih can have strong effects on both the ability of thalamocortical relay neurons to endogenously oscillate and the frequency of this oscillation (McCormick and Huguenard 1992
; McCormick and Pape 1990a
,b
; McCormick and Williamson 1991
). Partial reduction of Ih in thalamocortical relay neurons results in the enhancement of rhythmic oscillation, whereas complete block of Ih results in an abolition of endogenous oscillations (McCormick and Pape 1990a
).
Hyperpolarization of inferior olivary neurons results in a strong "depolarizing sag" (Yarom and Llinás 1987
) that is likely the result of activation of a hyperpolarization-activated cation current. This anomalous rectification in the hyperpolarizing range is blocked by extracellular application of harmaline and extracellular Cs+ (Yarom and Llinás 1987
). Together with previous results in thalamocortical relay neurons, these results suggest that Ih may also play an important role in the determination of the occurrence and frequency of oscillations in the inferior olive and that the reduction of this current may be the major mechanism by which harmaline induces rhythmic oscillations in this nucleus. In the present study, we demonstrate that Ih can potently modulate the presence and frequency of single and ensemble oscillations in the inferior olive.
 |
METHODS |
Male or female adult Hartley guinea pigs or ferrets, 2-9 mo old, were deeply anesthetized with pentobarbital sodium (30-40 mg/kg) and killed by decapitation. The brain was rapidly removed and the region of the brain stem containing the inferior olive was dissected free. Slices 400 µm thick were cut in the coronal plane with the use of a Vibratome (Ted Pella). A modification of the technique developed by Aghajanian and Rasmussen (1989)
was used to increase tissue viability. During preparation of slices, the tissue was placed in the normal slice bathing solution (5°C), except that NaCl was replaced with sucrose while an osmolarity of 307 mosM was maintained. After preparation, slices were placed in an interface-style recording chamber (Fine Sciences Tools) and allowed
2 h to recover. The normal bathing medium contained (in mM) 126 NaCl, 2.5 KCl, 1.2 MgSO4, 1.25 NaH2PO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose, aerated with 95% O2-5% CO2 to a final pH of 7.4. For the first 20 min the inferior olivary slices were in the recording chamber, the bathing medium contained an equal mixture of the normal-NaCl and the sucrose-substituted solutions. After the 2 h of recovery, extracellular multiple-unit recordings were performed from multiple locations in each slice to determine the prevalence of spontaneous spiking activity. Recordings were performed with the slices between the temperatures of 34.5 and 35.5°C.
Cesium acetate was dissolved in the bathing medium (1-2 mM) or was applied with the pressure-pulse technique (10-20 mM in the micropipette), in which a brief pulse of pressure (10-100 ms, 200-350 kPa) to a broken micropipette (tip diameter 2-10 µm) was used to extrude volumes of 2-20 pl. Extracellular multiple-unit recordings were obtained with tungsten microelectrodes (Frederick Haer, Brunswick, ME), whereas intracellular recordings were obtained with glass microelectrodes formed on a Sutter Instruments (Novato, CA) P-80/PC micropipette puller, filled with 1.2 or 4 M potassium acetate, and beveled to a final resistance of 70-100 M
. Beveled electrodes were found to be necessary in our experiments to obtain recordings from IO neurons that retained the ability to generate intrinsic and prolonged oscillations. Electrical stimulation was performed with the use of bipolar tungsten electrodes similar to those used for extracellular recordings. Tips were electroplated with gold in a 2% HAuCL4 solution with the use of a 1.5-V battery as a current generator. Stimulations (25-250 µA, duration 0.1 ms) were delivered as one to three shocks with an intershock interval of 10 ms. All data are presented as means ± SD.
 |
RESULTS |
Intracellular recordings were obtained from guinea pig (n = 48) and ferret (n = 17) inferior olivary neurons. In addition, simultaneous extracellular multiple-unit recordings were routinely performed to monitor neuronal population activity (e.g., see Fig. 1A). Recordings in both species revealed similar results and therefore were combined for analysis. Extracellular and intracellular recordings from IO neurons revealed that these cells were typically silent at rest (Figs. 1 and 2). In contrast, electrical stimulation of the slice near the IO resulted in a typical 6- to 10-Hz rhythmic bursting activity in both the extracellularly recorded multiple-unit activity and intracellularly recorded single neuron. These rhythmic oscillations could be either supra- or subthreshold for the generation of action potential activity. Simultaneous extracellular and intracellular recordings at neighboring sites revealed that these ensemble oscillations were synchronous for the cells recorded (Fig. 1A).

View larger version (41K):
[in this window]
[in a new window]
| FIG. 1.
Ensemble and single-cell oscillation in inferior olivary nucleus (IO). A: electrical stimulation of slice ( ) outside border of IO induces synchronous oscillatory and rhythmic bursting activity in intracellular and extracellular multiple-unit recordings. First few s of oscillation are expanded for detail ( ). Note synchronization of activity of single intracellularly recorded neuron and multiple-unit activity. Intracellular and extracellular electrodes were spaced ~100 µm apart. B: in a different cell, injection of a brief hyperpolarizing pulse of current induces an intrinsic single-cell oscillation, the duration and frequency of which are similar to those of the ensemble oscillation shown in A. Cells in A and B were slightly hyperpolarized (5-10 mV) by a holding current. Action potentials in intracellular recordings are not shown at full height. Recording is from a guinea pig IO cell.
|
|

View larger version (22K):
[in this window]
[in a new window]
| FIG. 2.
Oscillatory properties of IO neurons and presence of hyperpolarization-activated rectification. A: intracellular injection of a hyperpolarizing current pulse at resting membrane potential of 57 mV resulted in a rhythmic oscillation consisting of 3 presumed low-threshold Ca2+ spikes interspersed by afterhyperpolarizations (AHPs). Intracellular injection of same current pulse after hyperpolarization of the IO neuron to 60 mV through intracellular injection of DC resulted in a prolonged (4-s) sequence of rhythmic oscillation, the frequency of which decreased over time. Last 3 low-threshold Ca2+ spikes failed to reach action potential threshold. Hyperpolarization of IO neuron to 63 and 65 mV decreased frequency and duration of oscillation. At 70 mV, oscillation was suppressed altogether and only 1 low-threshold Ca2+ spike and AHP were generated. B: comparing response of neuron to hyperpolarizing current pulse at 60 and 70 mV reveals decrease in "depolarizing sag" at 70 mV. C: intracellular injection of hyperpolarizing current pulses of progressively larger amplitude resulted in progressively larger hyperpolarizations, depolarizing sags, and rebound low-threshold Ca2+ spikes. Data obtained from ferret IO.
|
|
In cells at resting membrane potential, intracellular injection of a hyperpolarizing current pulse 0.1-1 s (typically 200 ms) in duration was followed by a rebound low-threshold Ca2+ spike (Yarom and Llinás 1987
), which itself triggered between zero and two fast, Na+-dependent action potentials (Figs. 1B and 2, A and B). On occasion, the offset of the hyperpolarizing current pulse was followed by a brief period of damped oscillation (e.g., Fig. 2A,
57 mV). Hyperpolarization of inferior olivary neurons to the voltage range of approximately
60 to
67 mV with intracellular injection of DC resulted in 4- to 8-Hz oscillations being triggered by the offset of the same hyperpolarizing current pulses (Figs. 1B and 2A; n = 18). The frequency of these oscillations decreased over time and the oscillations terminated after several seconds (up to 30 s and 170 oscillation cycles) and were not critically dependent on the generation of fast Na+ spikes (Fig. 2A,
), although fast spikes were shown to facilitate the generation of the oscillation (see Fig. 9 below). Further hyperpolarization of inferior olive cells with DC to membrane potentials of
70 mV or more negative resulted in an abolition of rhythmic oscillations (Fig. 2A). Close examination of the rhythmic oscillations revealed that they were composed of low-threshold Ca2+ spikes that activated one or two fast Na+-dependent action potentials and that were followed by pronounced afterhyperpolarizations (AHPs) (e.g., Fig. 2A,
70 mV), as described previously (Benardo and Foster 1986
; Llinás and Yarom 1986
) (see also below). These results indicate that IO cells, although typically not spontaneously oscillating at rest in vitro, possess all the necessary currents required to endogenously oscillate as individuals and as a network (Llinás and Yarom 1981a
,b
).

View larger version (16K):
[in this window]
[in a new window]
| FIG. 9.
Contribution of tetrodotoxin (TTX)- and apamin-sensitive currents to generation of rhythmic oscillations in IO neurons. A: intracellular injection of hyperpolarizing current pulse into IO neuron after block of depolarizing sag with extracellular application of Cs+ is followed by a rhythmic sequence of low-threshold Ca2+ spikes interspersed with AHPs. B: block of voltage-dependent Na+ currents with local application of TTX (10 µM in micropipette) results in a decrease in duration of oscillation. C: local application of apamin (100 nM in micropipette), a potent antagonist of some Ca2+-activated K+ currents, results in abolition of AHP and rhythmic oscillations. D: overlaying response to hyperpolarizing current pulse under these conditions reveals that although electrotonic response to current pulse did not change, block of voltage-dependent Na+ currents with TTX resulted in a broadening of low-threshold Ca2+ spike and a small decrease in AHP (B). Application of apamin resulted in further broadening of low-threshold Ca2+ spike and complete abolition of AHP (C). Data obtained from guinea pig IO.
|
|
Evidence for the presence of Ih in inferior olivary cells
The enhancement of rhythmic oscillations in IO neurons with hyperpolarization suggests that currents activated by hyperpolarization, such as Ih, may contribute to this feature of IO electroresponsiveness. Indeed, examination of the response of IO neurons to the 200-ms hyperpolarizing current pulse revealed a substantial depolarizing sag (Fig. 2, A and B). The amplitude of this depolarizing sag decreased markedly on tonic hyperpolarization, as would be expected if this feature were generated by activation of Ih, because this current may be tonically activated at these membrane potentials (McCormick and Pape 1990a
).
To more closely examine the properties of the depolarizing sag activated by hyperpolarization, a family of hyperpolarizing current pulses was injected into inferior olivary neurons (Fig. 2C). Progressive hyperpolarization of IO neurons resulted not only in progressively larger hyperpolarizations of the membrane potential, but also in activation of a progressively larger depolarizing sag, as well as progressively larger rebound low-threshold Ca2+ spikes (Fig. 2C).
Further evidence for the presence of Ih in IO neurons was obtained with local application of cesium chloride, which is known to specifically block this current in other neuronal systems (e.g., see McCormick and Pape 1990a
). Local application of Cs+ (several 10- to 20-pl drops of 10-20 mM CsCl in micropipette) to the region near the recorded IO neuron resulted in a reversible block of the depolarizing sag (Fig. 3) (Yarom and Llinás 1987
). In addition, local application of Cs+ hyperpolarized the cell from rest by 10-18 mV (14.8 ± 3.1 mV, mean ± SD; n = 5). This hyperpolarization could bring the membrane potential of the IO neuron below the threshold for activation of the low-threshold Ca2+ current and therefore prevent rebound low-threshold Ca2+ spikes (Fig. 3, A and D). Depolarization of the IO neurons back to the pre-Cs+ membrane potential reinstated rhythmic rebound oscillations (Fig. 3). Comparison of the rebound oscillations in control versus Cs+ revealed that extracellular application of Cs+ resulted in a slowing of the frequency of oscillation (cf. Fig. 3, B and C). In addition, there was a slowing of the rate of rise of the offset of the AHP and on occasion an increase in the number of fast action potentials generated by the initial rebound low-threshold Ca2+ spike (from 1 to 2).

View larger version (35K):
[in this window]
[in a new window]
| FIG. 3.
Effects of extracellular application of Cs+ on membrane potential and rhythmic oscillations in IO neurons. A: intracellular injection of hyperpolarizing current pulses at a rate of once every 2 s results in generation of a rhythmic sequence of 2 low-threshold Ca2+ spikes, interspersed with an AHP (B). Extracellular application of Cs+ (20 mM in micropipette) resulted in hyperpolarization of membrane potential, abolition of depolarizing sag generated by hyperpolarizing current pulse (expanded in D), and abolition of rebound oscillations. Depolarization of cell back to 63 mV with intracellular injection of current (A, top trace) resulted in reinstatement of rhythmic oscillations, although depolarizing sag was completely blocked (expanded in C). Comparison of rhythmic oscillations generated on offset of current pulse before and after application of Cs+ revealed a block of the depolarizing sag in the electrotonic response to the hyperpolarizing current pulse, an increase in amplitude of the rebound low-threshold Ca2+ spike, and a decrease in oscillation frequency. Data obtained from ferret IO.
|
|
One complicating influence of the extracellular application of Cs+ is that there is also an increase in the amplitude of membrane hyperpolarization in response to the intracellular injection of the current pulse (not shown). However, compensating for both the changes in membrane potential, with intracellular injection of DC, and the increase in electrotonic membrane response to the current pulse at steady state, by decreasing the amplitude of this current pulse, still resulted in a slowing of the frequency of rhythmic oscillation in IO neurons (mean increase of the 1st oscillation cycle duration: 28.1 ± 6.4%; n = 6) and a decrease in the rate of rise of the offset of the AHP (Fig. 4, A, B, and D; n = 6). These effects were reversible on washout of Cs+ (Fig. 4C). Measurements of the duration of action potentials in the presence of Cs+ revealed that they were not significantly changed in comparison with values obtained in normal medium (measures were made at the threshold of the 1st action potential generated in sequences of oscillations in 3 cells: 0.99 ± 0.06 ms, normal; 1.06 ± 0.05 ms, cesium).

View larger version (18K):
[in this window]
[in a new window]
| FIG. 4.
Effect of cesium on oscillation frequency. A: intracellular injection of a hyperpolarizing current pulse results in a rebound rhythmic oscillation. Local application of Cs+ (20 mM in micropipette) results in suppression of depolarizing sag and slowing by 30% of frequency of oscillation (B, bottom, overlay of 3 traces), an effect that is reversible (C). D: overlaying 1st cycle of oscillation for control, cesium, and recovery conditions reveals decrease of slope of pacemaker potential that results from cesium application. Intensity of holding current and of hyperpolarizing current pulses was adjusted to keep amount of hyperpolarization preceding calcium spike and membrane potential of cell the same before and after application of Cs+. Data obtained from guinea pig IO.
|
|
The hyperpolarization of IO neurons in response to extracellular application of Cs+ suggests that Ih may contribute to the resting membrane potential of these cells. If this is the case, then depolarization of IO neurons should result in deactivation of Ih and removal of this depolarization would then lead to progressive (time-dependent) reactivation of Ih and thus give rise to an apparent AHP (e.g., McCormick and Pape 1990a
). Such an Ih-dependent AHP would contribute to the large AHP that terminates rebound bursts in IO neurons and that has previously been demonstrated to be generated through the activation of a Ca2+-activated K+ current (Llinás and Yarom 1981a
). Apamin, a potent blocker of some types of calcium-dependent potassium channels, applied locally (100 nM in micropipette), largely reduced the amplitude of the AHP, although a 2- to 4-mV AHP remained (Fig. 5A; n = 5); this residual AHP was reversibly blocked by local application of cesium with (Fig. 5B; n = 3) or without (not shown) compensation for the Cs+-induced hyperpolarization of the membrane potential. The apparent AHP was still present even in the absence of calcium spikes or Na+-dependent action potentials. These findings can be explained by taking into account the properties of Ih. The depolarization occurring during the generation of a low-threshold Ca2+ spike results in the deactivation of some portion of Ih. After the low-threshold Ca2+ spike, therefore, the membrane potential is allowed to achieve a more hyperpolarized level (owing to the deactivation of Ih) and generate an AHP. However, Ih once again activates and this slowly depolarizes the membrane potential, generating the rising phase of the AHP (see also McCormick and Pape 1990a
).

View larger version (20K):
[in this window]
[in a new window]
| FIG. 5.
Cesium- and apamin-sensitive AHPs are generated after a rebound burst. A: application of apamin (100 nM in micropipette) reduces amplitude of AHP, lengthens duration of low-threshold calcium spike, and suppresses rhythmic oscillations. B: extracellular application of cesium (20 mM in micropipette) on a different cell, in presence of apamin, reversibly blocks residual apamin-insensitive AHP and lengthens duration of low-threshold calcium spike. Large low-threshold calcium spikes in B were induced at offset of a 1-s hyperpolarizing current pulse. Intensity of holding current (in the 0.4-nA range) and of hyperpolarizing current pulses were adjusted to keep amount of hyperpolarization preceding calcium spike and membrane potential of cell constant. Data obtained from guinea pig IO.
|
|
If this hypothesis is true, the intracellular injection of a depolarizing current alone should result in the generation of an AHP. Indeed, removal of a prolonged depolarization of IO neurons was followed by the generation of an AHP, even if the prior depolarization was allowed to come to steady state and several seconds had passed since the generation of any low-threshold Ca2+ spikes (Fig. 6A; n = 10). Local application of Cs+ resulted in an abolition of this AHP (Fig. 6B), an effect that was reversible (Fig. 6C; n = 4). Examination of this Cs+-sensitive AHP revealed it to be ~100-400 ms in duration and 2-6 mV in amplitude (mean = 4.6 ± 1.2 mV; n = 4; Figs. 5 and 6). The depolarization-activated AHP remained unaltered after extracellular application of apamin (n = 2; Fig. 6D). The amplitude of this AHP was also dependent on the duration of the preceding depolarization (Fig. 6D; n = 2) and on its amplitude (not shown), as expected if Ih were involved in the generation of this AHP.

View larger version (18K):
[in this window]
[in a new window]
| FIG. 6.
Cesium blocks an AHP following a depolarizing current pulse. A: intracellular injection of a prolonged depolarizing current pulse is followed by generation of an AHP. B: local application of Cs+ (20 mM in micropipette) resulted in a complete suppression of this AHP, an effect that was reversible (C). Arrows: expansion of AHP before and after local application of Cs+. D: increasing duration of depolarizing current pulse results in a progressively larger AHP, presumably owing to progressive deactivation of Ih by the depolarization. Data obtained from ferret IO.
|
|
Contribution of Ih to rhythmic firing
Previous investigations in thalamocortical neurons revealed that extracellular application of Cs+ could induce rhythmic oscillations in nonoscillating neurons through reduction of Ih (McCormick and Pape 1990a
). The possibility that reduction of hyperpolarization-activated cation currents may also induce rhythmic oscillations in IO neurons was examined. In the example shown in Fig. 7, ensemble oscillations in the inferior olive were not present, as revealed with extracellular and intracellular recordings (Fig. 7A), although hyperpolarization of individual IO neurons could result in these cells generating sustained periods of rhythmic oscillation (Fig. 7A). Extracellular application of cesium chloride to the inferior olive, either locally via ejection from a micropipette placed near (<50 µm) the recorded cell (5-20 mM in micropipette) or in the bath (1-2 mM), resulted in robust rhythmic activity in multiple IO neurons recorded extracellularly (Fig. 7, B and C). Cells recorded intracellularly in the vicinity of the multiple-unit electrode (at ~20 and 100 µm for cells 2 and 3, respectively) demonstrated membrane potentials that were more negative than in the absence of Cs+, presumably resulting from the Cs+-induced hyperpolarization of these cells (e.g., Fig. 3A). In the presence of Cs+, intracellularly recorded IO neurons were found to oscillate at the same frequency as the ensemble oscillation (e.g., Fig. 7C, cell 3).

View larger version (28K):
[in this window]
[in a new window]
| FIG. 7.
Cesium induces synchronized ensemble oscillations in IO. A: extracellular recording from IO reveals absence of synchronized activity. Intracellular injection of a hyperpolarizing current pulse at resting potential typically did not result in rebound oscillations (cell 1), although these oscillations were evoked after hyperpolarization to 64 mV. Note that synchronized oscillations in extracellular multiple-unit recording were also absent during evoked generation of oscillations in a single cell. B: several minutes after addition of 2 mM Cs+ to bathing medium, synchronized oscillations were prevalent in both an intracellularly recorded neuron (cell 2) and the extracellular multiple-unit recording. C: these oscillations continued, appeared to be even more synchronized, and slowed down in frequency when Cs+ reached steady 2 mM concentration in bathing medium (1 h of infusion). Intracellular recordings revealed rhythmic generation of low-threshold Ca2+ spikes and subthreshold oscillations of membrane potential (cell 3). Phase of oscillation recorded in extracellular recordings and cells 2 and 3 differed. This may have resulted from the fact that cell 3 was recorded at an increased distance from the extracellular recording electrode in comparison with cell 2. D: plot of frequency of oscillation vs. time after switch to 2 mM Cs+ in bathing medium. Recordings were performed in an interface-style recording chamber where change of ion concentrations does not reach steady state for ~30-40 min. Data obtained from ferret IO.
|
|
If the low-threshold Ca2+ spikes failed to occur during the Cs+-induced ensemble oscillation (Fig. 7C), or were prevented through membrane depolarization or hyperpolarization (not shown), then rhythmic oscillations of the membrane were seen during these ensemble oscillations. These rhythmic oscillations were only seen when the extracellular multiple-unit recordings revealed rhythmic oscillations in the population of IO cells, and therefore presumably result from the electrotonically coupled activity of neighboring IO neurons, as reported previously (Benardo and Foster 1986
; Llinás and Yarom 1986
).
During the bath application of Cs+, the frequency of oscillation generated by the inferior olive progressively decreased from 5-7 Hz to ~3 Hz (n = 4), presumably representing the slowly rising concentration of extracellular Cs+ (Fig. 7D). These results indicate that extracellular application of cesium can both induce ensemble oscillations in inferior olive neurons and determine the frequency of oscillation. Because extracellular application of Cs+ is known to block Ih in many cell types (Denyer and Brown 1990
; DiFrancesco 1985
; Halliwell and Adams 1982
; Mayer and Westbrook 1983
; McCormick and Pape 1990a
; Spain et al. 1987
; Tabata and Ishida 1996
), these results suggest that this ionic current may play an important role in the determination of rhythmic oscillation in the inferior olive.
In other neuronal systems, hyperpolarization-activated cation currents have been demonstrated to be carried by both Na+ and K+ ions (Mayer and Westbrook 1983
; McCormick and Pape 1990a
; Tabata and Ishida 1996
). The depolarizing sag in IO neurons is present even at membrane potentials positive to the presumed equilibrium potential for K+ (approximately
100 mV), indicating that this feature is not mediated by the activation of a pure K+ current. Indeed, substitution of choline chloride for NaCl and choline bicarbonate for sodium bicarbonate in equimolar concentration in the bathing medium (n = 3) or of N-methyl-D-glucamine chloride (NMDG) for NaCl (n = 3), resulted in reduction of the depolarizing sag (Fig. 8, F and G) and in hyperpolarization of the cells by 10-24 mV (mean = 14.7 ± 5.3 mV; Fig. 8; n = 6). As the extracellular concentration of Na+ was reduced and the membrane potential of IO neurons became more negative, rhythmic "sine-wave" oscillations appeared indicating that the IO was generating an ensemble oscillation (Fig. 8, C and D; n = 6). These oscillations could trigger low-threshold Ca2+ spikes, which were then in phase with the sine-wave oscillation (Fig. 8, C and D). As the extracellular concentration of Na+ continued to decline, IO neurons hyperpolarized further and the ensemble oscillation ceased (Fig. 8E). These results are remarkably similar to those obtained with extracellular application of Cs+ (see above) and suggest that at these membrane potentials the depolarizing sag is largely mediated by the inward movement of Na+ ions.

View larger version (20K):
[in this window]
[in a new window]
| FIG. 8.
Effects of reduction of extracellular concentration of Na+ on IO cells. Before reduction of extracellular Na+ concentration, no spontaneous rhythmic oscillations in membrane potential were seen (A) and intracellular injection of a hyperpolarizing current pulse was associated with a depolarizing sag and the generation of a rhythmic sequence of low-threshold Ca2+ spikes (B; spikes are truncated). Only a portion of the rhythmic oscillation is shown. Changing extracellular Na+ concentration from 153.25 to 1.25 mM by substituting choline chloride for NaCl and choline bicarbonate for sodium bicarbonate in equimolar concentration in bathing medium resulted in steady hyperpolarization of neuron and appearance of rhythmic "sine-wave" oscillations in membrane potential (C) that could trigger generation of low-threshold Ca2+ spikes (D). After complete wash-in of low extracellular Na+ concentration, rhythmic oscillations were blocked (E) and cell no longer generated fast action potentials nor oscillations on offset of current pulses, either at 68 or 58 mV (F and G). Data obtained from ferret IO. [Na+], Na+ concentration.
|
|
Contribution of an apamin-sensitive K+ current and Na+ action potentials to the AHP
The presence of a large AHP following each low-threshold Ca2+ spike after blockade of Ih with extracellular Cs+ (Figs. 3 and 4) indicates that additional ionic currents are involved in the generation of this electrophysiological feature. Indeed, block of fast Na+ spikes activated by the low-threshold Ca2+ current with the local application of tetrodotoxin (TTX) (10 µM in micropipette) reduced the amplitude of the AHP (Fig. 9, B and D). Similarly, low-threshold Ca2+ spikes that did not generate fast action potentials were followed by an AHP that was smaller in amplitude than those that generate action potentials (not shown; n = 4). Interestingly, the application of TTX not only reduced the amplitude of the AHP, but also reduced the overall duration of the oscillation induced by the hyperpolarizing current pulse (Fig. 9B). Extracellular application of apamin (100 nM in micropipette) blocked the residual AHP remaining in the presence of Cs+ and TTX (Fig. 9, C and D). In addition, local application of apamin also completely blocked the rhythmic oscillations generated in response to a hyperpolarizing current pulse (Figs. 5A and 9C). Comparison of the rebound low-threshold Ca2+ spikes following the hyperpolarizing current pulse revealed a broadening of these with the application of either TTX or apamin accompanying the reduction of the AHP (Figs. 5A and 9D). This result suggests that the AHP considerably shortens the duration of the low-threshold Ca2+ spike.
 |
DISCUSSION |
The present results indicate that the activation of an inward current by hyperpolarization of the membrane contributes to the intrinsic and population oscillations of inferior olivary neurons. This current is sensitive to extracellular Cs+ and is at least partly carried by Na+ ions, and thus is likely to be Ih. The contribution of K+ or Cl
to Ih in inferior olivary neurons remains to be tested.
Ih generates the time-dependent rectification in IO neurons
The inward rectification first described in IO neurons by Yarom and Llinás (1987)
shares common features with the effects generated by a class of hyperpolarization-activated current, termed Ih, described in a variety of electrically excitable cells ranging from heart cells and axons (DiFrancesco and Noble 1989
; Eng et al. 1990
) to principal cells in the cerebral cortex, thalamus, and cerebellum (Crepel and Penit-Soria 1986
; Halliwell and Adams 1982
; McCormick and Pape 1990a
; Spain et al. 1987
). In all of these cell types, hyperpolarization of the membrane potential results in the slow activation of Ih, which is carried by both Na+ and K+ ions and therefore has a reversal potential of around
30 to
40 mV. At hyperpolarized membrane potentials, Ih is dominated by the entry of Na+ ions into the cell, and therefore generates a slow depolarizing sag. The half-activation point in the steady-state activation curve for Ih varies between cell types, but is often in the range of
70 to
80 mV (McCormick and Pape 1990a
). Because IO neurons are electrotonically coupled to one another, it is not possible to perform accurate voltage-clamp experiments on Ih in these cells. However, the block of the time-dependent inward rectification by extracellular application of Cs+ (Figs. 3 and 4) or by replacement of extracellular Na+ with choline+ or NMDG+ (Fig. 8) and the absence of block by extracellular Ba2+ (Yarom and Llinás 1987
) indicate that the depolarizing sag generated in these neurons on hyperpolarization is due to the activation of Ih.
Contribution of Ih to the resting membrane potential
In other cell types, such as thalamic relay neurons, Ih not only generates a depolarizing sag on hyperpolarization, but also contributes to the determination of the resting membrane potential (e.g., McCormick and Pape 1990a
). Similarly, Ih also appears to contribute strongly to the determination of the resting membrane potential and input conductance of IO neurons. In vitro, nonoscillating IO neurons have a resting membrane potential of
55 to
60 mV and are strongly hyperpolarized by the extracellular application of Cs+ (Fig. 3) or reduction of the extracellular concentration of Na+ ions (Fig. 8). Interestingly, the partial reduction of Ih can hyperpolarize the cell into the range of membrane potentials at which the cell can intrinsically oscillate through the generation of rhythmic low-threshold Ca2+ spikes (Fig. 7). Persistent activation of Ih can therefore result in tonic depolarization of IO neurons, resulting in inactivation of the low-threshold Ca2+ current and a block of spontaneous rhythmic oscillations. Because IO neurons are electronically coupled to one another, the exact value of the resting membrane potential of each cell will depend on that of the other cells in the network.

View larger version (19K):
[in this window]
[in a new window]
| FIG. 10.
Summary diagram indicating possible contribution of hyperpolarization-activated cation current Ih to rhythmic oscillations in IO. Rhythmic burst firing is created through generation of a low-threshold Ca2+ current (IT), which activates fast action potentials mediated by Na+, K+, and Ca2+ currents (INa, IK, and ICa, respectively). Entry of Ca2+ during generation of these fast action potentials and during generation of low-threshold Ca2+ spike results in generation of an AHP through activation of a Ca2+-activated K+ current. AHP activates Ih. Combined activation of Ih and decrease of a Ca2+-activated K+ current (IK,Ca) results in depolarization of neuron, which activates another low-threshold Ca2+ spike. In this manner, Ih modulates frequency of oscillation. In addition, tonic activation of Ih also contributes to average membrane potential, and therefore to presence, strength, and frequency of oscillation in these neurons. Data obtained from guinea pig IO.
|
|
The maintenance of the membrane potential of the cells in a "silent" region between rhythmic oscillations at hyperpolarized and depolarized membrane potentials may explain the general absence of spontaneous oscillatory activity in IO in vitro (Llinás and Yarom 1986
; present results). However, even in the absence of spontaneous oscillations, the network of IO neurons displays a marked propensity to oscillate. For example, local electrical stimulation could induce prolonged (1-40 s) periods of 4- to 8-Hz synchronous oscillations in the IO (Fig. 1).
Contribution of Ih to oscillations in the inferior olive
The pioneering work of Llinás and Yarom (1981a
,b
) demonstrated that inferior olivary neurons can generate two distinct intrinsic oscillations that differ in frequency. A 3- to 6-Hz oscillation occurs at depolarized membrane potentials and is generated through the activation of a fast combined Na+ and high-threshold Ca2+-mediated action potential that activates a large and long-lasting AHP generated through a Ca2+-activated K+ conductance. The rising phase of the AHP was proposed to then activate a low-threshold Ca2+ spike, which reinitiated the cycle by activating again the Na+/Ca2+ action potential. In hyperpolarized IO neurons, these cells generate a 9- to 12-Hz oscillation through similar mechanisms, although at a higher frequency owing to a decrease in the involvement of the dendritic high-threshold Ca2+ currents, resulting in a shortening of the duration of the AHP (Llinás and Yarom 1981a
,b
, 1986)
. Our data are consistent with this hypothesis for the generation of rhythmic oscillations in inferior olivary neurons, but also demonstrate an important role for Ih.
In our scenario, rhythmic oscillations at hyperpolarized membrane potentials in IO neurons are generated through the following sequence of events: activation of a somatic low-threshold Ca2+ spike, which generates one or two Na+- and K+-dependent action potentials, is followed by an AHP mediated largely by the activation of an apamin-sensitive Ca2+-activated K+ current (Fig. 10). In addition, during the low-threshold Ca2+ spike and the generation of action potentials, a portion of Ih is deactivated. This deactivation of Ih facilitates the generation of the AHP by allowing it to reach more negative membrane potentials (Fig. 10). The AHP subsequently results in two important effects: removal of inactivation of IT and the activation of Ih. Activation of Ih depolarizes the membrane potential toward the threshold for activation of IT and subsequently promotes the generation of a low-threshold Ca2+ spike and associated Na+- and K+-dependent action potential(s), and therefore reinitiates the oscillation. In our recordings, rhythmic oscillations in IO neurons occurred at frequencies of between 6 and 10 Hz, but may occur at frequencies as low as 3 Hz after block of Ih.
The reduction of the rate of rise of the pacemaker potential between low-threshold Ca2+ spikes in IO neurons by application of extracellular Cs+ gives experimental support to the hypothesis that Ih contributes to the generation of the pacemaker potential and the determination of oscillation frequency. Similar results have been obtained in pacemaker cells of the sinoatrial node of rabbit (Denyer and Brown 1990
). The robust spontaneous pacemaking activity of the sinoatrial node cells was reduced in frequency by ~30% after block of the hyperpolarization-activated current If (which is homologous to Ih) by the extracellular application of 2 mM Cs+. As in IO neurons, the reduction of If in sinoatrial cells was accompanied by a marked decrease in the slope of the pacemaker potential between action potential generation. Similarly, the activation of Ih also generates a pacemaker potential in single thalamocortical neurons and, through the interaction with the low-threshold Ca2+ current, can generate rhythmic burst firing in the frequency range of 0.5-4 Hz (McCormick and Pape 1990a
). However, in these cells, complete block of Ih results in the suppression of rhythmic burst firing owing to the lack of a prominent AHP following low-threshold Ca2+ spikes in these cells in the absence of Ih.
Contribution of Ih to ensemble oscillation
Simultaneous hyperpolarization of inferior olivary neurons seems to be a condition under which synchronous sine-wave oscillation and rhythmic firing can be generated among IO cells. Application of the alkaloid harmaline on IO cells, in addition to blocking the time-dependent inward rectification (sag) (Yarom and Llinás 1987
), also results in hyperpolarization of the cells and appearance of ensemble oscillation (Llinás and Yarom 1986
). In the present study we demonstrate that bath application of low concentration of cesium or removal of extracellular Na+ ions hyperpolarizes IO cells and results in generation of robust sine-wave oscillations that derive from the combined generation of rhythmic activity in the coupled network of IO neurons. These rhythmic oscillations of the membrane potential served to coordinate the rhythmic activity in the intracellularly recorded cell such that each low-threshold Ca2+ spike and subsequent action potentials were in synchrony with those of other neurons (Figs. 7 and 8). Therefore, by controlling the value of the membrane potential, Ih may play a key role in determining the generation and frequency of ensemble oscillations in the IO. Interestingly, in several different cell types including thalamic relay neurons (McCormick and Pape 1990b
), brain stem cells (Bobker and Williams 1989
), and heart neurons (DiFrancesco 1985
), the voltage dependence of Ih is modulated by the activation of a variety of receptor subtypes. A similar type of modulation of Ih in IO cells could play an important role in the generation of normal (e.g., movement-related) and abnormal (e.g., tremor-related) network oscillations in the IO.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the National Institutes of Health to D. A. McCormick, and from the Centre National de la Recherche Scientifique and the Fondation pour la Recherche Médicale to T. Bal.
 |
FOOTNOTES |
Address for reprint requests: D. A. McCormick, Section of Neurobiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06473.
Received 15 November 1996; accepted in final form 4 February 1997.
 |
REFERENCES |
-
AGHAJANIAN, G. K.,
RASMUSSEN, K.
Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices.
Synapse
3: 331-338, 1989.[Medline]
-
ARMSTRONG, D. M.,
ECCLES, J. C.,
HARVEY, R. J.,
MATTHEWS, P.B.S.
Responses in the dorsal accessory olive of the cat to stimulation of hind limb afferents.
J. Physiol. Lond.
194: 125-145, 1968.[Medline]
-
BELL, C. C.,
KAWASAKI, T.
Relations among climbing fibre responses of nearby Purkinje cells.
J. Neurophysiol.
35: 155-169, 1972.[Free Full Text]
-
BENARDO, L. S.,
FOSTER, R. E.
Oscillatory behavior in inferior olive neurons: mechanism, modulation, cell aggregates.
Brain Res. Bull.
17: 773-784, 1986.[Medline]
-
BOBKER, D. H.,
WILLIAMS, J. T.
Serotonin augments the cation current Ih in central neurons.
Neuron
2: 1535-1540, 1989.[Medline]
-
BOWER, J.,
LLINÁS, R.
Simultaneous sampling of the responses of multiple, closely adjacent, Purkinje cells responding to climbing fiber activation.
Soc. Neurosci. Abstr.
9: 607, 1983.
-
CREPEL, F.,
PENIT-SORIA, J.
Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells. An in vitro study.
J. Physiol. Lond.
372: 1-23, 1986.[Abstract]
-
DE MONTIGNY, C.,
LAMARRE, Y.
Rhythmic activity induced by harmaline in the olivo-cerebello-bulbar system of the cat.
Brain res.
53: 81-95, 1973.[Medline]
-
DENYER, J. C.,
BROWN, H. F.
Pacemaking in rabbit isolated sino-atrial node cells during Cs+ block of the hyperpolarization-activated current if.
J. Physiol. Lond.
429: 401-409, 1990.[Abstract]
-
DIFRANCESCO, D.
The cardiac hyperpolarizing-activated current, If, origins and developments.
Prog. Biophys. Mol. Biol.
46: 163-183, 1985.[Medline]
-
DIFRANCESCO, D.,
NOBLE, D.
The current If and its contribution to cardiac pacemaking.
In: Neuronal and Cellular Oscillators,
edited by
and J. W. Jacklet
. New York: Dekker, 1989, p. 31-57
-
ECCLES, J. C.,
LLINÁS, R.,
SASAKI, K.
The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum.
J. Physiol. Lond.
182: 268-296, 1966.[Medline]
-
ENG, D. L.,
GORDON, T. R.,
KOCSIS, J. D.,
WAXMAN, S. G.
Current clamp analysis of a time-dependent rectification in rat optic nerve.
J. Physiol. Lond.
421: 185-202, 1990.[Abstract]
-
FUKUDA, M.,
YAMAMOTO, T.,
LLINÁS, R.
Simultaneous recordings from Purkinje cells of different folia in the rat cerebellum and their relation to movement.
Soc. Neurosci. Abstr.
13: 603, 1987.
-
HALLIWELL, J. V.,
ADAMS, P. R.
Voltage-clamp analysis of muscarinic excitation in hippocampal neurons.
Brain Res.
250: 71-92, 1982.[Medline]
-
JAHNSEN, H.,
LLINÁS, R.
Electrophysiological properties of guinea-pig thalamic neurons: an in vitro study.
J. Physiol. Lond.
349: 205-226, 1984a.[Abstract]
-
JAHNSEN, H.,
LLINÁS, R.
Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurons in vitro.
J. Physiol. Lond.
349: 227-247, 1984b.[Abstract]
-
LLINÁS, R.
The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function.
Science Wash. DC
242: 1654-1664, 1988.[Medline]
-
LLINÁS, R.,
BAKER, R.,
SOTELO, C.
Electrotonic coupling between neurons in cat inferior olive.
J. Physiol. Lond.
37: 560-571, 1974.
-
LLINÁS, R.,
SASAKI, K.
The functional organization of the olivo-cerebellar system as examined by multiple Purkinje cell recordings.
Eur. J. Neurosci.
1: 587-602, 1989.[Medline]
-
LLINÁS, R.,
VOLKIND, R. A.
The olivocerebellar system: functional properties as revealed by harmaline-induced tremor.
Exp. Brain Res.
18: 69-87, 1973.[Medline]
-
LLINÁS, R.,
WELSH, J. P.
On the cerebellum and motor learning.
Curr. Opin. Neurobiol.
3: 958-965, 1993.[Medline]
-
LLINÁS, R.,
YAROM, Y.
Oscillatory properties of guinea-pig inferior olivary neurones and their pharmacological modulation: an in vitro study.
J. Physiol. Lond.
376: 163-182, 1986.[Abstract]
-
LLINÁS, R.,
YAROM, Y.
Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances.
J. Physiol. Lond.
315: 549-567, 1981a.[Abstract]
-
LLINÁS, R.,
YAROM, Y.
Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro.
J. Physiol. Lond.
315: 569-584, 1981b.[Abstract]
-
MAYER, M. L.,
WESTBROOK, G. L. A
voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones.
J. Physiol. Lond.
340: 19-45, 1983.[Abstract]
-
MCCORMICK, D. A.,
HUGUENARD, J. R. A
model of the electrophysiological properties of thalamocortical relay neurons.
J. Neurophysiol.
68: 1384-1400, 1992.[Abstract/Free Full Text]
-
MCCORMICK, D. A.,
PAPE, H.-C.
Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones.
J. Physiol. Lond.
431: 291-318, 1990a.[Abstract]
-
MCCORMICK, D. A.,
PAPE, H.-C.
Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones.
J. Physiol. Lond.
431: 319-342, 1990b.[Abstract]
-
MCCORMICK, D. A.,
WILLIAMSON, A.
Modulation of neuronal firing mode in cat and guinea pig LGNd by histamine: possible cellular mechanisms of histaminergic control of arousal.
J. Neurosci.
11: 3188-3199, 1991.[Abstract]
-
SASAKI, K.,
LLINÁS, R.
Dynamic electrotonic coupling in mammalian inferior olive as determined by simultaneous multiple Purkinje cell recording (Abstract).
Biophys. J.
47: 53a, 1985.
-
SOLTESZ, I.,
LIGHTOWLER, S.,
LERESCHE, N.,
JASSIK-GERSCHENFELD, D.,
POLLARD, C. E.,
CRUNELLI, V.
Two inward currents and the transformation of low frequency oscillations of rat and cat thalamocortical cells.
J. Physiol. Lond.
441: 175-197, 1991.[Abstract]
-
SPAIN, W. J.,
SCHWINDT, P. C.,
CRILL, W. E.
Anomalous rectification in neurons from cat sensorimotor cortex in vitro.
J. Neurophysiol.
57: 1555-1576, 1987.[Abstract/Free Full Text]
-
TABATA, T.,
ISHIDA, A. T.
Transient and sustained depolarization of retinal ganglion cells by Ih.
J. Neurophysiol.
75: 1932-1943, 1996.[Abstract/Free Full Text]
-
VALLBO, A. B.,
WESSBERG, J.
Organization of motor output in slow finger movements in man.
J. Physiol. Lond.
469: 673-691, 1993.[Abstract]
-
WELSH, J. P.,
LANG, E. J.,
SUGIHARA, I.,
LLINÁS, R.
Dynamic organization of motor control within the olivocerebellar system.
Nature Lond.
374: 453-457, 1995.[Medline]
-
YAMAMOTO, T.,
FUKUDA, M.,
LLINÁS, R.
Bilateral synchronization of climbing fiber activity.
Soc. Neurosci. Abstr.
12: 577, 1986.
-
YAROM, Y.,
LLINÁS, R.
Long-term modifiability of anomalous and delayed rectification in guinea pig inferior olivary neurons.
J. Neurosci.
7: 1166-1177, 1987.[Abstract]