1Department of Physiology and 2the Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024; and 3Departamento de Fisiología, Facultad de Medicina, Gral Flores 2125, Montevideo, Uruguay
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ABSTRACT |
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Pedroarena, Cristina M.,
Inés E. Pose,
Jack Yamuy,
Michael H. Chase, and
Francisco R. Morales.
Oscillatory Membrane Potential Activity in the Soma of a Primary
Afferent Neuron.
J. Neurophysiol. 82: 1465-1476, 1999.
In the present report, we provide evidence
that mesencephalic trigeminal (Mes-V) sensory neurons, a peculiar type
of primary afferent cell with its cell body located within the CNS, may
operate in different functional modes depending on the degree of their membrane polarization. Using intracellular recording techniques in the
slice preparation of the adult rat brain stem, we demonstrate that when
these neurons are depolarized, they exhibit sustained, high-frequency,
amplitude-modulated membrane potential oscillations. Under these
conditions, the cells discharge high-frequency trains of spikes.
Oscillations occur at membrane potential levels more depolarized than
53 ± 2.3 mV (mean ± SD). The amplitude of these oscillations increases with increasing levels of membrane
depolarization. The peak-to-peak amplitude of these waves is ~3 mV
when the cells are depolarized to levels near threshold for repetitive
firing. The frequency of oscillations is similar in different neurons (108.9 ± 15.5 Hz) and was not modified, in any individual neuron, by changes in the membrane potential level. These oscillations are
abolished by hyperpolarization and by TTX, whereas blockers of
voltage-dependent K+ currents slow the frequency of
oscillations but do not abolish the activity. These data indicate that
the oscillations are generated by the activation of inward
Na+ current/s and shaped by voltage-dependent
K+ outward currents. The oscillatory activity is not
modified by perfusion with low-calcium, high-magnesium, or
cobalt-containing solutions nor is it modified in the presence of
cadmium or Apamin. These results indicate that a calcium-dependent
K+ current does not play a significant role in this
activity. We postulate that the membrane oscillatory activity in Mes-V
neurons is synchronized in adjoining electrotonically coupled cells and that this activity may be modulated in the behaving animal by synaptic influences.
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INTRODUCTION |
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Trigeminal mesencephalic (Mes-V) neurons are
unique among primary afferent neurons in that their cell bodies are
located within the CNS. These cell bodies are contacted by synaptic
buttons in which different neurotransmitters and neuromodulatory
substances have been identified (Copray et al. 1990a,b
,
1991
; Hayar et al. 1997
; Inagaky et al.
1987
; Lazarov and Chouchkov 1995
;
Liem et al. 1993
, 1997
; Nagy et al. 1986
;
Tashiro et al. 1989
; Yamamoto et al.
1988
). Most Mes-V neurons are large unipolar cells that lack
dendritic processes. Peripheral branches of the axons of these neurons
innervate muscle spindles of the jaw closer musculature and periodontal
mechanoreceptors, whereas their central branches innervate jaw closer
motor nuclei, the supratrigeminal nucleus, the principal sensory
nucleus of the trigeminal nerve, and the parvocellular reticular
formation (Appenteng et al. 1985
, 1989
; Cody et
al. 1972
; Corbin and Harrison 1940
,
Dessem and Taylor 1989
; Harrison and Corbin
1942
; Jerge 1963
; Luo and Li
1991
; Luo et al. 1991
; Raapana and
Arvidsson 1993
; Shammah-Lagnado et al. 1992
). In
contrast to other primary afferent neurons, such as dorsal root
ganglion cells, the cell bodies of Mes-V cells receive projections from
other parts of the CNS, such as the reticular formation and the
hypothalamus, in which behavioral functions are integrated. This body
of evidence supports the notion that Mes-V neurons function as
interneurons in networks that control the orofacial musculature
(Del Negro and Chandler 1997
; Kolta et al.
1990
; Nagy et al. 1986
, Roberts and
Witkovsky 1975
; Shammah-Lagnado et al. 1992
;
Ter Horst et al. 1991
).
In the present work, we focused on a specific electrophysiological
property of Mes-V neurons, i.e., their ability to exhibit robust and
sustained membrane potential oscillatory activity on membrane
depolarization. This oscillatory activity may be significant for the
ability of these neurons to function as both primary afferent cells and
as excitatory interneurons within networks involved in jaw movement
control, as originally postulated by Roberts and Witkovsky
(1975) (see also Del Negro and Chandler 1997
;
Nagy et al. 1986
). The present report provides a
description of the basic electrophysiological properties of adult Mes-V
neurons, an analysis of the membrane potential oscillatory activity,
and the pattern of repetitive firing triggered by this oscillatory
activity. Preliminary results of this study have appeared in abstract
form (Pedroarena et al. 1994
).
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METHODS |
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Surgical procedures
Adult Wistar rats (200-250 g) were used in the present work.
The animals were first anesthetized with ether and ketamine (100 mg/kg)
and decapitated. A craniotomy then was performed and the cerebellum
excised. The brain stem was removed carefully and placed in a cold
(4°C) modified artificial cerebrospinal fluid (M-ACSF) in which
sucrose was substituted for NaCl to minimize hypoxic damage
(Aghajanian and Rasmussen 1989). This solution was
dripped continuously over the brain during the surgical procedures
required to remove the brain stem.
The brain stem was glued by its rostral end to the platform of a Vibroslice chamber and covered with cold M-ACSF bubbled with 95% O2- 5% CO2. Coronal slices (400- to 500-µm thick) were cut and placed in a holding chamber that contained M-ASCF at room temperature. The slices were kept in this chamber for 1 h while the M-ACSF was progressively replaced by normal-ACSF (N-ACSF).
A selected brain stem slice was then transferred to a interface-type recording chamber, placed on a piece of filter paper and continuously perfused at a rate of 0.8-1 ml/min with N-ACSF at 32°C.
Solutions
The composition of the N-ACSF solution (in mM) was: 126 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 2 HEPES, and 10 D-glucose. The pH was 7.4. In the M-ACSF, 252 mM sucrose was substituted for the 126 mM NaCl. All solutions were bubbled continuously with a 95% O2-5% CO2 gas mixture. In selected experiments after the properties of Mes-V neurons and the characteristics of their oscillatory activity were examined in N-ACSF, the perfusion solution was changed to one in which calcium was substituted for calcium conductance blockers such as magnesium (0.1 mM CaCl2, 1.9 mM MgCl2) or cobalt (0.1 mM CaCl2, 1,9 mM CoCl2). Tetrodotoxin (TTX, 1-3 µM,), 4-aminopyridine (4-AP, 100-200 µM), cesium chloride (CsCl, 5 mM), and cadmium chloride (CdCl2, 500 µM) were added to the perfusion medium. Tetraethylammonium (TEA, 5-30 mM) was substituted for equimolar amounts of NaCl. Apamin (100 µM) was applied in microdroplets by pressure ejection from a broken micropipette (8-12 µ tip diam) positioned on the surface of the slice in the vicinity of the recording electrode. In three experiments, the recording pipettes were filled with CsCl 3 M to permit intracellular injection of cesium ions (Cs+).
Recording and analysis
Intracellular recordings were obtained with glass micropipette
electrodes (filled with KCl 3 M, tip resistance: 50-100 M) using a
high-input impedance amplifier. These recordings were displayed on a
oscilloscope and on the monitor of a microcomputer and stored for
subsequent analyses on a video cassette recorder .
The electrophysiological properties of Mes-V neurons were measured in the following manner: The resting membrane potential was calculated as the difference between the recorded intracellular potential and that obtained after withdrawing the electrode from the cell. Many Mes-V neurons exhibited membrane potential oscillations on microelectrode penetration (see RESULTS). In these neurons, the membrane potential level was determined during short pauses in oscillatory activity (Figs. 1 and 4, - - -).
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Action potentials were evoked by threshold current pulses. Their
amplitudes were measured from the origin of the spike to its peak. The
spike half-width was the spike duration measured at half-amplitude. To
estimate the magnitude of the action potentials overshoot, the value of
the resting membrane potential was subtracted from the sum of the spike
amplitude plus the membrane potential displacement produced by the
threshold current pulse. These measurements were performed off-line
using software designed to correct for bridge imbalance by digital
methods (Engelhardt et al. 1995; Zengel et al.
1985
).
To analyze the action potential afterhyperpolarization (AHP), averages were obtained of 20 action potentials evoked by short (0.5 ms) current pulses. The AHP amplitude was defined as the difference between the membrane potential level immediately preceding the pulse and the voltage value at the peak of the AHP. The AHP duration was measured from the point in the downstroke phase of the spike that intersected the prespike baseline to the return of the membrane potential to prespike baseline.
The input resistance was measured from the average of 20 voltage
responses to a 100-ms, 1-nA constant current pulse using the
"direct" method wherein the maximal membrane potential change during the pulse is divided by the magnitude of the current
(Engelhardt et al. 1995
; Zengel et al.
1985
). The membrane time constant was measured using the
"peeling" method (Engelhardt et al. 1995
) in neurons
that were hyperpolarized by DC injection to avoid the distortions
produced by active responses of the cell membrane that cannot be
eliminated by the peeling method.
The presence of oscillatory membrane potential activity first was determined visually and then confirmed by an examination of the autocorrelation function of the membrane potential. The dominant frequency of the oscillations was estimated from the autocorrelation function. To obtain these autocorrelation functions, 0.5- to 1-s segments of recording epochs were filtered (500 Hz low-pass), digitized at a 2-kHz sampling frequency, and analyzed using SuperScope II software.
The reported amplitude of the membrane potential oscillations was that of the largest oscillation measured peak to peak during a 1-s period of recording at a membrane potential level just subthreshold for repetitive firing.
Location and identification of Mes-V neurons
In the rat, Mes-V neurons are distributed in a thin band that
extends from the rostral pons to the midbrain (Raapana and
Arvidsson 1993). The brain stem slices used in the present
experiments corresponded to pontine sections at the level of, or just
rostral to, the anterior pole of the trigeminal motor nucleus. The
relevant structures in the sections were located under a dissecting
microscope with the help of tangential illumination. Using this
technique, the locus coeruleus (LC), the fibers of the superior
cerebellar peduncle (SCP), and part of the Mes-V tract were easy to
distinguish. Mes-V neurons were found medial to the Mes-V tract and the
SCP and lateral to the LC.
Five neurons were injected intracellularly with biocytin (2% in KCl
0.5 M) by iontophoresis. Only one cell on each side of an individual
slice was injected. Slices then were submerged in a 4%
paraformaldehyde solution. Biocytin was developed using standard procedures (Horikawa and Armstrong 1988).
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RESULTS |
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The data included in the present report were obtained from 38 Mes-V neurons that exhibited persistent subthreshold membrane potential
oscillations either on impaling the cell ("spontaneous," n = 20) or when they were depolarized by current
injection (n = 18). For the pharmacological portion of
the present work, 30 additional neurons were examined. The recordings
were selected based on their stability and duration (>20 min), by the
amplitude of the spikes (60 mV), and the value of the membrane
potential, which was in all cases more negative than
45 mV.
The mean membrane potential of these neurons was 51.5 ± 3.3 mV
(mean ± SD for these and subsequent entries). Action potentials in Mes-V neurons were of large amplitude (75.1 ± 6.9 mV,
n = 38) and short-duration (width at half-amplitude:
0.32 ± 0.03 ms, n = 38) and exhibited prominent
overshoots (25.9 ± 5.3 mV, n = 26). Table
1 includes the values of other
electrophysiological variables.
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The characteristic membrane potential oscillatory activity exhibited by
one Mes-V neuron is illustrated in Fig. 1C. A
photomicrograph of this neuron, which was injected with biocytin, is
shown in Fig. 1B. Its location is indicated in the diagram
in Fig. 1A. Four other neurons which exhibited this same
kind of oscillatory activity (2 spontaneously and 2 on membrane
depolarization) also were labeled. These cells were large (30-40 µm
diam), spherically shaped neurons. The initial portion of the axon
arising from the cell body was clearly distinguishable; dendritic
processes were not observed. According to their location and general
morphology, these labeled cells are the large, pseudounipolar, Mes-V
neurons described by Luo et al. (1991).
Figures 2 and
3 illustrate the typical responses of
Mes-V neurons to depolarizing and hyperpolarizing current pulses. The voltage changes evoked by the injection of augmenting depolarizing current pulses in a Mes-V neuron at resting membrane potential (56
mV) are depicted in Fig. 2A1. With a pulse of 0.4 nA, a
damped oscillation was elicited (Fig. 2A2). An action
potential that originated from the first peak of the oscillation was
triggered by a 0.5-nA pulse. The time course of the responses was
different in hyperpolarized neurons. Figure 2B depicts the
responses of another neuron to 0.2-nA pulses applied either at resting
membrane potential (
53.7 mV in this example, trace 1in Fig.
2B) or during the injection of
0.9 nA of hyperpolarizing
current (trace 2 in Fig. 2B). During the application of this
hyperpolarizing DC bias, the depolarizing pulse did not elicit membrane
potential oscillations. Instead, the induced voltage drop exhibited a
typical "sag" and, at pulse break, a marked undershoot.
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The voltage changes evoked by hyperpolarizing pulses are illustrated in
Fig. 3A. Figure 3B is an amplified view of the
membrane responses at the end of the pulses. The responses to small
pulses (i.e., 0.1 or
0.2 nA) were clearly different from those
evoked by larger pulses (i.e., at least
0.5 nA; see, for example,
Fig. 3C, wherein the voltage changes to
0.2 and to
1.0
nA were normalized and superimposed). The voltage drop produced by the
0.2-nA pulse reached a minimum and rapidly repolarized to a steady
value; the response evoked by the
1.0-nA pulse reached its negative
peak later and then gradually "sagged." At the cessation of
hyperpolarizing pulses (Fig. 3B), there was a rebound
depolarization that increased in amplitude and developed into a damped
oscillation when larger pulses (
0.5 and
0.6 nA) were applied. An
action potential originated from the first peak of the oscillation at
the end of the largest current pulse (
1.0 nA).
The responses of a neuron to the same pulse (0.2 nA) at two different
membrane potential levels are shown in Fig. 3D. The voltage
drop produced by a
0.2-nA pulse applied at resting potential (
45 mV
in this example, trace 1 in Fig. 3D) rapidly reached its minimum and almost immediately repolarized to a steady value, whereas
when this Mes-V neuron was hyperpolarized (30-mV hyperpolarization, trace 2 in Fig. 3D), the response reached a negative peak
slowly and then gradually declined (sagged). The responses observed at the end of a pulse consisted either of damped oscillations or a slow
and prolonged rebound depolarization (Fig. 3D).
The abovementioned responses of hyperpolarized neurons to subthreshold
pulses (Figs. 2B and 3D) are characteristic of
the membrane rectification attributed to the effects of a
Ih type cationic current (Del
Negro and Chandler 1997; Hayar et al. 1997
; Hille 1992
; Khakh and Henderson 1998
);
indeed, this rectification was blocked by extracellular CsCl (5 mM, see
following text).
Characteristics of membrane potential oscillatory activity
The typical oscillatory activity displayed by Mes-V neurons is illustrated in Figs. 1, 4, and 5. It is apparent that the oscillatory cycles are composed of a sequence of alternating depolarizing and hyperpolarizing phases. Characteristically, this activity exhibited spindle-like amplitude modulation. One of these spindles is illustrated in Fig. 1, inset. The autocorrelation function of the membrane potential activity is illustrated in the top right panel of this figure. The mean dominant frequency of the oscillatory activity for all neurons was 108.9 ± 15.5 Hz (range: 83.3 -142.8 Hz, n = 35). The 10-90% percentile values were between 90.9 and 133 Hz. In terms of cycle duration, the latter values correspond to 11 and 7.5 ms, respectively; this indicates that the membrane potential of Mes-V neurons oscillates within a restricted frequency range. Indeed, in 10 of 35 neurons the activity had the exact same frequency, i.e., 100 Hz (or 10-ms cycle duration).
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In 20 Mes-V neurons, oscillations were present on microelectrode
impalement, but this activity was abolished when the cells were
hyperpolarized (Fig. 4). In another 18 neurons, sustained oscillations
were elicited by depolarization of the cell (as illustrated in the
example in Fig. 5A). The mean membrane potential of these latter neurons was 54.2 ± 2.5 mV, whereas the potential of
those with spontaneous oscillations was
50.2 ± 3.0 mV. The
difference between these two means (4 mV) was statistically significant
(P < 0.001). The mean membrane potential level at
which oscillatory activity was abolished by hyperpolarization, as
illustrated in the example in Fig. 4, was
53.4 ± 2.3 mV. The
level at which oscillations became apparent during depolarizing current
was
52.2 ± 2.4 mV. These values were not statistically
different (P > 0.2). Therefore the membrane potential
level beyond which Mes-V neurons exhibit sustained membrane potential
oscillations is apparently similar among different cells.
In a subset of these Mes-V neurons (n = 8) the input
resistance was measured at membrane potential levels where oscillations were present (51 mV) and again at levels where they had been abolished by experimental hyperpolarization (
60 mV). At these hyperpolarized levels there was an average 28% increase in input resistance (P < 0.05 in a paired t-test).
The amplitude of the oscillations depended on the level of membrane
depolarization. Recordings obtained from one neuron at increasingly
depolarized levels are illustrated in Fig. 5. In this neuron, at
resting potential (51 mV), the oscillatory activity was not present.
It became detectable, superimposed on a noisy baseline, at
49.5 mV,
during the injection of a 0.2-nA depolarizing current. With increasing
levels of depolarization, the amplitude of the oscillations increased
and became the dominant membrane activity. Figure. 5A, top,
illustrates a cluster of action potentials triggered by large
oscillations. Measured at these membrane potential levels, near
threshold for repetitive firing, the mean amplitude of the oscillation
was 3.2 ± 1.1 mV peak to peak. In spite of the membrane potential
dependence exhibited by the amplitude of the oscillations, their
frequency did not change with different levels of membrane potential.
This fact is illustrated in Fig. 5B, 1 and 2,
which includes the autocorrelation functions corresponding to the
recordings depicted in Fig. 5A.
Action potential characteristics and repetitive firing properties
Examples of action potentials evoked in Mes-V neurons by depolarization or at the break of hyperpolarizing pulses are illustrated in Fig. 6A, 1 and 2, respectively. The time courses of the AHPs that followed single spikes are illustrated in Fig. 6B, 1 and 2. These AHPs were obtained from the same cell which was recorded when the membrane potential exhibited oscillatory activity (Fig. 6B1) or during the injection of hyperpolarizing current that abolished this activity (Fig. 6B2). The AHP elicited at depolarized membrane potential levels was followed by damped oscillations, whereas that elicited at hyperpolarized levels was not. These AHPs were brief and their repolarizing phase exhibited an upward convexity. Overall, AHPs in Mes-V neurons that exhibited spontaneous membrane potential oscillations were of larger amplitude and shorter duration than those observed in neurons that did not exhibit spontaneous oscillations (12.9 ± 2.4 mV and 6.2 ± 2.2 ms vs. 9.8 ± 2.4 mV and 13.2 ± 7.1 ms; P < 0.002). The AHPs in Mes-V neurons lacked the long-lasting components that, in other neurons, are due to the activation of Ca2+-dependent K+ currents (K+(Ca2+) currents). As illustrated in Fig. 6B3, this was also the case for AHPs that followed trains of action potentials.
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Examples of the electrophysiological behavior of these neurons during suprathreshold membrane depolarizations are illustrated in Fig. 7. The four traces in the top row of this figure represent responses to current pulses of increasing intensities. In this example, the current threshold for eliciting a single spike was 0.2 nA. Larger pulses evoked a train of discharges the duration of which depended on the magnitude of the pulse. Although there was some degree of adaptation, the discharge frequency was high throughout the train.
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In response to sustained membrane depolarizations, as illustrated in Fig. 7B, Mes-V neurons discharged with recurrent trains of spikes. When the threshold for repetitive discharge (0.5 nA) was attained, a single train was evoked. The discharge ceased abruptly and did not recur in spite of the fact that the depolarizing bias was maintained. When increased levels of membrane depolarization (0.7 or 1 nA in this example) were applied, recurrent trains of spikes were present. The larger the magnitude of the depolarizing bias, the longer the duration of each train and the more frequent their occurrence. A train of spikes (Fig. 7B, *) is shown at an expanded time base and higher gain in Fig. 7C. Membrane potential oscillations of increasing magnitude preceded the first spike of this train. Characteristically, a high frequency of discharge was maintained for the duration of this train. This frequency was higher than the dominant frequency of the oscillation. For example, in this neuron the autocorrelation function of the membrane potential taken between trains indicates a dominant frequency for the oscillations of 111 Hz (9-ms interval), whereas the average frequency of discharge during the train was 181 Hz (5.5- ms interval; see Fig. 7C, top and bottom right).
Each train of spikes stopped abruptly and was not followed by slow AHPs
but rather by oscillations of decreasing amplitude. This behavior
contrasts with the activity of many other neurons which show
pacemaker-like discharge patterns (Hille 1992). The facts that AHPs were short (<10 ms) and were followed by rebound depolarizations may explain the ability of these cells to discharge at
high frequency.
Two different types of responses could be evoked by short current
pulses depending on the membrane potential level. Figure 8 illustrates the response to a 0.5-ms
suprathreshold (1.0 nA) depolarizing current pulse applied to a Mes-V
neuron when sustained oscillations dominated the membrane potential
activity (membrane potential, 50 mV). The pulse triggered a train of
spikes that outlasted the duration of the stimulus. When this neuron
was hyperpolarized to abolish membrane potential oscillations, the cell
responded to the same pulse with single spikes.
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Effects of TTX, K+ channel blockers and Ca2+ removal
These experiments were designed to examine the ionic basis of the
oscillatory activity. Figure
9A depicts the effects of the sodium channel blocker, TTX. In control conditions, large amplitude oscillations were evoked in this cell by experimental depolarization to
48.5 mV (A, top left). After TTX application (bottom
left), the same depolarization (or greater) did not evoke
oscillatory activity. The blocking effect of TTX on the rhythmic
membrane potential activity was also evident on examination of the
autocorrelation functions shown in Fig. 9A, right. Similar
blocking effects of TTX (1-3 µM) were observed in eight neurons in
eight different experiments.
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Figure 9B illustrates the effects of K+ channel blockers (TEA, 4-AP, and intracellular Cs ions) and of Ca2+ replacement in the bathing medium on membrane potential oscillations in four different Mes-V cells. The left traces correspond to membrane potential recordings with conspicuous oscillatory activity obtained in control and under the abovementioned experimental conditions. The corresponding autocorrelation functions are depicted in Fig. 9B, right. For each experiment, the autocorrelations are superimposed and are represented at normalized ordinates to facilitate the comparison of the dominant frequencies. Application of TEA (30 mM) in this example resulted in an increase in amplitude and a decrease in the frequency of the oscillation. TEA (5-30 mM) was applied in five cells from four animals. Overall there was a decrease in the mean frequency of spontaneous and evoked oscillations (113.3 vs. 90.3 Hz, 20% decrease, P < 0.02). In the example shown in Fig. 9B in which doses of 30 mM TEA were used, there was a 62.4% decrease in frequency (from 133 to 50 Hz). The amplitude of the oscillations (compared at similar levels of membrane potential) was increased from a mean value of 1.5 mV to a mean value of 2.7 mV by TEA (data from 4 cells of 3 animals; P < 0.05, see for example Fig. 10A, bottom). 4-AP application (200 µM in the example of Fig. 9B) also lowered the oscillatory frequency. In three cells from two animals, the mean frequencies were 126 versus 83.6 Hz (33.6% decrease, P < 0.05). Intracellular injection of Cs+ ions had a profound effect on membrane potential activity. The oscillatory activity became progressively slower and reached a minimum of 20 Hz in the example illustrated in Fig. 9B (the control frequency was 72 Hz). In this case, it should be noted that recordings obtained 7 min after cell impalement are compared with those obtained 42 min after cell impalement. The effects of intracellular Cs+ ions were studied in three cells from three animals. The frequency of membrane potential oscillations decreased from a mean initial value of 81.3 Hz (measured between 5 and 7 min after cell penetration) to a mean value of 24.2 Hz (70.2% decrease, P < 0.005). Extracellular CsCl (5 mM, 4 cells from 4 animals) had the well-known effect of blocking phenomena attributable to Ih, such as those illustrated in Fig. 3, but did not affect the characteristics of the oscillatory activity. None of the blockers of voltage-dependent K+ channels that we used abolished the oscillatory waves. All of them, however, induced a decrease in the frequency of both the oscillations and spontaneous firing. TEA, 4-AP, and intracellular Cs+ all prolonged the repolarization phase of the spikes recorded from these cells. The mean decay time was increased from 0.45 to 1.55 ms (71%).
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Figure 10A illustrates the effects of TEA (5 mM) on the repetitive firing of a Mes-V cell in response to 100-ms depolarizing pulses (Fig. 10A, top) and to sustained imposed depolarizations (Fig. 10A, middle and bottom). Whereas in control conditions there were only two action potentials in this cell in response to a 0.3-nA depolarizing pulse, during TEA application, the same pulse caused the cell to discharge with a train of eight action potentials. TEA also disrupted the repetitive firing pattern observed during long-lasting imposed depolarizations (compare Fig. 10A, middle and bottom). TEA caused the cell to fire repeatedly in an irregular fashion with shorter silent periods.
The effects of 4-AP (200 µM) on the firing of a Mes-V cell are shown in Fig. 10B. Comparison of the responses to 100-ms depolarizing current pulses of 0.6 nA (Fig. 10B, top) shows an augmented firing during 4-AP. Application of 4-AP also caused the cell to discharge spontaneously at a low frequency as seen in Fig. 10B, bottom. Interestingly, small biphasic potentials (see arrow) also were observed during brief silent periods. This activity may reflect action potentials in a nearby, electronically coupled Mes-V cell, which also became active during exposure to 4-AP. Full-sized action potentials in the recorded neuron emerged from the peak of an oscillatory wave or from these small biphasic wavelets.
Four Mes-V cells were recorded before and after switching the perfusion from N-ACSF to a solution in which CaCl2 was substituted for calcium conductance blockers such as MgCl2 (1 cell) or CoCl2 (3 cells). The recordings were maintained for an average of 40 min after changing solutions. In spite of the prolonged perfusion with a nominal Ca+-free medium, no changes were observed either in the frequency nor in the amplitude of the oscillations (see example in Fig. 9B). Application of apamin (a blocker of calcium-dependent K+ channels; 100 µM by pressure ejection) or CdCl2 (500 µM bath applied, 50 min) did not result in any modification of the oscillatory activity.
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DISCUSSION |
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The evidence obtained in the present work suggests that Mes-V neurons have different modes of operation according to the degree of their membrane polarization.
This conclusion is based on the following results. Mes-V cell somas
when depolarized to levels less negative than 53 mV exhibited sustained membrane potential oscillatory activity (Figs. 1, 4, and 5).
When the membrane potential was close to this value, damped oscillations were observed during or after membrane potential perturbations, i.e., damped oscillations occurred during small depolarizing pulses (Fig. 2), at the cessation of hyperpolarizing pulses, or after action potentials (Figs. 3 and 6). This kind of
activity was not observed when Mes-V cells were hyperpolarized. Brief
depolarizing pulses, which evoked single action potentials when Mes-V
neurons were hyperpolarized, evoked trains of spikes when the level of
membrane potential was close to
53 mV (Fig. 8). At more depolarized
membrane potential levels, these neurons discharged in a characteristic
pattern of repeated trains of spikes (Fig. 7). When taken together,
these data indicate that Mes-V neurons possess a set of conductances
responsible for the generation of membrane potential oscillations.
Because Mes-V neurons are connected by gap junctions (Hinrichsen
and Larramendi 1968
, 1970
; Liem et al. 1991
), it
is possible that oscillations originate from the interaction between
intrinsic membrane properties of these cells and their electrotonic
coupling (Draguhn et al. 1998
; Llinás
1990
; Mann-Metzer and Yarom 1999
; Sherman
and Rinzel 1992
). This coupling would contribute to the
synchronization of the oscillatory activity between connected neurons
(Draguhn et al. 1998
; Llinás 1990
;
Mann-Metzer and Yarom 1999
; Sherman and Rinzel
1992
). On the other hand, there are cases in which membrane
potential oscillations are generated by recurrent synchronized synaptic
activity (Bringuier et al. 1997
). For Mes-V neurons in
the in vitro preparation, this mechanism is unlikely because
oscillations were completely blocked by membrane hyperpolarization and
observed in Ca2+-free medium.
Oscillatory and action potential activity resembling that observed in
Mes-V neurons have been described in sensory ganglion cells
(Amir and Devor 1997; Puil and Spigelman
1988
; Puil et al. 1989
; Wall and Devor
1983
). Chemically mediated cross-excitation takes place between
these neurons (Amir and Devor 1996
). These observations
suggest that the ability to generate oscillations, to fire in trains,
and to communicate via chemical signals or gap junctions is common in
primary afferent neurons.
Clusters of Mes-V cells in close apposition have been described by
anatomists studying material sectioned in both the coronal and
rostrocaudal planes (Hinrichsen and Larramendi 1969,
1970
). Mes-V neurons are not only connected by gap junctions
(Hinrichsen and Larramendi 1968
, 1970
; Liem et
al. 1991
) but also by recurrent axon collaterals (Dessem
and Taylor 1989
; Luo and Dessem 1996
). Therefore
in the intact animal, if Mes-V neurons are depolarized by excitatory
synaptic drives, oscillatory, and/or action potential activity similar
to that described in the present report may be conducted between
populations of interconnected neurons. Inhibitory postsynaptic
processes may be needed, on the other hand, to avoid the generation of
somatofugal action potentials that otherwise would be transmitted to
their peripheral receptors and/or their central target neurons
(Baker and Llinás 1971
). In this regard and in
contrast to the results of early work (see Hayar et al. 1997
), Mes-V cells are now known to be responsive to glutamate (Pelkey and Marshall 1995
), GABA (Hayar et al.
1997
), and ATP (Khakh et al. 1997
). The
neuromodulators serotonin, histamine and dopamine also are known to
exist in fibers that innervate these neurons (Copray et al.
1990a
,b
, 1991
; Inagaky et al. 1987
; Lazarov and Chouchkov 1995
; Liem et al. 1993
,
1997
; Tashiro et al. 1989
).
TTX completely blocked the membrane potential oscillations, indicating
that this activity depends on the activation of TTX-sensitive Na+ current/s. In other neurons, TTX-sensitive
membrane potential oscillations have been attributed to the activation
of persistent Na+ currents (Alonso and
Llinás 1989, Llinás et al. 1991
). At
the present time, however, there is no direct information on the
kinetics of Na+ currents in Mes-V neurons
(Del Negro and Chandler 1997
). Two Na+ currents with relatively rapid activation and
inactivation kinetics have been described in the somata of large- and
medium-sized dorsal root ganglion (DRG) neurons (Caffrey et al.
1992
, Rizzo et al. 1994
), cells that are
homologues of Mes-V cells. Similar types of currents could participate
in the generation of the oscillatory activity observed in Mes-V
neurons. Rapid inactivation of these currents could contribute to the
repolarizing phase of the oscillatory waves.
Potassium channel blockers like TEA, 4-AP, and
Cs+ ions slowed the frequency of the oscillations
but did not abolish them, a fact that raises the question as to the
kind of outward currents that participate in the oscillatory activity
and suggests that more than one type may be involved. Recently,
sustained and transient voltage-dependent K+
outward currents were described by Del Negro and Chandler
(1997) in immature rat Mes-V neurons. According to the voltage
dependence of these currents and to the voltage range in which
oscillations occur, a sustained outward current sensitive to low
concentrations of 4-AP (50 µM) and a slow transient current less
sensitive to 4-AP (>500 µM) could conceivably be involved in the
oscillatory activity. In our experiments, application of 4-AP at a
concentration of 200 µM did not abolish the oscillations, which
suggests that the sustained 4-AP-sensitive current either does not play
an essential role or is not the only K+ current
involved. The slow transient current, which is sensitive to higher
doses of 4-AP, has a window component at the membrane potential levels
at which oscillations occur. Therefore on this basis, it is more likely
that this current underlies the hyperpolarizing phase of these waves.
We may have not blocked it because we used lower doses of 4-AP (200 µm) than those that are required according to Del Negro and Chandler
(>500 µM). Caveats against the present interpretation of our data
are that intrinsic properties, and even the morphology of neurons, are
known to change during development (Berger 1995
;
Spitzer 1979
) and also that experimental conditions of
the in vitro preparation can introduce differences in membrane responses. In this regard, previous work that examined the membrane properties of Mes-V neurons (Del Negro and Chandler
1997
; Yoshida and Oka 1998
) was carried out at
room temperature, whereas our experiments were performed at 32°C.
These different experimental conditions may explain the fact that
others have not observed sustained oscillatory activity in these cells.
The fact that the amplitude of the oscillatory activity was increased
by the application of K+ channel blockers could
be explained by postulating that voltage-dependent K+ channels are open at the resting membrane
potential. The blockade of these conductances may result in cell
depolarization, especially in the absence of a counter balance provided
by K+ currents for the depolarizing actions of
Na+ currents. Indeed, as we have shown in the
results section, application of TEA and/or 4-AP resulted not only in
the development of large oscillatory activity but also in the
development of "spontaneous" action potential activity (see Figs. 9
and 10). The closure of K+ channels at
hyperpolarized membrane potential levels could explain our observation
that cell input resistance increased when the membrane potential was
shifted from a level (51 mV) at which these cells oscillate to a
hyperpolarized level (
60.5 mV) at which oscillations did not occur.
The oscillatory activity was not modified by perfusion with
low-calcium, high-magnesium, or cobalt-containing solutions nor was it
modified in the presence of cadmium or apamin. Therefore it is unlikely
that K+ (Ca2+)
currents are involved in the oscillatory activity. Our data rather indicate that a combination of voltage-dependent
K+ conductances are involved in the oscillatory
mechanisms. These conductances, the nature of which has not yet been
determined, may be a target of neurotransmitters and/or neuromodulators
contained in fibers that innervate these neurons (see preceding text
and Del Negro and Chandler 1997).
In conclusion, in the present report we describe a robust, sodium-dependent fast oscillatory membrane potential activity that occurs in the soma of Mes-V sensory neurons when these cells are depolarized. We suggest that in the intact animal during behaviors that involve the coordination of the jaw musculature, similar membrane potential oscillations serve to synchronize the activity of these neurons.
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ACKNOWLEDGMENTS |
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We thank A. Roca for technical assistance and J. K. Engelhardt for useful suggestions during the revision of this manuscript.
This work was supported by National Institutes of Health Grants HL-61224, AG-04307, NS-09999, and MH-43362, and CONICYT-Fondo Clemente Estable N1082 to I. E. Pose.
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FOOTNOTES |
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Address for reprint requests: F. R. Morales, Dept. de Fisiología, Facultad de Medicina, Gral Flores 2125, Montevideo, Uruguay.
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 30 July 1998; accepted in final form 18 May 1999.
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REFERENCES |
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