Oscillatory Membrane Potential Activity in the Soma of a Primary Afferent Neuron

Cristina M. Pedroarena,3 Inés E. Pose,3 Jack Yamuy,1 Michael H. Chase,1,2 and Francisco R. Morales1,2,3

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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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Fig. 1. Typical membrane potential oscillatory activity in identified mesencephalic trigeminal (Mes-V) neurons. A: representative coronal section of the brain stem of the rat (LC, locus coeruleus; SCP, superior cerebellar peduncle). down-arrow , location of the stained Mes-V neuron. This diagram represents the level of the brain stem in which most recordings were obtained; at this level, Mes-V neurons are more numerous and aggregate in clusters. B: example of a Mes-V neuron that was labeled by immunostaining of intracellularly deposited biocytin. Process that arises from a globular soma is the axon. Note the absence of dendritic profiles. C: example of recordings obtained from the neuron shown in B that exhibited "spontaneous " membrane potential oscillations on penetration by the microelectrode. - - -, membrane potential value (-52 mV) during brief pauses of the oscillations. Note the characteristic AM of the oscillations. Oscillatory waves were composed of brief depolarizing and hyperpolarizing phases. Inset: spindle-like episode characteristic of the membrane potential activity of depolarized Mes-V neurons. Bottom right, autocorrelation function of this activity is shown; in this neuron the dominant frequency of oscillation was 109 Hz.

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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1. Electrophysiological variables of Mes-V neurons

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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Fig. 2. Voltage changes produced by 100-ms depolarizing current pulses. A: responses of a Mes-V neuron to progressively larger depolarizing current pulses. A1: superimposed averages of the responses to 3 pulses (100 ms, +0.1, +0.4, and +0.5 nA, respectively, traces in 3). - - -, resting membrane potential (-56 mV). A2: response to the 0.4-nA pulse. Note the damped oscillations at the initiation of the pulse. Traces corresponding to +0, 1, and +0.4 nA are averages of 20 individual responses; trace corresponding to +0.5 nA with the truncated action potential is an individual record. B: responses, shown superimposed, to the same 0.2-nA depolarizing pulse applied at the resting membrane potential level (-53.7 mV, trace 1) and during the injection of a -0.9-nA hyperpolarizing current (trace 2), (data correspond to another experiment similar to that illustrated in A). To compare their time courses, the responses were normalized. Traces are averages of 10 individual responses (upper voltage calibration bar corresponds to the trace obtained during hyperpolarization).



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Fig. 3. Voltage changes elicited by hyperpolarizing current pulses. A: responses of a Mes-V neuron to progressively greater 100-ms hyperpolarizing pulses. Top: superimposed averages of the responses to 6 pulses (-0.1, -0.2, -0.5, -0.6, -1.0, and -1.5 nA respectively, bottom). B: amplified view of the voltage responses at the cessation of 4 of the 6 pulses in the example in A. All traces are averages of 20 responses except for the trace that depicts the truncated action potential, this trace is an individual record. C: responses to the -0.2 and the -1.0-nA pulses of A shown superimposed and normalized to compare their time courses. (Upper voltage calibration bar corresponds to the response to -1.0 nA). D: responses shown superimposed to the same -0.2-nA hyperpolarizing pulse applied at the resting membrane potential (-45 mV, trace 1) and during strong membrane hyperpolarization (30 mV, trace 2). (Data from another experiment than those in A-C).

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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Fig. 4. Membrane hyperpolarization abolished the oscillatory activity. A, top: spontaneous membrane potential activity in a Mes-V neuron. - - -, resting membrane potential (-51 mV). Hyperpolarization to -57 mV completely abolished membrane potential oscillations (by injection of -0.5-nA continuous current: A, bottom). B: autocorrelation functions of the membrane potential activity recorded at -51 and at -57 mV (traces superimposed). Note the presence of a robust rhythmic activity at -51 mV and the absence of this activity after a hyperpolarization of 6 mV.



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Fig. 5. Imposed membrane depolarization evoked membrane potential-dependent oscillatory activity. A: representative recordings obtained from a Mes-V neuron that did not show membrane potential oscillations at the "resting" membrane potential (0 nA). Oscillations are observed during the injection of 0.2 nA of depolarizing current. Application of depolarizing currents of increasing magnitude (0.4, 0.5, 0.6 nA) resulted in progressively larger amplitude oscillations. A, top: train of action potentials that arose from one spindle-like episode. B1: autocorrelation functions of the membrane potential activity of each of the recordings in A. These traces indicate that as membrane depolarization increases, oscillatory activity becomes greater. In B2, selected autocorrelations are shown at normalized ordinates to illustrate the almost constant frequency of the oscillations at different membrane potential levels. The dominant frequency of oscillation was 90.9 Hz. Membrane potential on penetration: -51 mV.

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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Fig. 6. Characteristics of the action potential of Mes-V neurons. A: examples of action potentials evoked at the beginning (1) of a depolarizing pulse and at the cessation (2) of a hyperpolarizing pulse (see insets). Spikes were practically identical. B, 1 and 2: afterhyperpolarizing potentials (AHP) that follow spikes obtained from 2 different levels of membrane potential. Spikes were evoked by 0.5-ms depolarizing pulses. B1: AHP of a neuron in the oscillatory state (membrane potential: -50 mV). Note that the AHP is followed by rebound damped oscillations of the membrane potential. B2: AHP in the same neuron when it was evoked from a more hyperpolarized membrane potential level (-55 mV); in this case, the membrane potential of the neuron was not oscillating. AHP was of smaller amplitude and longer duration. Note the difference in time course of the repolarizing phase and the absence of damped oscillations. B3: series of repetitive discharges evoked by a 100-ms depolarizing pulse in the same neuron. A prolonged AHP was not observed.

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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Fig. 7. Repetitive firing of Mes-V neurons. A: examples of neuronal discharge elicited by current pulses (100-ms duration) of progressively greater magnitude. B: repetitive discharges and trains of action potentials evoked by long duration (5 s) current steps of increasing magnitude (0.5, 0.7, and 1.0 nA, respectively, bottom). Note the increase in duration of the first train of spikes with increasingly higher current intensities and the pattern of iterative trains with 0.7 and 1.0 nA. See text for further description. C: train of action potentials corresponding to that signaled in B (*) shown at higher magnification and expanded time base. C, top right: autocorrelation function of the membrane potential activity between trains during the 1.0-nA current step in B. Dominant frequency of the oscillation was 111 Hz (9 ms interval). Bottom right: plot of the intervals vs. time for the train in C (4.8, 6.6, and 5.5 ms, first, last, and mean interval duration, respectively).

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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Fig. 8. Firing evoked by short (0.5 ms) suprathreshold depolarizing pulses depends on the membrane potential level. Top: responses of a Mes-V neuron to a pulse (0.5 ms, 1.0 nA, bottom) obtained at -50-mV membrane potential level. A cluster of spikes followed the pulse. Middle: responses to the same pulse when the neuron was hyperpolarized by 5 mV. Pulse evoked only single spikes.

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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Fig. 9. Actions of TTX, K channel blockers, and low Ca2+ on membrane potential oscillatory activity. A: effects of TTX. Left: traces correspond to membrane potential recordings. Top left: corresponds to the recordings obtained before the application of TTX when this cell was experimentally depolarized to -48.5 mV. This neuron did not exhibit spontaneous membrane potential oscillations at the resting membrane potential level (-51 mV). Bottom left: membrane potential recording during application of TTX (3 µM) when the cell was again depolarized to -48.5 mV. Right: traces are the autocorrelation functions of the recordings shown at the left. Thick trace corresponds to TTX aplication. Note the absence of rhythmic activity. Action potential amplitude of this cell in control conditions was 73.4 mV. This potential also was blocked by TTX. B: effects of TEA (30 mM), 4-aminopyridine (4-AP; 200 µM), intracellular injection of Cs+ ions and perfusion with artificial cerebrospinal fluid (ACSF) in which calcium was replaced for magnesium (0.1 mM CaCl2, 1.9 mM MgCl2) in 4 different Mes-V cells. Left: membrane potential recordings; right: corresponding autocorrelation functions shown superimposed and at normalized ordinates. Thick traces correspond, in each case, to the autocorrelograms obtained during drug application. TEA experiment: the control membrane potential recordings were obtained during experimental depolarization to evoke membrane potential oscillations. Trace corresponding to TEA was obtained without experimental depolarization of the cell. TEA application in this case resulted in a 3-mV depolarization and in the appearance of oscillations. Frequency of the oscillations was 133 Hz in control conditions and 50 Hz during the application of TEA. 4-AP experiment: membrane potential recordings were obtained in control conditions as well as during 4-AP application while experimentally depolarizing the cell with current injection. Membrane potential level during 4-AP was depolarized by 3 mV compared with the control. Oscillation frequency was 125 Hz in control conditions and 77 Hz during 4-AP. Cs+ experiment: membrane potential recordings were obtained 7 min after cell impalement, and 42 min after penetration after active injection of Cs+ ions. Corresponding frequencies were 72 and 20 Hz. Ca2+ replacement experiment: membrane potential recordings were obtained in control conditions and 40 min after switching to a solution containing 0.1 mM CaCl2 and 1.9 MgCl2. In both cases, the cell was experimentally depolarized to -49 mV with current injection. Note that in contrast to the experiments with TEA, 4-AP, and intracellular Cs+, Ca2+ replacement did not result in a slower oscillation frequency.

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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Fig. 10. Effects of TEA and 4-AP on the repetitive firing of Mes-V neurons. A: effects of TEA (5 mM). Top: responses to rectangular depolarizing current pulses (100 ms, 0.3 nA) before (top left) and during (top right) TEA application. Middle and bottom: repetitive firing during similar long-lasting experimental depolarization before and during TEA application. In the middle and bottom traces, the full amplitude of the action potentials is not apparent. B: effects of 4-AP (200 µM). Top: responses to rectangular depolarizing current pulses (100 ms, 0.6 nA) before (top left) and during (top right) 4-AP application. Bottom: repetitive firing during 4-AP application. After 4-AP the cell fired spontaneously in absence of experimental depolarization. down-arrow , small biphasic potentials (see text). Membrane potential was -49 mV and stable for 40 min during 4-AP application, the action potential amplitude was 69 mV. Full amplitude of the action potentials is not apparent in this figure.

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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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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