Ionic Mechanisms of Intrinsic Oscillations in Neurons of the Basolateral Amygdaloid Complex
Hans-Christian Pape and
Robert B. Driesang
Institut für Physiologie, Medizinische Fakultät, Otto-von-Guericke-Universität, D-39120 Magdeburg, Germany
 |
ABSTRACT |
Pape, Hans-Christian and Robert B. Driesang. Ionic mechanisms of intrinsic oscillations in neurons of the basolateral amygdaloid complex. J. Neurophysiol. 79: 217-226, 1998. Ionic mechanisms underlying low-threshold (LTO) and high-threshold (HTO) oscillations occurring in a class of spiny neurons within the basolateral amygdaloid complex (see companion paper) were investigated in slice preparations of the guinea pig amygdala in vitro. LTOs were abolished through local application of tetrodotoxin (TTX, 10-20 µM) or a decrease in the extracellular sodium concentration ([Na+]o) from 153 to 26 mM, whereas HTOs were more readily elicited under these conditions. The effects of TTX and low [Na+]o were accompanied by a hyperpolarizing shift of the membrane potential by 3 ± 1 mV and a decrease in apparent input resistance by 14 ± 11 M
. LTOs were not observed during intracellular recording with QX 314 (50 µM) or Cs-acetate (2 M) containing micropipettes. At membrane potentials associated with LTO generation, voltage responses to sinusoidal current input with changing frequency between 0 and 10 Hz were characterized by a peak in the response (resonance) at 2.4 ± 1 Hz, largely corresponding to the frequency range of the LTOs. Resonance behavior was evident as a peak in the impedance amplitude plot (ZA-plot) and a maximum in the fast Fourier transformation (FFT). Resonance and LTOs were concomitantly reduced by TTX and barium (Ba2+;2-10 mM) and were preserved during action of extracellular cesium (Cs+; 10-30 mM) or tetraethylammonium chloride (TEA; 20-50 mM), although the peak in the frequency domain tended to shift to lower values in TEA. Application of carbachol (50-200 µM) significantly reduced or blocked LTOs, whereas 4-aminopyridine (4-AP; 10 mM), iberiotoxin (Ibtx, 10 µM), and apamin (20 µM) had no effect. Slow depolarizing/repolarizing current ramps (12.5-125 pA/s) evoked HTOs as rhythmic deflections in membrane potential at either phase of the current ramp. Substitution of extracellular calcium (Ca2+) by magnesium and addition of cobalt chloride (2-4 mM) blocked HTOs but had no measurable effect on the propensity of the cells to produce LTOs. HTOs were abolished within ~10 min after impalement of the cells with a bis-(2-aminophenoxy)-N,N,N
,N
-tetraacetic acid (BAPTA; 200 mM)-containing micropipette. Intracellular Cs+, extracellular Ba2+ (2-10 mM), or extracellular TEA (20-50 mM) induced an increase in amplitude of the rhythmic discharges and an increasingly slowed time course of repolarization at successive oscillatory events, until a steady depolarization was reached at
20 to
10 mV. Application of Ibtx (10 µM) reversibly abolished rhythmic activity during the repolarizing phase of the current ramp, whereas charybdotoxin (2-10 µM) and apamin (20 µM) had no effect. Changes in the chloride (Cl
) equilibrium potential by approximately +30 mV through intracellular recording with a KNO3 (3 M)-containing micropipette or lowering [Cl
]o from 128 to 4 mM, or blockade of Cl
conductances through niflumic acid (100 µM), did not significantly effect LTOs or HTOs. The generation of repetitive spike patterns on membrane depolarization was substantially influenced through removal of extracellular Ca2+ and associated blockade of HTOs, in that the initial high frequent discharge was abolished, frequency adaptation toward slow-rhythmic firing was delayed, and firing occurred at a more irregular pattern during strong depolarizing stimuli. It is concluded that a TTX-sensitive Na+ conductance and the M current contribute to generation of the LTOs, although their exact role in rhythmogenesisremains to be determined. HTOs seem to largely depend on a functional coupling between high-voltage-activated Ca2+ conductances, a Ca2+-activated K+ current presumably carried through BKCa channels, and additional voltage-dependent K+ conductances. In functional terms, the HTOs are important in determining spike frequency adaptation toward a slow-rhythmic firing pattern during maintained depolarizing influence.
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INTRODUCTION |
In the preceding paper, two types of slow intrinsic oscillations of the membrane potential at 2-10 Hz were described in neurons of the lateral (AL) and basolateral (ABl) amygdala. The oscillations, referred to as low- (LTO) and high-threshold oscillation (HTO), occurred at a range of membrane potentials negative and positive to the threshold for generation of fast spikes, respectively, thereby largely contributing to the generation of regular spike patterns in response to ongoing depolarizing influence. The present study was aimed at evaluating the ionic mechanisms underlying these intrinsic oscillations. Particular interest was paid to sodium (Na+) and calcium (Ca2+) membrane conductances, due to the significance of a noninactivating Na+ current for the generation of subthreshold slow oscillations (Alonso and Llinás 1989
; Amitai 1994
; Gutfreund et al. 1995
; Klink and Alonso 1993
; Leung and Yim 1991
), and the involvement of low-threshold- and high-threshold-activated Ca2+ currents in slow oscillatory activity at hyperpolarized and depolarized membrane potentials in other types of neurons (e.g., Llinás and Yarom 1981a
,b
, 1986
; McCormick and Pape 1990
). In light of conflicting results evolving from experimental and computational studies on the repolarizing mechanisms involved in subthreshold oscillations in different types of cells (Gutfreund et al. 1995
; Klink and Alonso 1993
), the possibility was investigated in detail that different components of K+ outward as well as chloride (Cl
)-dependent mechanisms may contribute to the different types of oscillatory activity.
 |
METHODS |
Experiments were performed in slices of the basolateral amygdaloid (BL) complex from guinea pigs (200-350 g). The procedure of preparation and maintenance of the slices, and the electrophysiological techniques that were used were similar to those described in the preceding paper. Glass microelectrodes from thin-walled capillaries (TW-100F, World Precision Instruments, Sarasota, FL) were prepared on a Flaming/Brown micropipette puller (Model P-87, Sutter Instruments, San Rafael, CA) and filled with 4 M K-acetate (resistances of 40-70 M
), 3 M KNO3 (resistances of 40-60 M
), or 1% biocytin in 2 M K-acetate (resistances of 50-90 M
). In some experiments, neurons were impaled with microelectrodes containing at the outermost tip 2 M K-acetate, 1% Biocytin and at more proximal parts 2 M cesium (Cs+) acetate (resistances of 50-70 M
), or 4 M K-acetate with either 50 µM N-ethylbromide lidocaine (QX 314; resistances of 40-70 M
) or 50-200 mM bis-(2-aminophenoxy)-N,N,N
,N
-tetraacetic acid (BAPTA; resistances of 30-60 M
).
Drug application
Changes in the ionic composition of the bathing medium were achieved by the following substitutions: 1) the extracellular Na+ concentration was decreased from 153 to 26 mM by replacing NaCl and NaH2PO4 with choline chloride, 2) Ca2+ was omitted from the solution by replacing CaCl2 with 2-4 mM MgCl2, 3) tetraethylammonium (TEA) was applied by equimolar substitution for NaCl, and 4) the extracellular Cl
concentration ([Cl
]o) was lowered from 128 to 4 mM by substitution of sodium isethionate for NaCl and KCl by K-acetate. DC offsets due to changes in junction potential at the Ag-AgCl reference electrode in different Cl
concentrations were compensated in the following way: the membrane potential at which Na+ spikes were elicited was determined in normal bathing solution. During introduction of a different Cl
solution, this firing threshold was repeatedly monitored, and its reading was kept constant by adding current to the input circuitry of the amplifier. Complete exchange of the solution was indicated by a constant reference potential. At the end of the experiment, the change in reference potential was controlled by measuring the DC offset of the electrode in normal bathing solution. Finally, when cobalt, cadmium, or Ba2+ were introduced, MgCl2 was substituted for MgSO4 and NaCl for NaH2PO4 to avoid precipitation. Substances were superfused on parts of the slice containing the AL and ABl through pipettes of a large tip diameter (10-20 µm) by constant low pressure (Picopritzer II, General Valve, Fairfield, NJ). Carbamylcholine chloride (carbachol, CCh) was locally applied in small volumes (5-15 pl) through pipettes (tip diameter 2-5 µm) that were either positioned on the exposed surface of the slice or lowered into the slice, until maximal responses were elicited. Substances were obtained from Sigma, except for
-conotoxin GVIA (Cgtx),
-agatoxin IVA (Agtx), iberiotoxin (Ibtx; from Research Biochemicals International, Natick, MA), and CGP 35348, which was a generous gift from Ciba Geigy (Basel). Cgtx, Agtx, and Ibtx were prepared as concentrated stock solutions, kept frozen, and diluted in slice solution immediately before each experiment.
Oscillation analysis
Evaluation of pharmacological influences on the LTO turned out to be difficult, because of the rather small amplitudes of the LTO (2-6 mV) and the contaminating effect of fast spikes. Therefore the frequency domain analysis (Gutfreund et al. 1995
; Hutcheon et al. 1996a
; Puil et al. 1986
) was used to estimate drug-induced alterations of the LTOs. In particular, a direct current (DC) was injected to hold the neurons near the desired membrane potential, followed by injection of an alternating current (AC) with constant amplitude and a linearly changing frequency between 10 and 0 Hz (Fig. 2C, left trace). This current input function was produced with the software Spike2 for Windows operating on a CED 1401 interface (Cambridge Electronic Design, Cambridge, UK). The amplitude of the current input function was adjusted to keep the resulting deflections in membrane potential at ~2-6 mV peak-to-peak. The input current and the voltage response were digitized at a sampling rate of 2 kHz, and filtered with anti-aliasing filter at 25 Hz upper corner frequency. This current function was repeatedly applied three to five times at each potential. ASCII files were read into the software Microcal Origin V4.01 (Microcal Software, Northampton, MA), data were averaged and transformed into the frequency domain through fast Fourier transformation (FFT). Part of the noise in the FFT curves resulted from the discrete transformation procedure and increased at the boundaries. Impedance (Z) is a complex number defined as Z = Zreal + Zimaginary = FFT(V)/FFT(I) (cf. Fig. 2). Because of the increased noise level at the boundaries, this calculation turned out to be useful for frequencies between 0.85 and 9 Hz. The curves were smoothed using a 10-point adjacent averaging algorithm to further reduce noise. The LTO was represented as a peak in impedance or amplitude at2.4 ± 1 Hz in the impedance amplitude plot (ZA-plot) and FFT, respectively (Fig. 2, A and C). The peak was determined by differentiating a third-order polynomial fit of the smoothed curve. Frequency domains were compared at different prevailing values of the membrane potential before and during action of pharmacologically active substances. To investigate the effects of CCh on LTOs, a slow depolarizing current ramp (3.5-7 pA/s) was injected from a potential of ~10 mV negative to spike threshold, and the amplitude of the current ramp was adjusted to move the membrane potential through the voltage range of LTO generation to firing threshold. Before calculation of a FFT, the slow current-induced voltage shift was subtracted from the original traces through the use of polynomial fits of first (control, wash) or third order (CCh). FFTs were then calculated over a period of 8.192 s ending 20 ms before generation of the first spike.

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| FIG. 2.
Resonance behavior: voltage dependence and TTX sensitivity. Voltage responses to an alternating current (AC) with constant amplitude and a linearly changing frequency (10 to 0 Hz, as shown in left panel of C) injected at different prevailing membrane potentials. A: at a membrane potential just subthreshold to Na+ spikes ( 53 mV), a peak in response amplitudes is reached at 13 s after current onset (left trace) and is represented as a peak at 2.5 Hz in the impedance amplitude plot (ZAP, right panel). Local application of TTX (20 µM) strongly reduces resonance behavior (middle trace) and peak in the ZA-plot (right). The ZA-plot is smoothed in this and following figures by 10-point adjacent averaging. Note that the amplitude of the current input function has been increased by +5 dB during action of TTX (cf. horizontal lines in C) to elicit amplitudes of voltage response comparable with those obtained under control conditions. B: the same current input function applied at resting membrane potential ( 69 mV). The maximum impedance is reached at lowest frequencies, as is expected for a passive resistor-capacitor (RC) circuit, with no measurable differences occurring before and during action of TTX. C: current input function, starting with a frequency of 10 Hz with continuous decrease to 0 Hz within 20 s (left trace). The fast Fourier transformation (FFT) plot of the current function (middle) reveals an almost equal distribution of the frequencies with increasing errors at the boundaries. FFT plot of the membrane potential at 53 mV (right) reveals a peak under control conditions and no clear peak in TTX.
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After blockade of the LTOs through tetrodotoxin (TTX), HTOs were analyzed in the following way. A slow depolarizing current ramp (25-125 pA/s) was injected at resting potential, whose amplitude was adjusted to move the membrane through the voltage range of HTO generation toward inactivation of the oscillatory mechanisms (Fig. 6; cf. companion paper Pape et al. 1998). The maximal current was maintained for 5 s, followed by a slow repolarizing current ramp (12.5-62.5 pA/s) toward the zero current level. The HTO was clearly discernible as rhythmic deflections in membrane potential during the depolarizing and repolarizing phase of the current ramp (Fig. 6). The amplitude of the current ramp was adjusted to reach similar final values of the membrane potential before and during drug action, and drug-induced changes in resting potential were compensated by DC injection. Data are presented as means ± SD.

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| FIG. 6.
Contribution of K+ conductances to generation of HTOs. All recordings were obtained during blockade of LTOs and Na+ spikes through TTX. Neurons are moved from rest through the potential range of HTO generation toward inactivation of the oscillatory mechanisms by injection of a slow depolarizing current ramp (bottom traces). The maximal current is maintained for 5 s, followed by a slow repolarizing current ramp back to resting potential. HTOs are seen as rhythmic deflections of the membrane potential (middle trace; numbers indicate resting potential) during the depolarizing and repolarizing phase of the current ramp. Top traces display periods (2 s) of HTOs at a faster time as indicated; dashed line indicates the respective value of the membrane potential. Small downward deflections are hyperpolarizing current pulses and resulting changes in membrane potential, indicating the apparent input resistance. Scale bars in C are for A-C. Intracellular recording with a Cs-acetate (2 M)-filled electrode (A), extracellular application of TEA (20 mM, B) or Ba2+ (2 mM, C), all result in an increase in amplitude and increasingly delayed repolarization of the HTOs, resulting in a typical shoulder with increasing duration at successive oscillatory events and, eventually, an interruption of HTOs and steady depolarization at 20 to 10 mV. The repolarizing phase of the current ramp is associated with a sudden hyperpolarizing drop in membrane potential of ~40 mV amplitude toward near resting level. Note that the amplitude of the current ramp was adjusted to reach similar final values of the membrane potential before and during drug action, and drug-induced changes in resting potential were compensated by injection of DC. Recordings in A were obtained 21 and 79 min after impalement of the neuron, respectively.
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 |
RESULTS |
The data are based on stable intracellular recordings in a sample of 90 neurons in the guinea pig AL and ABl that possessed electrophysiological properties similar to those described in the preceding paper. The characteristics included an average resting potential at
70 ± 5.5 mV, a resting input resistance at 98.6 ± 25.1 M
, overshooting action potentials, and the generation of LTOs and HTOs in response to maintained depolarizing stimuli (Figs. 1 and 5). The LTOs and HTOs were not significantly different in neurons of the AL and ABl, and therefore the results are pooled.

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| FIG. 1.
Na+ dependence of low-threshold oscillations (LTOs). A: replacement of extracellular Ca2+ by Mg2+ (0 [Ca2+]o) and addition of cobalt chloride (4 mM) has no effect on the propensity of a guinea pig lateral amygdala (AL) neuron to produce LTOs, whereas local application of tetrodotoxin (TTX; 20 µM) abolishes LTOs in the same cell. Note that removal of Ca2+ blocks the slow component of hyperpolarizing afterpotentials following spikes (inset, averaged from 5-7 trials). B: decreasing the extracellular Na+ concentration from 153 to 26 mM (low [Na+]o) blocks fast spikes and LTOs. Note the decrease in apparent input resistance in low [Na+]o, as indicated by neuronal responses to a hyperpolarizing current pulse. The hyperpolarizing shift of the membrane potential during action of TTX and low [Na+]o was compensated by intracellular injection of outward current (current traces not shown). Numbers near voltage traces indicate the prevailing membrane potential. Spikes in B and in A, inset, are truncated.
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| FIG. 5.
Ca2+ dependence of high-thresholds oscillations (HTOs). A: decreasing the extracellular Na+ concentration from 153 to 26 mM (low [Na+]o), or local application of TTX (20 µM), has no effect on the propensity of an amygdaloid neuron to produce HTOs. Note that in low [Na+]o, fast spikes are blocked and replaced by a high-threshold Ca2+ spike (insets a1 and a2), and HTOs occur in a more regular manner. B: replacing extracellular Ca2+ by Mg2+ and adding 4 mM cobalt (0 [Ca2+]o) abolishes HTOs and blocks slow hyperpolarizing afterpotentials following repetitive firing (insets b1 and b2). Reinstating extracellular Ca2+ partly restores the HTOs (wash). Numbers near voltage traces indicate the prevailing membrane potential; downward deflections are neuronal responses to hyperpolarizing current pulses (current traces not shown).
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Depolarizing mechanisms involved in the generation of LTOs
The interruption of LTOs after application of TTX (n = 30) (cf. companion paper Pape et al. 1998) suggested a contribution of Na+-dependent mechanisms. Indeed, decreasing the extracellular Na+ concentration ([Na+]o) from 153 to 26 mM resulted in a blockade of fast spikes and abolished the LTOs in all tested cells (n = 4; Fig. 1B). The effects of TTX and low [Na+]o were accompanied by a hyperpolarizing shift of the membrane potential by 3 ± 1 mV and a decrease in apparent input resistance by 14 ± 11 M
(n = 14), indicating reduction of a steady inwardly directed membrane conductance that is activated at the voltage range of LTO generation. The interruption of LTOs was not secondary to the shift in membrane potential and decrease in input resistance, because the initial value of the membrane potential was routinely restored through injection of depolarizing current, and an increase in apparent input resistance through local application of Mg2+ (10 mM) did not reinstate the LTOs (n = 6; data not shown). In addition, LTOs were not observed when neurons were impaled with micropipettes containing the lidocaine derivative QX 314 (50 µM), and which blocked fast Na+ spikes within ~5-10 min after impalement (n = 8; data not shown). Omitting Ca2+ from the bathing medium and adding cobalt chloride (2-4 mM) resulted in a decrease in amplitude and duration of hyperpolarizing afterpotentials succeeding fast spikes and blocked the HTOs (see below), but had no measurable effect on the propensity of the cells to produce LTOs (n = 8; Fig. 1A). In fact, the LTOs appeared more regular in low [Ca2+]o, presumably due to blockade of interacting synaptic influences. Local application of TTX (20-30 µM) abolished the LTOs in the same cells (Fig. 1A), thereby strongly indicating the contribution of a TTX-sensitive, Na+-dependent membrane conductance.
The ionic mechanisms underlying the LTOs were further examined using the frequency domain analysis. Voltage responses of 18 neurons to intracellular injection of current with changing frequency (range 10 to 0 Hz) were analyzed. At resting potential (i.e.,
69 mV), maximal voltage response to the sinusoidal current input occurred at lowfrequencies, and the response decreased monotonicallyat higher frequencies (Fig. 2B, left trace). The respectiveZA-plot demonstrated a monotonic decrease in impedance with increasing frequency, as is expected for a passive resistor-capacitor (RC) circuit (Fig. 2B). When the membrane was depolarized through DC injection into the range of LTO generation and subthreshold to generation of fast spikes, i.e., to
53 mV, a peak in the response (resonance) was reached at intermediate frequencies (Fig. 2A, left trace). The resonance behavior was evident as an impedance peak in the ZA-plot (Fig. 2A) and a maximum in the FFT (Fig. 2C). The maximum occurred at an average frequency of 2.4 ± 1 Hz (n = 18), i.e., well within the frequency range of the LTO (1-3.5 Hz; cf. companion paper). That this resonance behavior indeed represented the LTO was indicated by the finding that both phenomena, resonance and LTO, were substantially reduced or blocked by local application of TTX (20 µM; n = 3). During action of TTX, the resonance peak was reduced to ~30% in the ZA-plot (Fig. 2A), and frequency responses displayed no clear maximum in the FFT (Fig. 2C). The interruption of resonance behavior was not secondary to the shift in membrane potential and decrease in input resistance during action of TTX, because the initial value of the membrane potential was routinely restored through injection of depolarizing current, and the amplitude of the current input function was adjusted to keep the resulting deflections in membrane potential similar to the maximal deflections obtained under control conditions (Fig. 2A, middle column; Fig. 2C, left column). Moreover, TTX had no measurable effect on neuronal responses in the frequency domain at around the resting potential (Fig. 2B).
The hyperpolarization-activated cation current, termed Ih, reportedly contribute to generation of LTOs in neocortical neurons (Hutcheon et al. 1996a
,b
). In amygdaloid neurons, extracellular application of Cs+ at 10-30 mM, which blocks Ih in many preparations (as reviewed by Pape 1996
), had no measurable effect on the LTOs (n = 9) or the resonance behavior (n = 4). Moreover, there was very little, if any, anomalous membrane rectification in the hyperpolarizing direction in the population of oscillating amygdaloid neurons (data not shown).
Hyperpolarizing mechanisms of LTOs
During intracellular recording with Cs-acetate (2 M)-filled electrodes, LTOs were not observed (n = 5; data not shown), indicating a contribution of K+-mediated mechanisms. Therefore the effect of various types of K+ channel blockers on the generation of LTOs was examined using the frequency domain analysis. Local application of Ba2+ (2-10 mM) resulted in an increase in membrane impedance, an increase in duration of action potentials, and an interruption of LTOs and resonance behavior in all cells that were tested (n = 11; Fig. 3A). Application of TEA at concentrations of 20-50 mM resulted in a substantial increase in duration of Na+ spikes and high-threshold Ca2+ action potentials (see, e.g., Fig. 6). LTOs were preserved in all cells that were tested (n = 5), although the peak in the frequency domain sometimes shifted to lower values during action of TEA (n = 2; Fig. 3C). In addition, 4-AP (10 mM), a blocker of A-type K+ conductances (reviewed by Rudy 1988
; Storm 1993
) known to contribute to spike repolarization in amygdaloid neurons (Gean and Shinnick-Gallagher 1989
), did not significantly affect the generation of LTOs(n = 6; data not shown). Ibtx (10 µM; n = 3) and apamin (20 µM; n = 6), which block Ca2+-activated K+ channels of the BKCa- and SKCa-type, respectively (reviewed by Sah 1996
), had no measurable effect on the LTOs (data not shown). Finally, intracellular recording with a 3 M KNO3-filled micropipette and injection of NO
3, which readily passes through Cl
channels but does not support outward Cl
transport (e.g., Thompson et al. 1988
), resulted in a positive shift in the Cl
-equilibrium potential by approximately +30 mV, as was indicated by a respective shift in the reversal potential of strychnine-sensitive glycine-induced responses. By contrast, LTOs were not significantly influenced under these conditions (n = 3; data not shown).

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| FIG. 3.
Effects of extracellular Ba2+ (A and B) and TEA (C and D) on resonance behavior. Stimulus protocols as in Fig. 2. A: at 56 mV, resonance behavior occurs at 3.9 Hz under control conditions (left trace and ZAP), and is blocked on application of Ba2+ (2 mM; middle trace and ZAP). Partial recovery from Ba2+ is indicated by the impedance peak at 1.4 Hz in the ZAP. B: at resting membrane potential ( 70 mV), the maximum impedance is reached at lowest frequencies with no measurable differences occurring before and during action of Ba2+. Note the similar ZA-plots at 56 and 70 mV during action of Ba2+. C: at 52 mV, application of tetraethylammonium (TEA; 20 mM) results in a shift of the impedance peak from 4 Hz (control) to 2.5 Hz (TEA). D: at resting membrane potential, resonance behavior is lacking before and during action of TEA.
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The subthreshold activation range of the LTOs suggested an involvement of the M current, which differs from other types of K+ currents in that it both activates below the spike threshold and does not inactivate and is completely suppressed by muscarinic agonists (Brown and Adams 1980
; reviewed by Brown 1988
). The M current has been found to exist and to be suppressed by CCh in basolateral amygdaloid neurons (Womble and Moises 1992
). In the present study, local application of CCh (50-200 µM) resulted in an increase in input resistance and blockade of the slow hyperpolarizing afterpotentials following repetitive spike activity (Fig. 4B) (cf. Washburn and Moises 1992
; Womble and Moises 1993a
). Analysis of resonance behavior turned out to be difficult during action of CCh, due to the high-input resistance and strong tendency of the cells to fire action potentials on injection of sinusoidal current at low frequencies. Therefore the influence of CCh on oscillatory activity was investigated using FFT of LTOs in 14 cells. Injection of a slow depolarizing current ramp was used to move the membrane from resting level through the range of LTO generation toward the threshold for spike generation. LTOs were clearly discernible as rhythmic deflections of the membrane potential in a subthreshold range of membrane potential (Fig. 4A). After application of CCh, responses to the slow depolarizing current ramp consisted of a relatively fast depolarization of concave trajectory with no indication of LTOs (Fig. 4C). Calculation of FFT over a period of 8.192 s before generation of the first spike (see METHODS) verified the presence and absence of LTOs before and during action of CCh, respectively (Fig. 4, E and F). Moreover, during action of CCh, the membrane potential could not be stabilized (e.g., through injection of a holding current) in a region just subthreshold to spike generation due to a depolarizing response that was intrinsically generated by the cell. In the population of cells that were tested, LTOs were abolished in six cells and reduced by >50% in eight cells during action of CCh, as judged from the decrease in power of the resonance peak at ~2.3 Hz in the FFT. The effects of CCh were reversible (Fig. 4, D and G).

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| FIG. 4.
Sensitivity of LTOs to carbachol (CCh). Depolarizing current ramps are injected from predepolarized levels of the membrane potential and adjusted to move the membrane through the range of LTO generation toward firing threshold. A: under control conditions, LTOs occur in a voltage range before spike generation (indicated by horizontal bar). B: after local application of CCh (100 µM), the slow afterhyperpolarization (AHP) following a train of spikes (evoked by injection of a 0.8 nA, 500 ms current pulse) is blocked and replaced by a depolarizing afterpotential. C: during action of CCh, following the current ramp the neuron is relatively fast depolarized comparing with a concave trajectory with no indication of LTO. D: partial recovery of the LTOs occurs ~10 min after removal of CCh (wash). Note that the neuron is depolarized from -65 mV and the 1st spike is elicited 24 s after beginning of the current ramp. E-G: FFTs calculated for an 8.192-s interval before the occurrence of the 1st spike in the respective voltage traces in A, C, and D (cf. bar in A). Under control conditions, the peak in the FFT at 2.3 Hz indicates LTOs, is abolished during action of CCh, and partially recovers after wash. Spikes are truncated in all traces.
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Depolarizing mechanisms contributing to production of HTOs
Decreasing [Na+]o from 153 to 26 mM (n = 4) or applying TTX (20-30 µM; n = 45) blocked LTOs and fast Na+ spikes (Fig. 5A, a1 and a2), but did not abolish HTOs (Fig. 5A). Rather, the HTOs were more readily elicited by membrane depolarization in low [Na+]o or TTX, presumably due to the decrease in slope conductance at potentials negative to the range of HTO production associated with blockade of the Na+-dependent mechanisms (cf. Fig. 3 in the preceding paper). By contrast, omitting Ca2+ from the extracellular solution and adding cobalt chloride (2-4 mM) or cadmium chloride (0.5-1 mM) blocked HTOs in all cells that were tested (n = 8, Fig. 5B). In addition, hyperpolarizing afterpotentials following spike discharges were substantially reduced (Fig. 5B, b1 and b2). The notion that Ca2+-dependent membrane conductances are critically involved in the generation of HTOs was supported by the observation that HTOs were abolished within ~10 min after impalement of the cells with a BAPTA (200 mM)-containing micropipette (n = 11, data not shown). Local application of nifedipine (100 µM; n = 2), and the selective Ca2+ channel antagonists Cgtx (100 µM; n = 2) (Tsien et al. 1988
) and Agtx (50 µM; n = 1) (Mintz et al. 1992
) did not significantly affect the HTOs (data not shown).
Mechanisms involved in the repolarizing phase of HTOs
Repolarizing mechanisms of HTOs were analyzed during presence of TTX, which blocked fast spikes and LTOs. During intracellular recording with Cs-acetate (2 M)-filled electrodes, repolarization of the HTOs was increasingly delayed, resulting in a typical shoulder with increasing duration at successive oscillatory events and, finally, in an interruption of the HTOs (n = 9, not shown). The underlying mechanisms were further analyzed by slowly moving the membrane from rest through the potential range of HTO generation by injection of a slow depolarizing/repolarizing current ramp (see METHODS). The HTOs occurred as rhythmic deflections in membrane potential during both the depolarizing and the repolarizing phase of the current ramp (Fig. 6A). Intracellular Cs+ induced an increase in amplitude of the rhythmic discharges and an increasingly slowed time course of repolarization, until a steady depolarization was reached at approximately
20 to
10 mV (Fig. 6A). Depolarization was maintained during the initial repolarizing phase of the current ramp, followed by a sudden drop in membrane potential toward the activation threshold of the HTOs during further relieve of depolarizing current (Fig. 6A). Similar effects were observed after application to oscillating cells of TEA (20-50 mM; n = 7; Fig. 6B) or Ba2+ (2-10 mM; n = 3; Fig. 6C), thereby strongly indicating the involvement of K+ conductances in the repolarization of the HTOs. These conductances appear to include Ca2+-activated K+ channels, because local application of Ibtx (10 µM), a blocker of BKCa channels (reviewed by Kaczorowski et al. 1996
; Sah 1996
), reversibly abolished rhythmic activity during the repolarizing phase of the current ramp (n = 3; Fig. 7). Apamin (20 µM; n = 8) and charybdotoxin (2-10 µM; n = 4), by comparison, did not exert a measurable effect on the HTOs. Finally, changes in the Cl
equilibrium potential by about +30 mV (as indicated by a respective shift in the reversal potential of strychnine-sensitive glycine responses) through intracellular recording with a KNO3 (3 M)-containing electrode (n = 6) or lowering [Cl
]o from 128 to 4 mM (n = 3), or blockade of Cl
conductances through local superfusion of niflumic acid (100 µM; n = 2), did not significantly influence generation of HTOs (data not shown).

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| FIG. 7.
Effects of iberiotoxin (Ibtx) on HTOs. Same stimulus protocol as in Fig. 6. After local application of Ibtx (10 µM), repolarization of HTOs is slowed, and HTOs are abolished during the repolarizing phase of the current ramp and replaced by short periods of damped oscillations following short hyperpolarizing current pulses. Recovery occurs 15 min after wash of Ibtx.
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Influence of Ca+-dependent mechanisms on repetitive firing patterns
The results of the preceding paper indicated a contribution of intrinsic oscillatory properties to the patterning of repetitive firing during maintained levels of membrane polarization. To further evaluate the influence of Ca2+-mediated HTOs, long (>50 s) depolarizing current pulses of increasing amplitude were injected from resting levels, and firing patterns were analyzed using plots of instantaneous frequency versus time before and after blockade of HTOs by removing Ca2+ from the bathing medium and adding cobalt chloride (2-4 mM, n = 3). A typical experiment is illustrated in Fig. 8. As noted before (Fig. 5 in the preceding paper), responses of the cells in the control solution consisted of an initial high-frequency discharge of Na+ spikes, the frequency of which depended on the amplitude of the depolarizing current, followed by a rapid decline toward slow-rhythmic firing at 2-10 Hz. Removal of extracellular Ca2+ strongly reduced the initial high-frequent discharge (Fig. 8, A1 and B1) and significantly delayed the decline toward slow-rhythmic firing (Fig. 8B2), thereby resulting in an increase in firing frequency in an intermediate period of time between 0.3 and 40 s after onset of the depolarizing stimulus (Fig. 8, A and B). At increased depolarizing current strength, ongoing activity following the initial accommodating period was associated with a progressive decrease in amplitude and an increase in duration of the Na+ spikes, due to Na+ inactivation and recruitment of the mechanisms underlying HTOs (see also Figs. 4 and 6 in Pape et al. 1998). HTOs fully replaced Na+ spikes within 1-2 min, resulting in a constant frequency of electrogenic activity at 6 Hz determined by the HTOs (Fig. 8C3). During blocked influx of Ca2+, spike frequency adaptation was characterized by a slower decline compared with that under control conditions (Fig. 8C1), a sudden drop in frequency at ~10-20 s after onset of the depolarizing stimulus (Fig. 8C2), and maintained firing at slow frequency and irregular pattern (Fig. 8C3).

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| FIG. 8.
Firing patterns of an AL neuron in response to long depolarizing current pulses of increasing amplitude (A: +0.3 nA; B: +0.5 nA; C: +0.8 nA; current traces not shown) injected at resting potential ( 65 mV) before (control) and after removal of extracellular Ca2+ and addition of 4 mM cobalt. Left column (a) illustrates the membrane potential and plots of instantaneous frequency vs. time (conditions before and after removal of Ca2+ are represented by lines and dots, respectively). Numbered periods of neuronal activity are depicted at faster time scale in columns (b, control; c, 0 Ca2+). Note that removal of extracellular Ca2+ results in a blockade of the initial high-frequent discharge (A1, B1), a delayed decline toward slow-rhythmic firing (B2), and a sudden drop in frequency followed by a slow irregular firing pattern at stronger depolarizing influence (C2, C3). See text for further details.
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DISCUSSION |
In the present study, frequency domain analysis and current ramp protocols were combined with pharmacological approaches to understand the mechanisms underlying the two types of intrinsic oscillations operating at different ranges of membrane potential in amygdaloid neurons. A first point of discussion has to consider frequency domain analysis that was used to investigate the mechanisms underlying LTOs. The tendency of the neurons to generate subthreshold voltage signals at a certain frequency (low-threshold oscillatory activity) and to preferentially respond to inputs at a certain frequency (resulting in resonance in the frequency domain) are clearly related phenomena (see, e.g., Gutfreund et al. 1995
; Hutcheon et al. 1996a
,b
; Lampl and Yarom 1993
, 1997
). In amygdaloid neurons of the present study, resonant behavior and LTOs shared the same average frequency at 2.4 Hz, were concomitantly reduced by TTX and Ba2+, and were insensitive to local application of Cs+, thereby indicating that both phenomena recruit the same membrane mechanisms. Frequency domain analysis was only used in amygdaloid neurons that had proven to generate LTOs and therefore seemed an appropriate method for the investigation of the ionic mechanisms underlying oscillatory activity.
In the following, the data on the ionic mechanisms involved in the generation of LTOs and HTOs will be discussed in light of the repertoire of distinctive membrane conductances known to exist in amygdaloid neurons and the possible location and spatial segregation of the respective ion channels.
Contribution of a Na+ current and the M current to LTOs
The LTOs are blocked by intracellular injection of QX 314, application of TTX, and bathing solutions containing low concentrations of Na+, indicating the involvement of Na+ currents. The dominant Na+ current appears to rapidly activate in a voltage-dependent manner and to persist during maintained depolarization, as is indicated by the TTX-induced decrease in apparent input resistance and associated hyperpolarization, and the TTX-sensitive negative slope conductance obtained in the current versus voltage relationship under voltage-clamp conditions (Fig. 3 in the accompanying paper), which all occur in a region just subthreshold to spike generation and hence in a region of the LTOs. The subthreshold activation range and the pharmacological profile of the LTOs, i.e., sensitivity to intracellular Cs+, and to extracellular Ba2+ and CCh, suggested a critical contribution of the M-type K+ current (for a review on IM, see Brown 1988
). However, these substances are rather nonspecific in that they block more than one type of K+ conductance (for a review see Rudy 1988
; Storm 1993
). In basolateral amygdaloid neurons in particular, CCh blocks at least the M current (Womble and Moises 1992
), a voltage-independent "leak" K+ conductance (Womble and Moises 1992
), and a Ca2+-dependent K+ current underlying the slow spike afterhyperpolarization (AHP), and which has been termed slow IAHP (Womble and Moises 1993a
). The slow IAHP is not likely to contribute to the LTOs, because it activates on an increase in intracellular Ca2+ concentration (Sah 1996
) and is eliminated by perfusion of zero Ca2+ medium (Womble and Moises 1993a
), whereas the LTOs persisted under conditions that blocked Ca2+-dependent conductances. Similar considerations hold for other types of Ca2+-dependent K+ conductances, such as IC, known to exist in amygdaloid neurons (Meis and Pape 1997
; Womble and Moises 1993a
). The contribution of a leak K+ conductance to the LTOs cannot be completely ruled out, although the observation that an increase in apparent input conductance by ~30% through application of Mg2+ did not significantly alter the ability of the cells to oscillate (data not shown), argues against this possibility. An inactivating A-type K+ current (Gean and Shinnick-Gallagher 1989
; reviewed by Rudy 1988
; Storm 1993
) seems not to be involved, because LTOs could be evoked from predepolarized values of the membrane potential at which the conductance underlying IA is largely inactivated (data not shown), and LTOs were not affected by 4-AP. The relative insensitivity of the LTOs to TEA argues against a major contribution of the family of TEA-sensitive K+ conductances (as reviewed by Rudy 1988
; Storm 1993
). Finally, the population of oscillating amygdaloid neurons displayed very little anomalous membrane rectification in the hyperpolarizing direction (data not shown), and extracellular Cs+ did not affect LTOs or resonance behavior, thereby arguing against a contribution of the hyperpolarization-activated cation current, Ih (Womble and Moises 1993b
; reviewed in Pape 1996
). We are thus left with the conclusion that the M-type K+ current contributes to LTOs, although its exact role cannot be deduced from available data. Activation of IM may just help to maintain the membrane potential at a stable oscillatory level, with LTOs being generated by a Na+ current and an as yet undetermined type of outward current. As an alternative, a persistent Na+ current and the M-type K+ current may periodically interact with the membrane capacitance, thereby resulting in a sinusoidal deflection in membrane potential capable of triggering spike activity. Support of the latter hypothesis comes from guinea pig frontal cortical neurons, in which an interrelation between a persistent Na+ current and a slowly activating and noninactivating K+ current was found to produce subthreshold oscillations and resonant behavior at 4-20 Hz (Gutfreund et al. 1995
). Results from modeling experiments indicated that the magnitude of the K+ relative to the Na+ conductance critically determines spike patterns in these cells, in that a cell with a small K+ current tended to fire repetitively on depolarization, whereas a cell with a larger K+ current tended to generate oscillations (Gutfreund et al. 1995
). Indeed in amygdaloid neurons, reduction in K+ conductance through application of CCh resulted in blockade of LTOs and appearance of a slow membrane depolarization, most likely due to the persistent Na+ conductance, and which, in turn, triggered repetitive spike activity. Na+-dependent, Ba2+-sensitive subthreshold oscillations at an average frequency of ~8 Hz reportedly also occur in entorhinal cortical cells, and a low-threshold type of delayed rectifier K+ current of faster activation than IM was proposed to contribute to generation (Alonso and Klink 1993
; Klink and Alonso 1993
).
Involvement of Ca2+-dependent membrane conductances in HTOs
The HTOs were abolished after perfusion of a nominally Ca2+-free medium with added inorganic Ca2+ channel blockers, and during recording with a BAPTA-containing electrode, whereas Na+ channel blockers had no significant effect. These results indicate a critical involvement of Ca2+ currents and Ca2+-dependent mechanisms to the HTOs. Foehring and Scroggs (1994)
described the presence of various pharmacological subtypes of high-voltage-activated Ca2+ currents in amygdaloid neurons, namely L-, N-, and P-type currents in about equal proportions and an as yet uncharacterized type of current accounting for ~15% of the whole cell Ca2+ current. In the present study, local application of nifedipine and the selective Ca2+ channel antagonists Cgtx (Tsien et al. 1988
) or Agtx (Mintz et al. 1992
) did not significantly affect the HTOs. Although data from isolated cells suggest that the concentrations that were used were saturating (Foehring and Scroggs 1994
), we cannot exclude the possibility that an insufficient number of channels were reached by the substances in our multicellular preparation. An alternative explanation for the maintenance of HTOs is that a combination of different subtypes of Ca2+ currents, possibly containing at least one type of unclassified Ca2+ current, is involved, which seems feasible in light of the emerging diversity of amino acid sequences of Ca2+ channels and related number of channel subtypes (for a review see Hofmann et al. 1994
). In any case, the voltage range of activation positive to
40 mV of the HTOs argues against a contribution of the low-voltage-activated Ca2+ conductance known to exist in a subpopulation of amygdaloid neurons (Foehring and Scroggs 1994
). Calcium-sensitive rhythmic oscillations at 8-10 Hz were found to also occur at potentials positive to spike threshold in guinea pig cingulate cortical (Tanaka et al. 1991
) and rat pedunculopontine neurons (Takakusaki and Kitai 1997
), and proposed to shape repetitive firing behavior in these cells.
HTOs in amygdaloid neurons were effectively blocked by intracellular Cs+ as well as by application of TEA and Ba2+, thereby indicating a critical contribution of voltage-dependent K+ currents. The range of activation of HTOs positive to approximately
40 mV and the amplitude of the electrogenic events of up to 40 mV suggest the involvement of various types of K+ conductances of the "delayed rectifier" type (Rudy 1988
; Storm 1993
). In light of the rich diversity of K+ channel proteins that are likely to form the respective voltage-gated ion channels (as reviewed by Dolly and Parcej 1996
; Jan and Jan 1992
; Pongs 1992
), a further characterization appeared not feasible under the present experimental conditions, but will require future voltage-clamp and single-channel analysis. However, two assumptions may be made based on the present observations. First, members of the A family of K+ currents (Pongs 1992
; Rudy 1988
; Storm 1993
) are not contributing, as is indicated by the lack of influence of 4-AP and the activation of HTOs from predepolarized values of the membrane potentials at which A currents are inactivated. Second, K+ currents activated on the influx of Ca2+ associated with the depolarizing phase of the oscillatory cycle are likely to be involved, as is demonstrated by the reduction of the HTOs through the Ca2+ chelator BAPTA and by iberiotoxin, a blocker of BKCa channels (as reviewed by Kaczorowski et al. 1996
). In addition, various types of Ca2+-dependent K+-mediated AHPs following spike activity (Danober and Pape 1996
; Womble and Moises 1993a
) and a Ca2+-activated K+ current carried through BKCa channels (Meis and Pape 1997
) have been found to exist in neurons of the basolateral amygdaloid complex (Meis and Pape 1997
). The BKCa channels are voltage sensitive and require a relatively high activity of Ca2+ (1-10 µM) for activation at membrane potentials near rest (Reinhart et al. 1989
; for a review see Sah 1996
). Recruitment of a significant fraction of these channels may therefore depend on the Ca2+ influx during electrogenic depolarizations such as the Ca2+-mediated high-threshold action potentials. Another type of Ca2+-dependent K+ current, termed sIAHP, is known to mediate a "slow" AHP in amygdaloid as well as other types of neurons (Danober and Pape 1996
; reviewed by Sah 1996
; Womble and Moises 1993a
). The channels underlying the sIAHP are voltage insensitive and are thought to be gated by Ca2+ itself at concentrations of 100-400 nM (cf. Sah 1996
). The sIAHP current seems to be indirectly involved in the generation of the HTOs, as is indicated by the observation that amygdaloid neurons possessing a strong "slow" AHP and associated sIAHP did not generate HTOs, but readily oscillated after pharmacologically induced reduction of the slow AHP (unpublished observations). It thus seems reasonable to speculate that the relationship between Ca2+ influx and two different types of Ca2+-dependent K+ channels are important elements in the generation or prevention of the HTOs, and which may be determined by the limited diffusion of Ca2+, microdomains of high Ca2+ concentration and the spatial location of the respective ion channels (Llinás et al. 1992
). Since the conductance underlying the sIAHP is under control of a number of metabolic and transmitter systems (Danober and Pape 1996
; Pedarzani and Storm 1993
; Womble and Moises 1993a
; reviewed by Sah 1996
), it will represent an effective physiological mechanism for the regulation of HTOs in amygdaloid neurons. It is important to mention, however, that activation of as yet unclassified K+ conductances in addition to Ca2+-activated K+ channels are likely to also contribute to the repolarizing phase of the HTOs. This conclusion is supported by the following observation: the rather unspecific blockade of net membrane K+ conductance (through intracellular Cs+, or application of TEA or Ba2+) resulted in a replacement of HTOs by a depolarizing plateau potential at around
10 mV, whereas the more specific blockade of Ca2+-activated K+ currents (through intracellular BAPTA or application of Ibtx) abolished HTOs with no development of depolarizing plateau potentials, presumably indicating two different states of equilibrium between Ca2+-mediated inward current and K+-mediated outward current.
In functional terms, the HTOs are important in determining spike frequency adaptation toward a slow-rhythmic firing pattern during maintained depolarizing influence, as is indicated by the substantial delay in adaptation behavior and the less regular firing patterns obtained after reduction of HTOs through removal of extracellular Ca2+. The HTOs may then represent intrinsic mechanisms able to regulate ongoing spike activity and to reinforce the neuronal output at a particular frequency.
 |
ACKNOWLEDGEMENTS |
The authors thank L. Danober and R. Pott for participation and help in some of the experiments. A. Krieger and A. Reupsch provided expert technical assistance.
This work was supported by grants to H.-C. Pape from the Deutsche Forschungsgemeinschaft (SFB 426, TP B3) and from the Kultusministerium des Landes Sachsen-Anhalt.
 |
FOOTNOTES |
Address for reprint requests: H.-C. Pape, Institut für Physiologie, Medizinische Fakultät, Otto-von-Guericke-Universität, Leipziger Str. 44,D-39120 Magdeburg, Germany.
Received 18 March 1997; accepted in final form 25 August 1997.
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