Physiological Properties of Central Medial and Central Lateral Amygdala Neurons

Marzia Martina, Sébastien Royer, and Denis Paré

Laboratoire de Neurophysiologie, Département de Physiologie, Faculté de Médecine, Université Laval, Québec G1K 7P4, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Martina, Marzia, Sébastien Royer, and Denis Paré. Physiological Properties of Central Medial and Central Lateral Amygdala Neurons. J. Neurophysiol. 82: 1843-1854, 1999. Mounting evidence implicates the central (CE) nucleus of the amygdala in the mediation of classically conditioned fear responses. However, little data are available regarding the intrinsic membrane properties of CE amygdala neurons. Here, we characterized the physiological properties of CE medial (CEM) and CE lateral (CEL) amygdala neurons using whole cell recordings in brain slices maintained in vitro. Several classes of CE neurons were distinguished on the basis of their physiological properties. Most CEM cells (95%), here termed "late-firing neurons," displayed a marked voltage- and time-dependent outward rectification in the depolarizing direction. This phenomenon was associated with a conspicuous delay between the onset of depolarizing current pulses and the first action potential. During this delay, the membrane potential (Vm) depolarized slowly, the steepness of this depolarizing ramp increasing as the prepulse Vm was hyperpolarized from -60 to -90 mV. Low extracellular concentrations of 4-aminopyridine (30 µM) reversibly abolished the outward rectification and the delay to firing. Late-firing CEM neurons displayed a continuum of repetitive firing properties with cells generating single spikes at one pole and high-frequency (>= 90 Hz) spike bursts at the other. In contrast, only 56% of CEL cells displayed the late-firing behavior prevalent among CEM neurons. Moreover, these CEL neurons only generated single spikes in response to membrane depolarization. A second major class of CEL cells (38%) lacked the characteristic delay to firing observed in CEM cells, generated single spikes in response to membrane depolarization, and displayed various degrees of inward rectification in the hyperpolarizing direction. In both regions of the CE nucleus, two additional cell types were encountered infrequently (<=  6% of our samples). One type of neurons, termed "low-threshold bursting cells" had a behavior reminiscent of thalamocortical neurons. The second type of cells, called "fast-spiking cells," generated brief action potentials at high rates with little spike frequency adaptation in response to depolarizing current pulses. These findings indicate that the CE nucleus contains several types of neurons endowed with distinct physiological properties. Moreover, these various cell types are not distributed uniformly in the medial and lateral sector of the CE nucleus. This heterogeneity parallels anatomic data indicating that these subnuclei are part of different circuits.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Much evidence indicates that the amygdala transduces the affective value of sensory events into endocrine, visceral, and behavioral responses through divergent projections reaching functionally diverse brain structures (Amaral et al. 1992; Davis 1992; LeDoux 1995). Moreover, the amygdala appears to be the site of plastic synaptic changes that mediate a well characterized form of fear learning termed Pavlovian fear conditioning (LeDoux et al. 1990; Miserendino et al. 1990; Quirk et al. 1995; Rogan et al. 1997; Romanski and LeDoux 1992a,b).

The central (CE) amygdaloid nucleus plays a pivotal role in this respect as it is one of the main output structures of the amygdala. Indeed, it is the prime source of amygdaloid projections to the brain stem (Hopkins and Holstege 1978; Veening et al. 1984). Moreover, lesions of the CE nucleus (Gentile et al. 1986; Hitchcock et al. 1989; Iwata et al. 1986; Kapp et al. 1979; Zhang et al. 1986) or of its brain stem and hypothalamic targets (Francis et al. 1981; LeDoux et al. 1988) abolish conditioned fear responses.

Despite the important role of the CE nucleus, little is known about the physiological properties of its neurons. So far, in vitro electrophysiological studies have focused on the synaptic responses (Nose et al. 1991; Rainnie et al. 1992) and spike after-hyperpolarizations (Scheiss et al. 1993) of CE neurons. In these studies, little attention was paid to possible differences between the medial and lateral sectors of the CE nucleus (CEM and CEL). This is an important point because the CEL and CEM have different projection sites and contain morphologically distinct cell types (reviewed in McDonald 1992). For instance, the vast majority of CEM neurons project to the brain stem, whereas comparatively few CEL neurons do (Hopkins and Holstege 1978; Veening et al. 1984). On the input side, the CEL receives a dense projection from the lateral amygdaloid nucleus, whereas the CEM does not (Krettek and Price 1978; Pitkanën et al. 1995; Smith and Paré 1994). Moreover, the main cell type found in the CEL resembles the medium spiny neurons of the striatum (Hall 1972), whereas CEM cells tend to have a sparse to moderate spine density (Cassel and Gray 1989; Kamal and Tömböl 1975; McDonald 1982; Tömböl and Szafranska-Kosmal 1972).

Because the intrinsic membrane properties of neurons shape their spontaneous activity and constrain their synaptic responsiveness (Llinás 1988), we characterized the physiological properties of CEM and CEL neurons using whole cell recordings in brain slices kept in vitro. This study revealed that the CE nucleus contains multiple physiologically defined classes of neurons that are not distributed homogeneously in its lateral and medial sectors.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

Preparation of amygdala slices

Coronal slices of the amygdala were obtained from Hartley guinea pigs (approx 250 g) of either sex. Prior to decapitation, the animals were deeply anesthetized with sodium pentobarbital (40 mg/kg ip) and ketamine (100 mg/kg ip). The brain was rapidly removed and placed in a cold (4°C) oxygenated solution containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose. A block containing the amygdala was prepared and coronal sections (400 µm) were obtained using a vibrating microtome. The slices were stored for 1 h in an oxygenated chamber at room temperature. One slice was then transferred to a recording chamber (submerged type) perfused with an oxygenated physiological solution (2 ml/min). The temperature of the chamber was gradually increased to 32°C before the recordings began.

Electrophysiological recordings

Current-clamp recordings were obtained with borosilicate pipettes filled with a solution containing (in mM) 130 K-gluconate, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10 KCl, 2 MgCl2, 2 ATP-Mg, and 0.2 GTP-Tris. In some experiments, Neurobiotin (0.5%) was added to the intracellular solution. pH was adjusted to 7.2 with KOH and osmolarity to 280-290 mOsm. With this solution, the liquid junction potential was approx 10 mV and the membrane potential (Vm) was corrected accordingly after the experiments. The pipettes had resistances of 3-6 MOmega when filled with the above solution. Recordings with series resistance higher than 15 MOmega were discarded.

Using trans-illumination of the slice (Fig. 1), the outline of the CE nucleus as well as the border between its lateral and medial sectors could be identified easily. Recordings were obtained under visual control using differential interference contrast and infrared video microscopy (IR-DIC). Recordings were carried out with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) and a microcomputer equipped with pClamp software (Axon Instruments).



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Fig. 1. Identification of amygdala nuclei in trans-illuminated slices. A and B: two consecutive 400 µm thick slices, B being more caudal than A. The outline of the CE nucleus as well as the border between its lateral and medial sectors could be identified easily because of the presence of fiber bundles. AHA, amygdalohippocampal area; BL, basolateral nucleus of the amygdala; BM, basomedial nucleus of the amygdala; CEL, lateral sector of the central amygdaloid nucleus; CEM, medial sector of the central amygdaloid nucleus; EC, external capsule; GP, globus pallidus; L, lateral nucleus of the amygdala; ME, medial amygdaloid nucleus; P, putamen; rh, rhinal sulcus.

Analyses were carried out off-line with the software IGOR (Wavemetrics, Lake Oswego, OR) and home-made software running on Macintosh microcomputers. When studying current-voltage relations, voltage measurements were performed at fixed time intervals with respect to the onset of current pulses (see Results and figure legends), unless the signal was distorted by spontaneous synaptic potentials. In such instances, the voltage was measured immediately before the synaptic event. The input resistance (Rin) was estimated in the linear portion of current-voltage plots. The membrane time constant was derived from single exponential fits to voltage responses in the linear portion of current-voltage relations.

Morphological identification of recorded cells

When recorded cells were dialyzed with Neurobiotin, the slices were removed from the chamber and fixed for 1 to 3 days in 0.1 M phosphate-buffered saline (pH 7.4) containing 2% paraformaldehyde and 1% glutaraldehyde. Slices were then embedded in gelatin (10%) and sectioned on a vibrating microtome at a thickness of 60-100 µm. Neurobiotin-filled cells were visualized by incubating the sections in the avidin-biotin-horseradish peroxidase (HRP) solution (ABC Elite Kit, Vector Labs, Burlingame, CA) and processed to reveal the HRP staining (Horikawa and Armstrong 1988).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

A total of 161 CE neurons that had Vm >=  -60 mV and overshooting action potentials were recorded in the present study. Of these neurons, 80 were recorded in the CEM and 81 in the CEL. On the basis of their responses to intracellular current injection, these neurons were subdivided in several classes described below (see Table 1).


                              
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Table 1. Physiological properties of CEL and CEM amygdala neurons

CEM neurons

LATE-FIRING NEURONS. In the vast majority of CEM neurons (95%), there was a conspicuous delay between the onset of suprathreshold depolarizing current pulses and spike discharges (Fig. 2), hence the designation "late-firing neurons." During this delay, the Vm depolarized slowly, giving rise to a ramp lasting hundreds of milliseconds.



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Fig. 2. Physiological properties of late-firing CEM neurons. A and B: voltage response of two CEM neurons to graded series of intracellular current pulses. The current pulses were applied at rest (-85 and -80 mV, respectively). 1: actual voltage responses; 2: plots of current-voltage relations 220 ms (crosses) and 1920 ms (filled circles) after the onset of current pulses. In B1 (second trace from the top), note that the spike burst occurred after a delay of 1.6 s and from a Vm of approx  -53 mV, suggesting that low-threshold Ca2+ channels are not involved.

REPETITIVE-FIRING BEHAVIOR. In response to depolarizing current pulses, a continuum of repetitive-firing behaviors was observed among late-firing neurons (Fig. 2). The two examples of Fig. 2 represent the opposite poles of this continuum, where neurons generating single spikes were prevalent (Fig. 2A) and those generating high-frequency (>= 90 Hz) spike bursts (Fig. 2B) were infrequent (7.5%). The bursting and nonbursting firing patterns were not dependent on the initial membrane potential; applying suprathreshold depolarizing current pulses from Vms ranging from -60 to -90 mV did not transform bursting cells into nonbursting ones and vice versa.

The repetitive firing behavior of the cells depicted in Fig. 2 is contrasted in Fig. 3A, which plots their instantaneous firing frequency as a function of the interval number during depolarizing current pulses of various intensities. Whereas the instantaneous firing frequency of neurons generating isolated spikes (continuous lines in Fig. 3A) was stable during depolarizing current pulses near threshold (average of 4.9 ± 0.43 Hz, n = 10), it varied markedly in bursting cells (dashed lines in Fig. 3A). In these neurons, depolarizing current pulses just above threshold first elicited a high-frequency burst of three action potentials (average of 96.9 ± 7.8 Hz, n = 6). Although, these spike bursts were stereotyped at a given current intensity (Fig. 3B), the intraburst frequency diminished as the current amplitude was increased (Fig. 3A, dashed lines) and the spike latency decreased (see top two traces of Fig. 2B1). The initial burst was followed by a long pause of approx 0.5 s after which a second burst, of markedly lower frequency (average of 37.3 ± 4.18 Hz, n = 6), was elicited. Subsequently, these neurons generated only single spikes.



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Fig. 3. Repetitive firing properties of late-firing CEM neurons. A: plot of the instantaneous firing frequency as a function of the interval number in two late firing neurons generating high-frequency spike bursts (dashed lines) or repetitive single spikes (continuous lines) in response to depolarizing current pulses just above threshold. The amplitudes of the current pulses and corresponding symbols are indicated in the graph. Inset: a representative response of a bursting cell. B: superimposition of spike bursts elicited by nine current pulses of constant amplitude. C: in the presence of TTX, depolarizing current pulses elicit slow Ca2+ spikes in a bursting neuron. In the TTX condition, current pulses were applied from -65 mV, as determined by DC current injection (0.04 nA).

To determine the origin of the different repetitive firing properties of bursting and nonbursting neurons, we compared their passive and active physiological properties. Differences in Rin and membrane time constant between these two types of late-firing neurons did not reach statistical significance. Moreover, although late-firing neurons often displayed a time-dependent inward rectification in the hyperpolarizing direction (Fig. 2), there was no systematic relation between the magnitude of this phenomenon and the firing pattern of late-firing CEM cells. Finally, the shape and amplitude of spike afterhyperpolarizations were highly variable in both cell types.

However, bath application of tetrodotoxin (TTX; 0.5 µM) to bursting cells (n = 5) revealed that bursting neurons could generate slow Ca2+ spikes (Fig. 3C) identified as such by their sensitivity to Cd2+ (200 mM; not shown). Their voltage-dependence suggested that they were not mediated by low-threshold Ca2+ channels. Indeed, these TTX-resistant spikes were triggered at a more depolarized Vm than Na+ spikes (-44.6 ± 0.4 and -54 ± 1.30 mV, respectively), had a longer duration (118 ± 13.2 and 0.8 ± 0.02 ms at half-amplitude), and lower amplitude (18.8 ± 2.6 and 86 ± 5.8 mV). All these differences were statistically significant (two-tailed t-test, P < 0.05). By contrast, TTX application in late-firing cells generating single spikes (n = 6; not shown) revealed much smaller high-threshold Ca2+ spikes (9.3 ± 1.40 mV, P < 0.05) that were triggered at even more depolarized Vms (-38.3 ± 1.96 mV, P < 0.05).

ORIGIN OF THE DELAY TO FIRING. The characteristic delay observed between the onset of depolarizing current pulses and action potentials (Fig. 2A1 and 2B1) appeared to result from a marked voltage- and time-dependent outward rectification in the depolarizing direction. This is shown in Fig. 2, A2 and B2, where the voltage responses of late-firing neurons to graded series of subthreshold current pulses applied from rest is plotted as a function of the injected current. In these graphs, crosses and circles refer to the voltage responses measured 220 and 1920 ms after the pulse onset, respectively. Although the initial and late responses to negative current pulses were nearly identical, they differed for positive current pulses that brought the Vm beyond approx  -65 mV (average difference of 6.6 ± 0.68 mV for subthreshold current pulses of maximal amplitude; Figs. 2A2 and 2B2).

To examine the voltage dependence of this phenomenon, a graded series of depolarizing current pulses was applied at different Vms, as determined by steady current injection (Fig. 4A). Then, the difference between the early and late voltage responses to the subthreshold current pulse of highest amplitude was plotted as a function of the prepulse potential (Fig. 4B). The data plotted in Fig. 4B were obtained from 20 cells in which this test was carried out at one or more prepulse Vms, for a total of 38 data points. Note that the difference between the early and late part of voltage responses grew as the prepulse Vm was hyperpolarized (r = 0.67, P < 0.05).



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Fig. 4. Effect of membrane polarization on the response characteristics of late-firing CEM neurons. A: voltage response of the same neuron to graded series of current pulses applied from two different Vms (-70 in A1 and -90 mV in A2) as determined by intracellular current injection (0.03 and -0.1, respectively). Rest was -75 mV in this neuron. B: plot of the difference between the early and late voltage responses to subthreshold current pulses of maximal amplitude as a function of the prepulse potential. The data were obtained from 20 neurons tested at one or more prepulse potentials for 38 data points.

These features suggested that the delay between the pulse onset and spike discharges resulted from a slowly inactivating K+ current similar to the 4-aminopyridine (4-AP)-sensitive current previously described in several cell types (see Discussion). Thus, the effect of low concentrations of 4-AP (30 µM) was tested. In a first group of cells (n = 12), depolarizing current pulses bringing the Vm just below spike threshold were applied from a prepulse Vm of -80 mV. As shown in Fig. 5, bath application of 4-AP (30 µM) gradually increased the voltage response to the current pulses (Fig. 5, A and C), until the firing threshold was reached. Then, the spike latency gradually diminished (Fig. 5, A and B). However, bath application of 4-AP (30 µM) produced no change in Vm. Comparing the response of late-firing neurons to graded series of current pulses before/after 4-AP application (Fig. 6, A and B) revealed that this drug reversibly abolished the range of subthreshold current pulses where the voltage- and time-dependent outward rectification is apparent. It should be pointed out that in contrast with other cell types (see DISCUSSION), the outward rectification displayed by late-firing CEM neurons did not diminish when repetitive depolarizing current pulses were applied unless the interpulse interval was shorter than 1.5 s.



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Fig. 5. Bath application of 4-AP (30 µM) increases the Rin of late-firing cells and reduces the delay to firing. A depolarizing current pulse of constant amplitude just below firing threshold was applied every 6 s from rest (-74 mV). A: effect of 4-AP at a slow time base. One of every 2 current pulses shown in A is depicted with a faster time base in B from the control situation (lowest trace) to the full development of the 4-AP effects (top trace). C: plot of the voltage response to the current pulses as a function of time during this experiment. Only subthreshold responses were included in this graph.



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Fig. 6. Bath application of 4-AP abolishes the voltage- and time-dependent outward rectification displayed by late-firing cells in the depolarizing direction. A: voltage responses to series of depolarizing current pulses applied at -80 mV before (Control), during, and after (Recovery) application of 4-AP. B: plots of current-voltage relations 220 ms (crosses) and 1920 ms (filled circles) after the onset of current pulses in the three conditions.

In the previous experiment, the presence of Na+ spikes prevented us from examining the behavior of late-firing cells in the same range of injected currents before/after 4-AP application. Thus, the same tests were carried out in the presence of TTX (n = 8; Fig. 7). 4-AP markedly reduced the time- and voltage-dependent outward rectification that characterizes these cells in the depolarizing direction and produced a significant increase in Rin (44 ± 9.3%, P < 0.05, paired t-test; compare the I-V curve of Figs. 7B1 and 2). Another representation of this phenomenon is provided in Fig. 7C, where the difference between the early and late voltage responses is plotted as a function of the Vm reached at the end of the current pulses. However, note that low concentrations of 4-AP did not abolish the early component of the depolarizing ramp.



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Fig. 7. Effect of bath-applied 4-AP in the presence of TTX. A: graded series of current pulses applied at -90 mV before (A1) and after (A2) 4-AP application. B: plots of current-voltage relations 420 ms (crosses) and 1920 ms (filled circles) after the onset of current pulses in the two conditions. C: plot of the difference between the early and late current-evoked voltage responses as a function of the Vm reached at the end of the current pulses in the absence and presence of 4-AP (empty and filled circles, respectively).

CEL neurons

Two predominant types of CEL neurons were distinguished on the basis of their responses to intracellular current injection. A first class of cells, accounting for 56% of CEL neurons, displayed the late-firing behavior, which prevails in the CEM nucleus. A second class of CEL neurons, hereafter termed "regular spiking cells," lacked the characteristic delay to firing observed in CEM cells and generated single spikes in response to membrane depolarization. This class of neurons accounted for 38% of our sample.

Within each of these cell classes, there were large variations in the amplitude, shape, and duration of spike afterhyperpolarizations as well as in the amount of inward rectification (sag) in the hyperpolarizing direction. In other words, there was no systematic relation between these physiological features (afterhyperpolarizations, sags) and the above bipartite classification. In addition, late-firing and regular spiking neurons were intermixed in the CEL. They were not differentially distributed in particular sectors of this subnucleus.

LATE-FIRING NEURONS. An example of late-firing CEL neuron is shown in Fig. 8A. In response to depolarizing current pulses, these cells displayed the characteristic time-dependent outward rectification observed in most CEM neurons. As in CEM cells, the importance of this rectification increased as the prepulse Vm was hyperpolarized (cf. Fig. 8A1, at -75 mV and Fig. 8A2, at -90 mV), and it was abolished by bath application of 4-AP (30 µM, n = 4; not shown). However, in contrast to CEM cells, late-firing CEL neurons only generated single spikes in response to membrane depolarization. No late-firing bursting neurons were observed in the CEL nucleus. Although the membrane time constant of late firing neurons was significantly shorter in the CEL than that in the CEM (two-tailed t-test, P < 0.05), differences in Rin did not reach statistical significance (see Table 1).



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Fig. 8. Contrasting physiological properties of late-firing (A) and a regular spiking (B) CEL neurons. Voltage responses to graded series of current pulses applied from -75 mV (1) or -90 mV (2), as determined by steady current injection. 3: plots of current-voltage relations 220 ms (crosses) and 1920 ms (filled circles) after the onset of current pulses applied at rest. The two insets show magnified current-voltage relations just below firing threshold with prepulse potentials of -75 (1) and -90 mV (2).

REGULAR SPIKING NEURONS. Figure 8B illustrates the behavior of a regular spiking CEL neuron. In contrast to late-firing CEL neurons (Fig. 8A), these cells did not display outward rectification in the depolarizing direction (Fig. 8B3) and lacked the typical delay to firing (Fig. 8B1). These neurons generated only single spikes in response to depolarizing current pulses and did not display spike-frequency adaptation when repetitive spiking was elicited by depolarizing current pulses >=  0.12 nA. However, various degrees of spike frequency adaptation were seen when the cells were challenged with higher depolarizing currents (not shown). These neurons had a significantly higher Rin, a longer membrane time constant and a more depolarized resting potential than late-firing CEL cells (two-tailed t-tests, P < 0.05; see Table 1).

Rare types of CEM and CEL neurons

Other types of CE neurons were encountered too infrequently (approx 6%) to allow meaningful quantitative analyses. One class of cells, observed in both sectors of the CE nucleus, generated very brief action potentials and was able to sustain high firing rates with little or no spike frequency adaptation, hence the designation "fast-spiking neurons." A second class of CE neurons, also encountered in both sectors of the CE nucleus, had a behavior reminiscent of dorsal thalamic relay neurons (Deschênes et al. 1982; Llinás and Jahnsen 1982). Hereafter, they will be referred to as "low-threshold bursting neurons."

It should be emphasized that the low percentage of fast-spiking and low-threshold bursting cells was obtained despite the fact that a deliberate attempt was made to record from somatic profiles with atypical shapes and/or diameters.

FAST-SPIKING NEURONS. Three fast-spiking neurons were recorded in the CEM (Fig. 9B) and one in the CEL (Fig. 9A). These neurons were characterized by a very high Rin (ranging from 467 to 700 MOmega ), generated brief action potentials (ranging from 0.5 to 0.6 ms at half-amplitude), and large spike afterhyperpolarizations (range of 16 to 28 mV). A distinguishing feature of fast-spiking neurons was their ability to sustain firing rates as high as 100 Hz without spike frequency adaptation. However, during current-evoked spike trains, fast-spiking CEM neurons always exhibited short pauses in firing during which a low-amplitude Vm oscillation occurred (Fig. 9B). This phenomenon was not observed in the fast-spiking CEL cell.



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Fig. 9. Physiological properties of fast-spiking CEL (A) and CEM (B) neurons. Voltage response to graded series of current pulses applied from -70 mV (A) and -68 mV (B). Rest was -81 and -80 mV in A and B, respectively. C: plots of current voltage relations 50 ms (crosses) and 480 ms (filled circles) after the onset of current pulses for the neurons shown in A (C1) and B (C2).

LOW-THRESHOLD BURSTING NEURONS. Four low-threshold bursting neurons were recorded in the CEL and one in the CEM. At the break of hyperpolarizing current pulses applied from rest, these neurons generated rebound spike bursts whose latency decreased as the amplitude of the current pulses was increased (Fig. 10A2). The rebound spike bursts rode on a slow depolarizing potential (13.8 ± 2.40 mV) of variable duration (182 ± 79.2 ms; compare the cells depicted in Fig. 10, A1 and B1). The intraburst frequency varied markedly from cell to cell (from 50 to 200 Hz), but in all cases, the duration of successive interspike intervals increased as the burst progressed. Spike bursts with similar features could be elicited by depolarizing current pulses applied at Vms negative to -65 mV (data not shown), but pulses of similar amplitudes elicited accommodating trains of action potentials at depolarized Vms (Fig. 10B1, top trace).



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Fig. 10. Physiological properties of low-threshold bursting CEL neurons. 1: voltage responses to current pulses. Some of the low-threshold spike bursts shown in A are depicted with a faster time base in 2. The neuron shown in A was recorded at rest (-70 mV), whereas the cell depicted in B was steadily depolarized to -64 mV by steady current injection (0.04 nA; rest was -73 mV). The inset in A1 is a plot of current-voltage relations. Crosses and filled circles indicate Delta V measured at the hyperpolarizing peak of the voltage response and just before the end of the current pulses, respectively.

The ability to generate low-threshold spike bursts was observed in neurons that displayed varying degrees of inward rectification in the hyperpolarizing direction (compare the cells depicted in Fig. 10, A1 and B1).

Morphological properties of CE neurons

Intracellular injections of Neurobiotin were performed in 30 CE neurons belonging to the major physiologically defined cell classes described above. Twenty-one of these cells were recovered (CEM, 13 late-firing; CEL, 6 late-firing, and 2 regular spiking cells). The dendrites of all these cells remained within the confines of the nucleus where their soma was located.

Late-firing CEM neurons (Fig. 11, A1-A3) had an ovoid cell body (long axis, 24.1 ± 0.90 µM), collateralized axons bearing varicosities, and dendrites with a sparse to moderate spine density. Typically, they had two to three main dendritic trunks from which emerged a few secondary dendritic branches that rarely divided more than once. No consistent differences were found between the morphological properties of bursting (n = 4; Fig. 11, A2 and A3) and nonbursting (n = 9; Fig. 11A1) late-firing CEM cells.



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Fig. 11. Morphological properties of CE neurons. All panels except B3 are reconstruction of CE neurons from 2 to 3 consecutive sections. A: late-firing CEM neurons that, in response to depolarizing current pulses, generated repetitive single spikes (A1) or bursts of action potentials (A2 and A3). B1 and B2: late-firing CEL cells; B3: photomicrograph of a regular spiking CEL neuron. Calibration bar in A2 is valid for A1 and A3. Calibration bar in B1 is also valid for B2.

Compared with CEM cells, CEL neurons (Fig. 11, B1-B3) tended to have a more angular and smaller (long axis, 19.3 ± 1.02 µM; P < 0.05) soma from which emerged three to five primary dendrites. Most of these proximal dendritic segments branched several times within a short distance of the cell body (Fig. 11B1). The dendrites of late-firing CEL neurons, particularly their distal segments, were densely covered with spines. Local axonal ramifications of CEL neurons could be seen rarely, in contrast to CEM cells. In our admittedly limited sample of CEL neurons, no striking morphological difference was observed between late-firing (Fig. 11, B1-B2) and regular spiking (Fig. 11, B3) CEL cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

In the present study, the physiological and morphological properties of CE amygdala neurons were correlated using whole cell recordings and intracellular Neurobiotin injection in vitro. It was found that the CE nucleus contains several physiologically defined cell classes that are not distributed homogeneously in its lateral and medial sectors.

The most common type of CE neurons (95% of CEM and 56% of CEL cells) were late-firing cells. These neurons displayed a pronounced time- and voltage-dependent outward rectification in the depolarizing direction that was associated with a long delay to firing. In response to depolarization, late-firing CEM cells generated repetitive single spikes or high-frequency spike bursts. In contrast, only nonbursting late-firing cells were seen in the CEL. On the other hand, the CEL contained a second major class of cells (38% of our sample) that lacked the characteristic delay to firing observed in late-firing neurons and generated repetitive single spikes on membrane depolarization. Finally, two rare types of neurons, collectively representing <= 6% of our samples, were observed in both sectors of the CE nucleus: cells that had a behavior reminiscent of thalamocortical neurons ("low-threshold bursting cells") and fast-spiking neurons, which had a high Rin, a fast time constant, and the ability to sustain high firing rates without spike frequency adaptation.

Technical considerations

Filling recorded neurons with Neurobiotin precluded the use of the perforated-patch method. As a result, the intrinsic membrane properties of CE neurons might have been altered by exchanges between the intracellular compartment and the pipette solution. However, the electroresponsiveness and Vm of CE neurons remained stable over time, implying that potential artifacts introduced by the intracellular dialysis were immediate or too subtle for detection. Similar conclusions were reached in a recent analysis of the electrophysiological properties of hilar neurons (Lübke et al. 1998). Consequently, we ascribe the more polarized Vm of CE neurons described here, in comparison to previous studies using conventional intracellular recordings (Nose et al. 1991; Scheiss et al. 1993), to the damage caused to the membrane by the sharp micropipettes.

Another potentially confounding factor is the relatively young age of the animals used this study (2-4 wk old). Consequently, it is possible that the physiological properties of CE neurons change with maturation. Juvenile rather than adult animals were used because myelination increases with age, making it difficult to visualize neurons with IR-DIC.

Relationship between physiological and morphological features

Various parcellations of the CE nucleus have been proposed using Nissl and Golgi staining as well as peptide immunoreactivity (Brodal 1947; Cassel and Gray 1989; Krettek and Price 1978; McDonald 1982; Wray and Hoffman 1983). However, considering that there is disagreement regarding this issue and since we could only distinguish a lateral and a medial sector of the CE nucleus in transilluminated slices, we used this classical bipartite parcellation (Brodal 1947; Krettek and Price 1978).

Golgi studies (reviewed in McDonald 1992) have revealed that the CEM and CEL each contain one predominant morphological cell type (Hall 1972; Kamal and Tömböl 1975; McDonald 1982; Tömböl and Szafranska-Kosmal 1972). In the CEM, most cells have an oval cell body, a collateralized and varicose axon, and three to four poorly branched primary dendrites with a sparse to moderate spine density. In the CEL, the predominant cell type is termed "medium spiny neuron," in reference to the main class of cells found in the striatum (Hall 1972). These cells have a smaller soma than CEM neurons, a high-density of dendritic spines, and multiple primary dendrites with numerous branching points. These morphological features correspond well to those of the Neurobiotin-filled CEM and CEL cells recovered in the present study.

These considerations imply that morphologically uniform populations of CE cells can exhibit different physiological properties. For instance, CEL neurons with the typical medium spiny appearance could belong to the late-firing or regular spiking cell classes. Similarly, bursting and nonbursting late-firing CEM cells had similar morphological features. Conversely, our findings also show that morphologically different populations of CE cells can be endowed with similar physiological properties. The clearest example of this are the late-firing neurons of the CEL and CEM, which had distinct morphological properties but similar electroresponsive characteristics.

This equivocal relation between the physiological and morphological properties of CE amygdala neurons parallels the results of immunohistochemical studies (Cassel and Gray 1989), where it was found that there is no obvious morphological difference among medium spiny CEL neurons containing different neuropeptides. Conversely, the same neuropeptides were observed in different morphological types of CE neurons.

The lack of systematic association between morphological features and electroresponsive behavior was also noted in other structures of the CNS. In the cerebral cortex for instance, it was observed that aspiny local-circuit neurons could display regular spiking, intrinsically bursting, or fast-spiking discharge patterns (Thomson et al. 1996). Others noted that some physiologically identified corticothalamic neurons could behave like conventional cortical fast-spiking cells (Steriade et al. 1998), a behavior traditionally assumed to be expressed only by aspiny local-circuit neurons (McCormick et al. 1985).

Identity of the different physiologically defined cell classes

Even though we did not identify the projection site of late-firing CEM neurons, it is likely that most of them have brain stem projections. Indeed, it was reported that virtually all CEM neurons are retrogradely labeled after large retrograde tracer injections in the brain stem (Hopkins and Holstege 1978). On the other hand, little can be said about the projection site of CEL cells. This is due to the fact that late-firing and regular spiking CEL cells were intermingled and did not appear to be concentrated in particular regions of the CEL, thus preventing correlations with the results of tract tracing studies. As to the identity of fast-spiking and low-threshold bursting neurons found in both subnuclei, they might correspond to the rare intensely GABA immunoreactive cells found in both sectors of the CE nucleus (McDonald and Augustine 1993; Nitecka and Ben-Ari 1987; Paré and Smith 1993a). However, this idea awaits experimental confirmation.

Late-firing neurons are endowed with a slowly inactivating K+ conductance

Several factors suggest that late-firing neurons are endowed with a slowly inactivating K+ conductance similar to that previously described in striatal (Bargas et al. 1989; Nisenbaum et al. 1994), hippocampal (Storm 1988), thalamic (McCormick 1991), and cortical (Hammond and Crépel 1992) neurons. In voltage clamp analyses (Gabel and Nisenbaum 1998; McCormick 1991; Storm 1988), it was observed that this conductance activates around -65 mV, has relatively rapid activation kinetics, inactivates slowly, requires hyperpolarization beyond -100 mV for full deinactivation, and is sensitive to low µM concentrations of 4-AP.

These features are consistent with our current clamp observations. First, late-firing neurons exhibited a marked outward rectification in the depolarizing direction that increased as the prepulse Vm was hyperpolarized. Second, application of long depolarizing steps to near firing threshold gave rise to long depolarizing ramps, suggesting that the underlying conductance inactivated slowly. Last, the outward rectification and depolarizing ramps were sensitive to low µM concentrations of 4-AP. However, in contrast with previous descriptions in other cell types, this current did not seem to undergo cumulative inactivation when repetitive depolarizing current pulses were applied unless interpulse intervals shorter than 1.5 s were used. Finally, it should be noted that low 4-AP concentrations did not abolish the early component of the outward rectification, suggesting that late-firing neurons are also endowed with a fast A-type K+ conductance (Rudy 1988; Storm 1988).

The presence of these K+ currents is likely to have important consequences for the activity of late-firing neurons. As a result, depolarizing voltage transients caused by excitatory synaptic inputs will be attenuated. Moreover, in response to a sustained barrage of excitatory synaptic inputs, the Vm rise will be delayed. Of course, the magnitude of these alterations will be dependent on the Vm prior to the occurrence of the synaptic events, as the inactivation of the 4-AP sensitive conductance is steeply voltage-dependent and recovers slowly (McCormick 1991; Nisenbaum et al. 1994; Storm 1988).

These considerations imply that the slowly inactivating K+ conductance should tend to reduce the spontaneous and synaptically evoked discharges of late-firing CE neurons. Consistent with this, physiologically identified brain stem projecting CE neurons were reported to have extremely low spontaneous firing rates in conscious animals (Collins and Paré 1999; Pascoe and Kapp 1985). However, inhibitory synaptic inputs might also play an important role in this respect (Collins and Paré 1999; Nose et al. 1991; Paré and Smith 1993b; Sun et al. 1994).


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

In summary, our results indicate that the CE nucleus contains several physiologically defined cells classes. These different types of neurons are not distributed homogeneously in the lateral and medial sectors of the CE nucleus, thus paralleling their involvement in distinct circuits. The distinctive physiological properties of the various types of CEM and CEL neurons are likely to be major determinants of their activity in the intact brain.


    ACKNOWLEDGMENTS

We thank D. R. Collins and E. J. Lang for comments on an earlier version of this manuscript and P. Giguère and D. Drolet for technical support.

This research was supported by the Medical Research Council of Canada.


    FOOTNOTES

Address reprint requests to D. Paré.

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 April 12, 1999; accepted in final form June 1, 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society