1Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601; and 2Neuroscience Group and Discipline of Anatomy, University of Newcastle, Newcastle, NSW 2308, Australia
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ABSTRACT |
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Faber, E.S.L., R. J. Callister, and P. Sah. Morphological and Electrophysiological Properties of Principal Neurons in the Rat Lateral Amygdala In Vitro. J. Neurophysiol. 85: 714-723, 2001. In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology.
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INTRODUCTION |
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The amygdala has been implicated
in a variety of functions including memory and attention and in placing
emotional significance to sensory input to produce the appropriate
physiological response (Adolphis et al. 1994;
Aggleton 1993
; Kluver and Bucy 1939
;
LeDoux 1995
; Rogan and LeDoux 1996
). The
amygdala has also been shown to be involved in the generation of
temporal lobe epilepsy (Callahan et al. 1991
;
Danober and Pape 1998
) and to be important in auditory fear conditioning (LeDoux 1995
). Anatomically, the
amygdaloid complex is divided into a number of nuclei including the
basolateral complex (BL), which is made up of the lateral (LA),
basolateral (BLA), and basomedial nuclei (Brodal 1947
;
Pitkänen et al. 1997
). These nuclei can then be
further divided into subdivisions that have extensive interconnections
(Krettek and Price 1978
; Pitkänen et al.
1997
). The amygdala receives input from both cortical and thalamic regions (McDonald 1999
; Turner and
Herkenham 1991
). The LA is particularly important in mediating
fear conditioning as sensory inputs involved in this process have been
proposed to enter the amygdaloid complex via this nucleus
(LeDoux 1995
; Pitkänen et al. 1997
;
Romanski and LeDoux 1993
).
A complete understanding of information processing in the amygdala
requires detailed information about the properties of neurons comprising the various nuclei as well as their connections. Golgi studies have identified two types of neurons in the LA and BLA (McDonald 1984; Millhouse and DeOlmos
1983
; Price et al. 1987
). The principal cell
type has three to five primary dendrites that are invested with a
moderate density of spines and resembles cortical pyramidal neurons.
These cells are thought to be projection neurons and use glutamate as
their neurotransmitter (Carlsen 1988
; Smith and
Paré 1994
). The other cell type has dendrites that lack
spines and are thought to be local circuit cells that use GABA as their transmitter (McDonald 1984
; Paré and Smith
1993
).
Electrophysiologically, neurons within the BLA have been classified
into three groups with distinct morphologies (Paré et al.
1995; Rainnie et al. 1993
; Washburn and
Moises 1992
). In contrast, there have been few extensive
studies of the physiological properties of neurons in the LA. In one
study using microelectrode recordings from rat brain slices, three
classes of cell were identified based on the fast
afterhyperpolarization that followed single action potentials and their
responses to applied agonists (Sugita et al. 1993
).
Other studies have revealed two cell types in the LA based on the
properties of their action potentials. One type has relatively broad
action potentials, shows spike frequency adaptation, and has been
classified as pyramidal cells. The other type has faster action
potentials and shows no spike frequency adaptation in response to
prolonged current injection. The latter cells have been classified as
interneurons (Mahanty and Sah 1998
; Paré et al. 1995
). In the LA, in vivo and in vitro recordings from
projection (noninterneuronal) neurons from cat and guinea pig have
shown that these cells generate two types of intrinsic membrane
potential oscillations in response to somatic current injection
(Pape et al. 1998
; Paré et al.
1995
). A small proportion of cells did not generate these
oscillations, suggesting that there may be two types of pyramidal
neurons in the LA. In a recent in vitro study in the rat
(Faulkner and Brown 1999
), only neurons bordering the
external capsule in the LA were studied. In this study,
noninterneuronal cells were divided cells into four categories (see
DISCUSSION). However, a detailed analysis of the repetitive
firing properties of neurons in the LA has not been performed. In the
present study, we have characterized the noninterneuronal neurons in
the rat LA in vitro using electrophysiological and quantitative
morphological techniques. We find that these cells show a range of
repetitive firing properties that are best explained by their
afterhyperpolarizations (AHPs) and by the fact that their morphology
cannot predict their discharge properties.
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METHODS |
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Tissue preparation
All experiments were performed on rat brain slices maintained in vitro. Wistar rats (of either sex, 17- to 20-days old) were anesthetized with intraperitoneal pentobarbitone (50 mg/kg) and decapitated. These procedures were in accordance with the guidelines of the Institutional Animal Ethics guidelines. Brains were rapidly removed and placed in ice cold artificial cerebral spinal fluid (ACSF) containing (in mM) 118 NaCl, 2.5 KCl, 25 NaHCO3, 10 glucose, 1.3 MgCl2, 2.5 CaCl2, and 1.2 NaHPO4. Coronal slices (400 µm thick) containing the amygdala (see Fig. 1) were cut using a microslicer (Dosaka, DTK-1000). Slices were allowed to recover in oxygenated (95% O2-5% CO2) ACSF at 30°C for 30 min, then kept at room temperature for a further 30 min before experiments were performed. Slices were transferred to the recording chamber as required and were continuously perfused with oxygenated ACSF maintained at 28-30°C.
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Electrophysiological analysis
Whole cell recordings were made from neurons in the LA using the
"blind" or infrared differential interference contrast
(IR/DIC) techniques. Patch pipettes (3-6 M) were fabricated
from borosilicate hematocrit glass and filled with a solution
containing (in mM) 135 KMeSO4, 8 NaCl, 10 HEPES,
2 Mg2ATP, and 0.3 Na3GTP
(pH 7.3 with KOH, osmolarity 300 mOsm). On some occasions biocytin
(0.1%) was included in the internal solution. Access resistance was
5-30 M
and was monitored throughout the experiment. Signals were
recorded with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Responses were filtered at 5-10 kHz and digitized at 10 kHz (Instrutech, Greatneck, NY, ITC-16). All data were acquired, stored, and analyzed on a Power Macintosh 7500/100 using Axograph (Axon Instruments).
To investigate the firing properties of neurons, six to eight current
injection steps (600 ms in duration) were delivered in 100 pA
increments. AHPs were evoked in current clamp by applying a 50-ms,
400 pA current step from a holding potential of 70 mV. Currents
underlying the AHP (IAHP) were evoked
in voltage clamp by applying a 50-ms, 50-mV depolarizing step from a
holding potential of
50 mV. To compare AHPs across cells, their
properties were quantified by measuring the area under the curve from
baseline over the first 2 s of the response. Spike amplitudes were
measured from resting potential. The threshold for spike initiation was taken as the beginning of the upstroke of the action potential.
Tissue processing and histology
Following physiological recording slices were fixed overnight in
4% phosphate buffered formalin (0.1 M, pH 7.4) at 4°C. The slices
were rinsed in phosphate-buffered saline (0.1 M, pH 7.4; 3 changes, 20 min each) and permeabilized by overnight exposure to 0.5% Triton-X in
phosphate buffered saline. The slices were then soaked overnight in
avidin-horseradish peroxidase (Vectastain ABC Elite Kit, Vector
Laboratories, Burlingame, CA). After washing in Tris buffer
(0.1 M, pH 7.4), filled cells were visualized using the
diaminobenzidine (DAB) procedure (Adams 1977). Slices
were then mounted on albumin coated slides, dried overnight, dehydrated in an ascending series of alcohols, cleared in xylene, and coverslipped in Permount.
Morphological analysis
Biocytin-filled cells were drawn at a final magnification of
×650 using a camera lucida mounted on an Olympus BX50 microscope. These drawings were used for morphological analysis. All measurements were made from the drawings using a Bioquant Image Analysis system. Soma size was taken as the area enclosed by a smooth line made on the
drawings (Culheim et al. 1987; Kernell and
Zwaagstra 1981
). This line closely followed the soma's border
but excluded emerging dendritic processes. The length of each dendritic
segment was measured and corrected for its trajectory in the
z plane by using the depth values from the fine focus of the
microscope and the nominal section thickness (400 µm) and applying
Pythagoras' theorem. Using this approach, we were able to account for
shrinkage due to tissue processing in the z plane. No
allowance was made for shrinkage in the x and y
planes, so dendritic dimensions in these planes will have been slightly
underestimated. The extensive investment of dendrites with spines often
caused the surface of dendrites to appear blurred when viewed under the
light microscope. For this reason, we were unable to accurately measure
the diameters of dendritic segments. Emerging processes were classified
as axons if they showed no evidence of tapering, possessed "en
passant" boutons and lacked spines.
For analysis of dendritic branching patterns, we have used the
terminology originally proposed by Percherson (1979) and
subsequently modified by Larkman et al. (Larkman and Mason
1990
) for cortical pyramidal cells. According to this scheme,
dendritic trees branch at "branch points" and the portion of the
dendrite between each branch point is called a "dendritic segment."
The segment originating from the soma is called the "stem segment"
and the final dendritic segment terminates at a "dendritic tip."
Dendritic segments can be further classified as first order (the stem
segment), second order, and so forth.
Results are expressed as means ± SE. Student's t-tests were used for statistical comparisons between groups. Tetrodotoxin was obtained from Alomone and biocytin from Vector Laboratories.
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RESULTS |
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All recordings were made from dorsolateral, ventrolateral, or medial portions of the lateral amygdaloid nucleus (Fig. 1).
Electrophysiology
A total of 252 neurons were recorded from the LA that reached the
minimal criteria for health and stability. These cells had a resting
membrane potential more negative than 55 mV and access resistance
less than 15 M
. Of these, four were classified as interneurons and
are not included in this study. The remaining cells were classified as
projection neurons based on their action potential half width and
varying degrees of spike frequency adaptation in response to a 600 ms
depolarizing current injection. These cells had a range of firing
properties that varied from those that showed complete spike frequency
adaptation, firing at most a few action potentials, to those that fired
repetitively throughout the depolarizing current step with little or no
spike frequency adaptation. In a population of 73 cells recorded using
the blind method, 81% (59/73) of cells spiked only two to five times,
whereas 19% of cells (14/73) generated more spikes in response to a
600-ms current injection. For comparison, in a population of 66 neurons recorded using IR-DIC techniques, 79% (52/66) fired only two to five
spikes, whereas 21% (14/66) fired more than five spikes. These results
indicate that the differences in firing properties are unlikely to be
due to removal of dendritic processes in cells close to the surface of
the slice. Three representative examples of the extremes and middle of
this continuum are shown in Fig. 2. There
were no clear differences in the passive membrane properties in cells
showing different degrees of spike frequency adaptation. These
measurements have therefore been lumped together. The average resting
membrane potential was
66 ± 0.8 mV, input resistance was
150 ± 9 M
, and membrane time constant was 29 ± 1 ms
(n = 66). Application of the sodium channel blocker
tetrodotoxin (TTX, 1 µM) completely blocked action potentials in all
cells (Fig. 3A, 1 and
2). In the presence of TTX, depolarizing current injections did not initiate calcium spikes but revealed a sustained outward rectification in all cells at potentials more depolarized than
50 mV
(Fig. 3B).
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The distribution of firing properties formed a continuum and is plotted as the number of action potentials fired in response to a 400 pA, 600 ms current injection in Fig. 4. It can be seen that the most common cell type (151/248 cells; 61%) fired only one to five action potentials in response to increasing amplitudes of current injection. The remaining cells fired between 6 and 30 action potentials during the current injection. Of these cells, it was notable that some (70/248; 28%) showed clear spike frequency adaptation during the first 5-10 action potentials (e.g., Fig. 2B), while others (27/248; 11%) fired repetitively throughout the current injection with little or no spike frequency adaptation (e.g., Fig. 2C).
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In the BLA, pyramidal neurons have been classified into two types that
resemble the fully adapting and repetitively firing cells observed in
this study (Rainnie et al. 1993; Washburn and Moises 1992
). Neurons that showed little spike frequency
adaptation in the BLA were also described as late firing. These cells
showed a clear delay in the initiation of the first action potential when it was evoked from negative membrane potentials (Washburn and Moises 1992
). In the LA, we did not find any late firing
neurons, and hyperpolarization of the membrane potential did not delay the time to first spike initiation in either fully accommodating cells
or those that fired repetitively (data not shown).
In many cell types, the AHP that follows trains of action potentials is
largely responsible for spike frequency adaptation (Sah
1996). We therefore tested if the differences in spike
frequency adaptation was correlated with differences in the amplitude
and time course of the AHP following a train of action potentials. In
neurons that accommodated fully (Fig. 2A), action potentials evoked by a 100-ms depolarizing current injection evoked a large slow
AHP (Fig. 5A, left)
that had a mean integral of 4.3 ± 1 mV.s (n = 42). In contrast, cells that fired repetitively throughout the current
injection (Fig. 2C) had a significantly smaller slow AHP
with a mean integral of 2.3 ± 0.6 mV.s (P < 0.05, n = 12, Fig. 5A, right). The
differences in the integral of the AHP were also reflected in the
amplitude of the current underlying the slow AHP (Fig. 5B).
In cells that showed complete spike frequency adaptation, the peak
amplitude of the slow AHP current (measured at 500 ms after the voltage
step) was 108 ± 13 pA (n = 40) compared with
34 ± 7 pA (n = 10) in cells that fired
repetitively. These results suggest that the differential firing
properties in these cells may be due to differences in the amplitude of
the slow AHP. In agreement with this, in neurons that showed full spike
frequency adaptation, blockade of the AHP with cadmium (250 µM)
evoked repetitive spikes in response to the same current injection
(Fig. 6A).
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Broadening of action potentials during spike trains has been reported
in a number of cell types (Agmon and Connors 1992;
Kasper et al. 1994
; McCormick et al.
1985
; Shao et al. 1999
). Since calcium influx
largely occurs during the repolarizing phase of the action potential
(Llinás et al. 1982
), broadening of the action
potential would increase calcium influx leading to more activation of
calcium-dependent ion channels (Jackson et al. 1991
). We
therefore examined whether the differences in AHP amplitude in the
different cell types could be due to a contrast in spike broadening. In
LA neurons, action potentials during a train showed a clear change in
the rate of repolarization and half-width. Recordings from two cells
with distinct firing properties are shown in Fig.
7. Clearly spike broadening is similar in
both cell types. In both cases, the first action potential had a mean
amplitude of 90.1 ± 0.6 mV (n = 66), a mean
half-width of 1.4 ± 0.02 ms (n = 230), and a
spike threshold of
41 ± 1 mV (n = 66). The
second and subsequent action potentials had the same peak amplitude but
a significantly (P < 0.001) wider half-width of
1.9 ± 0.04 ms (n = 230; Fig. 6, B and
D).
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Apart from the firing properties described thus far, 7 of the 252 neurons (3%) spiked only once (Fig.
8A) despite attempts to raise
their excitability by either increasing the amplitude of depolarizing
current injection or by holding the cell at more depolarized membrane
potentials. These neurons had a mean resting membrane potential of
56 ± 1 mV, a mean input resistance of 158 ± 30 M
, and
a mean time constant of 24.7 ± 4 ms (n = 7). No
AHP could be evoked in any of these cells either in current clamp (Fig.
9B) or voltage clamp (Fig.
8C).
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Morphology
All cells characterized in this study had their somata located at least 100 µm below the surface of the slice (Table 1). These criteria ensured that much of a cell's complete dendritic arbor was represented in our drawings. However, despite these measures, more than 30% of the dendritic processes traveled out of the slice (see Table 1). Thus the data presented here underestimate the true size of LA neurons. However, this caveat will not affect comparisons between the morphology of different neurons because it likely applies equally to all types.
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One possible explanation for the extremes in electrophysiological
response described in the preceding text is that projection cells in
the LA fall into distinct morphological classes as has been suggested
in the BLA (Washburn and Moises 1992). With this in
mind, for morphological analysis, we divided cells into those that
showed marked spike frequency adaptation and fired one to five spikes
and those that showed little or no adaptation (more than 10 spikes in
the spike train). These will be referred to as type 1 and
type 2, respectively. Eighteen electrophysiologically characterized neurons were selected for detailed morphological analysis. An example of a recovered biocytin filled cell is shown in
Fig. 9. Selection for detailed analysis was based on the following criteria: cells were located deep in the slice (see preceding text),
were deemed to be well filled, had complete electrophysiological data
sets, and had dendritic processes that traveled to both the top and
bottom of the slice. Of these cells, nine were classed as type
1 and the other nine as type 2 (see Fig.
10).
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At first glance, our recovered cells appeared to form two classes based on the shape of their somata; those with a clear pyramidal morphology and those that appeared multipolar or stellate. On close examination, however, it became clear that cells oriented predominantly in the horizontal plane appeared to have rounded or stellate cell bodies, whereas cells that lay in the plane of section, i.e., coronal, could be described as pyramidal (see Fig. 9). In spite of the apparent differences in the shape of their somata, when viewed in two dimensions, all cells featured a dominant dendrite that had a greater number of branch points and dendritic membrane. These qualitative observations were confirmed by our detailed quantitative data (see Table 1). In all cells examined, one dendrite contained more dendritic segments and branch points and represented over 30% of the cell's total dendritic length. This dendrite was classed as the apical dendrite. For these reasons, we have classified all our cells as pyramidal-like.
Pyramidal-like cells have a dominant or apical dendrite and two to five
basal dendrites emerging from the base of the cell somata (Table 1).
Cells in either physiological class showed no preferred orientation or
location within the LA (see Fig. 1), which contrasts with pyramidal
neurons in other brain regions such as cerebral cortex
(Colonnier 1981). Of the type 1 neurons, 4/9
had their long axis orientated chiefly in the horizontal plane and the
remaining five cells in the coronal plane. Of the type 2 neurons, 6/9 were orientated primarily in the horizontal plane and the
other 3 in the coronal plane. In some cells, a number of dendritic
processes traveled into and even crossed the external capsule as
previously described (Millhouse and DeOlmos 1983
). The
dendrites of all cells in both classes were invested with spines (see
Fig. 9). These were located on all regions of the dendrites except on
the initial portion where they emerged from the somata. Axons (see Fig.
9) were noted on 6/9 of type 1 neurons and 7/9 of type
2 neurons. In most cases, the axon originated from the somata (5/6
and 5/7 for type 1 and 2 neurons, respectively). In the other cases, the axon originated from a basal dendrite.
Three examples of type 1 (left) and 2 (right) neurons and their dendritic trees are shown in Fig. 10. For each class, cells have been chosen according to increasing (top to bottom) complexity of their dendritic arbors. Dendrograms representing the branching pattern for each cell's primary dendrites are shown on the left and right of each cell (Fig. 10). Examination of all the cells and their dendrograms revealed no obvious qualitative morphological differences between the cells types. A detailed quantitative analysis of a variety of dendritic characteristics supports this initial observation (Table 1). The dendritic morphology of the two cell types is strikingly similar according to the parameters presented in Table 1. No significant difference (P > 0.05) was found among the mean soma size, number of primary dendrites, the mean length of second, third, and fourth, etc. segments and number of dendritic tips of the two cell types. The total dendritic length, which provides a crude estimate of cell size, suggests that type 1 and type 2 neurons are similar in overall size. Furthermore the dendritic morphology of apical and basal dendrites in terms of the number of branch points and total dendritic length in cells belonging to the two classes was not significantly different (P > 0.05). However, within both classes there were a significantly (P < 0.05) greater number of branch points in the apical dendrite compared with the basal dendrites (8.3 vs. 4.6 for type 1 and 9.2 vs. 3.6 for type 2 neurons). In both cell classes over 30% of the total dendritic tree length is contained within the apical dendrite (see Table 1).
In summary, there are no clear morphological distinctions between cells that differ markedly in their electrophysiological properties (as assessed by the response to a 600-ms current injection). In fact, the variation in dendritic complexity and the degree to which cells clearly exhibit apical versus basal dendrites is just as great within each physiological class.
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DISCUSSION |
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The present experiments have categorized spiny noninterneuronal neurons within the LA based on their electrophysiological properties. Cells showed firing properties that varied in a continuum from those that had marked spike frequency adaptation to cells that fired repetitively with little or no spike frequency adaptation. However, the most common cell type were those that accommodated fully in response to depolarizing current injections. In spite of the clear differences in firing properties between cells, our morphological analysis shows that all cells are pyramidal in shape with spiny dendrites and could not be clearly separated in terms of cell soma area or dendritic structure into distinct classes.
These differences in spike frequency adaptation between pyramidal cells
in the LA appear to be largely due to differences in the amplitude of
the AHP that followed a short train of action potentials. Neurons that
adapted fully have a large, long-lasting AHP, whereas neurons that
showed little adaptation have a shorter duration AHP. The AHP that
follows action potentials is mediated by activation of
Ca2+-activated potassium currents. Neurons are
known to express two types of these currents that have been called
IAHP and
sIAHP. IAHP underlies the medium AHP and is
responsible for a reduction in spike frequency. In contrast,
sIAHP underlies the slow AHP and its
activation leads to spike frequency adaptation (Sah
1996). The relatively smaller amplitude of
sIAHP in repetitively firing neurons
would explain the lack of marked spike frequency adaptation observed in
this cell type. Similar regulation of firing patterns by
sIAHP has also been reported in
neurons in the BLA (Washburn and Moises 1992
),
hippocampus (Madison and Nicoll 1984
) and neocortex (McCormick et al. 1985
). These differences in the size
of the AHP between pyramidal cells in the LA suggest that there
are likely to be differential expression patterns of either
voltage-dependent calcium channels or the potassium channels that
underlie the AHP or both. It is notable that in LA pyramidal cells
recorded in vivo, calcium-activated potassium currents have also been
suggested to be activated by subthreshold synaptic inputs (Lang
and Paré 1997
), suggesting that these neurons will also
respond differently in response to afferent stimulation.
At first inspection, most of our biocytin-filled cells in the LA could
be confidently classified as having pyramidal-like morphology (e.g.,
Fig. 9). However, the orientation of some cells in the slice made the
distinction between the basal and apical region of the cell difficult
(see also Danober and Pape 1998; Millhouse and
DeOlmos 1983
; Paré et al. 1995
). Thus LA
pyramids differ from classical pyramidal neurons (Bannister and
Larkman 1995
; Larkman and Mason 1990
) as there
is no rigid orientation of the pyramids in any one plane. LA pyramids
also differ from cortical pyramids in several other ways. First, there
are very clear differences in the branching patterns of apical and
basal dendrites in cortical pyramids. In LA pyramids, stem segment
length (distance from somata to first branch point; see Table 1) is similar for both apical and basal dendrites. This contrasts with classical layer-5 cortical pyramids as well as hippocampal pyramidal neurons (Bannister and Larkman 1995
; Larkman and
Mason 1990
) where the apical dendrite may extend for up to
hundreds of micrometers before major branches emerge to form a terminal
arbor. In addition, the apical dendrites of layer-5 cortical pyramids
taper very little as they project toward the pial surface. In contrast,
all dendrites in LA pyramids taper rapidly as they move away from the
soma. In hippocampal and cortical pyramidal neurons, basal dendrites are easily distinguished from apical because they branch repeatedly close to the soma (Bannister and Larkman 1995
;
Larkman and Mason 1990
). In LA pyramids, however, the
distinction between apical and basal dendrites is less clear using the
same criteria. Excluding orientation, we suggest that LA pyramids
resemble most closely those of layer-2/3 visual cortex neurons, where
the contribution of the apical dendrite accounts for about 40% of the
total dendritic length (see Fig. 1 in Larkmann and Mason
1990
for comparison) compared with approximately 32% in LA
pyramids. The classical layer-5 pyramids have a much greater proportion
of their dendritic length (49%) and surface area (53%) in their
apical dendrites. For these reasons, we propose that these spiny cells
in the LA are perhaps better classified as pyramidal-like neurons
rather than pyramidal to distinguish them from cortical type pyramidal neurons.
Previous morphological studies of the rat amygdaloid complex have shown
that spiny projection neurons comprise the majority of the total
neuronal population in the basal and lateral nuclei. The remaining
cells are smaller aspiny cells that are thought to be local circuit
interneurons (McDonald 1982; Millhouse and DeOlmos 1983
; Price et al. 1987
). In the BLA,
electrophysiological studies have divided the spiny noninterneuronal
cells into two distinct classes based on physiology and morphology
(Rainnie et al. 1993
; Washburn and Moises
1992
). Thus a clear correlation was made between morphology and
electrophysiology of these neurons in the BLA.
Our results in the LA failed to show such a correlation since all of
the morphologically characterized cells were clearly pyramidal-like.
The morphology of our recovered cells is consistent with that of
neurons classified as pyramidal using Golgi techniques (McDonald
1982). This is also in agreement with in vivo studies, which,
apart from fast firing interneurons, have seen no morphological differences between neurons with differing firing properties and AHPs
in the LA (Paré et al. 1995
). In the BLA, a small
fraction of cells were described as having stellate or multipolar cell bodies (Washburn and Moises 1992
). Such cells have also
been described in Golgi studies of the rat BLA (McDonald
1982
; Millhouse and DeOlmos 1983
). We have not
been able to identify cells that definitively fit into this category in
the LA. Furthermore three-dimensional reconstructions of dendritic
trees in studies on LA and BLA neurons (this study and others: Sah and
Callister, unpublished observations) (Danober and Pape
1998
; Millhouse and DeOlmos 1983
;
Paré et al. 1995
) have suggested that cells that
appear stellate are likely to be pyramidal because their apical
dendrite lies in the z plane of the section. The LA has been
divided into several subdivisions (e.g., see figures in
Pitkänen et al. 1997
). It is clear from the
dendritic arbors of filled cells (Paré et al.
1995
; this paper), however, that pyramidal-like neurons in
these subdivisions would likely extend their dendrites outside the
proposed limits.
Previous electrophysiological recordings from LA projection neurons
both in vivo and in vitro have described intrinsic voltage-dependent oscillations in membrane potential near action potential threshold (Pape et al. 1998; Paré et al.
1995
). We have not found these oscillations in recordings made
in acute brain slices. Furthermore while the properties of all cells
were not described in detail, these studies (Pape et al.
1998
; Paré et al. 1995
) have not reported the presence of fully adapting neurons. The reason for these
differences are not immediately apparent. However, these studies were
done using sharp microelectrodes at higher temperatures and different animal species (cat and guinea pig). It is therefore possible that
these differences may be one reason for the different results. In
addition, because of the use of sharp microelectrodes, the reported
input resistance of the cells was much lower. This difference in the
input resistance means that the impact of the AHP current activated by
action potential trains would be greatly reduced and may explain the
lack of accommodation.
In another recent investigation of cell physiology and morphology in
both the LA and perirhinal cortex of rats in vitro, neurons were
categorized into five groups based on their firing patterns in response
to prolonged depolarizing current injection (Faulkner and Brown
1999). In the LA, neurons were only recorded from regions adjacent to the external capsule and characterized as regular spiking,
fast spiking, or late spiking. The fast spiking neurons formed 7% of
the total population and probably correspond to interneurons described
previously (Lang and Paré 1998
; Mahanty and
Sah 1998
). The majority of the other neurons were classed as
regular firing. No fully accommodating neurons were described in the
LA. However, all the recordings in the Faulkner and Brown study were
performed at room temperature, with high concentrations of EGTA in the
internal solution. Inclusion of EGTA would significantly buffer
cytosolic calcium and thus calcium-activated potassium channels that
contribute to action potential repolarization and the AHP would have
been significantly less active. Furthermore differences in recording temperature are also likely to affect the firing properties of cells.
Thus cell location and these two methodological factors are likely to
explain the differences in cell types observed in this study versus
those of Faulkner and Brown.
The less frequently encountered (7 of 252) type of neuron, which fired
a single spike (Fig. 9), was not morphologically examined in the
present study. However, Faulkner and Brown (1999)
describe two neurons with electrophysiological properties similar to
these cells. One of these cells was recovered and was found to be
pyramidal in shape. The similarity of the passive membrane properties
of these neurons to the other pyramidal cells, together with the stability of these cells during recording, makes it unlikely that they
are simply "sick cells" and supports the idea that they represent a
distinct electrophysiological class. The precise role of this interesting electrophysiological class of cells is not clear and further studies are required to examine this more fully.
In summary, we have shown that the principal spiny cells within the LA
fall into a continuum with extremes that can be separated according to
their firing patterns. These different firing patterns are likely to be
determined by differential expression patterns of ion channels in
neurons. The morphological features of these cells place them in a
single class consistent with previous morphological studies
(McDonald 1982, 1984
; Millhouse and DeOlmos
1983
). The relative proportions of fully adapting cells
compared with the other types suggests that the predominant role of
neurons within the LA is to process sensory input by transforming tonic
excitatory input into phasic output. This assertion is supported by the
very low firing rates of LA neurons that have been recorded in vivo (Ben-Ari et al. 1974
; Bordi et al.
1993
; Paré and Gaudreau 1996
). In
contrast, neurons that have little spike frequency adaptation would
turn a tonic input into sustained output. Thus inputs arriving at these
two cell types would be differentially processed and suggest that they
may be receiving afferent inputs from different sources.
Finally, in comparison to other reasonably well-understood brain regions such as the cerebral cortex, hippocampus, and cerebellum, the amygdaloid nuclei do not show a rigid organization of its neuronal elements. Cells are not organized into layers nor do they have the striking preferred orientation of their major output neurons as described in these other brain regions. Thus it is not surprising that input-output mechanisms in the LA are regulated by the various ionic conductances in the membranes of their major output neurons rather than through mechanisms that rely on architectonics and differences in cell morphology.
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ACKNOWLEDGMENTS |
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We thank J. Bekkers for comments on the manuscript. P. Sah is a Sylvia and Charles Viertel Senior Medical Research Fellow.
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FOOTNOTES |
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Address for reprint requests: P. Sah, The Division of Neuroscience, The John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia (E-mail: pankaj.sah{at}anu.edu.au).
Received 8 June 2000; accepted in final form 24 October 2000.
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REFERENCES |
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