106 M
against NMDA and non-NMDA receptor mediated gluta- stellate neurons from our dataset are shown in Fig. 1.
matergic transmission, respectively. Picrotoxin 100 mM, Pyramidal and stellate neurons were subclassified based on
Sigma Chemical Co., St. Louis, MO, USA was used to their dendritic spine density spiny, slightly or sparsely
block GABA receptor-mediated inhibitory transmission. spiny and aspiny. Spine density was actually a principal
A
characteristic in the classification scheme of McDonald 2.4. Neurobiotin-labeling of single cells
[17]. Separately, recorded cells were classified based on their
For cells recorded with Neurobiotin-containing elec- electrophysiological properties. As in a previous study
trodes, after electrophysiological recording, Neurobiotin- [25], our principal characteristic was the appearance of
tracer was injected into these cells using 2–4 nA depolariz- afterpotentials following action potentials Fig. 2. We
ing rectangular current pulses 150 ms duration at 3.3 Hz distinguished two types of afterpotential that followed an
for 20–30 min. Post-injection survival times ranged from action potential initiated by current injection. One was a
10 to 60 min. Slices with Neurobiotin-injected cells were hyperpolarizing afterpotential appearing in 63 of re-
fixed in 4 paraformaldehyde and 0.2 picric acid in 0.1 corded cells. The other was a depolarizing afterpotential
M phosphate buffer pH 7.4 from overnight to 10 days. appearing in 37 of recorded cells.
Frozen sections 40–60 mm thick were cut from the fixed Neither of the two basic electrophysiological classes
tissue and kept in phosphate buffered saline PBS, pH 7.4. corresponded to a single morphological cell class. The
After rinses with PBS, these sections were treated with basic electrophysiological and morphological properties of
0.1 H O for 20 min and Triton-X100 0.4–0.5 in
the cell classes are given in Tables 1 and 2. Cells with
2 2
PBS for 2 to 3 h. They were rinsed in PBS and then hyperpolarizing afterpotentials were found to be both
incubated in the Vectastain ABC Reagent Vector Lab- pyramidal and stellate in shape, although most 19 22
oratories in PBS for 2 to 3 h. After rinses with PBS, were pyramidal cells. Cells with depolarizing afterpoten-
sections were reacted with diaminobenzidine DAB and tials were also found to be both pyramidal 8 13 and
H O 0.003 in PBS to visualize the injected cells. The stellate in shape 5 13, although the split was more
2 2
sections, which included the successfully stained cells, equitable.
were counter stained by Nissl staining. 3.2. Intrinsic electrophysiological properties
3. Results Both electrophysiological classes of cells discharged
several spikes at very short intervals at the onset of large Intracellular recordings were taken from 76 neurons in
depolarizing currents applied via the recording electrode the lateral nucleus of amygdala in 72 horizontal slices from
Fig. 2A2, B2. Cells with a hyperpolarizing afterpotential rat brains. In some experiments, multiple cells were
showed repetitive firing in response to depolarizing current recorded, but, in the vast majority of experiments, a single
injection without adaptation of the firing frequency Fig. neuron was recorded in each slice to permit unequivocal
2A2, A3, A4. Firing frequencies were stabilized to higher matching of morphological results with electrophysiologi-
rates in cells with a hyperpolarizing afterpotential. Hy- cal results. All recorded cells had overshooting action
perpolarizing current pulses revealed marked inward recti- potentials and resting membrane potentials in the range of
fication in this electrophysiological class of cells. 268 to 278 mV that were stable for 20 min to 1 h. Field
Cells with a depolarizing afterpotential showed slight potential recordings were taken from at least one location
adaptation of firing frequency in response to depolarizing deep layers of the retrohippocampal cortices or the lateral
current injection, but overall, their firing rates during nucleus of amygdala in each slice.
comparable current pulses were lower Fig. 2B2, B3, B4. Rectification of inward or outward currents was much less
3.1. Classification of cells in lateral nucleus for this electrophysiological class of cells. In three cells,
the depolarizing afterpotential led to the generation of Of the Neurobiotin-filled neurons, 35 cells were success-
spike doublets in response to current injection. fully recovered to permit morphological classification.
Labeled cells were classified as pyramidal 27 35; 77 or 3.3. Responses evoked by entorhinal stimulation
stellate cells 8 35; 23 based on the pattern of dendritic branching. Pyramidal cells had a clear ‘apical’ dendrite
Evoked responses were recorded in 40 lateral nucleus that was distinct from their multiple smaller ‘basal’
neurons to extracellular stimulation of the deep layers of dendrites. Stellate cells were characterized by multiple
the entorhinal cortex. Twenty-three of these cells were also dendrites of uniform diameter and with a uniform dis-
morphologically identified as pyramidal N517 or stellate tribution around the soma. Similar classification schemes
N56 neurons. have been employed in golgi studies of basolateral and
Responses by cells varied from a long duration event lateral nucleus [17,19]. Examples of labeled pyramidal and
with multiple action potentials resembling the responses
M . Funahashi et al. Brain Research 884 2000 104 –115
107
Fig. 1. Photomicrographs of Neurobiotin-labeled neurons in the lateral nucleus of the rat amygdala. A1 A low power image of a typical pyramidal cell with a thick primary ‘apical’ dendrite and ‘basal’ dendrites. This cell exhibited a hyperpolarizing afterpotential. A2 Higher magnification image shows
dendritic spines. B Low B1 and high B2 magnification images of a typical stellate neuron with several thin spiny dendrites uniformly distributed around the soma. This cell exhibited a depolarizing afterpotential. Calibration bar is 20 mm.
exhibited by deep layer retrohippocampal neurons Fig. 3, The second grade 2 of response was characterized by
to epsps with no associated firing. We graded responses an event duration totaling about 50 ms. The maximum
based on the pattern of evoked action potentials. Grade 1 firing frequency during grade 2 responses reached 100 Hz.
responses resembled responses of deep layer entorhinal Epsp amplitudes were considerably smaller than those in
neurons. These consisted of a brief 10–50 ms period of grade 1 responses. Grade 2 responses were recorded in
very high frequency 200 Hz firing followed by a variable 8 40 cells, including two identified pyramidal and one
period of sustained membrane depolarization without stellate neuron.
firing. A period of gamma 40–100 Hz firing occurred at Grade 3 responses were characterized by still smaller
the end of each compound event. The total event duration epsps and single action potentials 5 40 cells including
was over 100 ms in every cell of this grade. Grade 1 two identified pyramidal cells. The final grade 4 was
responses were seen in 10 40 cells, including four mor- characterized by small epsps with no associated firing
phologically identified pyramidal cells. 17 40 cells including nine pyramidal and four stellate
108 M
Fig. 2. Intrinsic membrane properties of morphologically identified pyramidal neurons of the lateral nucleus. Column A shows responses of pyramidal cells with hyperpolarizing afterpotentials. Column B shows cells with depolarizing afterpotentials. A1 Voltage responses by a cell with hyperpolarizing
afterpotentials to intracellularly injected current 100 ms duration, 10.3 to 21.05 nA, 0.15 nA steps. Voltages at the time marked by the arrow are plotted against the stimulus currents. Note inward rectification of injected currents and hyperpolarizing afterpotential enlarged in inset. Suprathreshold responses
of the same cell are shown in A2 400 ms duration, 10.6 to 1.8 nA, 0.4 nA steps. Note the initial high frequency discharge in response to larger depolarizing current pulses, followed by a stable firing frequency. Frequency–current relations for multiple cells are shown in A3. Plot of interval versus
interval number A4 shows that there was little spike frequency adaptation, especially at the higher stimulus currents. B1 Voltage responses by a cell with depolarizing afterpotentials to intracellularly injected current 100 ms duration, 10.15 to 20.9 nA, 0.15 nA steps. Membrane rectification of injected
currents was much less pronounced for these neurons. B2 Depolarizing pulses 400 ms duration, 10.2 to 1.0 nA, 0.2 nA steps triggered an initial burst of action potentials followed by a lower frequency train of single spikes. B3 Frequency–current relations for multiple cells. Spike frequency adaptation
was more pronounced in cells with depolarizing afterpotentials B4.
M . Funahashi et al. Brain Research 884 2000 104 –115
109 Table 1
a
Membrane properties of Neurobiotin-labeled neurons in the lateral nucleus of amygdala Afterpotential
Cell shape n
Resting Input
Time Spike
Spike potential
resistance constant
amplitude half-width
mV MV
ms mV
ms Hyperpolarization
Pyramidal 19
270.665.0 44.1611.1
12.764.6 72.7612.6
1.3860.4 Stellate
3 271.564.9
25.561.1 12.561.9
67.468.5 1.5260.2
Depolarization Pyramidal
8 268.863.3
44.6611.6 12.764.52
71.9615.2 1.3560.2
Stellate 5
268.861.8 39.8610.1
10.362.71 72.066.3
1.4260.3
a
Values expressed as mean6S.D.
cells. Representative responses of cells from each grade in these cells. Epsps were seen in relation to each of the
are shown in Fig. 4. afterdischarges in field potential recordings Fig. 6B.
Evoked responses of lateral nucleus neurons in all grades were associated with similar field potential dis-
3.5. NMDA and non-NMDA components of epsps charges in the entorhinal cortex and lateral nucleus of the
amygdala. Synchronous activation of lateral nucleus neu- CPP and CNQX were used to examine epsps in three
rons was also evidenced in pairwise recordings Fig. 5. lateral nucleus cells responding to entorhinal stimulation.
One example of a grade 4 cell is shown in Fig. 7. A long 3.4. Effects of GABA receptor blockade
sustained epsp was evoked by stimulation to the deep layer
A
of the entorhinal cortex. The amplitude and duration of To test the possibility that variations in the evoked
epsps were gradually decreased during the first 10 min responses were due to differences in the intensity of
following addition of CPP to the perfusate. The remaining concurrent inhibition that may be activated by propagating
component, stable in amplitude and duration after 10 min population events, we tested four cells with grade 3 epsp
of CPP exposure was completely suppressed by addition of plus one action potential and four epsp only evoked
CNQX to the perfusate. responses in the presence of 100 mM picrotoxin Fig. 6.
At resting membrane potential, cells that were classified as grade-3-evoked responses developed progressively more
4. Discussion
