JOURNALOFNEUROPHYSIOLOGY
Vol. 6 1, No. 5, May 1989. Printed in U.S.A.
Effects of Low Concentrations of 4-Aminopyridine
on CA1 Pyramidal Cells of the Hippocampus
PAUL PERREAULT AND MASSIMO AVOLI
Montreal Neurological Institute, Department of Neurology and Neurosurgery,
McGill University, Montr&al, Quebec H3A 2B4, Canada
SUMMARY AND CONCLUSIONS
I. Intracellular and extracellular recording techniques were
used to study the effects of bath application of 4-aminopyridine
(4.AP) on pyramidal cells of the CA1 subfield of rat hippocampal
slices maintained in vitro. The concentration of 4-AP used in
most experiments was 50 PM. However, similar results were ob-
tamed with a concentration ranging from 5 to 100 PM.
2. Following 4-AP application, cells impaled with K-acetate-
filled microelectrodes hyperpolarized by an average of 2.6 mV
(from -68.7 to -71.3 mV, P \( 0.01). This change was accompa-
nied by the appearance of high-frequency spontaneous hyperpo-
larizations. Conversely, when KCl-filled microelectrodes were
used, an average depolarization of 5.8 mV [from -73.1 to -67.3
mV, not significant (NS)] associated with the occurrence of repet-
itive depolarizing potentials was observed. In both cases, these
changes were concomitant with a small decrease in membrane
input resistance, which was statistically significant only for cells
impaled with K-acetate-filled microelectrodes. When synaptic
transmission was blocked by tetrodotoxin (TTX), 4-AP induced
in cells studied with K-acetate microelectrodes an average depo-
larization of 2.4 mV (from -62.8 to -60.4 mV, P < 0.01) accom-
panied by a small increase in input resistance (from 32.0 to 35.8
MQ, P < 0.05). High-frequency spontaneous potentials failed to
occur under these conditions. During 4-AP application, the
threshold and the latency of action potentials elicited by a depo-
larizing current pulse increased in 36% of the neurons studied
(n = 14).
3. The amplitude of the stratum (s.) radiatum-induced excit-
atory postsynaptic potential (EPSP) was augmented by 4-AP.
Both the early and late inhibitory postsynaptic potentials (IPSPs)
evoked by orthodromic stimuli were also increased in amplitude
and duration. In addition, a late (peak latency, 150-600 ms) and
long-lasting (duration, 600- 1,500 ms) depolarizing potential ap-
peared between the early and the late IPSPs and progressively
increased until it partially masked these hyperpolarizations. This
long-lasting depolarization (LLD) could also be induced by anti-
dromic stimulation, although in this case it was preceded by an
additional, fast-rising, brief depolarization.
4. A similar brief depolarization preceded the orthodromically
induced LLD in 69% of the neurons bathed in the presence of
4-AP. The average value of the peak latency of this potential was
62 k 27 (SD) ms for orthodromic and 110 t 70 ms for antidromic
responses. This brief depolarization increased in amplitude when
the membrane was hyperpolarized and was associated with a field
potential, indicating that it was a delayed EPSP generated
synchronously by CA1 pyramidal neurons. Focal application of
TTX to the CA2-CA3 area abolished this delayed EPSP, suggest-
ing that it probably resulted from the activation of CA2-CA3
neurons through collaterals of CA1 cells.
5. In 67% of the neurons, one or two (sometimes up to five)
late action potentials of variable amplitude (range, 9- 120 mV)
occurred after orthodromic stimulations at a latency of 20- 140
ms. These late action potentials arose either from the peak of the
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early IPSP or at the beginning of the rising phase of the LLD and
could not be abolished by hyperpolarizing the cell. Simultaneous
intracellular and extracellular recordings also indicated that these
late action potentials were not synchronously generated by CA1
neurons. The amplitude of these late action potentials fell into
two distinct groups: small- (9-20 mV) and large-amplitude ones
(90-120 mV). The latter displayed a marked inflection on their
rising phase similar to an initial segment-soma IS-SD fraction-
ation, whereas the small-amplitude ones were sometimes fused.
These observations suggest that they were generated in an area
remote from the soma of pyramidal neurons (probably the axons)
and that they had multiple sites of origin.
6. Spontaneous potentials appeared within a few minutes of
4-AP application. With K-acetate-filled microelectrodes, they
consisted of high-frequency, spontaneous hyperpolarizations and
low-frequency, depolarizing-hyperpolarizing sequences resem-
bling IPSPs or EPSP-IPSP sequences, respectively. In addition,
LLDs with a shape similar to those evoked by orthodromic stim-
uli also appeared spontaneously. They were usually preceded by
an initial EPSP-IPSP sequence and accompanied by late and vati-
able-amplitude action potentials and sometimes a delayed EPSP.
The LLDs occurred at a quite regular frequency varying between
0.008 and 0.14 Hz. Both spontaneous and stimulus-induced
LLDs were associated with a positive field potential in s. pyramid-
ale indicating that this activity was generated synchronously by
pyramidal cells.
7. The LLD lasted 600- 1,500 ms and reached an amplitude of
2- 15 mV (measured from base line). Its amplitude varied linearly
with membrane potential (I&), suggesting it was a postsynaptic
phenomenon. Its equilibrium potential was 5-20 mV positive to
rest(ll.0+4.9,n = 8). Even though in many cases this value was
above firing threshold, the shunting effect of the LLD produced
by its large increase in conductance usually prevented cell firing.
8. Bath application of bicuculline methiodide (BMI, 5-20 PM)
blocked spontaneous and stimulus-induced LLDs and disclosed
an underlying hyperpolarization. Whenever present, the delayed
EPSP (partly obscured by the LLD) increased in amplitude and
duration after BMI application. In all the neurons tested, the LLD
was consistently more sensitive to BMI than the early hyperpo-
larizing IPSP (n = 6).
9. Drop application of BMI (20 PM) onto the apical dendritic
field of the CA1 area blocked the spontaneous and stimulus-in-
duced LLDs without affecting the antidromic IPSP. Furthermore,
current-source density analysis performed by recording the field
potential at regular intervals along an axis perpendicular to s.
pyramidale, indicated the presence of a sink in the middle portion
of s. radiatum during this potential. These results suggest that
y-aminobutyric acid (GABA) receptors responsible for the LLD
are located on the dendrites.
10. When 50-86s of the Cl- ions in the perfusing medium
were replaced with the nonpermeant anions methylsulfate or
isethionate, a marked increase in the amplitude of the LLD was
observed. Intracellular ionophoresis of Cl- ions also produced an
953
954 P. PERREAULT
increase of the amplitude of the LLD, suggesting that an out-
wardly directed Cl- conductance was involved in this depolariza-
tion.
II. This conclusion was also supported by the application of
the Cl- pump blocker furosemide (OS-2 mM), which simulta-
neously blocked the early IPSP and the LLD evoked by ortho-
dromic stimulation. The antidromic IPSP, the high-frequency
spontaneous IPSPs, and the spontaneously occurring LLDs also
disappeared in the presence of furosemide. These effects were
associated with a shift of the reversal potential of the LLD and the
IPSP towards the resting vm of the cells. The increase in conduc-
tance associated with the LLD was not affected by furosemide,
indicating that only the electrochemical gradient of the response
was changed. The only spontaneous potentials recorded in the
presence of furosemide were long-lasting hyperpolarizations that
inverted near the IS+ equilibrium potential (-92.5 t 5.5 mV,
n = 3)
12. ‘Our data show that the effects of low concentrations of
4-AP on CA1 hippocampal pyramidal cells maintained in vitro
are characterized by an increase in both excitatory and inhibitory
synaptic potentials. This potentiation of synaptic transmission
allows GABA to activate dendritic receptors that generate a late,
long-lasting depolarization. In addition, 4-AP discloses a delayed
EPSP that originates from the CA2-CA3 areas and late action
potentials generated in an area remote from the recording site.
The significance of these observations is discussed with reference
to the well-known convulsant properties of 4-AP.
INTRODUCTION
In recent years 4-aminopyridine (4-AP) has attracted
much attention as a pharmacologic tool for analyzing re-
polarizing K+ conductances and synaptic transmission (for
review, see Refs. 13, 24, and 75). In a variety of prepara-
tions, 4-AP acts as a relatively selective blocker of the early
transient K+ current or A current. This effect can be seen in
most preparations with millimolar concentrations of the
drug although in some structures, particularly in the hip-
pocampus, micromolar concentrations of 4-AP have been
shown to be effective (18,25,68,76,82). In addition, it has
been shown that 4-AP in the millimolar range can also
block the delayed rectifying K+ channel in invertebrate (40,
46,83) and vertebrate axons (11, 78).
Concentrations in the micromolar range can also induce
a powerful and persistent enhancement of neurotransmit-
ter release from synapses in the peripheral (41,42, 50) and
central nervous system (15, 22, 3 1, 33,63). This effect has
been reported for both excitatory and inhibitory pathways
(15, 22, 38, 39, 63) The increase in neurotransmitter re-
lease appears to be related to an enhancement of Ca*+
influx during the depolarization of the nerve terminals
(51), a phenomenon that might be due to either a direct
effect of 4-AP on voltage-dependent Ca*+ channels (42,6 1,
67) or to a prolongation of the action potential caused by a
decrease of repolarizing K+ conductances (11, 28, 37).
4-AP and related aminopyridine compounds are also
powerful convulsant drugs, as reported in humans during
therapeutic trials (7 1) or following accidental consumption
(69). The epileptogenic action of aminopyridines has also
been documented by various investigators both in in vivo
(10,55,71,72) and in vitro experiments (22,32,63,79). In
the latter cases, synchronous epileptiform discharges were
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
observed when brain tissue was exposed to micromolar
concentrations of the drug.
Many compounds employed to produce epileptiform ac-
tivity in experimental models share the common property
of decreasing inhibitory GABAergic conductances (for re-
view, see Refs. 1 and 8). 4-AP probably acts through differ-
ent mechanisms, because it appears to increase the efficacy
of both excitatory and inhibitory (i.e., GABAergic) synap-
tic potentials. These features make 4-AP an interesting
agent for analyzing alternative basic mechanisms of epi-
leptiform activity. The present experiments were designed
to study, in the CA1 subfield of the in vitro hippocampal
slice, the changes induced by relatively low doses of 4-AP
on excitatory and inhibitory synaptic activity and some
intrinsic properties of pyramidal cells. A preliminary ac-
count of these results has been published (9).
METHODS
Preparation and incubation of the slices
Male Sprague-Dawley rats ( 150-300 g) were decapitated under
ether anesthesia and their brains quickly removed from the skull.
Hippocampi were dissected free and cut transversely into 400- to
450.pm-thick slices using a McIlwain tissue chopper. The slices
were then transferred into a tissue chamber where they laid at the
interface between oxygenated artificial cerebrospinal fluid
(ACSF) and humidified gas (95% 02, 5% CO*). The composition
of the ACSF was the following (in mM): 124 NaCl, 2 KCl, 1.25
KH2P04, 2 MgSO,, 2 CaC12, 26 NaHC03, and 10 glucose at pH
7.4. The temperature of the perfusing ACSF was maintained be-
tween 33 and 36”C, and the perfusion rate varied from 0.3 to 1
ml/min.
4-Aminopyridine was added to the ACSF to achieve a final
concentration of 5-100 PM, although most experiments were
performed using a concentration of 50 PM. In some experiments,
bicuculline methiodide (BMI, 3-50 PM), tetrodotoxin (TTX, 1
PM), 3,3(2-carboxypiperazine-4-yl)propyl- 1-phosphonate (CPP,
l-5 PM), or=2-amino-5-phosphonovalerate (APV, lo- 100 PM),
and nipecotic acid (1-2 mM), were also added to the ACSF. Furo-
semide was dissolved in dimethyl sulfoxide (DMSO) and sepa-
rated in aliquots that were kept frozen until the day of the experi-
ment. It was added to the ACSF to achieve a final concentration
of 0.5-2.0 mM. The maximal concentration of DMSO in the
perfusing medium reached 0.5%. In two experiments, DMSO
alone failed to reproduce the effects observed during furosemide
application. In some experiments, BMI (20 PM) or TTX ( 10 PM)
were dissolved in the ACSF and delivered into discrete regions of
the hippocampal slice by using a small, glass micropipette (tip
diameter, 5-10 pm) connected to a pressure generator. Low Cl-
solutions were prepared by replacing 62- 100 mM of -NaCl with
equimolar Na-isethionate or Na-methylsulphate. All the chemi-
cals were acquired from Sigma with the exception of Na-methyl-
sulfate and CPP, which were obtained from ICN and Tocris
Neuramine, respectively.
Recording and stimulation
Conventional intracellular recordings were made from CA1
pyramidal cells in the subregion b identified according to the
criteria of Masukawa et al. (45). For intracellular recordings, glass
microelectrodes were filled with 4 M K-acetate (resistance, 50-90
MS2) or 3 M KC1 (resistance, 60-80 Ma), whereas for extracellular
recordings, low-impedance microelectrodes (2- 10 MQ) were filled
with 2 M NaCl. The intracellular signals were fed to a high-im-
pedance-negative-capacitance direct current (DC) amplifier with
 4-AP AND SYNAPTIC TRANSMISSION 955

bridge circuit, which allowed current to be passedthrough the changes observed differed depending on the type of micro-
intracellular microelectrode. The bridge balance was carefully electrode used to impale the cells (see Table 1). In 72% (n =
checked throughout the experiment and adjusted if necessary. 13) of the cells impaled with K-acetate-filled microelec-
The intracellular data wereobtained from neuronsthat displayed trodes, 4-AP application caused a hyperpolarization of the
resting membranepotential (I&) more negative than -55 mV I&. This was accompanied by a decrease in input resis-
[-65.0 -t 4.5 (SD) mV, y2= 331,apparent input resistanceof at
least20 MQ (35.2 t 4.1 MQ, n = 30), and action potential ampli- tance (up to 20%) and the appearance of repetitive sponta-
tude >85 mV (104.3 t 9.5 mV, y1= 38). Intracellular and extra- neous potentials, mainly IPSPs, which rendered the base-
cellular data were displayed on an oscilloscope and/or on a line recording much noisier (Fig. 1A). In three other
GOULD pen writer. In somecasesdata were recorded on FM neurons, 4-AP did not cause any change in & whereas in
tape for later analysis.Most recordingswere stablefor periodsof the remaining two cells it produced a slight depolarization.
l-5 h. Conversely, in six of eight neurons impaled with KCl-
Orthodromic and antidromic stimulation (lo-500 PA, 30-90 filled microelectrodes, the application of 4-AP caused a
ps) were delivered through sharpenedand insulated monopolar depolarization of the resting F/mof 2-15 mV accompanied
tungstenor bipolar stainlesssteelelectrodesplacedin s. radiatum by the appearance of high-frequency depolarizing poten-
and alveus,respectively.Antidromic stimulation wasdeliveredto tials (Fig. 1B). The L& of two other cells did not change.
the outer portion of the alveus, and we used minimal current
intensity to avoid current spreadinto s. oriens. In two experi- Although the average changes observed were larger and of
ments,the fibersin the s. orienswereseveredwith a microknife to opposite polarity compared with those observed with K-ac-
prevent such contamination. The effectsof various drugson the etate-filled microelectrodes, they were not statistically sig-
responseof the neuronswere monitored by stimulating the cells nificant. This is due probably to the small sample size
continuously at low frequency (0.05-o. 1 Hz). (Table 1). As in cells impaled with K-acetate-filled micro-
All quantitative results are expressedas the mean t SD. All electrodes, the input resistance of these cells decreased
measurements were made by hand, and the paired-sampleStu- slightly in the presence of 4-AP (up to 15%).
dent t test was usedto compare values of resting potential and In five neurons impaled with K-acetate-filled microelec-
input resistancein controls and in the presenceof 4-AP. Compar- trodes, we blocked synaptic transmission by applying TTX
isonsof input resistance,firing characteristics,and synaptic po- in the bath. In four of these cells, 4-AP induced a depolar-
tentialsbefore and after drug application were madeat the same
ization of up to 4 mV that was accompanied by an increase
& Whenever necessary,a steady direct current was passed
through the recording electrodeto bring the I&, to control levels in input resistance of up to 23% (Fig. 1C). Repetitive spon-
beforemeasurements were made. taneous potentials failed to occur in this case, indicating
that they must have been generated by neurotransmitter
Laminar current source-density analysis released from spontaneously firing interneurons and/or
presynaptic terminals. In the absence of TTX, the small
In someexperiments,we usedthe current source-density(CSD) decrease in input resistance observed in cells impaled with
analysisto estimatethe location and the amplitude of current both KC1 and K-acetate microelectrodes resulted probably
sinks and sourcesassociatedwith extracellular field potentials from the shunting effect induced by this tonic synaptic
recorded in CAl. These field potentials were recorded sequen- activity. This shunting effect could also explain why in 50%
tially at regularintervals of 50 pm along an axis perpendicularto
the s.pyramidale. Another fixed electrodewaslocatedin s. pyra- of the cells studied (n = 14), more current was necessary to
midale,and the signalrecordedthrough it wasusedasa trigger for evoke firing after 4iAP application (Fig. WI). Interest-
the recordingsobtained from the other positions.Three to five ingly, 4-AP decreased the latency %andthe threshold of ac-
sampleswererecordedat eachlocation, and the amplitude of the tion potentials elicited by depolarizing current pulse, as
extracellularpotential measuredat a fixed time wasaveragedfor one would expect from a blockage of the A current (cf. Ref.
each location. These values were used to calculate the unidi- 70), only in cells in which this background synaptic activity
mensionalCSD profile according to formula 3 of Mitzdorf and was not prominent (36%, n = 6). One such example is
Singer(49). shown in Fig. 102 Note also in Fig. 1DZ that 4-AP appli-
cation decreased the anomalous rectification (29) seen
RESULTS when hyperpolarizing current pulses were passed through
the recording electrode. Although we did not quantify this
Eflects of 4-AP on resting membrane potential and input effect, it was observed in >50% of the neurons (n = 21).
resistanceof pyramidal cells The effects induced by 4-AP on membrane properties were
4-Aminopyridine had relatively small effects on resting not significantly dikerent with concentrations of 4-AP
I& and input resistance of pyramidal cells. However, the ranging from 5 to 100 PM.

TABLE 1. Eflects of 4-AP (50 PM) on resting potential (Vm) and input resistance (RI)

Kn RI

K-Acetate KC1 IS-Acetate + TTX K-Acetate KC1 K-Acetate + TTX

Control -68.7 t 7.5 -73.1 t 3.8 -62.8 t 3.3 32.0 zk 1.9 40.0 t, 9.9 32.0 AZ5.6
w (8) (5) (21) (8) (5)
4-AP -71.3 k 7.6t -67.3 t 8.0 -60.4 I!I 3.7t 30.0 t 2.1 t 38.7 t 11.5 35.8 Z?L9.1”
Values are means k SD; values in parentheses are number of pyramidal cells. Paired-sample Student t test was performed to compare values recorded
in control to the values obtained in the presence of 4-AP. *P < 0.05; tP < 0.01.

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956 P. PERREAULT AND M. AVOLI

A K Acetate D Control 4AP

10 mV
1 nA
4 40ms

2 I

C K Acetate + TTX

$zz!zy

c -I
J
2 min
10mV
540 ms
20 mV
1 nA

FIG. 1. Effects of 4-aminopyridine (4-AP) on resting membrane potential (I’,,,), input resistance, and repetitive firing of
pyramidal cells. A: chart records showing hyperpolarization induced by 4-AP on a cell impaled with a K-acetate-filled
microelectrode. Downwards deflections show membrane response to constant current hyperpolarizing pulse passed through
recording electrode to measure membrane input resistance before and during 4-AP application. The period of 4-AP
application is indicated by the bar below the record. B: chart records showing the depolarization induced by 4-AP in another
cell impaled with a KCl-filled microelectrode. C: chart records indicating the change induced by 4-AP in neuron impaled
with a K-acetate-filled microelectrode and bathed in the presence of tetrodotoxin [TTX (1 PM)]. D: oscilloscope records
showing the response of 2 different neurons impaled with K-acetate electrodes to hyperpolarizing and depolarizing current
pulses before and during 4-AP application. Repetitive spontaneous synaptic potentials appeared in the neuron shown in Dl,
but failed to occur in neuron shown in 02. DC current was passed in DI to maintain I’,,, at the original value. Calibration in
A and B as in C. Rest V,,, of the cell (in mV): in A, -11; B, -69; C, -51; Dl, -11; and 02, -16. The concentration of 4-AP
used in all neurons shown in this figure and all the following figures is 50 pm.

Efects of 4-AP on stimulus-inducedpostsynapticpotentials the soma (4, 7), was also enhanced in both amplitude and
As expected from previous data obtained in a number of
preparations, 4-AP application resulted in a potentiation of
synaptic transmission. As shown in Fig. 2A, 4-AP increased
the amplitude of the s. radiatum-induced EPSP and also
that of the following IPSP, which was not visible in control
following a lO-ps stimulus. The shorter duration of the
IPSP evoked by the 40-ps stimulus in the presence of 4-AP
was due to its faster repolarizing course. This was caused by
the progressive appearance of a late and long-lasting depo-
larization (LLD) that partially overlapped with it. This is
shown more clearly in Fig. 2B, in which the effects of 4-AP
on orthodromic responses of another neuron are shown at
a slower time base. Ten minutes after the application of
4-AP, the hyperpolarization that followed the EPSP took a
clear biphasic appearance due to potentiation of both the
early and late IPSPs. Later, a small.depolarizing compo-
ne’nt appeared between these two IPSPs (12’) and progres-
sively increased during the next few minutes to reach a
maximal steady state (I 6’). At this time, the amplitude of
the LLD varied according to the intensity of the stimula-
tion.’ Weak stimulations evoked a simple EPSP-IPSP se-
quence, whereas higher intensity stimulations evoked an
LLD of variable amplitude (not shown). The early and late
IPSPs appeared to be potentiated only transiently during
4-AP. application, because they were partly obscured by
the LLD.
The recurrent IPSP evoked by alvear stimulation, which
is due to a GABA-mediated increase in Cl- conductance at
duration by 4-AP (Fig. 2C). This suggested that inhibitory
potentials were also potentiated by 4-AP. Therefore, the
increase in the orthodromic, early IPSP was probably not
solely due to a potentiation of the preceding EPSP. More-
over, an LLD similar to the one observed with orthodromic
stimulation could also be induced by antidromic activa-
tion. The LLD seen in these cases appeared after an initial
IPSP and was preceded by a fast-rising, depolarizing hump
that occurred after alatency of 80 ms (Fig. 2C). This fast-
rising hump-LLD sequence was an all-or-nothing response;
i.e., either the antidromic stimulation evoked a prolonged
hyperpolarizing IPSP (Fig. 2B) or an initial IPSP followed
by this sequence of depolarizing potentials.
DelayedEPSP
In 69% of the neurons, orthodromic stimuli could also
evoke a delayed and short-lasting depolarizing potential
that followed the EPSP with an average peak latency from
the stimulus of 62 + 27 ms (range, 25-120 ms). This po-
tential usually appeared as a little hump at the beginning of
the LLD (Fig. 3A, ortho). The peak latency of a similar
potential seen after antidromic activation was 110 f 70 ms
and ranged from 40 to 300 ms (Fig. 3A, anti). This delayed
depolarization was characterized by a relatively long refrac-
tory period; i.e., it did not occur when the stimuli were
delivered at a frequency > 0.3 Hz. Furthermore, this po-
tential was more clearly observed in those neurons in
which the LLD had a slower rising phase and did not reach

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Copyright © 1989 the American Physiological Society. All rights reserved.
 4-AP AND SYNAPTIC TRANSMISSION 957

A
Control 4-AP, 30’
IO ps-i- 4

40 ms FIG. 2. Effects of 4-AP (50 PM) on stimulus-induced syn-

B Ortho. Anti
aptic potentials. A: orthodromic responses evoked by stimula-
tions at 2 different intensities. Both excitatory postsynaptic po-
tentials (EPSP) and inhibitory postsynaptic potentials (IPSP)
Control -[ appeared to be potentiated in presence of 4-AP (right). B: ortho-
dromic responses in another neuron (lej?) are shown at a slower
time base to demonstrate clearly the progressive appearance of a
4-AP 10’JJ - long-lasting depolarization (LLD) in the presence of 4-AP. In
comparison, the IPSP evoked by an antidromic stimulation
(rrght) is slightly increased and markedly prolonged. C ef&ts
t of 4-AP on the antidromic responses of another neuron. Note
12.4 that in this case, the LLD evoked by the antidromic stimulation
was preceded by a fast-rising, depolarizing hump (arrow). Volt-
age calibration in C also applies to A and B. Action potentials
16 I are truncated in A (bottom right) and B (ortho). Rest I& of the
cell (in mV): in A, -75; B, -66; and C, -64.

200 ms
C Control 4-AP
*
Anti v
l

100 ms

A D
_I 10mV
Ortho. 100 ms

Intra
Extra
1oqlv Alv. 0 --h--
100 ms
Anti 50 ms

-
- .. 110 mV
-I 10mv
50 ms
25Gs

FIG. 3. Characteristics of the delayed EPSP evoked in presence of 4-AP. A: delayed EPSP evoked by orthodromic and
antidromic stimulations (arrows) appeared as a little hump at the beginning of the LLD. Bottom trace in each pair shows the
initial part of the response that was expanded for clarity. B: latency of occurrence of the second EPSP varied with the
intensity of stimulation. The 3 superimposed orthodromic responses show that stronger stimulations decreased its latency.
Numbers indicate the intensity of the stimulus in microamperes. C orthodromic stimulations evoked at different I&. Note
increase in amplitude of both the early and the delayed EPSPs at hyperpolarized levels. D: simultaneous intracellular (top)
and extracellular recordings obtained along an axis perpendicular to s. pyramidale showing the similarities of the extracel-
lular profile of both the early and the delayed EPSPs. Action potentials are truncated in Cand D. Rest l& of the cell (in mV):
in A, -71; B, -68; C, -67; and D, -70.

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Copyright © 1989 the American Physiological Society. All rights reserved.
958 P. PERREAULT
a high amplitude, such as in the example shown in Fig. 3B.
In this example, we can also appreciate that the latency of
this depolarization shortened when the intensity of the
stimulation was increased. Hyperpolarizing the &, pro-
duced an increase in the amplitude of this potential and
failed to block it, indicating that it was generated by a
synaptic conductance; i.e., it was a delayed EPSP (Fig. 3C).
The duration of this delayed EPSP was always much longer
than the first EPSP, and it was associated with one or two
partial or full-blown action potentials in 20% of the cells in
which we could see this response. In Fig. 30, field poten-
tials corresponding to this delayed EPSP were recorded
sequentially with an extracellular electrode moved along
the neuron’s axis From alveus to s. pyramidale, a small,
positive field potential corresponded with the delayed
EPSP recorded intracellularly (top), and this extracellular
potential inverted to negativity in s. radiatum These ob-
servations indicate that the delayed EPSP was generated
synchronously by CA1 neurons. The similarities of the ex-
tracellular profile of both early and delayed EPSPs also
suggested that the two potentials were generated at the
same or closely adjacent sites along the dendritic tree.
From the above results it appeared that as the initial
EPSP, the delayed EPSP was generated by a synchronized
tierent volley traveling along the Schaffer collaterals. This
suggests that this potential resulted from a’ delayed syn-
chronous discharge of neurons located in the CA2-CA3
region. To investigate this hypothesis, we blocked the activ-
ity of the neurons in the CA2-CA3 region by applying TTX
(10 PM) focally onto this area (Fig. 4A). We used another
stimulating electrode located in the CA3 subfield to moni-
tor the effects induced by TTX (not shown). We considered
that TTX had effectively blocked the generation of action
potential in this area when supramaximal stimuli failed to
TTX
B I
Control V
1.
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
elicit a response in CAl. The results of one such experi-
ment are shown in Fig. 4B. In the control traces shown on
the left, the cell was activated at various vm by orthodromic
stimulations. Just the initial part of the response is shown
here, and therefore, only the rising phase of the LLD can be
seen. As shown in the traces on the right, TTX application
selectively abolished the occurrence of the delayed EPSP.
Similar potentials elicited by antidromic activation also
disappeared after this procedure. These results were also
confirmed in three other experiments, in which we found
that CA1 neurons in slices in which the connections be-
tween the CA2-CA3 and CA1 subfield had been severed,
did not show delayed EPSPs. We also assessed the possibii-
ity that the generation of the delayed EPSPs involved N-
methyl-D-aspartate (NMDA) receptors. This appeared un-
likely, however, because this potential was not affected by
the NMDA receptor antagonists APV ( 1O-100 PM, yt = 4)
and CPP (l-10 PM, n = 2).
Late action potentials
Intracellular and extracellular recordings obtained in the
CA1 area demonstrated that unlike pyramidal cells of the
CA3 area, CA1 neurons do not generate bursts in the pres-
ence of 4-AP (55, 62). However, in >67% of the cells
bathed in 4=AP, orthodromic stimulations could evoke
some additional late action potentials (n = 38). In those
cells in which these action potentials could be recorded,
they occurred in 72 t 32% of all the orthodromic responses
evoked (range, 5-100%; n = 20). Typically, these late ac-
tion potentials occurred with a variable amplitude and a
variable latency, although they were usually clustered
within 20-140 ms after the stimulus (47 t 19 ms, n = 20).
In any given cell, their latency also varied between stimula-
FIG. 4. Effects of focal application of
tetrodotoxin (TTX) ( 10 PM) in the CA2-
CA3 area on the delayed EPSP. A: sche-
matic drawing of a hippocampal slice
showing experimental procedure (see
text). B: in the control traces (Zefi), ortho-
dromic stimulations were delivered at
various membrane potentials by simulta-
neously passing hyperpolarizing and de-
polarizing current pulses. Traces on the
right show the abolition of the delayed
IOmV EPSP after focal application’of TTX onto
J I nA CA2-CA3 area. Action potentials are
20 ms truncated and the LLD is now shown, be-
cause only the initial portion of the re-
sponses is represented. Rest l/m of the
 4-AP AND SYNAPTIC
tions. In 75% of the neurons (n = 23) only one or two of
these late action potentials were recorded, but in seven cells
bursts of four to five full-blown or partial action potentials
appeared. The late action potentials were not a synchro-
nous phenomenon, as was shown by simultaneously re-
cording intracellular and extracellular signals from two
nearby sites in s. pyramidale. Thus, in the example shown
in Fig. 5A, the three late action potentials recorded intra-
cellularly did not correspond to any negative-going popula-
tion spike, as was the case for the initial action potential
evoked by the EPSP. Depolarizing current pulses injected
between spontaneous potentials readily evoke firing of the
neurons but failed to generate this type of action potential.
As shown in Fig. 5B (top), the late action potentials
usually arose abruptly from the hyperpolarized level of the
IPSP. In 22% of the cases, however, they appeared on the
late EPSP (n = 23). In either case, we could not block the
occurrence of these action potentials by hyperpolarizing
the I&. During this procedure they were merely replaced
by partial action potentials (Fig. 5B, -30 mV). In Fig. 5C, a
depolarizing current pulse was applied before the ortho-
dromic stimulation evoked at the resting and at a hyperpo-
larized & At the resting &, (Fig. SC, top), the current
pulse elicited two action potentials and the orthodromic
stimulus evoked one late partial action potential that arose
from the IPSP. When the neuron’s membrane was held 20
mV negative relative to rest by passing negative steady cur-
rent, the depolarizing current pulse failed to evoke action
potentials, but the orthodromic EPSP was still followed by
a late partial action potential. The insensitivity of these late
action potentials to current injection suggested that they
originate in an area remote from the recording site (which
is presumably the soma).
The amplitudes of these late action potentials varied be-
tween 9 and 120 mV (68 t 42 mV, n = 20), but interest-
ingly, they appeared to fall into two distinct groups: small-
++ 15 mV
;I
B
RL
200 mV/ ns
100 mV
E fractionation. Note the greater delay between the IS and the SD
\u^.7
---JkL-
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Copyright © 1989 the American Physiological Society. All rights reserved.
TRANSMISSION 959
amplitude ones ( lo-20 mV) and large-amplitude or full-
blown ones (90-120 mV, see Fig. 5B). In Fig. 5Da, three
late action potentials of maximal amplitude (90 mV) that
occurred at the beginning of an LLD are expanded in time.
As seen on the top trace, the rising phase of these late full
blown action potentials displayed an inflection reminiscent
of the initial segment-soma (IS-SD) fractionation seen on
motoneuron action potentials (16). This fractionation was
not observed on action potentials occurring on top of the
initial EPSP or on top of the LLD (not shown). The differ-
entiated recording in Fig. 5Da shows more clearly the pro-
gressive delay in the generation of the large component of
the action potential: this inflection is more pronounced on
the action potential that occurred later after the onset of the
spontaneous potential. This is emphasized in Fig. 5Db, in
which the three action potentials and their corresponding
differentiated traces are superimposed. The variable am-
plitude of the late action potentials appeared, therefore, to
be related to the difficulty of the IS component to invade
the SD area during the IPSP. When it failed, only a partial
action potential appeared, and when it succeeded, a full-
blown action potential was recorded. In some cells, one or
two low-amplitude brief depolarizations appeared on the
IPSP (Figs. 2C and lOA). These potentials were reminis-
cent of the M spike of motoneurons that represent the
somatic invasion of the axonal action potential (16). These
observations suggest that the late action potentials have an
axonal origin and invade the soma antidromically.
The fact that in some cells the amplitude of the late
action potentials could be either partial or full-blown even
if they occurred at the same latency and at the same I&
raised the possibility that they might have multiple sites of
origin. This was further suggested by the occurrence in
many cells of “fused” partial action potentials (Fig. 5E).
Assuming that these action potentials were followed by a
refractory period, it appears then very likely that this fusion
FIG. 5. Characteristics of the late action potentials observed
in the presence of 4-AP. A: simultaneous intracellular and ex-
tracellular recordings obtained in s. pyramidale following an
orthodromic stimulus. Unlike the action potential occurring on
top of the EPSP, the late full-blown action potentials are not
reflected by population spikes. B: orthodromic stimuli deliv-
ered at various I& Only the initial part of the response is
shown, and therefore, only the rising phase of the LLD appears.
200 mV/ms Note the presence of partial and full-blown action potentials
40 mV
near the peak of the IPSP and at the beginning of the rising
IOms phase of the LLD (top). When the I& was hyperpolarized, these
late action potentials were replaced by partial ones. C: action
potentials elicited by the depolarizing current pulse are abol-
ished when the cell is held at -20 mV, but the late action
potentials evoked by the orthodromic stimulus remain present.
Same cell as in B. Da: 3 late action potentials that occurred at
the onset of a spontaneous LLD are shown on a fast time base to
demonstrate the inflection on their rising phase. Their time
derivative shown in the lower trace demonstrates this inflection
more clearly. Db: 3 late action potentials shown in Da (bottom)
and their time derivative (top) are superimposed to show clearly
the 2 components reminiscent of initial segment-soma (IS-SD)
component on the 3rd action potential compared to 1st one. E:
fusion of 2 partial-amplitude, late action potentials (asterisk) in
the same neuron as shown in D. Arrow in inset points at portion
of recording displayed at faster time base in the trace below.
Calibrations in B apply to C. Full-blown action potentials in
300 ms40 mV A-C and inset in E are truncated. Rest V, (in mV): in A, -72; B
10ms and C, -66; D and E, -66.
960 P. PERREAULT
was due to action potentials traveling along different path-
ways and, therefore, originating from different areas.
Spontaneouspotentials in the presence of 4-AP
In cells impaled with IS-acetate-filled microelectrodes,
the application of 4-AP induced the appearance of repeti-
tive spontaneous potentials composed mainly of small hy-
perpolarizations, although some depolarizing potentials
also occurred (Fig 6A). The occurrence of these potentials
was not modified by varying the membrane potential, and
they could be blocked by the application of TTX, suggest-
ing that these events resulted from an action potential-de-
pendent release of neurotransmitters. Furthermore, as
shown in Fig. 6B, when the GABA antagonist BMI was
added to the bathing medium, the occurrence of these hy-
perpolarizations was abolished. These potentials were,
therefore, probably caused by the release of GABA from
spontaneously active interneurons. In agreement with this
conclusion, the spontaneously occurring depolarizing po-
tentials recorded with KCl-filled microelectrodes in the
presence of 4-AP (Fig. 1B) were also blocked by the appli-
cation of BMI or TTX (n = 2, not shown).
The LLD also occurred spontaneously after 15-30 min
of 4-AP perfusion (Fig. 6, A and B). This potential ap-
peared at the same time and developed the same way as the
one evoked by orthodromic stimulations (Fig. 6C). At first,
we noted the appearance of spontaneous slow hyperpolar-
A Control
4-AP + TTX
d 10 mV fi
J
1s
C
SpontaneouS
t;
D
aafl
L the same neuron). In each example, inset shows the initial part
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
izations that could be clearly distinguished from high-fre-
quency IPSPs because of their longer duration (Figs. 6C
and 12’). Later, these hyperpolarizations increased in am-
plitude and acquired a typical biphasic shape (Figs. 6C and
15’) when a depolarizing component appeared between the
early and late phase of this hyperpolarization. This slow
depolarization progressively increased and eventually
masked most of the hyperpolarization at the time it
reached a steady state (Figs. 6C, 18’, and 20’). This sponta-
neous potential occurred at a quite regular frequency that
varied among slices between 0.008 and 0.14 Hz and was
very similar to the response evoked by orthodromic stimu-
lation (Fig. 6C, bottom).
In 60% of the cells (n = 27) the spontaneously occurring
LLD was always preceded by an EPSP. As shown in the
example of Fig. 6Da, the shape of this EPSP was often quite
complex, not smooth as in an evoked EPSP, and usually
did not reach the threshold for action potential generation.
Furthermore, these EPSPs were quite long lasting, the
average duration being 40 t 35 ms (range, 20-l 15 ms, n =
6). In another 33% of the pyramidal cells (n = 9), the onset
of spontaneous LLD appeared quite variable and was
sometimes preceded only by an IPSP (Fig. 6Db). This was
seen in 22 -t 19% of all the spontaneous LLD recorded in
these neurons (range, 2- lOO%, n = 9). In other cases the
onset of the spontaneous potentials appeared to be a com-
plex mixture of both EPSP and IPSP (Fig. 6Dc). These
observations suggest that the generation of spontaneous
FIG. 6. Spontaneous potentials in the presence of 4-AP. A:
effects of TTX on spontaneous potentials seen in the presence
of 4-AP. Control recording obtained in a neuron impaled with a
K-acetate-filled microelectrode showing the relative absence of
spontaneous activity in normal artificial cerebrospinal fluid
(ACSF). Traces in middle show the spontaneous activity in-
duced by 4-AP. Note the presence of numerous IPSPs and of an
IPSP-LLD sequence (top). TTX (1 PM) completely abolished
these spontaneous events (bottom). B: effects of bicuculline
methiodide [BMI (20 PM)] on spontaneous potentials seen in
presence of 4-AP. Traces in top show spontaneous potentials
recorded in a neuron impaled with a K-acetate-filled microelec-
trode and bathed with 4-AP. BMI abolished IPSPs and LLD
(middle). This effect was reversible (bottom). C: 4 upper traces
show the appearance and progressive increase of the amplitude
of the LLD on spontaneous potentials (20’). Note the similar-
2.5 mV
ities between the evoked response (ortho) and the spontaneous
1s activity. D: 3 examples of spontaneous potentials associated
with LLDs in 2 different neurons are shown (b and c are from
of the corresponding potential at faster time base. Note the
variability of the initial part of the potentials. Voltage calibra-
tion in C applies to D. Action potentials in C and DC are trun-
cated. Rest V, (in mV): in A, -56; B, -65; C, -72; Da, -75; Db
and DC, -72.
 4-AP AND SYNAPTIC TRANSMISSION 961

LLDs does not necessarily require a preceding afferent ex- values were obtained for the spontaneously occurring and
citatory input. In agreement with this hypothesis, sponta- stimulus-induced LLDs. In both cases the LLD was asso-
neous LLDs could be recorded in the CA1 region even ciated with an extracellular field potential indicating that
when the connections between CA3 and CA1 were severed they were generated synchronously by the population of
(P. Perreault and M. Avoli, in preparation). pyramidal cells in CA1 subfield (see Fig 9). The frequency
Late- and variable-amplitude action potentials with of occurrence of the spontaneous LLDs did not vary during
characteristics similar to those described previously for the the imposed changes in &,, further supporting their syn-
evoked responses also occurred regularly with the sponta- aptic nature (not shown).
neous events in 85% of the cells (n = 16; Figs. 6C, 18’; Da The LLD was associated with an increase in membrane
and DC). In addition, a delayed EPSP occurring with a conductance that prevented depolarizing current pulses
latency similar to the one evoked by orthodromic stimula- from generating action potentials. In fact, action potentials
tion (40-l 20 ms) was associated with the spontaneous were rarely elicited during the LLD despite its clear depo-
LLD in 37% of the neurons. The spontaneously occurring larizing effect: they were noted in only 29% of the neurons
LLD could be observed even after prolonged exposure to analyzed (n = 27). Moreover, whenever action potentials
4-AP (up to 10 h). These spontaneous potentials disap- appeared, they were never generated synchronously as in-
peared after 30-60 min following 4-AP washout. Sponta- dicated by the absence of population spike in simultaneous
neous potentials also appeared in other areas of the hippo- extracellular recordings. The LLD appeared, therefore, to
campal slice exposed to 4-AP. These extracellular poten- be clearly different from the paroxysmal depolarization
tials occurred synchronously with the activity recorded in shifts evoked by other convulsants such as penicillin, bicu-
the CA1 subfield (not shown). culline, or picrotoxin (1, 59) both by its longer duration
and its inhibitory properties.
Characteristics of the long-lasting depolarization
Efects of bicuculline methiodide (BMI,) on spontaneous
The characteristics of the LLD have been described in a and evoked synaptic potentials
previous communication and will only be briefly men-
tioned here (9). The amplitude of the LLD, measured from Several features of the LLD observed in the presence of
the base line, varied between 2 and 15 mV (peak latency, 4-AP are similar to the depolarizing potentials observed
100-600 ms) with an average duration of 980 ms (range, during iontophoretic application of GABA onto the den-
450.1,500 ms). We have shown earlier that the amplitude drites of pyramidal neurons (5,6,74, 81) or when pyrami-
of the LLD varied linearly with l&, and the calculated dal cells are orthodromically activated in the presence of
reversal potential was 5-20 mV positive from the resting pentobarbital (4). To investigate the possibility that the
V,(11.0+4.9mV,n= 8), a value that should, however, be LLD that occurred in the presence of 4-AP was mediated
interpreted with caution because of the occurrence of hy- by GABAA receptors, we studied the effects of the antago-
perpolarizing IPSPs that overlapped with the LLD. Similar nist BMI on this response. As shown in Fig. 7A, bath appli-

A Ortho. Spontaneous

Control

FIG. 7. Effects of BMI on spontaneous and evoked LLDs. A:

application of BMI (5 PM) blocked both spontaneous and
evoked LLDs. Note 2nd EPSP in evoked response that was
markedly potentiated during BMI application. Wash samples
were obtained 1 h after BMI washout. B: selective blockade of
Wash spontaneous LLD by 5 PM BMI applied in bath. C: BMI (5 PM)
application markedly depressed LLD that occurred following
orthodromic stimulation (a) or spontaneously (b), whereas anti-
dromic IPSP (c) was not changed. Rest V;n of the neuron (in
BBM,_;_
Control - p(IIxmf& zs mV): in A, -70; B, -65; and C, not available.

+I*’ v
+12’r b&- k
Wash 1”--; CB worn” . . . . . . . . . . . . ..*..................

10mV
1 s (a,W
1s 20 ms (c)

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Copyright © 1989 the American Physiological Society. All rights reserved.
962 P. PERREAULT
cation of BMI (5 PM) reversibly blocked both the stimu-
lus-induced and the spontaneous LLD and gave rise to the
appearance of bursts of action potentials followed by a
long-lasting hyperpolarization. In addition, this procedure
caused an increase in the amplitude and duration of the
delayed EPSP, which appeared only as a little depolarizing
hump in control and which could eventually trigger two or
more action potentials during BMI perfusion. These obser-
vations suggest that the delayed EPSP is normally shunted
by the LLD, a finding that further supports the inhibitory
role of this potential. The involvement of GABA in gener-
ating the LLD was also supported by experiments in which
nipecotic acid, a GABA uptake inhibitor, enhanced the
amplitude and the duration of the LLD by 22.89% (n = 3).
The LLD had a greater sensitivity to BMI than the hy-
perpolarizing IPSP. As shown in Fig. 7B, BMI could block
the spontaneously occurring LLD while uncovering a pre-
viously masked, long-lasting hyperpolarizing IPSP. This
IPSP finally disappeared to be replaced by bursting activity
during prolonged application of BMI (not shown). Further-
more, during BMI washout the hyperpolarizing IPSP
always reappeared before the depolarizing component. The
greater sensitivity of the LLD to BMI was further demon-
strated by studying the recurrent IPSP induced by alvear
stimulation. During perfusion with low concentrations
of BMI, the antidromic IPSP remained present, whereas
the depolarizing component was markedly depressed
(Fig. 1OC).
Previous studies have indicated that the depolarizing ef-
fect of GABA is due to synaptically released GABA mole-
cules acting on dendritic receptors (4). We investigated this
possibility by applying BMI focally onto the dendritic field
of pyramidal neurons. To make sure that BMI molecules
were not spreading towards the soma of the cells, we moni-
tored the effect of dendritic application of BMI on the
antidromic IPSPs, a potential that is known to be generated
at the soma and that is also BMI-sensitive (7, 53). The
results of one such experiment are shown in Fig. 8. BMI
selectively depressed the LLD while uncovering a hyperpo-
larization in both spontaneous and stimulus-induced re-
sponses (Fig. 8, bottom). These effects were not accompa-
nied by any change of the antidromic IPSP.
Alveus , S. Radiatum
Control
-IF----
BMI + dendrites
IT--
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
The dendritic location of the GABA receptors responsi-
ble for the LLD was further confirmed by the CSD analysis
of the spontaneous field potentials associated with the
LLD. As shown in Fig. 9A, extracellular potentials were
recorded at a regular interval of 50 pm along an axis per-
pendicular to s. pyramidale and their amplitude measured
at the latency of 15 ms (arrow B), which corresponded to
the initial EPSP, and 500 ms (arrow C), which corre-
sponded roughly to the peak of the LLD. In Fig. 9B, the
CSD profile corresponding to the initial EPSP (open cir-
cles) indicates the presence of a sink located in s. radiatum,
suggesting that the spontaneous potentials recorded in CA 1
were triggered by an input traveling along the Schaffer col-
laterals. In Fig. SC, the unidimensional CSD profile corre-
sponding to the LLD (open circles) shows also the presence
of a current sink located in the middle portion of s. radia-
turn (400-550 pm) flanked by two probably passive sources
on each sides. Similar results were obtained with stimulus
induced potentials (not shown). These results therefore fur-
ther support the view that the receptors generating the LLD
are located on the dendrites of CA1 pyramidal neurons.
Ionic mechanism of the long-lasting depolarization
It has been previously suggested that the depolarizing
effect of GABA on pyramidal neurons is, at least in part,
mediated by an increase in the permeability to Cl- ions (5,
19). To investigate the role of a Cl- conductance in the
genesis of the 4-AP-induced LLD, we replaced 50-86% of
Cl- ions in the ACSF with the imperrneant anions iseth-
ionate or methylsulfate (n = 6). As shown in Fig. lOA, this
procedure resulted in a clear and reversible increase of
350.550% in the amplitude of the spontaneous and stimu-
lus-induced LLD. This substitution also induced a small
shift in the depolarizing direction of the early IPSP.
Similar changes were observed when we increased the
[Cl-], by impaling neurons with KCI-filled microelec-
trodes. In the example shown in Fig. lOB, the slice was first
exposed to 4-AP for 45 min, and the appearance of the
LLD was verified with extracellular recordings. A neuron
was then impaled, and negative DC current (-0.8 nA)
passed to ionophorese Cl- ions while delivering an ortho-
dromic stimulus every 30 s. Although the whole response
Spontaneous
FIG. 8. Effects induced by focal appli-
cation of BMI onto dendritic area on
LLD. Top traces show response evoked in
the presence of 4-AP by antidromic
J il (alveus) and orthodromic (s. radiatum)
stimuli, as well as 1 example of a sponta-
neously occurring LLD. Note the selective
depression of evoked and spontaneous
LLD, whereas the antidromic IPSP re-
mained present. Amplitude of the action
20 mV potentials is reduced by slow response
100 ms time of the pen recorder. Rest vm of
neuron, -76 mV.
n
 4-AP AND SYNAPTIC TRANSMISSION 963

A B Sink Source C Sink source

400 200 0 200 400 mV/mm2 100 50 0 50 100 mV/mm2
11 I I1 11 ‘1’ 11 1 I i 11 ‘1’
B c -6 -5 -4 -3 -2 -1 12 3 4 mV -3.0
I
4.5
1
-2.0
I
-1.5
1
-1.0
,
-.!j
I
.5
I
1
I
1.5
u
2
*
2.5
'
mV

4 s
Alveus OA-------
S.oriens 100 -b--------

S.pyr. 200 h---c---

3m-v-+---
S.rad. in;

S.lac- mot 700 yl-

Fissure
z-\Kirnv l Field ?otentlal
200ms . Field Potential oCSD
0 CSD

FIG. 9. Current source-density (CSD) analysis of spontaneously occurring LLDs observed with 4-AP. A: example of
some recordings obtained by moving the electrode sequentially along an axis perpendicular to s. pyramidale. B: field
potential amplitude (filled circles) measured at a latency of 15 ms (arrow B in A) and corresponding CSD (open circles)
calculated from the average value of 3 recordings obtained at each location. Note the presence of a pronounced sink in s.
radiatum with 2 sources on each side. C: as in B except that the data were obtained at the latency of 500 ms from the onset of
the spontaneous potential (arrow C in A). Note the presence of sink located in the middle portion of s. radiatum flanked by 2
sources on each side that further support the dendritic origin of the LLD.

of the cell appeared depolarizing even immediately follow- late part of this long depolarization differed: the initial part
ing impalement, the amplitude and the duration of this (which probably represents the inverted Cl--mediated hy-
depolarization progressively increased as Cl- ions diffused perpolarizing IPSP) did not change, whereas the late phase
into the cell. However, the changes of the initial and the of the depolarization (latency of 500 ms) slowly increased.

A
Control
Spontaneous

e FIG. 10. Effects of shifting Cl- gradient on LLD. A: Cl- ions

in the extracellular medium were decreased of 50% by replace-
ment with the impermeant anion methylsulfate. This resulted
in a marked increase of the LLD amplitude that appeared spon-
taneously or after an orthodromic stimulus. Bottom traces
(wash) show the recovery after reintroduction of Cl- ions. B:
superimposed orthodromic responses obtained in a neuron im-
paled with a WI-filled microelectrode, First response (labeled

-k--;:-_
1) was obtained after beginning of Cl- ionophorcsis by passing
negative DC current through’recording electrode (-0.8 nA) and
3 other traces are orthodromic responses obtained at 30-s inter-
Wash val after the first stimulus. Note the progressive increase in the
slow depolarization as Cl- was difising in cell. Early part of the
response corresponding presumably to the inverted IPSP re-
110mV mained stable during this time. Action potentials are truncated
ip A. Rest Vm, -67 mV.
200 ms

20 mV
200 ms

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Copyright © 1989 the American Physiological Society. All rights reserved.
964 P. PERREAULT
This was probably due to the time needed for Cl- ions to
reach the remote sites where the LLD is generated.
These experiments demonstrate that changing the Cl-
gradient by either decreasing its concentration in the ACSF
or by increasing its concentration inside the cell caused an
increase in the amplitude of the LLD, suggesting that the
permeability to Cl- ions is increased during this potential.
It is likely, however, that at least part of the increase seen
during those experiments was due to the ionic shift of the
overlapping Cl--mediated hyperpolarizing IPSP. Further-
more, Cl- substitutes also have the disadvantage of induc-
ing a decrease of other cations in the perfusing solution,
which further complicates the interpretation of their ef-
fects (4 1).
To further clarify the role of Cl- ions in this response we
investigated in 10 neurons the effect induced by bath ap-
plication of the Cl- pump blocker furosemide (14, 17,48).
In all cases, furosemide (2 mM) blocked the early IPSP and
B 500 ms
Control
Furosemide
IS
1 set
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M, AVOLI
the LLD evoked by orthodromic stimulation (Fig. 11Aa) as
well as the antidromic IPSP. In the presence of furosemide
the EPSP, although slightly broader, was not affected (Fig.
1lAa, b), whereas the late orthodromic IPSP, which in
control was partially obscured by the LLD, became very
prominent (Fig. 1 lAa, middle). Spontaneous LLDs and
IPSPs also disappeared when furosemide was added (Fig.
11B). Spontaneous events in this case consisted of single,
slow hyperpolarization with an amplitude of 6.5 t 2.1 mV
(n = 4) and a duration of 780-1,000 ms, which were very
similar in shape and time course to the late IPSP evoked by
orthodromic stimulation. The mean reversal potential of
this spontaneous hyperpolarization calculated at a latency
of 400 ms was -92.5 t 5.5 mV (n = 3), a value that is close
to the K+ equilibrium potential in these cells (Fig. 11, C
and D). These results suggest that the slow hyperpolariza-
tion recorded in the presence of furosemide could be due to
the activation of GABAB receptors.
FIG. 11. Effects of furosemide on spontaneous and
evoked responses seen in presence of 4-AP. A: furose-
mide application (2 mM) abolished early IPSP and LLD
induced by orthodromic stimulations (a) as well as the
antidromic IPSP (b). Note the occurrence of the late
IPSP evoked by orthodromic stimulation in the presence
of furosemide. Wash traces were taken 45 min after furo-
semide washout. B: control traces (top)show a sampling
of spontaneous potentials in the presence of 4-AP. Furo-
semide application abolished the high-frequency sponta-
neous IPSPs and the only spontaneous events recorded
in this case consisted of slow hyperpolarizations very
similar to the late IPSP induced by orthodromic stimu-
lus (in the experiment shown in A). Traces in A and B are
from the same neuron. C spontaneous potentials occur-
ring at various V,s imposed by constant current injec-
tion. D: reversal potential of the late IPSP has been calm
10mV culated by plotting amplitude of the response shown in C
as a function of vm (dotted line in C). Rest & of the
neuron (in mV): in A and B, -6 1; and C, -66.
-12
- 8
-46
 4.AP AND SYNAPTIC
Control Furosemide
Response (mV)
1Ar b 4APcontaining
. ... --. .
A 10mV
2oom8
400
Time (ms)
The application of furosemide did not have a significant
effect on the neuron’s resting vm and input resistance (data
not shown) but shifted the reversal potential of the IPSP
and the LLD towards the resting & A depolarizing shift
of the equilibrium potential of the early IPSP from -3.3 t
1.2 mV relative to rest in control to -0.2 t 0.3 mV after
the addition of furosemide (n = 3) was noted, whereas for
the LLD a hyperpolarizing shift from 10.0 t 8.7 mV rela-
tive to rest to -5.5 + 4.7 mV after the addition of furose-
mide was observed (Fig. 12, A and B). For the LLD the
value became more negative than resting vm due to the
presence of the overlapping late IPSP. As shown in Fig. 12,
C and D, the depression of these responses was not accom-
panied by any significant effect on their associated mem-
brane conductance. The decrease in membrane resistance
measured at the peak of the IPSP (latency, 30 ms) was
63.7 t 20.3% bef ore and 61.8 t 27.7% after furosemide,
whereas for the LLD (latency, 420 ms) the values were
49.5 t 23.8% bef ore and 47.5 t 28.8% after furosemide
(n = 3). These results indicated that the release of GABA,
the binding to the receptors, and the opening of the chan-
nels were not impaired by furosemide and that only the
electrochemical gradient of these potentials was changed.
DISCUSSION
The experiments reported in this paper were designed to
examine the effects of low doses of 4-AP on CA1 hippo-
campal pyramidal cells. Our results indicate that 4-AP in-
duces the appearance of high-frequency synaptic poten-
tials, mainly IPSPs, which induce a slight hyperpolariza-
tion of the neuronal membrane and a small decrease in
input resistance. Synaptic potentials evoked by ortho-
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Copyright © 1989 the American Physiological Society. All rights reserved.
TRANSMISSION 965
Wash
n&. 12. Effects of furosemide on the reversal potential and
conductance of the LLD. A: orthodromic responses evoked at
several I&s achieved by pa&g intracellular current pulses in
solution (lef2), after the addition of 1.8 mM
furosemide (middle), and after washout of tiosemide (r&q&).
Ba: amplitude of the LLD measured at a time latency of 420 ms
in the presence of 4AP (o), tier the addition of furosemide (0),
and after washout of furosemide ,(A) plotted against Vm (see
symbols in A). Bb: as in 9Q except the amplitude of the response
was calculated at a latency of 30 ms corresponding to the early
IPSP (see symbols in A). C: response to orthodromicstimula-
tion in the presence of 4-AP (top), after the addition of 1.8 mM
furosemide (middle), and after wa&out of furosemide (bottom).
Downward deflection rep&e& the responses to constant cur-
rent hyperpolarizing pulses injected to monitor membrane r+
sistance during the r&ponse. D: amplitude of the response to
the hyperpolarizing current pulses (shown in C) plotted in
function of time before and after the orthcxlromic stimulus
(atrow). Note the similarities of the response in pr&ence of
4AP (o), after the addition of Wosemide (o), and after washout
of furosemide (A). Same neuron in A and C. Rest V,, -66 mV.
6a
aromic and antidromic stimulations are also markedly po-
tentiated. A GABA-mediated LLD generated in the den-
drites also appears spontaneously or after stimulation. This
LLD is often preceded by a delayed EPSP dependent on the
activation of CA2-CA3 neurons and is usually associated
with late action potentials originating in an area remote
from the recording site (presumably the soma).
Effects of 4-AP on passive membrane properties -
Our results have shown that 4-AP at concentrations
varying from 5 to 100 PM has relatively small effects on
passive membrane properties. An average hyperpolariza-
tion of 2.6 mV was observed in cells impaled with a K-ace-
tate-filled microelectrode, whereas cells impaled with KC1
electrodes depolarized by 5.8 mV. Buckle and Haas have
also reported a clear depolarizing effect of 4-AP on cells
impaled with KCl-filled microelectrodes (15). These
changes were correlated with the appearance of repetitive
spontaneous potentials that were mainly hyperpolarizing
in cells impaled with K-acetate-filled recording microelec-
trodes and depolarizing with KCl-filled ones. The blocking
effect of BMI indicates that these potentials are caused by
the activation of GABA* receptors in response to sponta-
neously released GABA. The opening of Cl- channels by
the tonic release of GABA progressively shifted the-resting
vm toward the equilibrium potential of Cl- ions, which is at
a slightly more negative level compared to the resting value
in cells impaled with K-acetate-filled microelectrodes and
at a depolarized level in cells impaled with KCl-filled mi-
croelectrodes. The possibility that these IPSPs were the re-
sult of an increased excitatory input of CA 1 pyramidal cells
onto interneurons was ruled out, because none of the cells
966 P. PERREAULT
impaled in this study was spontaneously firing during this
activity. The blockade of these IPSPs by TTX is indicative
of spontaneous activity of intemeurons, but whether it re-
sults from an increased firing of interneurons and/or a fa-
cilitated release process at their terminals would require
paired recordings of pyramidal neurons and interneurons
to be answered. Note that spontaneous IPSPs are also pres-
ent in normal conditions, and 4-AP application merely
results in a potentiation of this tonic inhibition (3).
Previous work has shown that 4-AP has variable effects
on input resistance and V#,,of mammalian brain neurons.
Buckle and Haas (15), using the same preparation and
concentrations of 4-AP similar to those used in the present
experiment, have reported variable effects on these two
parameters, although membrane hyperpolarizations ac-
companied by a conductance increase of up to 30% were
seen in many cases when microelectrodes were filled with
K-acetate. Some investigators have reported no significant
changes in the olfactory cortex (22) or in the hippocampus
(32, 63), whereas others have found a marked increase in
input resistance accompanied by a depolarization, an effect
that was attributed to a blockage of K+ conductance (neo-
striatum, Ref. 34; hippocampus, Ref. 66). In our experi-
ments, it is likely that the changes observed in the presence
of 4-AP depended on the relative contribution of a block-
age of K” current(s) and the shunting effect induced by the
synaptic bombardment. This could explain why the firing
induced by depolarizing current pulses was increased, as
one would expect from a blockage of the A current (cf. Ref.
70), only in those neurons in which this tonic synaptic
bombardment was not very prominent (36% of the cells,
n = 6). This could also account for the fact that when
synaptic transmission was blocked by TTX, 4-AP applica-
tion led to a depolarization accompanied by a slight in-
crease in input resistance. The present data, unfortunately,
do not allow one to draw any firm conclusion regarding the
type(s) of K+ current involved. A blockage of the A current
is likely to contribute to these effects, because in the hippo-
campus this current can be blocked by concentrations sim-
ilar to those used in our study, and it is known to be par-
tially activated at resting level (30, 83).
Effects of 4-AP on synaptic potentials
It is well known that 4-AP enhances synaptic transmis-
sion in many areas of the central and peripheral nervous
system (see Ref. 75 for review). Our demonstration that
4-AP enhanced transmitter release at both excitatory and
inhibitory synapses is in line tith these previous findings
and agrees with other results reported in the same prepara-
tion (15, 63). Interestingly, the late IPSP, which is due to
the opening of a IS+ conductance linked to GABAB recep-
tors (2, 20, 52, 53), was also potentiated by 4-AP. In the
olfactory cortex, similar doses of 4-AP appear to block the
late IPSP (22), and in the hippocampus, Inoue et al. (32)
have reported that low concentration of 4-AP blocked the
K+ conductance activated by the GABAB agonist Baclofen.
However, in agreement with our results, others have re-
ported that 4-AP prolonged the late IPSP (2) or increased
the synaptic current associated with it (63) In addition,
Dutar and Nicoll (20) have recently reported that the re-
sponse of hippocampal pyramidal cells to Baclofen is not
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
sensitive to 49AP, a finding that further supports the pre-
synaptic origin of the potentiation we observed.
Delayed EPSP
4-Aminopyridine application gave rise to the appearance
of a delayed EPSP that was usually partly masked by the
LLD and became very prominent when BMI was added to
the bathing medium or applied focally onto the dendrites.
This delayed EPSP was most often recorded following or-
thodromic responses, although it was also observed follow-
ing antidromic stimuli and with spontaneously occurring
potentials. Kita et al. (34) observed a similar phenomenon
in cells of the neostriatum exposed to 4-AP.
It has been shown previously that during pharmacologic
blockade of GABAA-mediated inhibition, a single stimulus
to afferent fibers can produce a second EPSP and a burst in
CA1 cells (23, 80). The delayed burst in these cases ap-
peared to be caused by a ‘freactivation” of CA1 neurons by
a preceding burst in CA2-CA3. Several lines of evidence
suggest that a similar mechanism is operating with 4-AP as
well: I) the delayed EPSP appeared after orthodromic and
antidromic stimulations as an all-or-nothing event with a
variable and relatively long latency varying between 25 and
300 ms, 2) the extracellular profile of the initial and the
delayed EPSP were very similar, and 3) focal application of
TTX onto the CA2-CA3 areas selectively blocked its occur-
rence, However, the delayed EPSP observed during 4-AP
application was not associated with a burst of action po-
tentials as described in the presence of penicillin or BMI.
As demonstrated in experiments in which BMI was used,
this difference is probably due to the shunting effect ex-
erted on the delayed EPSP by the LLD (combined with the
underlying IPSPs), It is likely then that 4-AP application
uncovers connections that are not usually functional under
normal conditions. This effect is reminiscent of what has
recently been reported for picrotoxin, which can uncover
previously silent, recurrent, polysynaptic, excitatory inter-
actions among CA3 pyramidal cells (47). It should be
mentioned that the axonal ramifications from CA1 pyra-
midal cells have been shown to bifurcate in the alveus and
project toward the subiculum and also the fimbria (35).
During their course, these axonal branches in turn send
many collaterals going down into s. oriens and pyramidale,
which could provide the neuroanatomic basis for our elec-
trophysiological findings. The functional consequences of
this delayed EPSP in CA1 cells are not clear. However, this
potential is related to the relative strength of the overlap-
ping inhibition: in those cells in which the LLD was less
pronounced, the second EPSP often could trigger action
potentials. Although we cannot exclude the possibility that
it could be mediated by a different neurotransmitter or
receptor than the initial EPSP, this appears unlikely. Our
results indicate that both the early and the late EPSPs are
not mediated by NMDA receptors.
Long-lusting depolarization
An interesting finding of our experiments is the appear-
ance of a GABA-mediated LLD in 4-AP-treated slices. Re-
cently, Kita et al. (34) also described in the neostriatum a
GABA-mediated depolarization evoked in the presence of
4-AP. Focal application of BMI in the dendritic field and
 4-AP AND SYNAPTIC
current source-density analysis of the spontaneous and
evoked LLD support the hypothesis that the receptors gen-
erating this depolarization are located on the dendrites (5,
6, 74). The ability of antidromic stimulation to elicit the
LLD is not in disagreement with the hypothesis that it
involves the participation of a subset of interneurons acti-
vated through a feedforward pathway (4), because in our
experiments the antidromic stimulus appeared to trigger a
delayed EPSP originating from the CA2-CA3 areas, which
in turn provided the orthodromic input necessary to initi-
ate the LLD.
Alger and Nicoll (5) have also obtained pharmacologic
evidence that hyperpolarizing and depolarizing GABA re-
sponses reflect the activation of two different receptors, the
depolarizing responses being mediated by extrasynaptic
sites. The present data do not allow us to make any firm
conclusion on this issue. Based on that view, however, the
LLD seen in the presence of 4-AP would result from an
increase in the amount of GABA released and thus avail-
able to reach these extrasynaptic sites. This is different
from the postsynaptic effects of pentobarbital, the only
agent previously known to induce the appearance of the
depolarizing response following an “endogenous” release
of GABA. The fact that 4-AP does not change the response
of pyramidal neurons to GABA application further
strengthens the hypothesis that it acts mainly via presynap-
tic mechanism (P. Perreault and M. Avoli, in preparation).
Our results also demonstrate clearly the greater sensitivity
of the GABA-mediated depolarization to BMI. We could
selectively depress this depolarization while minimally af-
fecting GABA-mediated hyperpolarization by using 5 PM
BMI, which is very similar to the sensitivity of the response
elicited by iontophoretic application of GABA (5).
The determination of the apparent reversal potential of
the LLD was complicated by the presence of underlying
IPSPs. Although its actual value must be interpreted with
caution, our results of approximately -55 mV, i.e., - 10
mV more positive than the resting &, are similar to pre-
vious data obtained with pentobarbital(4) and with GABA
iontophoresis (6, 74). The ionic mechanisms of the
GABA-mediated depolarizing response have always been a
matter of debate, because its equilibrium potential does not
correspond to any known ion. Two major hypotheses have
been suggested. One proposes that hyperpolarizing and de-
polarizing responses are both mediated by Cl- ions, thus
requiring a reversed electrochemical gradient in the den-
drites. The other hypothesis that has received more support
in the case of the hippocampus, states that in addition to
Cl-, other ions like Na+ or Ca2+ are also involved in the
depolarizing response (5, 19). In our study, and probably in
others as well, the investigation of the ionic basis of this
depolarizing potential is complicated by the overlapping
IPSPs. Despite this limitation, the respective changes ob-
served on the orthodromic response following the replace-
ment of [Cl-], by the impermeant anions isethionate or
methylsulfate and the effects induced by injecting Cl- ions
inside the cell suggest that the permeability to Cl- ions is
increased during the LLD (4, 6, 19).
More direct evidence for the involvement and the rela-
tive contribution of a Cl- conductance in the GABA-me-
diated LLD was obtained by testing the effects of furose-
mide, which blocks Cl- transport without affecting passive
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TRANSMISSION 967
Cl- permeability (14, 17, 48). Furosemide application
(0.5-2.0 mM) resulted in a blockage of the early IPSP
evoked by orthodromic stimulation, the antidromic IPSP
and also the spontaneously occurring IPSPs. This blockage
was due to a positive shift of the reversal potential of the
IPSP toward the resting vm until both were finally equal. In
addition, evoked and spontaneous LLDs also disappeared
in the presence of furosemide due to a negative shift of the
equilibrium potential of the LLD toward resting & The
fact that the membrane conductance accompanying these
synaptic potentials was not significantly affected by furose-
mide is a further indication that only the electrochemical
gradient of these responses was changed and that the other
steps involved (release of GABA, binding to receptors,
opening of ionophores) remained unaffected.
The existence of a Cl--extruding mechanism that main-
tains low [Cl-]r and, therefore, accounts for hyperpolariz-
ing IPSPs has been demonstrated in a variety of prepara-
tions including spinal motoneurons (44), hippocampal
CA3 neurons (48), and cortical neurons (60,77). This out-
wardly directed Cl- pump appears to be sensitive to both
ammonium and furosemide (77). In addition, Misgeld et
al. (48) demonstrated recently in CA3 pyramidal neurons
that the depolarizing responses to GABA is also reduced in
the presence of furosemide suggesting the presence of an
additional furosemide-sensitive inward Cl- pump. In
agreement with these results, our data indicate that the
hyperpolarizing and depolarizing effect of GABA on CA1
pyramidal neurons are due to the existence of two opposite
Cl- gradients: an inward Cl- gradient in the soma main-
tained by an outward Cl- pump, and an outward Cl- gra-
dient in the dendrites that is maintained by an inward Cl-
Pump-
Although the LLD could sometimes elicit action poten-
tials, the large increase in membrane conductance asso-
ciated with it usually prevented cell discharge. As shown by
BMI experiments, this shunting effect also significantly re-
duced the excitatory influence of the delayed EPSP. An-
dersen and his co-workers (6) suggested that the dendritic
GABA-mediated depolarization could play a discrimina-
tive inhibitory role by shunting neighboring synaptic
inputs and thus facilitating more distant excitatory syn-
apses. This conclusion was obtained, however, by studying
slow depolarizations induced by GABA ionophoresis that
were very similar to the responses we have recorded with
4-AP. Unfortunately, this kind of response cannot be re-
corded in pyramidal cells under normal conditions, i.e., in
the absence of any pharmacologic manipulation. In fact,
we have reported recently that in the presence of a normal
medium, high-intensity afferent stimulations can activate
the dendritic GABA receptors producing only a low-am-
plitude late depolarizing potential (5 7).
Lute action potentials
In 67% of the neurons analyzed, late action potentials of
variable amplitude were recorded in association with the
spontaneous or stimulus-induced LLD. Although only one
or two late action potentials were usually recorded, bursts
of four to five late action potentials could be seen in some
cases. Interestingly, they were generated at a membrane
level negative to resting level, usually near the peak of the
968 P. PERREAULT
early IPSP or during the initial part of the rising phase of
the LLD. This observation, together with their apparent
insensitivity to current injection, indicates that they were
generated in an area remote from the recording site. Sev-
eral observations suggest that these late action potentials
have an axonal origin: I) they could be elicited by antidro-
mic activation; 2) their clear segregation into high- (90- 120
mV) and low-amplitude (lo-20 mV) action potentials,
which is reminiscent of IS and IS-SD action potentials of
motoneurons (16, 2 1); 3) the inflection seen on the rising
phase of the high-amplitude ones that is also reminiscent of
an IS-SD fractionation; and 4) the presence of very small
action potentials (2-3 mV) reminiscent of axonal or M
action potentials. However, we cannot rule out the possi-
bility that a dendritic area functionally equivalent to the
initial segment (IS) might be induced by 4-AP and produce
similar effects. The absence of any clear fast prepotential
preceding these action potentials would, however, not sup-
port a dendritic origin (27). A collision test, unfortunately
very difficult to perform in this case, would provide a defi-
nite answer on that issue.
We can only speculate about the mechanisms by which
these late action potentials are triggered. One possibility is
that the A current and/or some other 4-AP-sensitive K+
current(s) are playing a critical role in controlling axon
terminal excitability (36). For instance, it has been reported
that in the presence of 4-AP, demyelinated (73) or myelin-
ated sciatic nerve (37) as well as dorsal root fibers ( 12)
display broader action potentials and respond to stimula-
tion by generating a sustained depolarization and/or burst-
ing activity. The marked increase in [K’]* that occurs dur-
ing the evoked or spontaneous LLD in our experiments (P.
Perreault, C. Drapeau, and M. Avoli, in preparation),
could further depolarize the terminals and bring them to
threshold for action potential or burst generation. How-
ever, the LLD is not involved in their generation, because
its blockage by focal application of BMI did not prevent
their occurrence. The delayed EPSP that has a similar time
latency is also unlikely to be involved, because the blockage
of this response by TTX application over the CA2-CA3
areas did not affect them either.
Whatever the mechanism(s) involved, these late action
potentials are not generated in a synchronized manner.
The functional consequences of these late action potentials
might be, assuming they also propagate orthodromically,
to amplify the release of neurotransmitters at the nerve
terminals. Their occurrence at the onset of the LLD sug-
gests that they could participate in the generation of this
potential. They could increase the excitatory drive onto
interneurons or further facilitate the release of inhibitory
neurotransmitter if they occur in interneurons as well. Fur-
thermore, in a different cortical circuit, they might partici-
pate in recruiting and synchronizing the cells to generate
paroxysmal depolarization shifts or PDS (56).
Spontaneous potentials and possible mechanisms of
epileptogenesis
In the presence of 4=AP, spontaneous potentials con-
sisted of a tonic activity mainly composed of IPSPs, and
less frequent EPSP-IPSP sequences regularly associated
with LLDs. These spontaneous LLDs occurred at intervals
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Copyright © 1989 the American Physiological Society. All rights reserved.
AND M. AVOLI
varying from 7 s to 2 min, were very similar to the ortho-
dromic response, and behaved in the same way during all
the experimental procedures. Voskuyl and Albus (79) also
reported the occurrence of spontaneous field potentials in
the hippocampus in the presence of 4-AP. They distin-
guished two types based on their rate of occurrence, speed
of propagation, and sensitivity to blockers of synaptic
transmission. Both differed strikingly from the spontane-
ous potentials described in this paper by their constant
association with multiple population spikes. Spontaneous
epileptiform activity has also been reported in the CA3 area
(32, 56, 63) and a type of seizure-like activity following
prolonged exposure to 4-AP, has been described in the CA 1
subfield ( 15). Spontaneous or stimulus-induced seizure-
like discharges have also been described in the olfactory
cortex (22). Recently, by applying 4-AP focally onto the
CA1 area, Segal(66) reported the occurrence of spontane-
ous inhibitory potentials that appeared to be mediated by
the activation of GABAB receptors. These potentials were
very similar to the spontaneous slow hyperpolarizations
that appeared early after 4-AP application, before the LLD
became prominent. They also resembled the spontaneous
slow IPSPs seen when furosemide was added. The reason
for these discrepancies is not clear, but it might be related
to certain technical aspects ([K+],, strain of rat, and mode
of drug application).
The spontaneous LLDs were usually preceded by an
EPSP-IPSP sequence, which indicates that these events are
triggered by an excitatory input coming from the CA3 re-
gion. This was confirmed by the CSD analysis that indi-
cated the presence of an important sink located in the s.
radiatum. In certain cases, however, they were preceded by
a simple IPSP, suggesting that spontaneously active local
interneurons are capable of generating this activity (66).
This is further supported by the fact that CA1 neurons are
capable of generating spontaneous LLDs while being dis-
connected from CA2-CA3 areas (P. Perreault and M.
Avoli, in preparation).
An important question to be addressed is the significance
of our findings with reference to the well-known convul-
sant effects of 4-AP and other aminopyridines (10, 55, 69,
72, 73). A common finding in most models of focal epi-
lepsy is the presence of large, phasic depolarizations of the
membrane associated with multiple spikes called the PDS
(1, 59). However, the spontaneous and synchronous LLD
disclosed by 4-AP differed markedly from well-known
PDSs by its morphology and inhibitory nature. One signifi-
cant finding related to the epileptogenicity of 4-AP that we
recorded in the CA1 neurons was the appearance of addi-
tional late action potentials that sometimes occurred in
bursts. The relevance of this observation stems from the
fact that in acute (26, 27, 54, 62, 63, 65) and chronic (58)
focal epileptogenesis, bursts of action potentials are known
to occur in axons or axon terminals projecting to the focus.
The modulatory role of the A current at this level might,
therefore, play a significant role in preventing axon termi-
nal hyperexcitability.
Although 4-AP does not initiate the generation of typical
epileptiform activity in the CA1 region, our results clearly
show that unlike other convulsant drugs that act through
disinhibitory mechanisms, 4-AP clearly increased inhibi-
tory synaptic potentials. It is interesting to note that in
 4-AP AND SYNAPTIC
comparison to neurons of the CA1 region, CA3 pyramidal
cells readily produced bursts (often followed by a similar
LLD) in the presence of similar concentrations of 4-AP
(56). Analysis of the differential effects of 4-AP on these
two regions might, therefore, allow us to gain further in-
sight into the critical factors involved in the generation of
in vitro and in vivo epileptiform activity.
NOTE ADDED IN PROOF
Recently Storm (70a) reported that low concentrations of 4-AP prefer-
entially block a slowly inactivating potassium current called ID, which
might be responsible for the effects of 4-AP on synaptic potentials reported
in this paper.
We thank Dr. P. Gloor for reading an early version of the manuscript
and Dr. V. Tancredi for participating in some preliminary experiments.
The secretarial assistance of G. Robillard is gratefully acknowledged.
This work was supported by MRC of Canada Grant MA-8 109 to M.
Avoli. P. Perreault is an MRC Fellow; M. Avoli is an FRSQ Scholar.
Address for reprint requests: Dr. M. Avoli, Montreal Neurological Insti-
tute, 3801 University St., Montreal, Quebec H3A 2B4, Canada.
Received 25 April 1988; accepted in final form 27 December 1988.
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