Brain Research 846 Ž1999.

112–121
www.elsevier.comrlocaterbres

Research report

Polyunsaturated fatty acids modify mouse hippocampal neuronal excitability

during excitotoxic or convulsant stimulation
a,b,) c
Yong-Fu Xiao , Xiangyang Li
a
The Charles A. Dana Research Institute and The HarÕard-Thorndike Laboratory, CardioÕascular DiÕision, Beth Israel Deaconess Medical Center,
Boston, MA 02215, USA
b
Department of Medicine, HarÕard Medical School, Boston, MA 02215, USA
c
Department of Psychiatry, HarÕard Medical School, Boston, MA 02215, USA
Accepted 17 August 1999

Abstract

The n y 3 polyunsaturated fatty acids ŽPUFAs. reduce cardiac membrane excitability and prevent cardiac arrhythmias in animals and
probably in humans. In this study, we assessed the effects of n y 3 PUFAs on membrane excitability in mouse hippocampal neurons with
both whole-cell current and voltage-clamp methods. Extracellular application of 20 mM eicosapentaenoic acid ŽEPA, C20:5n y 3.
significantly reduced the frequency of electrical-evoked action potentials in CA1 neurons of hippocampal slices from 3.8 " 0.7 Hz of
control to 2.1 " 0.5 Hz. In addition, EPA significantly hyperpolarized the resting membrane potential and raised the stimulatory threshold
of action potentials in CA1 neurons. Another n y 3 PUFA, docosahexaenoic acid ŽDHA, C22:6n y 3., had effects on membrane
excitability similar to those of EPA. In contrast, EPA ethyl ester, oleic acid ŽOA, C18:n y 9., and stearic acid ŽSA, C18:0. did not alter
the membrane excitability in CA1 neurons. Bath application of pentylenetetrazole ŽPTZ. or glutamate reduced the stimulatory threshold
and increased the frequency of action potentials of hippocampal neurons. EPA restored PTZ- or glutamate-enhanced neuronal excitability
to the control level. EPA also suppressed glutamate-activated inward currents. Furthermore, EPA and DHA significantly inhibited the
frequency of action potentials without effecting the stimulatory threshold of CA3 neurons. These data demonstrate that n y 3 PUFAs
modify neuronal membrane excitability under control and drug-stimulated conditions. The sensitivity to these effects of PUFAs varies
from neurons of different hippocampal regions. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Eicosapentaenoic acid; Neuronal excitability; Seizure; Pentylenetetrazole; Glutamate

1. Introduction tion of FeCl 3 ; and Ž4. audiogenic seizure-prone prepara-

The effects of polyunsaturated fatty acids ŽPUFAs. on
membrane excitability have been examined in excitable
tissues, including neurons, heart, and skeletal muscles.
Recent studies of animal models of seizures reveal that
PUFAs have anticonvulsant actions. For example, the anti-
convulsant properties of a mixture of non-esterified a-lino-
lenic acid and linoleic acid with a ratio of 1:4 were
evaluated in four rat models of epileptic seizures: Ž1.
intraperitoneal injection of a single convulsant dose of
pentylenetetrazole ŽPTZ.; Ž2. repeated injections of sub-
convulsant doses of PTZ; Ž3. intraventricular administra-
) Ca2q currents in cardiac myocytes w28,29x. However, the
Corresponding author. Cardiovascular Division, Beth Israel Dea-
coness Medical Center, Harvard Medical School, 330 Brookline Avenue,
Boston, MA 02215, USA. Fax: q1-617-667-4833; e-mail:
yxiao@bidmc.harvard.edu
tion. Treatment with the mixture of the fatty acids reduced
seizure activity in each of these experimental models. The
authors postulated that the anticonvulsant effects of the
fatty acid mixture are related to stabilization of neuronal
membrane w31x. In addition, Voskuyl et al. w25x found that
the in vivo anticonvulsant effect of PUFAs persisted for
several hours after the plasma free fatty acid concentra-
tions returned to control.
Vreugdenhil et al. w26x recently reported that extracellu-
lar application of PUFAs caused a significant shift of the
steady state inactivation toward hyperpolarizing direction
for both Naq and Ca2q currents in isolated rat hippocam-
pal CA1 neurons. PUFAs also significantly inhibited these
currents w26x. PUFAs have similar effects on both Naq and
concentrations of eicosapentaenoic acid ŽEPA. and DHA,
to cause 50% inhibition of the currents, are much lower in
cardiac myocytes than in neurons w28,29x.

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 9 7 - 6
 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121
The effects on cardiac myocytes provide an electrophys-
iological basis for the anti-arrhythmic effects of PUFAs
w12x. In cultured neonatal rat heart cells, PUFAs, but not
the monounsaturated and saturated fatty acids, significantly
increased the amount of the depolarizing current for initia-
tion of action potentials. There was a concomitant prolon-
gation of the refractory period. Current clamp analysis
showed that these effects on cardiac action potentials were
due to hyperpolarization of the membrane resting potential
and an increase in the threshold potential. In this study, we
investigated the effects of n y 3 PUFAs on the membrane
excitability of mouse hippocampal neurons.
Time-limited ‘‘storm’’ bursts of neuronal firing in the
brain lead to seizures. A deficiency of effectively in-
hibitory mechanisms may be responsible for such seizures.
Epileptogenic neurons appear to have an increased mem-
brane permeability and are susceptible to endogenous or
exogenous stimuli w1x. To determine whether n y 3 PUFAs
protect against seizure-associated activities, we externally
perfused mouse hippocampal neurons with PTZ to increase
their frequency of action potentials. Since the excessive
release of the excitotoxic neurotransmitter glutamate dur-
ing brain ischemia markedly increases postsynaptic cal-
cium concentrations which account for certain neuronal
damages, we also evaluated the effects of PUFAs on
glutamate-enhanced action potentials and inward currents.
2. Materials and methods
2.1. Brain slices and electrophysiology
Male mice were purchased at 15–20 days of age ŽSwiss
Webster, Toconic Farms, Germantown, NY. and kept in
our institute animal facility for 4–6 days before study.
Hippocampal slices were prepared from the brains of
isoflurane-anesthetized mice as described previously w15x.
Briefly, the isoflurane-anesthetized mice were decapitated
and the hippocampi were sliced ŽVibratome 1000, TPI, St.
Louis, MO. to 250–300 mm thick slices in an oxygenated
iced artificial cerebrospinal fluid ŽCSF.. Prior to study,
slices were incubated at 21–238C for 60 min in artificial
CSF bubbled with 95% O 2 and 5% CO 2 . Next, one slice
was transferred to a submerged-slice recording chamber
with 500 ml of the bath solution and continuously perfused
with the artificial CSF at a rate of 2 mlrmin. Electrophysi-
ological data were acquired from individual neurons with
the whole-cell ‘‘blind-patch’’ recording technique w5x. Ac-
tion potentials were recorded under current clamp. Mem-
brane potential was depolarized to approximately y45 mV
by injecting constant currents Ž3.4 " 0.8 pA, n s 10.. The
threshold for action potential generation was determined
by injecting a series of depolarizing currents. Spontaneous
action potentials were found in some CA3 neurons under
zero-current clamp. Two protocols of ramp voltage clamp
were applied: Ži. from y100 to y35 mV for 370 ms Ž175
113
mVrs.; and Žii. from y100 to 0 mV for 400 ms Ž250
mVrs.. The holding potential was y60 mV. The pulse
interval was 5 s.
2.2. Materials and solutions
Glutamate and PTZ were obtained from Sigma ŽSt.
Louis, MO.. Glutamate and PTZ were freshly dissolved in
the perfusion solution at the concentrations used in the
experiments. Fatty acids were obtained from Cayman
Chemical ŽAnn Arbor, MI. and dissolved weekly in ethanol
at 10 mM and stored under a nitrogen atmosphere at
y208C before use. The experimental concentration of fatty
acids was obtained by dilution of the stocks. When the
fatty acid stocks were diluted to 20 mM, the concentration
of ethanol was 0.16% Žgr100 ml.. Control studies re-
vealed that this concentration of ethanol had no effect on
neuronal membrane excitability Ž n s 4.. The artificial CSF
for brain slice recordings contained Žin mM.: NaCl, 124;
KCl, 2; MgCl 2 , 1.3; CaCl 2 , 2.5; KH 2 PO4 , 3; NaHCO 3 ,
26; and glucose 10, pH 7.35. The pipette solution for brain
slice recordings contained Žin mM.: K gluconate, 120;
KCl, 10; MgCl 2 , 3; MgATP, 2; Na 2-GTP, 0.2 and HEPES
10, pH 7.2. External solutions containing different chemi-
cals were exchanged through a fast perfusion system hav-
ing a multi-barreled pipette as described previously w28x.
Experiments were conducted at 21–238C.
2.3. Data collection and statistical analysis
Action potentials and ion currents were recorded from a
patched neuron before, during and after drug treatment
with Axopatch-1D amplifier and Pclamp 5.5.1 software
ŽAxon Instruments, Foster City, CA.. Washout data were
obtained for all test agents to verify the effects of treat-
ment. The amplitude of inward currents was measured and
compared at the voltage of y90 mV. Data are presented as
mean " standard error of the mean ŽS.E.M... Paired or
unpaired Student’s t-test was used to evaluate differences
between control and experimental values. One-way analy-
sis of variance ŽANOVA. was used for multiple group
data. A level of p - 0.05 was considered statistically
significant.
3. Results
3.1. EPA decreases the frequency of eÕoked action poten-
tials in CA1 neurons
To test the effects of n y 3 PUFAs on neuronal activity,
we superfused mouse hippocampal brain slices with solu-
tion containing EPA. After forming a whole-cell configura-
tion, the resting membrane potential recorded in CA1
neurons was y59.3 " 0.6 mV Žmean " S.E.M., n s 36..
These neurons did not produce spontaneous action poten-
114 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121
tials at their resting membrane potentials. Injection of
currents Ž3.4 " 0.8 pA, n s 10. to depolarize the neurons
to approximately y45 mV consistently initiated action
potentials. Fig. 1 exemplifies evoked action potentials
when the membrane potential of a CA1 neuron was depo-
larized to y45 mV. Extracellular application of 20 mM
EPA significantly reduced the frequency of action poten-
Fig. 1. The inhibitory effect of EPA on the frequency of action potentials
in CA1 neurons. The membrane potential was depolarized to y45 mV by
intracellular injection of a constant current. Evoked action potentials for
control were continuously recorded 10 s after the membrane potential
depolarized to y45 mV ŽControl.. Extracellular application of 20 mM
EPA significantly reduced the frequency of action potentials ŽEPA.. The
frequency restored to the pretreatment level after washout EPA with 0.2%
delipidated BSA solution ŽWashout.. Bottom panel shows the averaged
frequency of evoked action potentials of the CA1 neurons Ž ns10. in the
absence ŽControl. and presence ŽEPA. of 20 mM EPA. UU p- 0.01 vs.
control.
tials ŽFig. 1, EPA.. The frequency of evoked action poten-
tials returned toward the pretreatment level after washout
of EPA with the bath solution containing 0.2% of fatty-
acid-free bovine serum albumin ŽBSA, Fig. 1, Washout..
The average frequency of evoked action potentials was
3.8 " 0.7 Hz at the membrane potential of y45 mV in the
CA1 neurons. External application of 20 mM EPA inhib-
ited the frequency by 45% to 2.1 " 0.5 Hz Ž n s 10, p -
0.01, bottom panel of Fig. 1.. Thus, EPA modulates the
membrane excitability of hippocampal CA1 neurons.
3.2. Effects of EPA on the action potential properties of
CA1 neurons
To examine whether EPA decreased the frequency of
action potentials in CA1 neurons by changing the thresh-
old potential, we injected a series of depolarizing current
pulses in increments of 1 or 2 pA into neurons in the
absence and presence of 20 mM EPA. Fig. 2 shows that 20
mM EPA increased the stimulation strength for initiation
of action potentials by 2.2 " 0.5 pA Žright panel at the
bottom, n s 9, p - 0.01.; i.e., from 6.0 " 0.6 pA for
control to 8.2 " 1.0 pA for EPA. The effect of EPA on the
action potential threshold reversed after washout of EPA
with 0.2% BSA solution ŽFig. 2, Washout..
Detailed analysis of action potentials revealed that the
EPA-induced increase in stimulatory current might result
from the changes in the threshold and the resting mem-
brane potential. Table 1 shows that 20 mM EPA shifted
the threshold potential by 3.1 " 0.9 mV in the depolarizing
direction Ž p - 0.05, n s 10.. Furthermore, 20 mM EPA
hyperpolarized the resting membrane potential by 1.5 " 0.2
mV, i.e., from y61.2 " 0.1 mV for control to y62.7 " 0.2
mV for EPA treatment Ž p - 0.01.. These effects of EPA
reversed after washout with superfusion solution contain-
ing delipidated BSA Ž2 mgrml.. EPA also significantly
reduced the value of dVrdt Žcalculated as the amplitude
divided by the time to reach maximal peak of the action
potential.. In contrast, 20 mM EPA had no significant
effect on amplitude and duration of action potentials.
Fig. 3 demonstrates hyperpolarization of resting mem-
brane potential and greater afterhyperpolarization in the
presence of 20 mM EPA. The small, but significant,
hyperpolarization of resting membrane potentials after EPA
treatment might result from inhibition of an inward cur-
rent. Therefore, we examined membrane currents in re-
sponse to ramp voltage-clamp pulses. EPA Ž20 mM.
slightly suppressed the membrane current over the entire
range of voltage ramp ŽFig. 4..
3.3. Effects of other fatty acids on neuronal actiÕity in CA1
neurons
Table 2 compares the effects of EPA with those of
docosahexaenoic acid ŽDHA, C22:6n y 3., EPA ethyl es-
ter, oleic acid ŽOA, C18:n y 9., and stearic acid ŽSA,
 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121 115

Fig. 2. Effect of EPA on the threshold and the current for initiation of action potentials in CA1 neurons. The resting membrane potential was y60 mV. A
series of depolarizing current pulses with 150 ms duration in 1 pA increments Žsee inset of the protocol. was applied to the neuron and generated action
potentials in the absence ŽControl and Washout. and presence ŽEPA. of 20 mM EPA. The bottom-right panel summarized the averaged currents for
initiation of action potentials in the absence ŽControl. and presence ŽEPA. of 20 mM EPA in CA1 neurons Ž n s 9.. UU p - 0.01 vs. control.

C18:0. on the threshold current for initiation of action
potentials and the frequency of evoked action potentials in
CA1 neurons. Clearly, 20 mM EPAEE, OA, and SA had
no significant effects on these membrane parameters. In
contrast, DHA at the same concentration as EPAEE, OA,
and SA significantly increased the threshold current. DHA
also inhibited the frequency of evoked action potentials.
Thus, the effects of DHA on neuronal membrane excitabil-
ity are similar to those of EPA.
3.4. Modulation of membrane excitability by PUFAs in
CA3 neurons
It has been shown that during ischemic injury, the
release of arachidonic acid is greater in CA1 than in CA3
field of rat’s hippocampus w27x. Therefore, we compared
the effects of n y 3 PUFAs on excitability of CA3 neurons
with the same methods applied to CA1 neurons. The
resting membrane potential was y57.8 " 1.1 mV Ž n s 8.,
which is very similar to the resting membrane potential of
CA1 neurons Žy59.3 " 0.6 mV, n s 36.. Four out of
eight CA3 cells exhibited spontaneous action potentials
without intracellular injection of depolarizing current. The
other four cells, like CA1 neurons, produced action poten-
tials only after intracellular injection of depolarizing cur-
rents Ž5.4 " 1.4 pA.. Fig. 5 illustrates spontaneous action
potentials in a CA3 neuron under the zero-current clamp
mode. Compared to control ŽFig. 5, Control., 20 mM EPA
significantly inhibited the frequency of spontaneous action

Table 1
Effects of EPA on the action potential of mouse CA1 neurons
Values are expressed as mean " S.E.M. Ž n s 10.. EPA, eicosapentaenoic acid.
Threshold ŽmV. Amplitude ŽmV. dVrdt ŽVrs. Duration Žms. Repolarization Ž75%, ms.
Control y49.3 " 2.2 82.8 " 7.5 67 " 8 4.71 " 0.33 3.54 " 0.21
EPA y46.2 " 1.9U 74.2 " 6.5 52 " 6U 4.90 " 0.46 3.50 " 0.31
U
p - 0.05, significant difference between control and treatment.
116 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121

Table 2
Effects of fatty acids on neuronal activities in mouse CA1 neurons
Values are expressed as mean"S.E.M. n, the number of individual
neurons recorded from different brain slices. DHA, docosahexaenoic
acid; EPAEE, eicosapentaenoic ethyl ester; OA, oleic acid; SA, stearic
acid. The concentration of all fatty acids used in experiments was 20 mM.
Fatty acid n Threshold Firing rate
Žpercentage of control. Žpercentage of control.
EPA 10 136"8UU 54"9UU
DHA 8 145"9UU 42"6UU
EPAEE 5 96"2 96"3
OA 4 97"7 115"9
SA 5 95"10 105"9
UU
p- 0.01, significant difference between corresponding control and
fatty acid treatment.

Fig. 3. Comparison of single action potentials in the absence ŽControl.

and presence ŽEPA. of 20 mM EPA in a CA1 neuron. The action
potentials were elicited by 150-ms superthreshold pulses. Note hyper- tion of action potentials was significantly increased by
polarization of the resting membrane potential and a greater afterhyperpo-
2.2 " 0.5 pA ŽFig. 1, n s 10, p - 0.01. for CA1 neurons
larization after application of 20 mM EPA Žsolid line..
after exposure of EPA.

potentials ŽFig. 5, EPA.. The frequency of action potentials
recovered to the pretreatment level after washout of EPA
with 0.2% delipidated BSA solution Ždata not shown.. The
averaged frequency of spontaneous action potentials was
reduced by 55 " 11% in CA3 neurons Ž n s 4, p - 0.01. in
the presence of 20 mM EPA. It is interesting that the
threshold potential of CA3 neurons with spontaneous ac-
tion potentials was not significantly affected by EPA treat-
ment. In CA3 neurons without spontaneous action poten-
tials, the threshold current for initiation of action potentials
was slightly decreased after exposure to EPA Ž D s y0.41
" 0.36 pA, p ) 0.05, n s 4.. This result differs from the
above finding that the threshold current required for initia-
3.5. Effects of EPA on membrane excitability during PTZ
stimulation of CA1 and CA3 neurons
Seizures may result from an increase in neuronal mem-
brane permeability which causes bursts of action potentials
in some brain areas. To explore the effects of EPA on
chemical-induced seizure-like activity, we applied PTZ to
CA1 neurons. Fig. 6 shows that the frequency of evoked
action potentials significantly increased during superfusion
with 200 mM PTZ. In four CA1 neurons, PTZ increased
the frequency of evoked action potentials by 5 " 1 Hz
Ž p - 0.05.. Addition of 20 mM EPA to the bath solution
containing 200 mM PTZ suppressed the PTZ-enhanced

Fig. 4. Effect of EPA on inward currents in a CA1 neuron. Inward currents were elicited by ramp voltage-clamp pulses from y100 to y35 mV for 370 ms
Ž175 mVrs.. The portion of the currents from y90 to y50 mV was displayed. Extracellular application of 20 mM EPA ŽEPA. caused an inhibition of the
current.
 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121 117

Fig. 5. EPA-induced reduction of spontaneous action potentials in a CA3 neuron. This neuron exhibited spontaneous action potentials at its own resting
membrane potential ŽControl.. Bath perfusion of 20 mM EPA markedly decreased the frequency of its spontaneous action potentials ŽEPA..

frequency of action potentials ŽFig. 6, PTZ and EPA.. and PTZ with 0.2% delipidated BSA solution ŽWashout..
These effects completely reversed after washout of EPA In addition, extracellular application of 200 mM PTZ

Fig. 6. Inhibition of PTZ-augmented action potentials by EPA. Action potentials of the CA1 neuron were evoked by 600-ms depolarizing current Ž6 pA.
pulses from a resting membrane potential of y60 mV. Compared to control ŽControl., PTZ Ž200 mM. significantly enhanced the frequency of action
potentials. Addition of 20 mM EPA blocked PTZ-induced enhancement of action potentials ŽPTZ and EPA.. After washout with 0.2% BSA bath solution,
the frequency of action potentials returned to the control level ŽWashout.. The inset under the control column is the experimental protocol.
118 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121

significantly decreased the threshold current for initiation
of action potentials by 4.3 " 1.2 pA Ž n s 6, p - 0.05..
This effect was abolished after addition of 20 mM EPA.
To test the effects of PTZ on spontaneous action poten-
tials, we recorded CA3 neurons with spontaneous action
potentials. Fig. 7A shows that under the zero-current clamp
mode, 200 mM PTZ increased the frequency of sponta-
neous action potentials by 474 " 107% of control Ž n s 5,
p - 0.05.. Co-application of 20 mM EPA suppressed the
PTZ-enhanced frequency of spontaneous action potential
back to 157 " 77% of control. PTZ depolarized the maxi-
mal resting membrane potential from y59.4 " 2.3 mV for
control to y49.2 " 1.9 mV for 200 mM PTZ Ž n s 5,
p - 0.05.. Co-application of 20 mM EPA restored the
maximal resting membrane potential to y57.0 " 1.7 mV.
In contrast, 200 mM PTZ had no significant effect on
inward currents activated by ramp voltage clamp pulses
and measured at y90 mV ŽFig. 7B.. However, it is very
interesting that 200 mM PTZ caused a negative shift of
clamp potential to initiate the first burst of an inward
current ŽFig. 7C.. Most portions of this burst current are
probably composed of Naq influx. Since space clamp is
inadequate for recording fast ion currents in brain slices,
we qualitatively tested the effects of PTZ on the number of
the bursts and the potential at which first burst current
occurred during ramp voltage-clamp pulses. The clamp
potentials, at which the first bursts of the inward current
occurred, were y42.5 " 1.9 mV for control, y49.2 " 1.9
mV for 200 mM PTZ Ž n s 5, p - 0.05, vs. control. and
y46.5 " 2.3 mV for 200 mM PTZ plus 20 mM EPA,
respectively. Fig. 7C also shows that PTZ increased the
number of bursts during ramp voltage-clamp pulses and 20
mM EPA abolished the PTZ’s effect. The same result was
confirmed in another four CA3 neurons. Under zero-cur-
rent clamp, PTZ with 1 Ž n s 4. or 5 mM Ž n s 2. did not
cause spontaneous action potentials in CA1 neurons Ž n s 3.
and also did not produce time-limited ‘‘storm’’ bursts in
CA3 neurons with spontaneous action potentials Ž n s 3..
3.6. Inhibition of glutamate-enhanced neuronal excitability
by EPA
Data derived from experimental animals demonstrate
that excessive release of endogenous excitatory amino
acids, particularly glutamate, plays a critical role in is-
chemic brain damage w8x. Neurons in the CA1 zone of the
horn of Ammon in the hippocampus are particularly vul-
nerable during ischemia and reperfusion w11,14x. There-

Fig. 7. Effect of EPA on PTZ-induced membrane excitability of CA3 neurons. ŽA. Spontaneous action potentials of the CA3 neuron were recorded under
zero-current clamp. The frequency of action potentials was normalized by control ŽControl, 100%.. PTZ at 200 mM ŽPTZ. significantly enhanced the
frequency of spontaneous action potentials and 20 mM EPA abolished the PTZ’s effect ŽPTZ and EPA.. ŽB. Inward currents were activated by ramp
voltage-clamp pulses from y100 to 0 mV for 400 ms. The amplitude of currents was measured at y90 mV. PTZ Ž200 mM. alone and plus EPA Ž20 mM.
had no significant effect on the inward current. The membrane holding potential was y60 mV. ŽC. Original current traces were elicited by ramp
voltage-clamp pulses from y100 to 0 mV for 400 ms. The displayed portion of the currents was from y90 to 0 mV in the absence ŽControl. and presence
of 200 mM PTZ ŽPTZ., as well as 200 mM PTZ plus 20 mM EPA ŽPTZ and EPA.. Note the changes in the clamp potential for initiation of the first burst
of an inward current in the presence of PTZ and plus EPA. Furthermore, note the changes in the number of bursts of the inward current. Several bursts of
the current were saturated under the recording condition.
 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121
fore, we explored the possibility that acute application of
free, long-chain n y 3 PUFAs could inhibit glutamate-
activated cation currents and reduce the frequency of
glutamate-augmented action potentials. Fig. 8 shows that
application of 200 mM glutamate doubled the frequency of
evoked action potentials ŽFig. 8A, control; B, 200 mM
glutamate.. Addition of 20 mM EPA reduced the fre-
quency of evoked action potentials to the control level
ŽFig. 8C.. All these effects reversed after washout of the
compounds with 0.2% delipidated BSA solution ŽFig. 8D..
Similar effects were found in another four CA1 neurons of
different slices. The threshold current for initiation of
action potentials was 6.8 " 0.5 and 3.2 " 0.7 pA Ž n s 5,
p - 0.01. for control and glutamate treatment Ž200 mM.,
respectively. Under the stimulation of 200 mM glutamate,
addition of 20 mM EPA restored the threshold current
back to 7.2 " 2.0 pA. With ramp voltage-clamp pulses, we
found that 200 mM glutamate produced a profound en-
hancement of the inward current ŽFig. 9, Glutamate.. EPA
at 20 mM almost completely blocked the glutamate-en-
hanced current ŽFig. 9, Glutamate and EPA.. The increase
in inward currents measured at y90 mV was from y153
" 16 pA for control to y334 " 59 pA for 200 mM
glutamate Ž n s 8, p - 0.05.. Co-application of 20 mM
EPA restored this current to y163 " 17 pA Ž p - 0.05, vs.
glutamate.. Therefore, EPA blockade of the glutamate-en-
Fig. 8. EPA-induced reduction of glutamate-augmented action potentials.
Action potentials of the CA1 neuron were evoked by 700-ms depolarizing
current pulses from a resting membrane potential of y60 mV. ŽA.
Control; ŽB. 200 mM glutamate; ŽC. 200 mM glutamate plus 20 mM
EPA; ŽD. washout with 0.2% BSA bath solution.
119
Fig. 9. EPA blockade of the glutamate-activated inward current in a CA1
neuron. Inward currents were elicited by ramp voltage-clamp pulses from
y100 to y35 mV for 370 ms Ž175 mVrs.. The portion of the currents
from y90 to y50 mV was displayed. Bath application of 200 mM
glutamate activated an inward current ŽGlutamate. which was inhibited
by addition of 20 mM EPA ŽGlutamate and EPA..
hanced current may be the basis of its suppression of
glutamate-augmented action potentials in CA1 neurons.
4. Discussion
Our present data show that free, long-chain n y 3 PU-
FAs modify the membrane excitability of hippocampal
neurons. It is very possible that unbalanced PUFAs in the
brain may cause some diseases. Evidence from animal
studies has shown that n y 3 fatty acids are essential to
both development and function of retinal and brain w23x.
Thus, some neurological disorders of man may arise from
deficiencies in the tissue levels of n y 3 fatty acids. For
example, the degeneration of the CNS seen in ‘‘kinky hair
disease’’ w20x and in Zellweger’s and pseudo-Zellweger’s
syndrome w18x is associated with a reduction of n y 3 fatty
acids without concurrent reductions in long-chain n y 6
PUFAs. In young patients with neuronal ceroid-lipo-
fuscinosis, the degree of severity of the neurological symp-
toms correlates with the degree of deficiency of DHA w21x.
Moreover, epidemiological research reveals that popula-
tions ingesting a diet with high n y 3 fatty acids exhibit a
much lower incidence of multiple sclerosis than those
having a diet with lower levels w3,13x. These studies
indicate that changes in n y 3 PUFAs and their metabolism
in the central nervous system may be associated with
certain cases of severe neuropathology. Further experi-
ments are needed to elucidate whether these neuronal
diseases mentioned above involve alterations of n y 3
PUFAs’ effects on neuronal electrical activities.
In the nervous system, the primary mode of intracellular
signaling is the action potential. Sodium influx through
opening Naq channels is responsible for initiation and
propagation of action potentials. Our finding, that n y 3
120 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121
PUFAs reduced the frequency of action potentials and
increased the action potential threshold, suggests a sup-
pressant effect on ion channels of hippocampal neurons.
Voltage-gated fast Naq channels are highly expressed in
the membrane of neurons and cardiac myocytes. Our pre-
vious data showed that free, long-chain PUFAs reduced
membrane electrical excitability of ventricular cardiomy-
ocytes via an inhibition of voltage-gated Naq and Ca2q
channels w28,29x. These n y 3 fatty acids also significantly
blocked voltage-activated Naq channels in human embry-
onic kidney ŽHEK293t. cells transfected with the a-sub-
unit of the human cardiac sodium channel w30x. In contrast,
EPA ethyl ester and the saturated fatty acid SA had no
effect on neuronal excitability. This result is consistent
with our previous findings in cardiomyocytes that esteri-
fied EPA, saturated and monounsaturated fatty acids did
not affect the membrane excitability and Naq channels
w2,26,28–30x. The structure requirements for the effects of
PUFAs on neuronal or cardiac membrane excitability are,
therefore, a long acyl or hydrocarbon chain, two or more
C5C unsaturated bonds and a free carboxyl group at one
end.
Multiple cDNAs encoding brain Naq channel a-subunit
isoforms have been cloned and sequenced w7,19x. Further-
more, several distinct a-subunit genes are expressed in the
brain. The different a-subunit structures and their localiza-
tion in different tissue type have been described w9,16,17x.
Clearly, neurons from different brain areas express differ-
ent ion channels or different subtypes of the same channel.
These channels may differ in their response to n y 3
PUFAs. In the present study, we found that PUFAs not
only reduced the frequency of action potentials, but also
affected the action potential threshold of CA1 neurons. In
contrast, EPA and DHA inhibited the frequency without
altering the action potential threshold of CA3 neurons.
These differences may result from different characteristics
of ion channels expressed in CA1 and CA3 neurons. In
fact, the effects of n y 3 PUFAs on cardiac and brain Naq
currents differ. Compared to cardiac voltage-gated Naq
channels, brain Naq channels are much less sensitive to
EPA and DHA w26,28x.
One of the striking findings of this study is that EPA
suppressed the PTZ-enhanced frequency of action poten-
tials in hippocampal neurons. PTZ significantly shifted
membrane potential of the first burst of inward currents to
the hyperpolarizing direction during ramp voltage-clamp
pulses ŽFig. 7C.. PTZ also enhanced the number of bursts
of the inward current during a ramp voltage-clamp pulse.
These changes in membrane excitability were probably
due to PTZ’s effects on the kinetics of ion channels.
Therefore, PTZ caused an increase in the frequency of
action potentials of hippocampal neurons. Since EPA in-
hibits both Naq and Ca2q channels in cardiac and neu-
ronal cells, the suppression of PTZ-enhanced membrane
excitability by EPA may result from EPA block of these
channels. The effects of n y 3 PUFAs on drug-enhanced
membrane excitability in neurons have clinical implica-
tions. For example, experiments in vivo demonstrated that
essential fatty acids raised the seizure threshold in rats
w31x. Furthermore, intravenous application of EPA or DHA
increased the seizure threshold by 25% in a cortical stimu-
lation model of seizures w25x. It is conceivable that the
traditional empirical usefulness of the ‘‘ketogenic diet’’
for control of some juvenile epileptics may result from
incorporation of PUFAs in these very high fat diets. Thus,
if PUFAs increase the seizure threshold, they would be
useful in the therapy of patients with convulsive disorders.
Another important finding of this study is that EPA
blocks the glutamate-activated inward current ŽFig. 9..
Cerebral ischemia for relatively brief periods results in
irreversible brain damage and subsequent loss of neuronal
functions. There is no proven efficacious treatment for this
condition which represents one of the major causes of
morbidity and mortality in western societies w6x. Two
major hypotheses, the excitotoxic neurotransmitter hypoth-
esis during ischemia and the free radical hypothesis during
reperfusion, have emerged from attempts to account for the
phenomenon of selective vulnerability occurring in neu-
ronal soma. During ischemia, selectively vulnerable cell
bodies receive afferent projections that release large
amounts of amino acid neurotransmitter Ži.e., glutamate
w24x.. Excessive release of glutamate increases post-
synaptic calcium concentration via activation of inotropic
receptors. Importantly, dietary supplementation with n y 3
fatty acids reduces edema formation and the fall in cere-
bral blood flow after temporary carotid artery occlusion in
the gerbil w4x. Compared to the control-fed group, an n y 3
fatty acid supplemented diet reduced ischemic brain dam-
age in rats following middle cerebral artery occlusion and
excitotoxic brain damage induced by infusion of the selec-
tive NMDA agonist Ž1-aminocyclobutane-cis-1,3-dicarbo-
xylic acid w22x.. Intra-amniotic injection of DHA produced
a protective effect from oxidative damage after post-
ischemic oxidative stress in fetal brain w10x. Thus, there is
a growing body of literature suggesting the possibility that
PUFAs may protect against stroke-induced ischemic and
excitotoxic brain damage. Effects of n y 3 PUFAs on
neuronal activities are also likely to be relevant to other
neurologic disorders, such as depression, schizophrenia,
and bipolar behaviors.
Acknowledgements
We gratefully acknowledge Dr. James P. Morgan for
his encouragement and support, and Dr. Joseph J. McArdle
for his helpful and constructive comments on the
manuscript. This work was supported, in part, by an
American Heart Association grant Ž9930254N to Y.F.X...
 Y.-F. Xiao, X. Li r Brain Research 846 (1999) 112–121
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