V . Mendoza-Fernandez et al. Brain Research 885 2000 14 –24
15
generally accepted that activation of several kinases are was held at 2100 mV by injecting hyperpolarizing current.
essential for the development of LTP and this is seen with Cell input resistance and time constant were measured
stimuli that produce relatively high concentrations of from electrotonic potentials induced by intracellular in-
intracellular calcium. An increase in the activity of several jection of constant current pulses 2100 pA and 100 ms
protein phosphatases, on the other hand, appear to be part applied at the resting membrane potential. After the gEPSP
of the biochemical events responsible for LTD, which is amplitude was stable for 20 min, the hyperpolarizing
induced with stimuli that produce a low to moderate current was removed and either LTP or LTD was induced.
increase in the intracellular calcium concentration. A For LTP two 1-s trains of electrical pulses 100 Hz were
relatively low-frequency stimulation of the glutaminergic applied to the Schaffer collateral-commissural afferents at
fibres that innervate CA1 neurons is known to induce LTD, an interval of 10 s. After this tetanic stimulation, the
whereas their stimulation at higher frequency induces LTP. membrane potential was again current-clamped to 2100
In the present study, we characterized the effects of mV and gEPSPs recorded over the next 40–120 min. LTD
IFN-a on the electrophysiological properties of CA1 was induced by 5 Hz stimulation to the Schaffer collateral-
neurons using intracellular recordings. Our evidence indi- commissural afferents for 5 min.
cates that this cytokine inhibits specifically LTP and Unless otherwise stated, drugs were applied by superfu-
glutamate excitatory postsynaptic potentials without LTD, sion. Because IFN-a responses did not desensitize, this
membrane excitability, or synaptic potentials mediated by cytokine was applied at progressively higher concentration
GABA -receptors. to obtain the concentration–response curve. In some
A
experiments, one pipette tip diameter 3–6 mm was filled with glutamate 5 mM, pH 7.4 or NMDA 10 mM, pH
2. Materials and methods 7.4 and a few nanoliters of these solutions were pressure
ejected typically 160 psi for 10–250 ms onto CA1 Rat hippocampal slices were prepared as previously
stratum radiatum. To avoid leakage effects the pipette tip described [14]. After decapitation, the brain was quickly
was always placed |500 mm from the ejection site and removed, and a block of tissue containing the hippocampus
advanced to the desired position just prior to ejection. was prepared and placed in an ice-cold artificial cere-
Glutamate application was carried out in cells current brospinal fluid aCSF with the following composition in
clamped at 2100 mV. NMDA application was performed
21
mM: 124 NaCl, 5 KCl, 1.2 NaH PO , 1.3 MgSO , 2.4 in extracellular medium without Mg
and at resting
2 4
4
CaCl , 26 NaHCO , 10 glucose. Coronal slices were cut membrane potential.
2 3
with a vibratome Campden Instruments at 400 mm and were incubated at room temperature in aCSF bubbled with
2.2. Drugs 95 O 5 CO mixture.
2 2
The following drugs were used: human recombinant 2.1. Intracellular recordings
IFN-a-2b IFN-a; Schering-Plough, N-methyl-
D
-aspartate NMDA, Sigma,
D
-2-amino-5-phosphonovalerate APV; A single slice was placed in the recording chamber and
RBI, Natick, MA,
L
-glutamate Sigma, picrotoxin RBI, superfused continuously with heated 34–358C aCSF at
kynurenic acid Sigma, and genistein RBI. Stock solu- 1.5–2.5 ml min. Intracellular recordings were made with
tion of genistein 50 mM was prepared with DMSO glass micropipettes filled with 2 to 3 M KCl resistance
whereas stock solutions of all other drugs were prepared in 40–60 mV. Membrane potential was measured with an
nanopure water and kept at 48C. NMDA was applied using
21
Axoclamp-2A preamplifier Axon Instruments, Foster City, a Mg
-free aCSF to prevent NMDA channel block. CA. The output of this preamplifier was displayed on an
oscilloscope TDS 210; Tektronics and recorded with a 2.3. Statistical analysis
PC and Axotape or pClamp software Axon Instruments. An intracellular impalement of a CA1 pyramidal cell was
Data are expressed as mean6S.E. The paired Student’s judged satisfactory if the resting membrane potential was
t-test was used to evaluate differences between mean 255 mV and action potentials were 60 mV in am-
values obtained in the same cell, whereas the unpaired plitude. Glutamate-mediated excitatory postsynaptic po-
Student’s t-test was used to compare data collected from tentials gEPSPs were evoked by an electrical pulse 20–
different cells; two-tailed P values 0.05 were considered 100 ms applied at 0.1 Hz to the Schaffer collateral-
statistically significant. commissural afferents, using a bipolar electrode made by
twisting tungsten wires of a diameter of 20 mm Teflon- coated. In the presence of 30 mM picrotoxin, five con-
3. Results
secutive gEPSPs were averaged in each experimental condition and the mean amplitude was calculated. To
3.1. General observations minimize voltage-dependent changes in gEPSP amplitude
and overriding actions potentials, the membrane potential Results were obtained from 110 CA1 pyramidal cells.
16 V
Electrophysiological properties of these neurons were antagonist picrotoxin was used to block synaptic potentials
similar to those published previously using patch-clamp mediated by these receptors. These potentials were reversal
and intracellular recordings [6,13,14]. The mean resting depolarizing at 2100 mV due to the high concentration
membrane potential was 26162 mV range, 256 to 265 of chloride ions inside the recording electrode 2 M KCl.
mV, mean cell input resistance was 5067 MV range, In 40 analyzed experiments, picrotoxin superfusion di-
35–65 MV, and mean time constant was 1360.9 ms minished the amplitude of fast synaptic potentials to an
range, 11–14 ms. The mean action potential amplitude average of 4164 from its control values.
was 7263 mV range, 64–85 mV as measured at resting To evaluate IFN-a effects on the GABA-mediated fast
membrane potential. synaptic potential, kynurenic acid 100 mM was used to
Unless otherwise stated, 30 mM of the GABA receptor block glutamate ionotropic receptors. Kynurenic acid
A
Fig. 1. IFN-a inhibited the excitatory postsynaptic potential EPSP in CA1 pyramidal neurons without significantly affecting the resting membrane potential or the cell input resistance. A Voltage–current VI relationships before and in the presence of IFN-a. Symbols are mean6S.E. n55. The cell
input resistance, calculated from these VI relationships, was 5762 and 6162 MV before and in the presence of IFN-a, respectively. B Electrotonic potentials induced by application of outward and inward current pulses of 100 pA with a duration of 100 ms, before upper tracings and then in the
presence of IFN-a lower tracings. Duration, amplitude, and number of spikes induced by depolarizing pulses were not significantly modified see Table 1. C Concentration–response curves to show the inhibitory effects of IFN-a on control glutamate GABA; n56 and glutamate-EPSPs n57. These
effects were concentration dependent with an IC 5151 U ml and IC 5148 U ml, respectively. Examples of such recordings are shown in D and E,
50 50
recorded 30 min after starting the superfusion of the indicated concentrations of IFN-a. Glutamate-EPSPs were recorded in the presence of 30 mM of picrotoxin.
V . Mendoza-Fernandez et al. Brain Research 885 2000 14 –24
17 Table 1
diminished the amplitude of the synaptic potentials to an
Parameters means6S.E., n55 of the action potentials induced by the
average of 6263 n59 of control values.
intracellular application of inward current pulses, in CA1 neurons before
a
Control and then in the presence of IFN-a 300 U ml
3.2. IFN-a effects on the excitability of CA1 pyramidal
Treatment Control
IFN-a
cells
Duration ms 1.5960.2
1.5960.3 Amplitude mV
8362 8262
Superfusion of IFN-a 300 U ml for 30 min had no
Number of spikes evoked by a 100-ms pulse 460.5
560.2
effect on the resting membrane potential of pyramidal
Rate of rise of the onset V s 234646
228621
neurons. Control and experimental values were 26361
Rate of rise of the offset V s 10169
9165
and 26261 mV, respectively n512. IFN-a failed also to
a
The observed changes were not significant.
alter the voltage-current relationships indicating that cell input resistance is not altered by this cytokine Fig. 1A;
which component was inhibited by IFN-a. Superfusion of n55. The action potential waveform was also not modify
this cytokine had no effect on the GABA-mediated EPSP, by the presence of this cytokine, as shown in Fig. 1B and
which was recorded in the presence of 100 mM kynurenic Table 1 n55.
acid and abolished by 30 mM picrotoxin. This EPSP was always completely inhibited by 30 mM picrotoxin.
3.3. IFN-a effects on the fast postsynaptic potentials The glutamate mediated EPSP gEPSP was inhibited by
mediated by glutamate and GABA IFN-a in a concentration-dependent manner with an IC 5
50
148 U ml and a maximal inhibition of 1662 Fig. 1C Superfusion of IFN-a 300 U ml had a small 963
and E; n57. The onset of this inhibition was between 6 inhibitory effect on EPSPs mediated by both GABA and
and 10 min and reached a maximum about 20–25 min glutamate see below. Fig. 1C and D show the effect of
after starting the superfusion of IFN-a. This was not different cumulative concentrations IC 5151 U ml of
reversible even after 30–60 min of washing n56.
50
IFN-a on these EPSPs. This inhibitory effect was present gEPSPs were recorded in the presence of 30 mM picrotox-
in all tested neurons n56 and irreversible. in and were abolished by 100 mM kynurenic acid. The
Because these EPSPs, as mentioned above, were me- inhibitory effect of IFN-a 300 U ml; n54 on the gEPSP
diated by both glutamate and GABA, we investigated was prevented by pre-treating for 10 min the slices with
Fig. 2. IFN-a inhibits the depolarization induced by glutamate but did not by NMDA. Pressure ejection of glutamate 5 mM; upper panel or NMDA 10 mM; lower panel was at the time indicated by arrows. Depolarization was recorded before Control, 20 min after adding IFN-a, and after 20 min of
IFN-a wash-out. Similar observations were obtained in four upper panel and six lower panel identical experiments. Only one neuron per slice was recorded. Glutamate experiments were carried out in cells current clamped at 2100 mV. NMDA experiments were performed in extracellular medium
21
without Mg and at resting membrane potential.
18 V
100 mM genistein not shown which specifically inhibits stimulation Fig. 3A. After reaching its maximal values, a
tyrosine kinase activity in hippocampal tissue [16]. transitory decay in EPSP amplitude was observed during
IFN-a 300 U ml also reduced the depolarization the first 10–15 min. After that, stable potentiated gEPSPs
induced by local application of glutamate Fig. 2A. 17067 of control values were observed for as long as
Control values were 1663 mV and in presence of IFN-a they were recorded up to 2 h. In accordance with others
these were 1163 mV P,0.01; n54. IFN-a, however, did [15], we will to the first portion of this response as
not modify the depolarization induced by local application short-term potentiation STP and to the second portion as
of NMDA n56; Fig. 2B. These observations indicate that the maintenance phase of long-term potentiation LTP.
at least part of the inhibitory effect of IFN-a on the gEPSP This potentiation was prevented by the presence of APV
is at the postsynaptic level involving non-NMDA channels see below indicating that it is mediated by NMDA
and tyrosine kinase activity. channels, as it has been previouly described [11,12].
Superfusion of IFN-a 300 U ml for 20 min before tetanus prevented the maintenance phase of LTP but a STP
3.4. IFN-a actions on synaptic plasticity in the was still observed Fig. 3. The average gEPSP amplitude
hippocampus reached during the peak of STP was 12464 of control
values. After STP, the EPSP amplitude decreased and was Following tetanic stimulation a twofold increase in the
maintained at 5665 of control values indicating that gEPSP amplitude 19666 of control values; n55 was
IFN-a unmasked a depression of this potential. observed, with the first recording performed 5 min after
Application of IFN-a after STP Fig. 4; n54 also
Fig. 3. IFN-a prevents long-term potentiation in neurons of the CA1 region. A, relative amplitude of glutamate mediated excitatory postsynaptic potential gEPSP taken before and after tetanic stimulation at time 0 arrow. Symbols are mean6S.E. of five untreated and five IFN-a-treated neurons. Only one
neuron per slice was recorded. IFN-a was applied before and during tetanic stimulation, as indicated by the bar. Tetanic stimulation consisted of two pulse-trains of 1 s each, applied at 100 Hz, and with a 10-s interval between trains. B Typical gEPSPs of two different neurons taken before and after
tetanic stimulation, at the times indicated by letters.
V . Mendoza-Fernandez et al. Brain Research 885 2000 14 –24
19
Fig. 4. IFN-a inhibits long-term potentiation in CA1 region. A Relative amplitude of glutamate-mediated excitatory postsynaptic potentials gEPSPs taken before and after tetanic stimulation see Section 2 at time 0 arrow. Both treatments were started during the maintenance phase of LTP, as indicated
by bar. Symbols are mean6S.E. of four IFN-a-treated neurons or of four neurons treated with heat-inactivated IFN-a solution. Only one neuron per slice was recorded. B Typical gEPSPs from two different neurons treated with either heat-inactivated IFN-a solution or with 300 U ml of IFN-a, recorded at
the indicated times.
inhibited the maintenance LTP. Indeed, in the presence of As expected, inhibition of NMDA receptors with APV
this cytokine the gEPSP amplitude reached a new steady- 50 mM during the tetanus stimulation prevented LTP but
state value of only |60 of the control values. This effect STP was still present Fig. 7A; n54. This treatment also
was abolished by boiling IFN-a solution for 30 min Fig. prevented the effects of IFN-a on the gEPSPs. Application
4; n54. of APV after STP neither affected the maintenance phase
Tetanic stimulation also potentiated the depolarization of LTP nor the inhibitory effect of IFN-a on LTP Fig. 7B;
induced by pressure ejection of glutamate Fig. 5; n54 n54. APV by itself did not alter the EPSP amplitude in
but this potentiation was smaller than that observed for control conditions before tetanus; Fig. 7A or after STP
gEPSPs Fig. 3A. Application of this cytokine also Fig. 7B, indicating a null contribution of NMDA chan-
reverted this potentiation Fig. 5; n54, suggesting that the nels for these potentials during such conditions.
IFN-a inhibitory effect on LTP is, at least in part, After applying a low-frequency tetanic stimulation see
postsynaptic. Section 2 a decrease in gEPSP amplitude was observed,
Pretreatment of brain slices with 100 mM genistein which lasted for as long as we recorded 60 min. This
prevented IFN-a inhibitory effects on the maintenance well-characterized phenomenon is long-term depression
phase of LTP Fig. 6; n55. In agreement with previous LTD [9,11]. Fig. 8 shows that the gEPSPs amplitude
observations [16], this concentration of genistein by itself during the LTD steady state was only slightly reduced by
did not modify LTP when it was added after STP. superfusion of IFN-a n54.
20 V
Fig. 5. IFN-a inhibits the potentiated depolarization induced by local application of glutamate. A Relative amplitude of the depolarization induced by local application of glutamate before and after tetanic stimulation at time 0, recorded in CA1 neurons. Symbols are mean6S.E. of four untreated and four
IFN-a-treated neurons. IFN-a was applied as indicated by bar. Only one neuron per slice was recorded. B,C Typical glutamate-induced depolarizations from two different neurons, one untreated and one treated with IFN-a. These depolarizations were recorded before and after the tetanus, at the indicated
times.
4. Discussion high tetanic stimulation widely used by others to induced a
