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
