28 C
2.3. Applications of chemicals The solutions containing known concentrations of the
drugs were ejected by gravity with a multiple-barrel pipette with a total tip diameter ,2 mm placed at a distance of
less than 0.2 cm from the patched cell. Ejection of each chemical was made using a gravity perfusion system
controlled by electrically-controlled valves. The ionic currents were induced by ejection of kainate or AMPA at
the soma of the recording neurons. Other chemicals were assigned randomly to one of the multiple barrels. Their
effects on kainate or AMPA-induced currents were tested by extracellularly perfusing the cell for 20–180 s. During
the experiment, the cells were continuously perfused with the control solution at a flow-rate of 0.5–1 ml min.
Kainate and AMPA were first prepared as a concentrated stock solution and diluted in standard extracellular bath
solution prior to use at their final concentration. BB, suramin, PIT, PPADS were dissolved in water and then
diluted in a standard extracellular bath solution at different concentrations.
GTPgS was stored in aliquots at 2708C and diluted to 500 mM in a standard internal solution, immediately before
use. In experiments involving cholera toxin ChTx, the
neurons were incubated at 378C in the extracellular solu- tion containing ChTx 20 mg ml for 3–4 h. Control
preparations were obtained with the same protocol in a toxin-free extracellular solution.
All chemicals were purchased from Sigma USA except ChTx Calbiochem, USA.
2.4. Data analysis Analysis of recordings was performed with the Ax-
opatch data analysis programs. Fitting was performed with
Fig. 1. Kainate-induced currents are recorded in rat cortical neurons in culture. A Inward current were induced by bath application of kainate
Origin Microcal.
All results
are expressed
as
100 mM in a 6-day-old cortical neuron at a holding potential of 260
mean6S.E.M. n5number of neurons analysed under each
mV. For this and the following figures, bars indicate the time of drug
experimental procedure. Differences between experimen-
application. B Plot of the normalized current amplitude as a function of
tal groups were statistically analyzed by Student’s paired
the kainate concentration. The sigmoidal curve was drawn according to
h
t-test and were considered significantly different if P,
the equation: I51 11EC [kainate] , with EC 5106 mM and h5
50 50
1,38. C Effect of CNQX 10 mM on whole-cell response of cortical
0.05. Statistical analysis was performed using SPSS 6.0 for
neuron to kainate 100 mM applied with the perfusion system. The
Windows.
inward current induced by kainate was reversibly blocked by 10 mM of CNQX. The holding potential was 260 mV and the cell was the same of
A.
3. Results
When cortical neurons in primary culture were voltage- [21,38,45]. The currents induced by kainate were reversib-
clamped at 260 mV, bath perfusion of 100 mM kainate for ly abolished by bath application of the non-NMDA re-
2–8 s induced inward currents in almost all tested neurons ceptor
antagonist 6-cyano-7-nitroquinoxaline-2,3-dione
Fig. 1A. The kainate-induced responses did not desensit- CNQX, 10 mM, indicating that the currents were me-
ize even with applications as long as 10 s and their initial diated by non-NMDA receptor activation Fig. 1C.
amplitude varied less than 10 during the recording In cortical neurons, PPADS 100–300 mM, a selective
period. Currents gated by kainate were concentration-de- antagonist of P2X receptors, was ineffective n58, Fig.
pendent with EC 5106 mM Fig. 1B. The behaviour of 2A on the kainate-induced currents while suramine 100
50
these receptors can be referred to as AMPA kainate mM, a non-selective antagonist of P2 receptors, reversibly
C . Zona et al. Brain Research 882 2000 26 –35
29
Fig. 2. Effects of purinergic antagonists on the kainate-induced currents in cortical neurons in culture. A PPADS 100 mM did not change the amplitude of the kainate-induced currents in cortical neurons while suramine 100 mM B and PIT 20 mM C decreased the currents reversibly and irreversibly
respectively. The neurons were 8, 13 and 9-day-old in culture respectively and were voltage-clamped at 260 mV.
decreased the current induced by kainate 47.5613.6, manner n557, Fig. 3B. The EC
of the reducing effect
50
n56, Fig. 2B. In addition, PIT 20 mM, an antagonist of of BB was 34 mM. The minimal effective concentration
P2Y receptors, decreased by 1165.7 n58 the currents was 5 mM and maximal inhibition was obtained with 1000
induced by kainate in cortical neurons and it was not mM BB Fig. 3B.
observable the recovery of the currents from these values, To study whether the effect of reduction of the kainate-
in all recorded cells, still with subsequent applications of induced current by BB was also observed in other areas of
kainate Fig. 2C. Higher concentrations than 20 mM PIT the central nervous system, we studied the current induced
were not tested because in previous work it has been by kainate 100 mM in cerebellar granule Fig. 4A and
reported to be toxic for neurons [40]. To test further if hippocampal neurons Fig. 4B in primary culture. In
preferring P2Y receptors antagonist were involved in the granule as well as in hippocampal cells, the current
effect of reduction of the kainate-induced current, we induced by kainate was comparable in amplitude and size
tested BB 5–1000 mM, Fig. 3A, a preferring P2Y with that recorded in cortical neurons. In the presence of
receptor antagonist, also known in literature as reactive 50 mM BB, the reduction of the current was 5365
blue 2. BB did not induce inward or outward currents n59 and 4866 n58 respectively.
during the perfusion but it decreased the current induced To test if BB was also effective on the AMPA-activated
by kainate in all tested neurons. The shape of the responses receptors, we recorded the AMPA-induced currents in
were not modified, while the amplitude of the currents cortical cells when the holding potential was of 260 mV.
induced by kainate was reduced in a dose dependent Current induced by AMPA 30 mM increased rapidly and
30 C
Fig. 3. BB reduces the currents activated by 100 mM kainate in a concentration-dependent manner in cultured cortical cells. A Currents induced by 100 mM kainate on 12, 13 and 15-day-old cortical cells in culture were recorded in the absence and in the presence of BB 10, 50 and 300 mM respectively. The
amplitudes of the currents were reduced during perfusion of BB at the three different concentrations and all the cells presented a full recovery. B Dose–response curve of the effect of BB on the amplitude of the current induced by kainate in 39 cortical cells in culture 9–15 days in culture. The EC
50
of the curve was 34 mM.
then decreased to a steady-state value. In all tested cortical BB by about 5566 n56, Fig. 6A. The kainate-induced
neurons n515, the current induced by AMPA decreased currents had apparent rate of activation and deactivation
or was almost abolished in the presence of BB 100–300 similar to those observed in untreated culture. Moreover,
mM. This effect was completely reversible Fig. 5. BB when cortical neurons were intracellularly loaded with
also decreased the amplitude of the current at all potentials, GTPgS 500 mM, a non-hydrolysable GTP analogue, the
but it did not change the reversal point of the AMPA perfusion of BB 100 mM still decreased the amplitude of
current not shown. the current induced by 100 mM kainate and there were no
To determine whether the effects of BB on the kainate- obvious changes in these Fig. 6B, 7368, n55. GTPgS
induced currents involve the activation of G-proteins, we in the electrode had no observable effect on kainate-
first analysed the ability of this compound to decrease induced currents.
kainate-induced currents in cortical neurons that had been Fig. 7A shows the kainate-induced current obtained by
previously incubated with cholera toxin ChTx, 250 ng changing the membrane potential from 280 mV to 180
ml, 3–4 h at 378C. In ChTx-treated cortical neurons, the mV in 5 s. in control condition and during the perfusion 50
kainate-induced currents were still decreased by 50 mM mM of BB. The current and the total conductance of the
C . Zona et al. Brain Research 882 2000 26 –35
31
Fig. 4. Effect of 50 mM BB on the kainate-induced currents in cerebellar granule and hippocampal neurons. A BB 50 mM decreased the kainate-induced currents in cerebellar granule cell 9-day-old and hippocampal neuron 12-day-old B. This effect of reduction was completely
reversible. The neurons were voltage-clamped at 260 mV and the concentration of the agonist was 100 mM.
membrane were significantly reduced while the reversal In n 527 cortical neurons, the total conductance was
potential of the kainate-induced current did not change. 10.6963.47 nS for the kainate induced current 100 mM
Using the equation: and 5.0562.35 nS in the presence of 50 mM BB P ,0.01,
t-test, Fig. 7B. G 5 I E 2 E
k k
m rev
In order to establish if BB acts as an antagonist of the where G is the total conductance of the membrane, I is
AMPA kainate receptors, different concentrations of kain-
k k
the amplitude of the currents elicited by kainate, E is the ate were used to elicit the current in cortical neurons in
m
membrane potential and E is the reversal potential for
control condition and with 20 mM BB. Concentration–
rev
currents elicited by kainate, using the experimental curren- response analysis revealed that 20 mM BB reduced the
t-voltage relation induced by kainate in control condition response of kainate but did not alter the EC
of kainate
50
and during perfusion of 50 mM BB, we could obtain the 117 mM vs. a control value of 106 mM; P .0.05, n 59.
kainate conductance for the neurons G , where the slope of These data indicated a non-competitive effect of BB on the
k
the regression fit curve indicates the total conductance. kainate action site.
Fig. 5. BB reduces the AMPA induced currents. Total current induced in a 12-day-old cortical neuron in culture by 30 mM AMPA before, during and after perfusion with 100 mM BB. The effect was reversible and the holding potential was 260 mV.
32 C
Fig. 6. Effects of BB on kainate-induced currents in cortical neurons treated with ChTx and with GTPgS in the electrode solution. A Kainate-induced current in a 12-day-old cortical neuron treated with ChTx 250 ng ml, 3–4 h at 378C. BB 50 mM still decreased the kainate induced current. The
membrane potential was 260 mV. B BB 100 mM reversibly decreased the current induced by 100 mM kainate in an 8-day-old cortical neuron recorded with GTPgS 500 mM in the electrode solution. The holding potential was 260 mV. The effect of reduction of the current was comparable to that
observable on the neuron recorded with the standard internal solution.
4. Discussion tors antagonist, in particular BB, exerts its protecting
