In order to examine the pre and post synaptic effects of NMDA on Purkinje cells NMDA was applied in the bath solution in the absence of external Mg2+. The response to NMDA was characterized firstly in Purkinje cells recorded from organotypic slice cultures at a holding potential of −40 mV, and secondly in Purkinje cells recorded from acute slices at a holding of −60 mV. In slice cultures, data were expressed as a function of age of Purkinje cells (for the postsynaptic effect) or the culture (for the presynaptic effect), whereas in acute slices the data were reported as a function of the postnatal age of the animal.
Extra synaptic NMDA receptors are expressed in old (more than 28 day old) Purkinje cells recorded from slice cultures.
In the presence of TTX, application of NMDA 5×10−5
M during 30 s induced a long lasting (~2 minutes, n
8) inward current in about 50% of Purkinje cells older than 28 days (). The inward current had a mean amplitude of 77±17 pA, n
8 (). The NMDA-induced current was still observed in the presence of the specific AMPA receptor antagonist NBQX (see , n
4), but was blocked in the presence of 2×10−3
M external Mg2+
(data not shown, n
3). The direct evidence of the presence of extra synaptic NMDA receptors in Purkinje cells older than 28 days is illustrated in . Two outside-out patches were obtained from two Purkinje cells responding to NMDA with an inward current. NMDA was then applied in the bath and after 1–2 minutes single channels with a conductance of 45 pS were detected. The channel activity was blocked by external Mg2+
A postsynaptic response to NMDA in old Purkinje cells.
NMDA induces an increase in the frequency of mEPSCs in 17–22 day old slice cultures.
mEPSCs were recorded in isolation in the presence of TTX and Gabazine, a specific blocker of γ-aminobutyric-acid (GABA)A
receptor. NMDA was applied for 60 sec at a concentration of 10−5
M. A typical example for a 17–22 day old slice culture is illustrated in . After a delay of 30 sec (see , upper part) NMDA induced an increase of mEPSC frequency (compare the cumulative distribution of inter-event intervals before and during NMDA, bottom left) without changing the holding current and the peak of the mEPSC amplitude distribution (compare the two amplitude distribution histograms, bottom right). When the frequency of mEPSCs was relatively low, with a majority of individual mEPSCs, an average of events detected in control and in presence of NMDA could be compared (). The average current in both conditions shared the same kinetic properties that are typical for mEPSC. Miniature EPSCs were blocked by the AMPA receptor antagonist NBQX, and in this condition NMDA did not increase current noise or affect the holding current. ( n
3). summarizes the data obtained on 55 Purkinje cells. The facilitatory effect of NMDA on AMPA mediated mEPSCs was observed on about 50% of Purkinje cells recorded in 17–22 day old slice cultures, and was never detected in Purkinje cells recorded in older cultures (histogram on the left). In 17 Purkinje cells recorded in 17–22 day old slice cultures the cumulative inter-event interval distribution was significantly different after the first application of 10−4
M NMDA, with a reduction of the mean inter-event interval indicating an increased frequency. NMDA increased the mEPSC frequency from 1.3±0.4 Hz to 6.4±1.1 Hz (n
17, ± SE, p
0.00012, , middle). Probably because of the presence of overlapping mEPSC, mean amplitude of mEPSCs in the presence of NMDA was significantly larger in 7/21 Purkinje cells. To summarize, the mean amplitude was 11.6±0.1 pA in control and 13.3±0.7 pA in the presence of NMDA (, left).
NMDA increases the frequency of mEPSCs in Purkinje cells recorded in a young (P18) slice culture.
The effect of NMDA is specific for mEPSC.
Summary of NMDA effects on mEPSCs recorded in Purkinje cells recorded from slice cultures.
Based on the observation that in a majority of Purkinje cell the amplitude of mEPSC is not changed by NMDA, we can proposed that the effect of NMDA is probably presynaptic, however we can not exclude any other more complicated effect.
The next series of experiments illustrated in were performed to determine if the effect of NMDA on mEPSC frequency was produced via the activation of NMDA receptors. As illustrated in the effect of NMDA on mEPSC frequency was almost abolished (p<0.0001) when NMDA was applied in the presence of 2 mM extracellular Mg2+
3) or in the presence of NMDA receptor antagonists such as MK801 (, n
3) and AP5 (, n
3). These experiments demonstrate that NMDA exerts its effect on mEPSC frequency via activation of a pharmacologically identified NMDA receptor sensitive to external Mg2+
NMDA binds a NMDA receptor and increases mEPSC frequency.
In all cases (n
4 out of 4), the NMDA dependent increase in mEPSC frequency was significantly promoted (p<0.005) at the second application of the agonist (). Furthermore the effect of NMDA was detected at a concentration of 5×10−6
Repetitive applications of NMDA promote the facilitatory effect on mEPSC frequency.
The presynaptic effect of NMDA on mEPSCs is also observed in acute slices prepared from juvenile mice but requires a specific protocol of application.
The use of cerebellar organotypic slice cultures clearly shows an age dependent facilitatory effect of NMDA on spontaneous glutamate release by granule cells on Purkinje cells. However, to exclude the possibility that this effect of NMDA is due to modifications of native properties on neurons introduced by the culture system, we also investigated the effect of NMDA on isolated mEPSCs recorded in Purkinje cells from acute slices prepared from juvenile (P14–17) or adult (P40–50) mice.
Interestingly, in juvenile mice whereas NMDA (10−4
M) applied for 2 minutes did not modify significantly the frequency of mEPSCs (n
4), a facilitatory effect on mEPSC frequency was revealed after three successive applications of NMDA with an interval of 2 minutes (, n
3) or after a single long lasting (at least 10 minutes) application of the agonist (, n
4). The age-dependent presynaptic effect of NMDA on glutamate release was confirmed using acute slices. No effect of long duration applications of NMDA on mEPSC frequency was observed in Purkinje cells recorded from adult mice (P40–50, n
NMDA increases the frequency of mEPSCs in Purkinje cells recorded in acute slices prepared from P15–16 mice.
The effect of NMDA on mEPSC frequency is seen in juvenile but not adult mice.
summarizes the effects of NMDA on mEPSC frequency recorded in Purkinje cells from juvenile (left) and adult (right) mice. In juvenile mice the frequency of mEPSCs increased significantly from 4.8±0.3 Hz to 17.±2.1 Hz in the presence of NMDA (n
4, ± SE, .P
0.0013), whereas in adult mice the frequency was almost unchanged, (1.4±0.3 Hz in control and 1.2±0.2 Hz in the presence of NMDA, n