M-type potassium channels modulate Schaffer collateral-CA1 glutamatergic synaptic transmission

Abstract

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents – Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

Figure 1. M1 muscarinic agonist increased the frequency of mEPSCs recorded from CA1 pyramidal neurons

A, representative traces of mEPSCs recorded from CA1 pyramidal neurons before (left) and after (right) the application of 10 μm McN-A-343. B, representative averaged traces of mEPSCs before (black) and during (grey) the application of McN-A-343 along with the overlay of the two traces. C and D, cumulative probability plots of mEPSC frequency (C) and amplitude (D) obtained by pooling data from 7 neurons before (continuous lines) and after (dashed lines) application of McN-A-343.

Figure 2. M-channel blocker XE991 increased mEPSC frequency in CA1 neurons

A, representative mEPSC recordings obtained from a CA1 pyramidal neuron before (left) and after (right) the application of 10 μm XE991. B and C, cumulative probability plots of mEPSC frequency (C) and amplitude (D) obtained by pooling data from 8 neurons before (continuous lines) and after (dashed lines) the application of XE991.

Figure 3. The effect of linopirdine and flupirtine, and the occlusion of XE991 to the effect of McN-A-343 on mEPSC in CA1 neurons

A, and B, cumulative probability plots of mEPSC frequency by pooling data from neurons before (continuous lines) and after (dotted lines) the application of linopirdine (A), flupirtine (B). C, cumulative probability plot of mEPSC frequency by pooling data from neurons before (continuous lines) and after (dashed lines) the application of McN-A-343 after 25 min incubation in XE991.

Figure 4. The effect of XE991 on mEPSC frequency was eliminated in calcium-free ACSF

A and C, representative mEPSC recordings obtained from a CA1 pyramidal neuron before (top) and after (bottom) the application of 10 μm XE991 after 20 min incubation in thapsigargin (2.5 μm) (A) or in calcium-free ACSF (C). B and D, cumulative probability plots of the frequency of mEPSCs obtained by pooling data from 8 neurons before (continuous lines) and after (dashed lines) the application of XE991 in thapsigargin incubation (B) or in calcium-free ACSF (D).

Figure 5. P/Q-type calcium channel blocker on the effect of XE991 on mEPSC frequency

A and B, representative mEPSC recordings obtained from a CA1 pyramidal neuron in which the blocking of P/Q-type of calcium channels by 200 nmω-agatoxin TK did (A) or did not (B) prevent the effect of 10 μm XE991. The left panels include data from recordings in the presence of ω-agatoxin TK before the application of XE991, and the right panels include data from recordings after the application of XE991. C and D, cumulative probability plots of mEPSC frequency obtained by pooling data from CA1 pyramidal neurons before (continuous lines) and after (dashed lines) the application of XE991 in the presence of ω-agatoxin TK that did (C) or did not (D) prevent the effect of XE991.

Figure 6. N-type calcium channel blocker on the effect of XE991 on mEPSC frequency

A and B, representative mEPSC recordings obtained from a CA1 pyramidal neuron in which the blocking of N-type calcium channels by ω-conotoxin GVIA (1 μm) did (A) or did not (B) prevent the effects of 10 μm XE991. Data in the left panels are from recordings in the presence of ω-conotoxin GVIA before the application of XE991, and data in the right panels are from recordings after the application of XE991. C and D, cumulative probability plots of mEPSC frequency obtained by pooling data from CA1 pyramidal neurons before (continuous lines) and after (dashed lines) the application of XE991 in the presence of ω-conotoxin GVIA that did (C) or did not (D) prevent the effect of XE991.

Figure 7. Combination of P/Q- and N-type calcium channel blockers on the effect of XE991 on mEPSC frequency

A, representative mEPSC recordings obtained from a CA1 pyramidal neuron in the presence of both ω-agatoxin TK and ω-conotoxin GVIA before the application of XE991 (top), and after the application of XE991 (bottom). B, cumulative probability plots of mEPSC frequency obtained by pooling data from CA1 pyramidal neurons before (continuous lines) and after (dashed lines) the application of XE991 in the presence of ω-agatoxin TK and ω-conotoxin GVIA.

Figure 8. M1 agonist McN-A-343 depolarized and increased action potential firing in CA3 pyramidal neurons

A, representative traces of the membrane potential of a CA3 pyramidal neuron before, during, and after a wash of McN-A-343. Note the profound depolarization and the repetitive action potentials. B, a representative trace of the depolarizing effect of McN-A-343 on the membrane potential of a CA3 pyramidal neuron with action potentials blocked by TTX (1 μm). C and D, quantification of the effect of McN-A-343 on the firing frequency (C) and membrane potential (D) of CA3 pyramidal neurons (means ± SEM).

Figure 9. M-channel blocker and opener on membrane potential and action potential firing in CA3 pyramidal neurons

A, representative effect of XE991 on the membrane potential of CA3 pyramidal neurons. B and C, representative recordings of the effects of XE991 (B) and flupirtine (C) on the membrane potential of CA3 pyramidal neurons in the presence of TTX (1 μm). D, quantification of the effect of XE991 on the firing frequency of CA3 pyramidal neurons (means ± SEM). E and F, quantification of the effect of XE991 (E) and flupirtine (F) on the membrane potential of CA3 pyramidal neurons (means ± SEM).

Figure 10. McN-A-343 and XE991 increased input resistance in CA3 neurons

A, top, representative recordings of current injections from −40 pA (in 5 pA steps) before (left) and after (right) the application McN-A-343. Bottom, I–V relationship before (open circles) and after (filled circle) the application of McN-A-343 in CA3 neurons (n = 6). B, top, representative recordings of current injections from −40 pA (in 5 pA steps) before (left) and after (right) the application of XE991. Bottom, I–V relationship before (open circles) and after (filled circles) the application of XE991 in CA3 neurons (n = 7).