High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN)

Abstract

The pedunculopontine nucleus (PPN) is part of the Reticular Activating System, and active during waking and REM sleep. Previous results showed that all PPN cells plateau at gamma frequencies and intrinsic membrane oscillations in PPN neurons are mediated by high-threshold N- and P/Q-type Ca(2+) channels. The present study was designed to determine whether some PPN cells have only N-, only P/Q-, or both N- and P/Q-type Ca(2+) channels. We used patch-clamp recordings in PPN cells in slices from anesthetized rat pups in the presence of synaptic receptor blockers (SB) and Tetrodotoxin (TTX), and applied ramps to induce intrinsic membrane oscillations. We found that all PPN cell types showed gamma oscillations in the presence of SB+TTX when using current ramps. In 50% of cells, the N-type Ca(2+) channel blocker ω-Conotoxin-GVIA (ω-CgTx) reduced gamma oscillation amplitude, while subsequent addition of the P/Q-type blocker ω-Agatoxin-IVA (ω-Aga) blocked the remaining oscillations. Another 20% manifested gamma oscillations that were not significantly affected by the addition of ω-CgTx, however, ω-Aga blocked the remaining oscillations. In 30% of cells, ω-Aga had no effect on gamma oscillations, while ω-CgTx blocked them. These novel results confirm the segregation of populations of PPN cells as a function of the calcium channels expressed, that is, the presence of cells in the PPN that manifest gamma band oscillations through only N-type, only P/Q-type, and both N-type and P/Q-type Ca(2+) channels.

Keywords: Arousal; N‐type; P/Q‐type; gamma oscillations; rats; waking.

Figure 1

Cells in the PPN mediate gamma band activity through N- and P/Q-, N only, and P/Q only type channels. (A) Membrane oscillations recorded in a PPN cell during 1-sec long ramps in the presence of SB + TTX (left record, black). Following superfusion with ω-CgTx for 10 min, oscillation amplitude was reduced (middle record, red). Thereafter, ω-Aga was superfused for 10 min blocking the remaining oscillations (right record, blue). At right is a power spectrum of the records from the neuron shown in A displaying amplitude and frequency of ramp-induced oscillations before (black record, beta/gamma range), after ω-CgTx (red record, reduced oscillations), and following ω-Aga (blue record, blocked remaining oscillations). (B) Membrane oscillations recorded in a different PPN cell during 1 sec long ramps in the presence of SB+TTX (left record, black). ω-Aga applied into the bath for 10 min caused no significant effect on the membrane oscillations (middle record, blue). ω-CgTx was then superfused for 10 min, causing a complete blockade of the membrane oscillations (right record, red). At right is a power spectrum of the record from this cell shown in B displaying amplitude and frequency of ramp-induced oscillations before application of toxins (black record, beta/gamma range), after ω-Aga (blue record, no effect), and ω-CgTx (red record, blocked oscillations). (C) Membrane oscillations recorded in a third PPN cell during 1 sec long ramps in the presence of SB + TTX (left record, black). ω-CgTx was applied for 10 min causing no significant effect on the oscillations (middle record, red). ω-Aga then was superfused for 10 min causing a complete blockade of the membrane oscillations (right record, blue). At right is a power spectrum of the record of the cell shown in C displaying amplitude and frequency of ramp-induced oscillations before application of toxins (black record, beta/gamma range), after ω-CgTx (red record, no effect), and ω-Aga (blue record, blocked oscillations).

Figure 2

Reduction by toxins on the mean peak oscillation amplitude of each channel type. The bar graph shows the mean and standard error of the peak oscillation amplitude of cells within each channel type (left, N+P/Q n = 15; middle, N only n = 9; right, P/Q only n = 6) calculated by measuring the three highest amplitude oscillations after filtering to derive mean amplitude. Light Gray bars indicate the percentage of reduction caused by the addition of ω-CgTx. Dark gray bars indicate the percentage of reduction caused by the addition of ω-Aga. On the left, N+P/Q cells, there was a significant reduction in amplitude after ω-Aga, and an additional significant reduction after ω-CgTx. In the middle, N only cells showed a significant reduction after ω-CgTx, but no further reduction after ω-Aga. On the right, P/Q only cells showed a significant reduction after ω-Aga, but no further reduction after ω-CgTx, *P < 0.05.

Figure 3

Ca²⁺ current recordings showing cells with N- and P/Q, only N-type, and only P/Q-type channels. (A) Using a depolarizing step at −50 mV, Ca²⁺ currents were recorded in the presence of SB+TTX+TEA-Cl from a cell with both N and P/Q- type channels (left panel) before (black record), after ω-CgTx (blue record, reduction) and ω-CgTx (red record, reduction). (B) Ca²⁺ currents recorded from a cell with only N-type channels (middle panel) before (black record), after ω-Aga (blue record, no effect) and ω-CgTx (red record, reduction). (C) Ca²⁺ currents were recorded from a cell with only P/Q- type channels (right panel) before (black record), after ω-CgTx (red record, no effect), and ω-Aga (red record, reduction). (D) Representative currents obtained using a depolarization protocol with multiple steps (see Methods), showing only the 0 mV step. Initial currents were recorded in the presence SB+TTX+TEA-Cl (left record). ω-Aga was superfused in the aCSF bath for 10 min causing a reduction in the current (second record). ω-CgTx (third record) was then superfused for 10 min causing a further reduction in the current, followed by the addition of Cadmium (right record) for 10 min leading to a complete block of the Ca²⁺ currents.

Figure 4

Average current-voltage (I–V) curve from PPN neurons. (A) Representative currents obtained in the presence of SB+TTX+TEA-Cl using a multiple step depolarization protocol (see Methods). (B) Average current-voltage (I–V) curve showing both low voltage- (T-type) and high voltage-activated currents in PPN neurons (n = 5). Note that high-threshold currents peaked at ∽−10 mV.