Mechanism behind gamma band activity in the pedunculopontine nucleus

Abstract

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta-gamma band range (~20-80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8-14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above -30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

Figure 1. All PPN neuron types exhibited gamma band oscillations when depolarized

A) Representative membrane potential responses to depolarizing 2 sec square pulses obtained in the presence of synaptic blockers and TTX in type I (top records), type II (middle records) and type III (bottom records) PPN neurons. B) Representative membrane potential responses to depolarizing 2 sec-long ramps for the same neurons shown in A. C) Overlapping curves comparing power spectrum amplitudes for oscillations obtained using square pulses vs. ramps. All PPN neurons were recorded in current-clamp configuration combining high-K⁺ intracellular solution and synaptic blockers + TTX (see Methods). The respective resting membrane potentials were −48 mV, −49 mV and −51 mV for these type I, II and III neurons.

Figure 2. PPN neurons exhibited gamma oscillations under both current and voltage clamp recording configurations

A) Histogram of the relative frequencies (in percentage) of gamma band oscillation amplitudes recorded from type I (left histogram), type II (middle histogram) and type III PPN neurons (right histogram) using 2 sec current clamp ramps. B) Representative current records obtained from PPN neurons voltage-clamped to holding potentials of −30 mV (left record), −20mV (middle record), and −10 mV (right record). C) Power spectra corresponding to the recordings shown in B. Note the increase in power of several frequencies at −10 mV. Both current and voltage-clamp recordings were performed combining high-K⁺ intracellular solution and synaptic blockers + TTX.

Figure 3. N- and P/Q-type calcium channels mediated the depolarizing phase of gamma band oscillations in the PPN

A&B) Representative membrane potential oscillations (A) and power spectrum (B) obtained using 2 sec-long ramps before (top record), and after bath application of ω-CgTX (2.5 μM; bottom record). Note the reduction of the amplitude of the oscillations after perfusion with the N-type channel blocker ω-CgTX. C&D) Representative membrane potential oscillations (C) and power spectrum (D) obtained using 2 sec-long ramps before (top record), and after bath application of ω-Aga (200 nM; bottom record). Note the elimination of oscillations by the P/Q-type channel blocker ω-Aga. Resting membrane potentials were −48 mV and −52 mV for panels A and C, respectively.

Figure 4. Delayed rectifier-like potassium channels mediated the repolarizing phase of gamma band oscillations in PPN

Gamma band oscillations induced by 2 sec-long current clamp ramps were blocked by the delayed rectifier K⁺ channel blocker α-Dendrotoxin (2 μM). A) Membrane oscillations in control (black record) recordings were blocked by α-Dendrotoxin (gray record). B) Power spectrum showing that power amplitude at gamma band frequency was blocked by α-Dendrotoxin. C) 2 sec-long ramps during control (black record) and after r-Charybdotoxin (200 nM) application (gray record). D) Power spectrum showing power amplitudes of ramp-induced oscillation during control (black peak), and during r-Charybdotoxin application (gray peak). Note the slight increase in amplitude and small shift towards higher frequencies after r-Charybdotoxin application.

Figure 5. Both N-type and P/Q-type calcium channels are present in PPN neurons

A) Representative calcium currents obtained using a depolarization protocol (see text). B) Average current-voltage (I–V) curve showing both low voltage- (T-type) and high voltage-activated calcium currents in type I and III PPN neurons (n=12). C) Representative calcium currents obtained at −10 mV holding potential (see above protocol) before (black records), and after either ω-CgTX (2.5 μM; blue record), or ω-Aga (100–200nM; red record). Control calcium current records were obtained from two different type I or III PPN neurons. D) Calcium current density values (measured at a holding potential −10 mV) in control condition (black bar), and in the presence of either ω-CgTX (blue bar), ω-Aga (red bar), or both (dashed bar). **, P<0.01, Student’s t-test, comparing channel blocker groups vs. control group.

Figure 6. Carbachol increased the maximum frequency of gamma oscillations in all three types of PPN neurons

A) Example of oscillating PPN neuron (11 day old) in the presence of synaptic blockers (APV 40 μM, CNQX 10 μM, STR 10 μM, and GBZ 10 μM) and TTX during 2 sec ramp protocol on the left, and power spectrum on the right. B) Example of oscillating PPN neuron (12 day old) in the presence of synaptic blockers, TTX, and CAR (30 μM). Note the almost doubling in frequency on the power spectrum on the right (~40Hz, gray) compared to PPN neuron recorded in the presence of only synaptic blockers and TTX (~21Hz, black). C) Bar graphs of average power amplitudes at different frequency ranges. Note the reduction in power amplitude at lower frequencies (theta, alpha and beta) but not at gamma band under CAR perfusion. ***, P<0.001, ANOVAs comparing no CAR group vs. CAR group

Figure 7. P/Q-type and N-type Ca²⁺ channel blocker effects on CAR-induced population responses in the PPN

A) Power spectrum of population responses recorded during control (black record and line), 4 min after superfusion of synaptic blockers (APV 40 μM, CNQX 10 μM, STR 10 μM, and GBZ 10 μM, brown record and line), 9 min after CAR (50 μM) superfusion in the presence of synaptic blockers (green record and line), and 10 min after ω-CgTx, in the presence of CAR and synaptic blockers (blue record and line). Note the decrease in amplitude of population responses at lower frequencies, while there was little effect on the higher frequencies (>20 Hz). B) Power spectrum of population responses during control (black record and line), 4 min after superfusion of fast synaptic blockers (brown record and line), 8 min after superfusion of CAR in the presence of fast synaptic blockers (green record and line), and 8 min after superfusion of CAR, fast synaptic blockers, and ω-Aga (200 nM, red record and line). Note the elimination of frequencies higher than theta by ω-Aga. C) Graph of percent of CAR response after specific calcium channel blockers vs. frequency. Note how ω-Aga (red line) blocked higher frequencies (12–47 Hz) and not lower ones (3–11 Hz), while ω-CgTx (blue) more effectively blocked lower frequencies rather than the higher ones.