Spatiotemporal properties of high-speed calcium oscillations in the pedunculopontine nucleus

Abstract

The pedunculopontine nucleus (PPN) is a component of the reticular activating system (RAS), and is involved in the activated states of waking and rapid eye movement (REM) sleep. Gamma oscillations (approximately 30-80 Hz) are evident in all PPN neurons and are mediated by high-threshold voltage-dependent N- and P/Q-type calcium channels. We tested the hypothesis that high-speed calcium imaging would reveal calcium-mediated oscillations in dendritic compartments in synchrony with patch-clamp recorded oscillations during depolarizing current ramps. Patch-clamped 8- to 16-day-old rat PPN neurons (n = 67 out of 121) were filled with Fura 2, Bis Fura, or OGB1/CHR. This study also characterized a novel ratiometric technique using Oregon Green BAPTA-1 (OGB1) with coinjections of a new long-stokes-shift dye, Chromeo 494 (CHR). Fluorescent calcium transients were blocked with the nonspecific calcium channel blocker cadmium, or by the combination of ω-agatoxin-IVA, a specific P/Q-type calcium channel blocker, and ω-conotoxin-GVIA, a specific N-type calcium channel blocker. The calcium transients were evident in different dendrites (suggesting channels are present throughout the dendritic tree) along the sampled length without interruption (suggesting channels are evenly distributed), and appeared to represent a summation of oscillations present in the soma. We confirm that PPN calcium channel-mediated oscillations are due to P/Q- and N-type channels, and reveal that these channels are distributed along the dendrites of PPN cells.

Keywords: P/Q channels; arousal; calcium imaging.

Fig. 1.

Localization and morphology of PPN neurons. A: the PPN is a wedge-shaped cellular region overlapping the superior cerebellar peduncle (SCP). Hybrid brightfield and fluorescence image showing the location of a single recorded neuron in the PPN pars compacta dorsal to the SCP. B: wide-field fluorescence image of the same neuron identified with intracellular injection of Alexa Fluor 594. Note the multipolar shape with multiple primary dendrites. C: maximum projection confocal image of the same Alexa Fluor 594-filled neuron. D: wide-field image of a different Alexa Fluor-filled neuron with brain nitric oxide synthase immunofluorescence labeling (green) of cholinergic PPN neurons. Note the red/green (yellow nucleus) colocalization indicating that the recorded neuron was a cholinergic cell.

Fig. 2.

Depolarizing current ramps generated oscillations and measureable intracellular calcium transients. A: representative oscillations of a PPN neuron (black records), and in the presence of the nonspecific calcium channel blocker Cd²⁺ (red records in top and third rows) or specific calcium channel blockers AgA and CgTx (red records in second and fourth rows) recorded during 2-sec-long ramps. Note that calcium oscillations were eliminated by Cd²⁺, AgA, and CgTx, indicating fluorescence (C) was mediated by N-type and P/Q-type calcium channels. B: overlapping recordings comparing power spectrum amplitudes for oscillations present in A, before and after channel blockers. C: somatic fluorescent transients induced by the current ramp (black record) recorded simultaneously with the oscillations present in A. Note how both membrane oscillations and intracellular calcium fluorescence signals were eliminated after blockers were applied (red record). D: bar graph showing the average integrated somatic curve area for the fluorescence curves before Cd²⁺ [black bar: Fura 2, 51 ± 7 (340/380)*msec, n = 12; Bis Fura, 29 ± 3 (340/380)*msec, n = 12] and after Cd²⁺ [red bar: Fura 2, 8 ± 1 (340/380)*msec; Bis Fura, 7 ± 1 (340/380)*msec]. Fluorescence curves before AgA and CgTx [black bar: Fura 2, 36 ± 6 (340/380)*msec, n = 8; Bis Fura, 64 ± 12 (340/380)*msec, n = 8] and after AgA and CgTx [red bar: Fura 2, 12 ± 1 (340/380)*msec; Bis Fura, 10 ± 2 (340/380)*msec]. E: bar graph showing the average integrated proximal dendritic curve area before Cd²⁺ [black bar: Fura 2, 21 ± 3 (340/380)*msec, n = 14; Bis Fura, 39 ± 4 (340/380)*msec, n = 10] and after Cd²⁺ [red bar: Fura 2, 7 ± 1 (340/380)*msec; Bis Fura 2, 11 ± 1 (340/380)*msec]. Fluorescence curves before AgA and CgTx [black bar: Fura 2, 35 ± 1 (340/380)*msec, n = 5; Bis Fura, 28 ± 3 (340/380)*msec, n = 16] and after AgA and CgTx [red bar: Fura 2, 16 ± 2 (340/380)*msec; Bis Fura, 9 ± 1 (340/380)*msec].

Fig. 3.

N-type and P/Q-type calcium channel-mediated calcium oscillations in the PPN visualized with OGB1/CHR. The two rows above the horizontal line used both synaptic blockers (SB) and TTX; however, the lower row used only synaptic blockers. A: patch-clamp recorded calcium oscillations before (black record) and after (red record) specific and nonspecific calcium channel blockers. B: power spectra of records in A. C: fluorescence recordings acquired simultaneously with electrical recordings in A, with (red record) and without (black record) calcium channel blockers. D: bar graph showing the average integrated area under the curve for the fluorescence recordings before Cd²⁺ [black bar: SB only 105 ± 14 (OGB1/CHR)*msec, n = 7; with TTX 301 ± 81 (OGB1/CHR)*msec, n = 10] and after Cd²⁺ [red bar: SB only 5 ± 2 (OGB1/CHR)*msec; with TTX 43 ± 19 (OGB1/CHR)*msec]. Before AgA and CgTx [black bar: 137 ± 46 (OGB1/CHR)*msec, n = 10] and after [red bar: 12 ± 4 (OGB1/CHR)*msec]. E: bar graph showing the average integrated area under the curve for proximal dendritic fluorescence before Cd²⁺ [black bar: SB only 44 ± 7 (OGB1/CHR)*msec, n = 8; with TTX 100 ± 15 (OGB1/CHR)*msec, n = 21] and after Cd²⁺ [red bar: SB only 9 ± 2 (OGB1/CHR)*msec; with TTX 15 ± 5 (OGB1/CHR)*msec]. Before AgA and CgTx [black bar: 52 ± 8 (OGB1/CHR)*msec, n = 17] and after [red bar: 10 ± 1 (OGB1/CHR)*msec]. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Oscillations present in the electrical signal were also evident in the recorded dendritic calcium signal. A: records of elicited calcium oscillations recorded simultaneously with both electrical recording (black record) and high-speed fluorescence imaging (red, blue, and purple records). The oscillations in the somatic fluorescence recording (red record) closely matched those in the electrical record. The blue dendritic record also followed the electrical record, but with more variation. Note that fluorescence was mostly eliminated by the addition of Cd²⁺ in the purple record. B: power spectrum and cross-correlation graph of the electrical record (black record) and the somatic fluorescence record (red record). C: two dendritic branches (b1 green, b2 pink) originating from the proximal dendrite in A (blue record). Both branches were summed (b1+b2 orange record) to yield a signal closely matching the oscillations in the proximal dendrite (blue in A and D). D: image from the inverted scope of the recorded cell indicating fluorescence sampling locations (red square, soma; blue square, main dendritic trunk; pink and green squares, b1 and b2 dendritic branches).

Fig. 5.

Wide-field calcium imaging with OGB1. A: image from the peak of a calcium transient showing an increase in calcium levels. The increase was present continuously across the length of the dendrites. Inset: fluorescence zoom of the filled cell. B: AgA and CgTx eliminated all calcium transients. C: image from the peak of a calcium transient showing an increase in calcium levels. Inset: fluorescence zoom of the filled cell. D: AgA blocked P/Q-type channels and eliminated the majority of the fluorescence with some fluorescence remaining in the soma (N-type calcium channel-mediated). Note that the color map on the left of each image was scaled to match the peak fluorescence of each cell, and that A and B used different scales compared with C and D.