Electrical coupling: novel mechanism for sleep-wake control

Abstract

Study objectives: Recent evidence suggests that certain anesthetic agents decrease electrical coupling, whereas the stimulant modafinil appears to increase electrical coupling. We investigated the potential role of electrical coupling in 2 reticular activating system sites, the subcoeruleus nucleus and in the pedunculopontine nucleus, which has been implicated in the modulation of arousal via ascending cholinergic activation of intralaminar thalamus and descending activation of the subcoeruleus nucleus to generate some of the signs of rapid eye movement sleep.

Design: We used 6- to 30-day-old rat pups to obtain brainstem slices to perform whole-cell patch-clamp recordings.

Measurements and results: Recordings from single cells revealed the presence of spikelets, manifestations of action potentials in coupled cells, and of dye coupling of neurons in the pedunculopontine nucleus. Recordings in pairs of pedunculopontine nucleus and subcoeruleus nucleus neurons revealed that some of these were electrically coupled with coupling coefficients of approximately 2%. After blockade of fast synaptic transmission, the cholinergic agonist carbachol was found to induce rhythmic activity in pedunculopontine nucleus and subcoeruleus nucleus neurons, an effect eliminated by the gap junction blockers carbenoxolone or mefloquine. The stimulant modafinil was found to decrease resistance in neurons in the pedunculopontine nucleus and subcoeruleus nucleus after fast synaptic blockade, indicating that the effect may be due to increased coupling.

Conclusions: The finding of electrical coupling in specific reticular activating system cell groups supports the concept that this underlying process behind specific neurotransmitter interactions modulates ensemble activity across cell populations to promote changes in sleep-wake state.

Figure 1

Modulation of electrical couplings in the pedunculopontine nucleus (PPN). A. Whole-cell patch-clamp recordings from a pair of electrical coupled PPN neurons under voltage clamp. Hyperpolarizing pulses (top 2 records show current pulses) injected to 1 cell induced a current in the other cell in the presence of 1 μM tetrodrotoxin (TTX) to block sodium channels and thus action potential generation. The coupling ratio was calculated using the current amplitude in the injected cell divided by the response current in the coupled cell. For this pair, the coupling ratio of cell 1 to cell 2 was 1.6%, and of cell 2 to cell 1 was 2%. The gray line represents the average of 20 sweeps after a 3-minute superfusion of TTX. B. No significant activity was present in this cell during superfusion of fast inhibitory and excitatory synaptic blockers (CAGM = 6-cyano-7-nitroquinoxaline-2, 3-dione [CNQX] 10 μM, (±)-2-amino-5-phosphopentanoic acid [APV] 10 μM, gabazine 10 μM, and mecamylamine 10 μM) (top record). Carbachol (CAR, 50 μM) induced oscillations in this PPN cell in the presence of fast inhibitory and excitatory synaptic blockers (CAGM) (second record), which was blocked by 300 μM carbenexolone (CBX), a putative gap junction blocker (bottom record). C. Power spectrum histogram of the oscillations induced by fast synaptic blockers (no discernible synchronization), CAR in the presence of fast synaptic blockers (theta frequency oscillations), and their blockade by CBX in the same cell shown in B. Each histogram was obtained from a 1-minute recording sample. D. An example of a PPN cell whose input resistance was decreased by fast synaptic blockers (CAGM), then decreased further by the superfusion of modafinil (MOD, 150 μM) in the presence of fast synaptic blockers (CAGM). The decrease in resistance was partially reversed by the putative gap junction blockers mefloquine (MEF, 25 μM). The cell was recorded under voltage-clamp mode. A ramp protocol was applied in order to test the change of membrane resistance, such that a higher current was required to compensate for the voltage change in the presence of modafinil, indicating a decrease in resistance. E. The ramp protocol used in the recording shown in D. The voltage was held at −60 mV during baseline and then was held at −105 mV for 500 milliseconds. to test the compensatory current. A 1000-millisecond ramp from −105 mV to −35 mV was then applied. F. The membrane resistance change during 50-minute recording from the same cell shown in D. The bars indicate the period when drugs were applied (black: 1 μM TTX + 10 μM CAGM; maroon: 150 μM MOD + TTX + CAGM; green: 25 μM MEF + TTX + CAGM). The resistance was calculated by dividing voltage change by the compensatory current.

Figure 2

Synchronous activity in 2 pedunculopontine nucleus (PPN) cells, effects of carbachol. Data in this figure were obtained from the same paired recording. A. Simultaneous extracellular “loose patch” recordings (1-second samples) were made from 2 cells (red and blue records) in the PPN under different experimental conditions. The occurrence of action potentials in both cells was correlated somewhat in the control, untreated condition (top record), which increased after superfusion with carbachol, and persisted in the presence of fast synaptic blockers (6-cyano-7-nitroquinoxaline-2, 3-dione [CNQX] 10 μM + (±)-2-amino-5-phosphopentanoic acid [APV], 50 μM + gabazine, 10 μM; second record). When carbachol was administered in the presence of these blockers, it induced much higher cross-correlation (third record), but the effect was blocked by adding carbenoxolone (300 μM, bottom record), which desynchronized the cells. B. Photograph of the sagittal slice (2× objective) from a 10-day rat showing the location of the PPN where the dual recording was performed. Inferior colliculus is at top right, basis pontis at bottom left. C. Sliding 3-D cross-correlogram of action potentials indicated significant synchronous activity throughout 50 minutes of recording. Note that the cross-correlation coefficient peak was near zero time lag and the significant correlation window was around 25 milliseconds (i.e.. the occurrence of action potentials in both cells tended to coincide within a 25-msec interval). Each cross-correlogram was obtained using 2 minutes of data with 1-minute intervals between consecutive cross-correlograms. D. Same cross-correlogram as in C but the 3-D graph is tilted in order to view the effect of carbachol on the peaks of coefficient of correlation. The neurons were somewhat correlated at the start, and their correlation increased during carbachol application at 3-7 and 15-18 minutes. The first 2 applications of carbachol were in control aCSF and the third was made in the presence of CNQX+APV+gabazine as in E. Note the sharp increases in correlation with carbachol, especially after fast synaptic blockade. E. Upper panel represents a frequency histogram of both cells (red and blue records) throughout the 50 minutes of experiment. Carbachol produced multiphasic responses on the first cell (red) and mainly inhibition on the second cell (blue). Lower panel is a scatter cross-correlation. Each dot represents the interval between an action potential in cell #1 and a given action potential in cell #2 during a time window of + 125 milliseconds. The color-coded superimposed matrix represents the density of dots and is equivalent to a sliding cross-correlogram. Note that the cross-correlation peak increased in magnitude after each application of carbachol (dark blue indicates higher coefficient of correlation according to the color-coded scale on right). Note also that the peak of correlation (black horizontal line) remained close to center (represented by the horizontal red line) except after application of carbenoxolone, which gradually reduced the activity of the cells and desynchronized them (at 47-50 min). The persistence of a significant cross-correlation coefficient with carbachol in the presence of synaptic blockers and its reduction by carbenoxolone suggest that these cells were coupled by gap junctions.

Figure 3

A. Spikelets and excitatory postsynaptic currents (EPSC) in the subcoeruleus. Top row, voltage clamp record (holding potential, HP = −50 mV) with spontaneous EPSC. Second row, the same neuron exhibiting spikelets after fast synaptic transmission was blocked (CNQX+APV+gabazine). Third row, overlays (20 sweeps) and averages (gray line) of spontaneous EPSC (left) and spikelets (right) are shown. Note the distinct monophasic shape of the EPSC and the biphasic shape of the spikelets, presumed to represent action potentials filtered by high-resistance gap junctions. B. Electrical coupling in the subcoeruleus. During application of tetrodotoxin (TTX, 1 μM), hyperpolarizing current was injected in simultaneously recorded neurons revealing direct electrotonic coupling. A hyperpolarizing step delivered to cell #2, lower record, induced an inward current in cell #1, upper record. Conversely, a hyperpolarizing pulse delivered to cell #1 induced an inward current in cell #2. The coupling coefficient, the response amplitude in the coupled cell divided by the amplitude in the injected cell, for these cells was ~2%. C. Carbachol-induced oscillations in subcoeruleus neurons. Simultaneously recorded cells (left side, top records) show little tendency to fire at particular frequencies as evidenced by the power spectrum for each cell (right side). During carbachol superfusion (left side, bottom records), however, both cells showed increased frequency of inhibitory postsynaptic currents (IPSC) as evidenced by the power spectrum for each cell (right side). Note that both cells showed oscillations in the theta frequency but at 8 Hz for cell #1 and 6 Hz for cell #2. On occasion, cell #2 showed doublet IPSC (bottom left), indicating inputs from multiple inhibitory neurons. The inset at bottom right shows the patched neurons fluorescing due to Lucifer yellow infusion from the recording pipettes.

Figure 4

A. Location of recorded subcoeruleus neurons. The background is a fluorescence photomicrograph of a slice processed for neurobiotin immunocytochemistry showing the locations of the recorded cells in the subcoeruleus pars D (SubCD, bottom left), the central gray (CtG) and the dorsal tegmental nucleus (DTg) (40X). The inset is a 400X confocal image of the same subcoeruleus cells (calibration bar 50 um). B. IV plot of a subcoeruleus cell. Overlayed voltage steps in voltage clamp revealed a putative low threshold spikes (LTS) current and an outward Ia current in this subcoeruleus cell. C. Sample recordings revealed a decrease in resistance during modafinil (MOD) application (red record at time marked by red arrow in D), compared to the control condition (black record at time marked by black arrow in D). D. Graph of the changes in resistance during modafinil exposure. During application of modafinil, input resistance in the cell shown in B changed from 550 MΩ (black arrow) to 440 MΩ (red arrow), followed by a partial increase during application of mefloquine (MEF). These values are expressed as a ratio of initial resistance.

Figure 5

Connexin 36 (Cx36) protein levels in punches of pedunculopontine nucleus (PPN) and subcoeruleus (SubC) at 10 and 30 days (4 rats, 8 bilateral punches of each nucleus pooled/age). Top row shows images of 400-μm sagittal slices punched (1 mm) in the SubC (left, note hole ventral to the locus coeruleus around the seventh nerve), and the posterior PPN (right, note hole ventral to the colliculi). The gels shown are (top row) Cx36 protein from each region in 10-day and 30-day slices and (bottom row) β-tubulin control for protein loading. The bottom graph shows Cx36 protein levels normalized to β-tubulin at 10-days and 30-days for SubC (left 2 bars), and PPN (right 2 bars). Note that the amount of protein (a) decreased by about 75% between 10 and 30 days, and (b) both regions showed a decrease, but (c) the absolute amount of Cx36 was higher in PPN and lower in SubC, paralleling the percentage of cells found to manifest spikelets in each nucleus, as described above.