Study objectives: Recent evidence suggests that certain
anesthetics decrease electrical coupling while the stimulant modafinil
appears to increase electrical coupling. Meijun is investigating
the potential role of electrical coupling in the cholinergic arm of the
reticular activating system (RAS), the pedunculopontine nucleus (PPN),
which has been implicated in the modulation of arousal via ascending
cholinergic activation of intralaminar thalamus and descending
activation of pontine regions to generate some of the signs of rapid eye
movement (REM) sleep.
Design: Meijun uses 6-30 day 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 PPN. Recordings in pairs of PPN revealed that some of these
were electrically coupled with coupling coefficients ~2%. After
blockade of fast synaptic transmission, the cholinergic agonist
carbachol (CAR) was found to induce rhythmic activity in PPN neurons, an
effect eliminated by the gap junction blockers carbenoxolone or
mefloquine. The stimulant modafinil was found to decrease
resistance in PPN neurons after fast synaptic blockade, indicating that
the effect may be due to increased coupling.
Conclusions: The finding of electrical
coupling in specific RAS 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.
Modulation of electrical couplings in the PPN.
A. Whole cell patch-clamp recordings from a pair of electrical
coupled PPN neurons under voltage-clamp. Hyperpolarizing pulses
(top two records show current pulses) injected to one cell induced a
current in the other cell in the presence of 1uM TTX to block sodium
channels and thus action potential generation. The coupling ratio
(CR) was calculated using the current amplitude in the injected cell
divided by the response current in the coupled cell. For this
pair, the CR 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 3 min
superfusion of TTX.
B. No significant activity was present in this cell during
superfusion of fast inhibitory and excitatory synaptic blockers
[CAGM=CNQX 10uM, APV 10uM, Gabazine 10uM and mecamylamine (MEC) 10uM]
(top record). Carbachol (CAR, 50 uM) induced oscillations in this
PPN cell in the presence of fast inhibitory and excitatory synaptic
blockers (CAGM) (second record), which was blocked by 300uM
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 min 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 uM) in the presence of fast synaptic blockers (CAGM).
The decrease in resistance was partially reversed by the putative gap
junction blockers mefloquine (MEF, 25 uM). 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 the current
was required to compensate for the voltage change in the presence of
MOD, 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 msec to test
the compensatory current. A 1000 msec ramp from -105 mV to -35 mV
was then applied. F. The membrane resistance change during
50 min recording from the same cell shown in D. The bars indicate
the period when drugs were applied (black: 1 uM TTX + 10 uM CAGM;
maroon: 150 uM MOD + TTX + CAGM; green: 25