Changes in frequency and amplitude of PSCs produced by CAR application. “Inward” and “Outward” indicate cells that responded to CAR by an inward or an outward current, respectively. “Increase” and “Decrease” indicate cells that responded to CAR by an increase or a decrease in IPSC frequency, respectively. A. CAR-induced Change in the frequency of spontaneous EPSCs in the presence of GBZ and STR. B. Change in the amplitude of spontaneous EPSCs in the presence of GBZ and STR. C. Change in the frequency of spontaneous IPSCs in the presence of CNQX and APV. D. Change in the amplitude of spontaneous IPSCs in the presence of CNQX and APV. E. Change in the frequency of miniature EPSCs in the presence of TTX. F. Change in the amplitude of miniature EPSCs in the presence of TTX. *P < 0.05, **P < 0.01.

Modulation of spontaneous PSCs by CAR. A. Membrane current record (HP = −50 mV) of a SubCD neuron showing the effects of CAR on miniature EPSCs in the presence of TTX. Note the outward current and the decrease in miniature EPSC frequency during CAR application. B. Membrane current record (HP = −50 mV) of a SubCD neuron in the presence of GBZ and STR. CAR decreased the frequency of spontaneous EPSCs and induced an outward current in this cell. C. Voltage clamp recording (HP = 0 mV with QX-314, cesium, and BAPTA in the intracellular solution) showing the effect of CAR on spontaneous IPSCs in the presence of blockers of fast excitatory synaptic transmission (CNQX and APV). CAR produced a large increase in the frequency of IPSCs in these conditions.

Pharmacological characterization of evoked PSCs in the SubCD. A. Superimposed EPSCs of variable amplitude were elicited using constant intensity stimulation at intervals of 5 sec. B. Underlying evoked IPSCs were revealed during blockade of fast synaptic transmission with CNQX and APV. C. Evoked IPSCs were reduced in amplitude in the presence of GBZ. D. The application of both GBZ and STR was necessary to completely block evoked IPSCs. E. The evoked EPSCs partially recovered after 25 min of drug washout. Ten superimposed records of evoked PSCs are shown in each panel. The stimulus artifact was truncated for clarity. These results established that electrical stimulation of the SubCD induced both excitation and inhibition in SubCD neurons consistent with the presence of glutamatergic and GABAergic inputs to these cells. The CAR effects on these evoked responses could be mimicked by endogenously released acetylcholine (see Figures 5-7).

Inward currents induced in SubCD neurons. A. Membrane current records (HP = −50 mV) revealed that CAR induced-inward currents persisted in the presence of the M1 receptor antagonist PRZ. Additional application of MEC was necessary to completely block the CAR-induced inward current. B. Voltage ramps revealed a parallel shift in the current-voltage record after TTX, before CAR (1), at the peak of the CAR effect (2) and after washout (3), that rectified above −45 mV. Inset shows the difference between CAR and before and after records. C. Averages with corresponding SEMs of CAR-induced inward current in the presence of TTX and PRZ revealed that a similar current was induced across all cells tested (n = 6). D. Membrane current record (HP = −50 mV) showed that the CAR induced-inward current also persisted in the presence of TTX and MEC but was blocked with the addition of PRZ. E. Current-voltage records revealed a CAR-induced inward shift (2) that partially returned to control (1) after washout (3) of drugs. Inset. Subtractions of control and CAR records revealed an inward current throughout the voltage range tested. F. Average of CAR-induced current-voltage records with corresponding SEMs in the presence of TTX and MEC (n = 5). Note the continued parallel shift across all cells tested. These results suggest that CAR excites some SubCD neurons via M1 muscarinic and nicotinic receptors.

Location of recorded SubCD neurons. A. Coronal section of a rat pup brain (−9.3 mm posterior to bregma equivalent). Two recorded neurons are evident within the dashed area. Calibration bar is 500 μm. B. Lucifer yellow labeling of the neurons in A. C. Traced diagrams displaying the location of the SubCD at the anatomical equivalents to adult slices in mm posterior to the bregma. Marked squares within the SubCD designate the location of all of the recorded neurons. These diagrams include the region sampled, which consisted of a ∼500 μm diameter sphere located immediately anterior to the 7th nerve. We targeted the small region that others have determined will induce REM sleep signs when CAR is injected there.⁷ This region did not contain nitric oxide synthase (NOS)-positive neurons, NOS is a selective label for mesopontine cholinergic neurons, suggesting that the neurons sampled were not part of the PPN/LDT cell groups. 7^th N = 7th nerve, DTg = dorsal tegmental nucleus, Me5 = mesencephalic 5 nucleus, MO5 = motor 5 nucleus, mlf = medial longitudinal fasciculus, py = pyramidal tract, SCP = superior cerebellar peduncle, SubCD = dorsal subcoeruleus.

Outward currents induced by CAR in SubCD neurons. A. An outward current could be induced in SubCD neurons in the presence of TTX. Application of the M2 receptor antagonist, MTO, blocked the effect of a subsequent CAR application. B. A voltage ramp protocol (−110 to −40 mV) revealed the current-voltage relationship in different conditions: control (1), peak CAR effect (2), and washout (3). There was an almost complete recovery to control state shortly after the cessation of the outward current. C. The CAR-induced current was obtained by subtracting the current-voltage record induced at the peak CAR effect (2) minus that obtained in control (1). This relationship revealed an outward current that reversed around −78 mV (near the potassium equilibrium when corrected for junction potential). D. Prior application of MTO (4) was able to completely block the CAR-induced outward current (5). E. The CAR-induced outward current could also be blocked by the application of the potassium channel blocker BaCl₂. F. Current-voltage relationship was changed during CAR application and recovered after washout. Inset depicts the current-voltage record of the CAR-induced current by subtracting record (2) from (1) as in C. G. There was no change in current-voltage record after the application of BaCl₂ (4, 5). H. Average of current induced by CAR minus control in TTX with corresponding SEM (n = 8). Note the reversal potential of −78.1 ± 0.6 mV. These results suggest that CAR inhibits some SubCD neurons via a M2 muscarinic receptor.

SubCD responses following electrical stimulation. A. Average of 5 evoked EPSCs in GBZ and STR control (dashed) and after addition of CAR (solid). Note records were shifted to the same baseline to allow amplitude comparison. B. The PPF ratio increased during application of CAR. CAR preferentially decreased the first evoked EPSC, suggesting a presynaptic effect. The first CAR-evoked EPSC was scaled to match the first control EPSC. Vertical calibration is 20 pA and 5.25 pA for the control and CAR records, respectively. C. The PPR was significantly increased (*P < 0.05) from control by the application of CAR in all cells tested (n = 22). This increase in the PPR implies that CAR preferentially decreased the amplitude of the first evoked EPSC. These data suggest that CAR may modulate the probability of transmitter release by activating cholinergic receptors on presynaptic glutamatergic terminals. The half-width duration did not change significantly between groups (n = 22). D. In the presence of GBZ and STR, CAR induced an outward current (top record) in a cell recorded in voltage clamp at a HP = −50 mV. Lower scatter plots show the amplitude of EPSCs evoked by electrical stimulation every 5 sec and a 25-point smoothed average of the EPSC amplitude. The effects of CAR on the membrane current and the evoked EPSCs persisted in the presence of the nicotinic receptor antagonist, MEC. E. In another SubCD cell, CAR induced an outward current (top record) and decreased the evoked EPSCs (bottom plot), and these effects were blocked by ATR (10 μM). F. In the presence of GBZ, STR, and MEC extracellulary, and GDP-β-S in the intracellular solution, CAR decreased the evoked EPSC amplitude without changing the input resistance or holding current.