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INTRODUCTION |
The state of active sleep is characterized by the appearance of tonic physiological events such as muscle atonia and electroencephalographic (EEG) desynchronization, as well as phasic events such as rapid eye movements (REM) and ponto-geniculo-occipital (PGO) waves. Muscle atonia results from tonic postsynaptic inhibition of motoneurons that is mediated by an active sleep-specific motor inhibitory system (Chase et al. 1989
).
During active sleep, in addition to sustained hyperpolarization of the membrane of motoneurons, a phasic exacerbation of inhibition of brain stem and spinal cord motoneurons occurs in conjunction with PGO waves (López-Rodríguez et al. 1992
; Pedroarena et al. 1994
). These phasic inhibitory events occurring in motoneurons have been called PGO-inhibitory postsynaptic potentials (IPSPs) (López-Rodríguez et al. 1992
; Pedroarena et al. 1994
). It was concluded that these PGO-IPSPs result from phasic enhancement of activity of the same motor inhibitory system that is responsible for the muscle atonia of active sleep (López-Rodríguez et al. 1990
).
The function of PGO-IPSPs occurring during active sleep is unknown; however, they may serve to preserve the state of active sleep from PGO-related disruptive influences. In this regard, Morrison and Bowker (1975)
proposed that PGO waves are a central component of a startle response produced by internally generated signals. They further refined their hypothesis to suggest that PGO waves during any behavioral state reflect central activation of alerting mechanisms that, during wakefulness, might be accompanied by orienting responses and/or acoustic startle responses (Ball et al. 1989
; Bowker and Morrison 1976
; Sanford et al. 1992b
). In fact, it has been demonstrated that PGO waves are accompanied by an increase in motor activity during states other than active sleep (see DISCUSSION). The PGO-IPSPs of active sleep may function to prevent motor excitation and movements from occurring as a result of a concomitant active motor excitatory drive (Kohlmeier et al. 1996
, 1997
).
If the spontaneous production of a PGO wave during active sleep reflects internal activation of alerting mechanisms, then an external stimulus capable of activating not only alerting mechanisms but also the startle network during active sleep could be associated with a phasic increase in inhibitory drive on motoneurons. The presence of PGO waves would be presumptive evidence of activation of mechanisms involved in CNS alerting.
We previously reported that somatosensory and auditory stimuli induce IPSPs in masseter motoneurons in
-chloralose-anesthetized cats after the injection of carbachol into the pontine reticular formation; IPSPs were never elicited in motoneurons before the induction of the carbachol state (Kohlmeier et al. 1994
, 1995
) We have reported data that suggest that IPSPs elicited by sensory stimuli result from phasic activation of the same motor inhibitory system that mediates atonia during spontaneously occurring active sleep (Kohlmeier et al. 1997
).
Therefore, we decided to investigate whether the sensory stimuli-induced increase in inhibitory drive on motoneurons during activation of the active sleep-specific motor inhibitory system was associated with activation of circuitry involved in mechanisms of alerting, as evidenced by the production of PGO waves. We have presented evidence that suggests that the active sleep-specific motor inhibitory system can be activated by injection of carbachol into the pontine reticular formation in cats anesthetized with
-chloralose (Kohlmeier et al. 1996
). Accordingly, the intracellular responses of masseter motoneurons and activity in the lateral geniculate nuclei (LGNs) in conjunction with the presentation of two types of sensory stimuli, somatosensory and auditory, were examined before and after the induction of motor atonia by injection of carbachol into the nucleus pontis oralis (NPO) of
-chloralose-anesthetized cats.
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METHODS |
Surgical procedures
The present experiments were performed on eight chronic cats that were prepared according to procedures that have been described previously (Chase et al. 1980
). Briefly, each cat was anesthetized with sodium pentobarbital (40 mg/kg); screw electrodes were inserted in the frontal bone over the sigmoid gyri to monitor electroencephalographic (EEG) activity and into the orbital portion of the frontal bone to record eye movements. These electrodes were soldered to a connector that then was cemented to the calvarium with acrylic resin. Two hollow plastic tubes with laterally directed openings also were placed within the acrylic resin mound. The tubes were designed to receive four calibrated steel bars that were fixed during experimental sessions to the stereotaxic apparatus by a Kopf 880 chronic head-holder attachment; this allowed head restraint during recording sessions (Chase et al. 1980
).
After a 2-wk period of recovery from the preceding procedures, permanent electrodes were implanted in both LGN during spontaneously occurring episodes of active sleep to optimize placement of the electrodes that were used to record PGO waves (for details, see López-Rodríguez et al. 1992
). In addition, by performing the LGN electrode implant in the unanesthetized animal, we could compare the waveforms of the spontaneously occurring PGO waves with those recorded later with the same chronic electrode when the animal was anesthetized with
-chloralose.
During each experimental session, pin electrodes were placed acutely into the dorsal neck musculature to monitor electromyographic (EMG) activity.
Intracellular recording
Electrodes used for intracellular recording from motoneurons were glass micropipettes filled with 2 M K-citrate (tip resistances: 10-15 M
). A reference Ag-AgCl electrode was placed subcutaneously in the neck. Fine-wire electrodes were inserted into the belly of the masseter muscle to stimulate nerve terminals for the intracellularly derived antidromic identification of masseter motoneurons (Chandler et al. 1980
).
A high-input impedance Dagen (Model 8100) amplifier was used to record membrane potential activity at a low (×10) and high gain (×100). These data, as well as recordings of the EEG, EMG, electrooculogram, and activity in both LGN, were stored for subsequent analysis on magnetic tape.
-Chloralose administration
In previous studies it has been reported that animals anesthetized with
-chloralose exhibit hyperexcitability and associated movements (Balis and Monroe 1964
; Hanriot and Gautier 1896
, 1897
). However, we found that when a solution (2%) of
-chloralose was filtered and injected as a bolus at a dose of 40 mg/kg through a catheter inserted in the cephalic vein, cats are anesthetized deeply but are not hyperreactive and do not exhibit spontaneous movements (Kohlmeier et al. 1996
). Therefore, filtered
-chloralose was administered during experimentation to maintain the cat under deep anesthesia, which was determined after a pinch of the cat's forepaw pads by the absence of a pupillary response and the lack of reflex withdrawal of the leg.
Carbachol administration
Carbachol was applied by microiontophoresis into a region of the NPO at the stereotaxic coordinates P3-P3.3, L2-L2.5, H-4 to H-4.5. The most efficacious site for the injection of carbachol was determined in each cat before experimental sessions during the nonanesthetized state (i.e., the site with the shortest latency to carbachol-induced motor atonia, 3-5 min). Five-barrel glass micropipette assemblies, with tips broken under a microscope to a total diameter of 30-40 µm, were used for microiontophoretic drug ejection. Four side barrels were filled with carbachol (200 mM). The central barrel of the drug ejection assembly was filled with NaCl (2 M) for automatic current balancing. Microiontophoretic currents of 300-500 nA were used. Injections of carbachol were carried out for a period of 3 min. Each carbachol injection was performed in the NPO contralateral to the motor five (V) nucleus in which intracellular recordings of motoneurons were obtained.
Histology
A metal microelectrode was lowered into the site of the injection of carbachol. This site was marked by a DC anodal current of 70 µA. The animal then was administered a lethal dose of pentobarbital sodium (Nembutal) and immediately perfused with saline followed by 10% formaldehyde with 2% potassium ferrocyanide in saline to obtain a Prussian blue reaction at the brain stem site where DC current was applied. To determine the placement of the LGN electrodes, the brain was removed with the electrodes intact; the location of the tips of the electrodes was determined histologically in Nissl-stained sections.
Data analysis
All electrophysiological data were obtained from intracellular recordings of identified masseter motoneurons with antidromic spike potentials
65 mV. Data from these motoneurons were converted to a digital format (50 µs/bin) with a microcomputer and the following membrane properties were analyzed with the procedures presented below.
ANTIDROMIC ACTION POTENTIAL.
The amplitude of the action potential was determined by measuring the difference in the DC potential recorded at the base and peak of the action potential.
MEMBRANE POTENTIAL.
Membrane resting potential values were determined by measuring the difference between the DC potential recorded intracellularly and that measured immediately after the microelectrode was withdrawn from the cell.
SYNAPTIC ACTIVITY.
The presence, frequency, and waveform characteristics of excitatory and/or inhibitory potentials were examined using specially devised software, which is described in Morales et al. (1985)
and López-Rodríguez et al. (1992)
.
Sensory stimuli
Excitation of two sensory pathways was conducted in this study: somatosensory (nerve stimulation) and auditory. Three types of studies with three different sets of masseter motoneurons were conducted to examine the response of the LGN and the membrane potential of these motoneurons. Initially, we wished to determine if the intensity of sensory stimuli that elicited a response in the membrane of masseter motoneurons also elicited a response from the LGN. If this were found to be so, we wished to determine the relative thresholds of the response in motoneurons and the LGN. After this, we wished to examine the response of the membrane of motoneurons and the LGN to suprathreshold intensities of stimulation.
Accordingly, in the first set of studies, the intensity of the stimulus was set at a fixed level (see below) at which it had been demonstrated previously that a synaptic response was elicited in motoneurons after the injection of carbachol. For the second set of experiments, in which the threshold of the response of the membrane potential to these two types of stimulation was examined, the intensity of stimulation was reduced from that in the first set of experiments. For the third set of studies, in which the graded nature of the responses in the LGN and masseter motoneurons was examined, the intensity of the sensory stimuli was increased sequentially and was always of a greater intensity than that used in the first set of experiments.
Somatosensory stimulus
Sciatic nerve stimulation was performed via a nerve cuff that was implanted around the sciatic nerve. During experimental trials, after a masseter motoneuron was impaled with a micropipette, the sciatic nerve was stimulated between 10 and 14 times, and the synaptic responses in masseter motoneuron and the electrical potentials in the LGN were averaged. The frequency of stimulation of the sciatic nerve was one stimulus every 30 s.
For the first set of experiments, the sciatic stimulus was set at an intensity that elicited a slight twitch of the leg muscle before the injection of carbachol. Preliminary data suggest that this intensity of stimulation excites not only group I fibers but also cutaneous afferents. For the second set of experiments, the intensity of stimulation of the sciatic nerve was reduced to determine the threshold intensity of a synaptic response in each motoneuron and that necessary for the elicitation of a PGO wave. The minimum stimulus intensity required to elicit a response in 50% of the trials was defined as threshold. For the third set of experiments, the intensity of stimulation was further increased
25% from that utilized in the first set of experiments.
Auditory stimulus
The auditory stimuli used in this study were clicks, 1.5 ms in duration that were presented every 30 s. A Grass AM 8 audio monitor and a Grass S-88 stimulator were used to produce the auditory stimuli. During impalement of a masseter motoneuron, between 10 and 14 clicks were presented to the cat and the responses recorded in the LGN, and masseter motoneurons were averaged. The frequency of the auditory stimulus was one click every 30 s. The intensity of the stimulus was monitored with a sound level meter placed next to the ear of the cat closest to the source of the stimulus. For the first series of experiments, the intensity of the stimulus was adjusted to 95 dB. It should be noted that clicks of 95 dB have been demonstrated extensively to elicit not only alerting mechanisms but also the acoustic startle response in cats (Morrison and Bowker 1976; Wu 1989). For the second set of experiments, the intensity of the stimulus was reduced to determine the threshold intensity for each motoneuron and for the evoked response in the LGN. The minimum stimulus intensity required to elicit a response in 50% of the trials was defined as threshold. For the third sets of experiments, the intensity of the stimulus was increased
100 dB.
Analysis of LGN activity
For the analyses of the effects of stimulation of the sciatic nerve and clicks in eliciting wave activity in the LGN and in motoneurons, both contra- and ipsilateral extracellular LGN activity and the intracellularly recorded membrane potential activity (high gain) were digitized (50 µs/bin), and 1,000-ms periods of LGN activity and the membrane potential were analyzed. In accord with previous publications, PGO waves were separated into three categories
isolated waves, double waves, and clusters of waves (Laurent et al. 1974
; Pivik et al. 1982
). In this study only single waves, which are isolated PGO waves that are not preceded or followed within 300 ms by another PGO wave, were analyzed. Isolated PGO waves that appear almost simultaneously in both lateral geniculate nuclei also were classified as primary and secondary (Laurent et al. 1974
; Nelson et al. 1983
). The wave in the LGN with the largest amplitude was called the primary PGO wave; the smaller wave, which also arose slightly later than the primary wave, was called the secondary PGO wave. The period of time elapsing from the foot of the primary PGO wave to the onset of the IPSP was determined. Data were obtained from both LGNs regardless of whether or not the primary PGO wave was recorded in the LGN contra- or ipsilateral to the masseter motoneuron pool from which intracellular recordings were obtained.
Statistics
Statistical levels of significance were evaluated using the two-tailed Student's t-test (P < 0.05) and the Mann Whitney U-test.
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RESULTS |
Eight chronically implanted cats were used for the analyses detailed in this report. A total of 20 antidromically identified masseter motoneurons were recorded before and 102 after the injection of carbachol into the NPO. The latency to carbachol-induced motor atonia was 3.3 ± 0.3 min (mean ± SE).
Before the induction of motor atonia, stimulation of the sciatic nerve at an intensity that elicited a slight twitch of the leg and clicks of 95 dB failed to elicit a carbachol-dependent inhibitory response in masseter motoneurons (for detail, see Kohlmeier et al. 1994
, 1995
). These stimuli did, however, elicit PGO waves. Figures 1A and 2A are illustrations of PGO waves and the corresponding synaptic activity in motoneurons elicited by stimulation of the sciatic nerve and clicks before the injection of carbachol.

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| FIG. 1.
A: Ponto-geniculo-occipital (PGO) wave recorded in the lateral geniculate nuclei (LGN) and the corresponding synaptic activity in a masseter motoneuron elicited by stimulation of the sciatic nerve before carbachol injection in the nucleus pontis oralis (NPO). B: PGO wave and the simultaneously occurring membrane potential activity elicited in a masseter motoneuron by stimulation of the sciatic nerve after the injection of carbachol. Each trace represents the average of 14 consecutive responses to stimulation of the sciatic nerve. Stimulation of the sciatic nerve elicited PGO waves before and after the injection of carbachol. During carbachol-induced motor inhibition, stimulation of the sciatic nerve elicited PGO waves and inhibitory postsynaptic potentials (IPSPs) in masseter motoneurons. Similar IPSPs were never elicited by stimulation of the sciatic nerve before carbachol-induced motor atonia. Spontaneously occurring PGO waves and simultaneously occurring membrane potential activity recorded in the same masseter motoneuron as the trace illustrated in B during carbachol-induced motor inhibition (C). Each trace in C represents the average of 67 spontaneously occurring PGO waves and the corresponding activity in the membrane. Changes in motoneuron membrane potential that were present in conjunction with spontaneously occurring PGO waves during carbachol-induced motor inhibition were the PGO-IPSPs. The sciatic nerve stimulation-elicited IPSP demonstrated a latency from the foot of the elicited PGO that was similar to that of the spontaneously occurring PGO-IPSP (Kohlmeier et al. 1996 ; Pedroarena et al. 1994 ).
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| FIG. 2.
A: PGO wave recorded in the LGN and the corresponding activity in the membrane potential of a masseter motoneuron elicited by auditory stimuli (95 dB clicks) before carbachol injection. B: PGO wave and the simultaneously occurring membrane potential activity elicited in a masseter motoneuron by clicks after the injection of carbachol. Each trace represents the average of 12 consecutive responses to clicks. Note that clicks elicited PGO waves before and after the injection of carbachol. Clicks elicited IPSPs in masseter motoneurons during carbachol-induced motor inhibition; similar IPSPs never were elicited by clicks before carbachol injection. Spontaneously occurring PGO waves and simultaneously occurring membrane potential activity recorded in the same masseter motoneuron as in the traces shown in B during carbachol-induced motor inhibition (PGO-IPSP). Each trace in C represents the average of 85 spontaneously occurring PGO waves and the corresponding activity in the membrane. The click-elicited IPSP demonstrated a latency from the foot of the elicited PGO that was similar to that of the spontaneous PGO-IPSP.
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A total of 65 motoneurons were recorded in the first set of experiments after the induction of carbachol-induced motor atonia. The synaptic response elicited by stimulation of the sciatic nerve and clicks was recorded in 28 and 37 motoneurons, respectively. Stimulation of the sciatic nerve at an intensity that elicited a slight muscle twitch before carbachol consistently elicited a PGO wave after the injection of carbachol. This intensity of stimulation elicited IPSPs in masseter motoneurons (for details, see Kohlmeier et al. 1995
). Figure 1B is an example of PGO waves and the corresponding synaptic response in a masseter motoneuron elicited by stimulation of the sciatic nerve after the administration of carbachol (elicited PGO-IPSP). It is interesting to note that stimulation of the sciatic nerve can elicit primary PGO waves in both the ipsi- or contralateral LGN in different trials. When sciatic stimulation-elicited primary PGO waves appeared in the ipsilateral LGN, the latency from the onset of the stimulus to the foot of the PGO wave was 21.6 ± 1.2 ms and the latency to the IPSP was 29.6 ± 0.7 ms. The time elapsed between the foot of the PGO and the IPSP was 8.0 ± 0.1 ms. There was no significant difference in latencies when the primary PGO wave appeared in the contralateral LGN (latency to PGO, 21.7 ± 1.4 ms; latency to IPSP, 30.5 ± 0.6 ms; latency from the foot of the PGO to the IPSP, 8.8 ± 0.2 ms; P > 0.05).
The presentation of 95 dB clicks also consistently elicited PGO waves after the injection of carbachol. Clicks at this intensity also elicited IPSPs in masseter motoneurons (for detail, see Kohlmeier et al. 1994
). Figure 2B is an example of a PGO wave and the corresponding synaptic response in a masseter motoneuron elicited by clicks after the administration of carbachol (elicited PGO-IPSP). When the click-elicited primary PGO wave appeared in the ipsilateral LGN, the latency from the onset of the click to the foot of the PGO wave was 21.2 ± 0.6 ms, the latency to the IPSP was 30.0 ± 0.3 ms, and the time elapsed between the foot of the PGO wave to the IPSP was 8.8 ± 0.3 ms. There was also no significant difference in these latencies when the primary PGO wave appeared in the contralateral LGN (latency to PGO, 21.5 ± 1.0 ms; latency to IPSP, 31.8 ± 0.4 ms; latency from the foot of the PGO wave to the IPSP, 10.3 ± 0.2 ms; P > 0.05).
As previously reported, during the carbachol-induced state, spontaneously occurring IPSPs in motoneurons are associated with PGO waves (PGO-IPSPs) (Kohlmeier et al. 1996
). In 10 motoneurons the click-elicited PGO-IPSP, the sciatic nerve stimulation elicited PGO-IPSP, and spontaneous PGO-IPSPs were recorded. A statistical difference was not found in the mean elapsed time between the foot of the primary ipsilateral PGO wave and the onset of the corresponding click-elicited IPSP, sciatic nerve stimulation-elicited IPSP, and the spontaneous IPSP (latencies to onset in ms, respectively, 8.6 ± 0.4; 8.2 ± 0.3; 8.4 ± 0.3). These three values were also not significantly different when the primary PGO wave was recorded in the contralateral LGN (latencies to onset in ms, respectively, 9.0 ± 0.3; 8.6 ± 0.5; 8.9 ± 0.3; P > 0.05). There was, however, a significant difference between the amplitudes of sciatic nerve stimulation-elicited, click-elicited, and spontaneously occurring PGO-IPSPs regardless of whether the primary PGO wave was recorded in the ipsi- or contralateral LGN (amplitudes in mV, respectively, for ipsilateral PGO-IPSPs: 1.8 ± 0.8; 2.5 ± 0.7; 1.0 ± 0.6; amplitudes in mV, respectively, for contralateral PGO-IPSPs: 2.0 ± 0.5; 2.4 ± 0.3; 1.0 ± 0.6). The spontaneous PGO-IPSPs shown in Figs. 1C and 2C were recorded from the same masseter motoneuron as the elicited IPSPs illustrated in Figs. 1B and 2B, respectively.
Pre-PGO hyperpolarization
PGO-IPSPs elicited by clicks and stimulation of the sciatic nerve occasionally were preceded by hyperpolarization that occurred before the elicited PGO wave (pre-PGO hyperpolarization). In 25% of the motoneurons (7 motoneurons) there was a pre-PGO hyperpolarization elicited by stimulation of the sciatic nerve: the latency to onset of the pre-PGO hyperpolarization from stimulation when the primary PGO wave was recorded in the ipsilateral LGN was 10.3 ± 0.8 ms and 10.6 ± 0.7 ms when the primary PGO wave was recorded in the contralateral LGN. An example of the pre-PGO hyperpolarization elicited by stimulation of the sciatic nerve is illustrated in Fig. 3A.

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| FIG. 3.
A: PGO wave and IPSP in one masseter motoneuron elicited by stimulation of the sciatic nerve. Each trace represents the average of 10 consecutive responses to stimulation of the sciatic nerve. B: PGO wave and IPSP elicited by clicks in another masseter motoneuron. Each trace represents the average of 13 consecutive responses to clicks. Occasionally, the stimuli-elicited PGO-IPSP was preceded by hyperpolarization of the membrane as shown in this recording. This hyperpolarization was similar to the hyperpolarization that precedes spontaneously occurring PGO-IPSPs after the injection of carbachol in -chloralose-anesthetized cats (pre-PGO hyperpolarization) (Kohlmeier et al. 1996 ).
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In 22% of the motoneurons (8 motoneurons), there was a pre-PGO hyperpolarization elicited by clicks. The latency to onset of the pre-PGO hyperpolarization from the onset of the stimulus when the primary PGO wave was recorded in the ipsilateral LGN was 16.2 ± 1.4 ms, and 16.9 ± 0.3 ms when the primary PGO wave was recorded in the contralateral LGN. An example of the pre-PGO hyperpolarization elicited by clicks is shown in Fig. 3B.
It appears, on average, that in many of the neurons there was also a trend for a post-PGO-IPSP hyperpolarization of the membrane in response to stimulation of the sciatic nerve and clicks. The membrane potential of masseter motoneurons was indeed characterized by a long-lasting hyperpolarization after the stimulation. Additionally, discreet IPSPs also typically followed the PGO-IPSP.
Depolarization elicited by stimulation of the sciatic nerve and clicks
Stimulation of the sciatic nerve and clicks elicited depolarization of the membrane potential that preceded the elicited PGO-IPSP. In four neurons, the sciatic nerve stimulation elicited a depolarization of the membrane potential: average latency to onset, 13.6 ± 2.0 ms; average amplitude, 1.6 ± 0.3 mV. Figure 4A illustrates depolarization of the membrane elicited by stimulation of the sciatic nerve. Clicks elicited depolarization of the membrane in 11 of the motoneurons with a mean latency to onset of 17.0 ± 1.1 ms and a mean amplitude of 1.8 ± 0.4 mV. Figure 4B illustrates depolarization of the membrane elicited by clicks. There was no difference in this depolarization with respect to whether the primary PGO wave was recorded in the ipsi- or contralateral LGN.

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| FIG. 4.
A: PGO wave and IPSP in one masseter motoneuron elicited by stimulation of the sciatic nerve. B: PGO wave and IPSP elicited by clicks in another masseter motoneuron. Each trace represents the average of 14 consecutive responses to the stimulus. Occasionally, stimulation of the sciatic nerve and clicks elicited a depolarizing potential before the PGO-IPSP as is shown in the response of these motoneurons. Depolarizing potential showed a latency to onset that was very similar to that of the elicited PGO.
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Short-latency IPSP that preceded the sciatic nerve stimulation-induced IPSP
As has previously been reported, both before and after the injection of carbachol into the NPO, stimulation of the sciatic nerve also, on occasion, elicited a relatively short-latency IPSP (Kohlmeier et al. 1995
). In eight of the motoneurons, stimulation of the sciatic nerve elicited a short-latency IPSP after the injection of carbachol that had similar characteristics to the short-latency inhibitory potential that has been seen in previous studies (latency to onset, 14.6 ± 0.7 ms; average amplitude, 1.7 ± 0.2 mV) (Kohlmeier et al. 1995
). It should be noted that clicks never elicited this short-latency synaptic response. The short-latency IPSP was distinguishable from the pre-PGO hyperpolarization on the basis of its waveform; the short-latency IPSP demonstrated a much shorter latency to onset and a greater amplitude than the pre-PGO hyperpolarization.
Stimulus response curves
For the second analysis, a total of 37 motoneurons were recorded after the injection of carbachol: in 20 motoneurons, the effects of varying of the intensity of stimulation of the sciatic nerve were examined, and in 17 motoneurons, the effects of varying the intensity of the clicks were determined.
The threshold for eliciting PGO waves and inhibitory synaptic responses in masseter motoneurons was different. The intensity of stimulation of the sciatic nerve necessary to elicit a PGO wave was slightly (15-20%) lower than that necessary to elicit an IPSP in masseter motoneurons (P < 0.05). Figure 5 illustrates the response in the LGN and the same masseter motoneuron to two levels of intensity of stimulation of the sciatic nerve. In this motoneuron, at the lower intensity (0.7 V), which induced a PGO wave, a synaptic response was not elicited. However, when the level of stimulation intensity reached 0.9 V, an inhibitory synaptic response was recorded in the motoneuron. The decreased intensity of stimulation of the sciatic nerve was subthreshold for eliciting any discernible movement of the leg.

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| FIG. 5.
A: PGO wave and the synaptic response in a masseter motoneuron elicited by stimulation of the sciatic nerve at an intensity of 0.7 V. B: PGO wave and the synaptic response of the same motoneuron elicited by an increase in intensity of stimulation. Each trace represents the average of 12 consecutive responses to stimulation of the sciatic nerve. "Subthreshold" and "suprathreshold" are relative terms defined by the intensity of the stimulus required for the elicitation of a response in the membrane of the motoneuron. Intensity of stimulation of the sciatic nerve required to elicit PGO waves was lower than that necessary to elicit the carbachol-dependent synaptic response in motoneurons.
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Auditory stimuli at an intensity as low as 60 dB elicited PGO waves; a membrane potential response was not elicited until the intensity of the stimulation reached an average of 85 dB. Figure 6 illustrates the response in the LGN and the same masseter motoneuron to two intensity levels of clicks. At the lower intensity (63 dB), which induced a PGO wave, a synaptic response was not elicited in the motoneuron. However, when the level of stimulation intensity reached 90 dB, an inhibitory synaptic response was recorded.

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| FIG. 6.
A: PGO wave and the synaptic response in a masseter motoneuron after 63 dB clicks. B: PGO wave and the synaptic response of the same motoneuron elicited by an increase in the intensity of stimulation. Each trace represents the average of 12 consecutive responses to clicks. "Low intensity" and "high intensity" are relative terms defined by the intensity of the stimulus required for the elicitation of a response in the membrane of the motoneuron. Intensity of clicks required to elicit PGO waves was lower than that necessary to elicit the carbachol-dependent synaptic response in motoneurons.
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Graded response
The third analysis focused on the response characteristics of the LGN and masseter motoneurons to graded stimuli. The response recorded in the LGN (PGO waves) and the corresponding motoneuron membrane potential of eight neurons to stimulation of the sciatic nerve was recorded as the intensity of the stimulation was increased to supramaximal for elicitation of an IPSP. This intensity was, maximally, 125% of the stimulation intensity utilized in the first group of experiments. At this level of stimulation, a large movement of the leg was elicited.
The response of the LGN and six motoneurons to a 10% increase in the intensity of the clicks was examined. The amplitude of the elicited PGO wave did not vary significantly as the intensity of either stimulation of the sciatic nerve or clicks was increased (P > 0.05). However, the amplitude of the response of the membrane potential to these stimuli was graded; that is, as the intensity of the stimulus increased, the amplitude of the elicited IPSP was significantly increased(P < 0.05). Figure 7 is an example of the increase in synaptic response of the same masseter motoneuron as the intensity of stimulation of the sciatic nerve was elevated. Figure 8 is an illustration of the increase in synaptic response ofanother masseter motoneuron as the intensity of the clicks was increased. At the lowest intensity, the mean amplitude of the IPSPs elicited by stimulation of the sciatic nerve was 2.2 ± 0.6 mV and that of the click-elicited IPSPs was 1.4 ± 0.3 mV. At the higher intensity, the amplitude of the sciatic nerve stimulation-elicited IPSPs was 3.8 ± 0.7 mV and the click-elicited IPSPs was 2.5 ± 0.5 mV. The increase in amplitude of the IPSP with the increase in stimulus intensity was significant (P < 0.05).

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| FIG. 7.
Response of the LGN (A) and a masseter motoneuron (B) to 2 intensities of stimulation of the sciatic nerve. Traces are superimposed. Note that as the intensity of stimulation was increased, the amplitude of the elicited synaptic response also increased; however, the amplitude of the elicited PGO remained constant.
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| FIG. 8.
Response of the LGN (A) and a masseter motoneuron (B) to 2 intensities of auditory stimuli. Traces are superimposed. As the intensity of stimulation was increased, the synaptic response elicited by clicks also increased in amplitude; however, the amplitude of the elicited PGO remained constant.
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DISCUSSION |
At the present time, the function of PGO waves occurring spontaneously during active sleep remains unknown. It, however, has been hypothesized that PGO waves occurring during this state are neuronal signatures of activation of the startle network by endogenous mechanisms (Morrison and Bowker 1975
). This hypothesis has been modified to suggest that PGO waves are a sign of CNS alerting. Therefore, it is possible that startle stimuli, which result in behavioral signs of alerting during wakefulness, would result during all behavioral states in the generation of PGO waves. This hypothesis is supported by several findings. During wakefulness, the presentation of "startling" sensory stimuli, such as loud auditory tones, elicits PGO waves in conjunction with orienting responses and/or the auditory startle reflex (Sanford et al. 1992a
). As a consequence of paravermal lesions, spontaneous flexion limb jerks occur; these jerks are associated with PGO spikes, especially at the transition to active sleep (Morrison and Bowker 1973
). Cats treated with parachlorophenylalanine (PCPA), a serotonin synthesis inhibitor that releases spontaneously occurring PGO waves into wakefulness, display orienting movements in conjunction with PGO waves (Ferguson et al. 1970
); because these treated cats display these behaviors in the absence of external stimuli, it is thought that the responses are triggered by internal cues.
Many studies have been conducted that have analyzed the relationship between active sleep-specific and startle stimuli-elicited PGO waves and phasically occurring motor system-related events. Pivik and Dement (1970)
reported that after spontaneous PGO waves of active sleep, there is a reduction of monosynaptic spinal reflexes. Orem (1980)
found that a decrease of diaphragm muscle activity can be cross-correlated with spontaneously occurring PGO waves. These decreases of diaphragm muscle activity have been called fractionations, and they occur spontaneously during active sleep. Kline et al. (1988
, 1990)
found that one major effect of tones that produce a "startle" during wakefulness is the production of a profound depression of diaphragm muscle activity if presented during active sleep. Startle tones elicited a mixed pattern of dorsal neck musculature EMG activity overlaid with an excitatory component when presented during wakefulness and quiet sleep (Wu et al. 1989
). The motor response to tones was associated with an elicited PGO wave (Wu et al. 1989
). The same stimuli presented during active sleep elicited PGO waves but failed to elicit any response in the EMG; on rare occasions, the animal reacted with a muscle jerk and awoke (Wu et al. 1989
). If presented during the transition to active sleep, the identical tones elicited EMG suppression (Wu et al. 1989
). Suppression of the EMG during the transition to active sleep was associated with elicited PGO waves (Wu et al. 1989
).
The synaptic basis for the spontaneous PGO-related phasic suppression of muscle and somatic reflex activity during active sleep was examined. It was demonstrated that postsynaptic inhibition of lumbar (López-Rodríguez et al. 1992
) and trigeminal (Pedroarena et al. 1994
) motoneurons occurs in conjunction with PGO waves during active sleep (PGO-IPSPs). The data suggest that PGO-IPSPs are generated by the same state-dependent mechanisms that are responsible for the decrease in excitation of motoneurons during spontaneously occurring active sleep (López-Rodríguez et al. 1990
).
Our laboratory recently reported that stimulation of a somatosensory nerve and auditory stimulation elicited IPSPs in masseter motoneurons during carbachol-induced muscle atonia in the
-chloralose-anesthetized animal (Kohlmeier et al. 1994
, 1995
). These responses were never elicited in motoneurons prior to the injection of carbachol (Kohlmeier et al. 1994
, 1995
). We believe, based on preliminary data from our laboratory, that the IPSPs elicited in masseter motoneurons by stimulation of the sciatic nerve after the injection of carbachol were induced by the excitation of cutaneous afferents of the skin (M.-C. Xi, personal communication). Therefore, we suggest that inhibitory synaptic responses in motoneurons were elicited by excitation of two different sensory pathways.
We wished to determine if the elicitation of IPSPs in motoneurons by sensory stimuli after the injection of carbachol was dependent on activation of alerting and/or startling mechanisms by these stimuli or whether sensory stimuli could elicit such a response independent of activation of this network. The presence of elicited PGO waves were indicators that alerting mechanisms of the CNS had been excited (Bowker and Morrison 1976
; Morrison and Bowker 1975
). In the first series of analyses presented in this report, we demonstrated that PGO waves can be elicited by auditory stimuli (95 dB clicks) in
-chloralose-anesthetized cats both before and after the injection of carbachol in the NPO. After the injection of carbachol, 95 dB clicks elicited IPSPs in masseter motoneurons. The intensity of stimulation used in this study to elicit PGO waves has been reported to be sufficient to evoke the auditory startle reflex when presented to cats during wakefulness (Morrison and Bowker 1975
). We also found that stimulation of the sciatic nerve at an intensity sufficient to evoke a slight twitch of the leg elicited PGO waves before and after the injection of carbachol. Sciatic nerve stimulation at this intensity only consistently elicited IPSPs after the injection of carbachol. These data indicate that the startle processing system is functional in the
-chloralose-anesthetized animal. This conclusion is supported by the findings of Pellet (1990)
who reported evidence that the sensory and motor paths of the startle reflex arc are preserved functionally under chloralose anesthesia.
The second and third sets of experiments in this paper focused on a determination of the relative thresholds for elicitation of PGO waves in the LGN and IPSPs in masseter motoneurons and the effects of stimulation intensity on these elicited events. Our results indicate that PGO waves can be induced without the induction of IPSPs in motoneurons. At increased intensities of stimulation, the amplitude of the elicited PGO wave did not significantly vary within the parameters used in this study. An all-or-none nature of the elicited-PGO wave during active sleep has been reported previously (Wu et al. 1989
). However, we found that with increases in stimulus intensity, the amplitude of the stimuli-elicited IPSP was graded and increased in conjunction with increases in stimulus intensity. The graded nature of this IPSP can be explained by a recruitment and/or an increase in frequency of firing of inhibitory interneurons in response to high intensities of stimulation. In this regard, it is interesting to note that the amplitude of the spontaneous PGO-IPSP was smaller than the PGO-IPSP induced by high-intensity sensory stimuli.
In the present study, we found that the sensory stimuli-elicited IPSP in masseter motoneurons was time-locked to the sensory stimuli-elicited PGO wave. The temporal relationship between the onset of the IPSP from the foot of the elicited PGO wave was not significantly different from the temporal relationship of spontaneous PGO-IPSPs in the
-chloralose-anesthetized cat after the injection of carbachol into the NPO (Kohlmeier et al. 1996
). In addition, the temporal relationship was not different from that of spontaneously occurring PGO-IPSPs during natural episodes of active sleep (Pedroarena et al. 1994
).
The time-locked relationship between PGO waves and internally generated PGO-IPSPs recorded in motoneurons during spontaneously occurring active sleep suggests that both phenomenon share common mechanisms of generation. The existence of a temporal relationship between elicited PGO waves and PGO-IPSPs in the present study also suggests that the networks involved in the generation of PGO waves and IPSPs are interconnected. Additionally, the similarity in temporal relationship shared by the spontaneously occurring and elicited PGO waves and PGO-IPSPs further indicates that the processes of generation of these two events is related; the circuitry and mechanisms by which generation of these two events may be connected is undetermined at present. However, it should be noted that, because in the present study, both PGO waves and IPSPs were generated by external stimuli, we cannot discard the possibility that the mechanisms of generation of PGO waves and IPSPs in motoneurons are completely independent.
In this regard, although the aforementioned data indicate that networks involved in the generation of PGO waves and motoneuron IPSPs are related, different data sets in the present study do indicate that they are functionally independent. The levels of intensity of the sensory stimuli required for elicitation of PGO waves and IPSPs in motoneurons were different. After the presentation of startle stimuli, the membrane potential of some motoneurons was characterized by hyperpolarization that preceded the onset of the elicited PGO wave (pre-PGO hyperpolarization). Although the amplitude of elicited PGO waves remained constant with increases in intensity of the stimulus, the amplitude of elicited IPSPs was graded. Additionally, the findings that the amplitudes of elicited and spontaneously occurring PGO-IPSPs are significantly different indicates that it is not generation of a PGO wave that determines the magnitude of the response at the level of the motoneuron, but rather other mechanisms must be involved in the excitation of inhibitory interneurons that synapse on motoneurons.
It has been suggested that although the mechanisms responsible for the generation of the acoustic startle response and PGO waves may share some components, this sharing may be limited (Sanford et al. 1992b
). Although the underlying mechanisms of PGO wave and acoustic startle response generation are not understood, one interpretation of the data in the present report is that sensory stimuli below a certain intensity elicit alerting mechanisms (as evidenced by the production of a PGO wave). As the intensity increases, circuitry involved in startle, which may involve the production of IPSPs in motoneurons during the atonia of active sleep, become excited. It has been demonstrated that neuronal populations that are excited by startle stimuli send projections to the region of the NPO that has been shown to be critically involved in motor atonia during active sleep (Henley et al. 1974
; Pellet 1990
; Wu et al. 1988
).
We believe that both the generation of spontaneous PGO-IPSPs of active sleep and stimuli-elicited PGO-IPSPs reflect mechanisms that act to prevent movement of the animal during, respectively, an internally generated component of alerting and/or a startle stimulus and an external startle stimulus during active sleep. This hypothesis is supported by the aforementioned data regarding the relationship between PGOs and phasic motor systems events. Further support for this hypothesis is the finding that the spontaneous PGO-IPSPs of active sleep are accompanied by depolarization of the motoneuron membrane, which is revealed when these IPSPs are blocked by strychnine (Kohlmeier et al. 1996
, 1997
; López-Rodríguez et al. 1990
). Additionally, supporting this hypothesis are the findings, in this report, that startle stimuli occasionally elicited depolarization of the motoneuron membrane and that this depolarization occurred at an earlier latency than that of the PGO wave or the PGO-IPSP. We also have reported previously that when the stimuli-elicited IPSP is blocked by strychnine, a slow depolarizing potential is revealed (Kohlmeier et al. 1997
). Depolarization of the membrane of motoneurons indicates an excitatory drive that potentially could result in excitation of the muscle and movement of the animal. Therefore, it is parsimonious to conclude that the excitation of the motor system would be suppressed by the relatively large inhibitory drive directed to motoneurons in response to the stimuli.
We have hypothesized that IPSPs produced by sensory stimuli and spontaneous IPSPs during active sleep are mediated by the identical motor inhibitory system (Kohlmeier et al. 1996
, 1997
). We suggest that IPSPs induced in motoneurons by startle stimuli are due to activity in the same premotor inhibitory interneurons as those that mediate spontaneous IPSPs. If this is true, then the premotor neurons should be ones that tonically increase their discharge during the carbachol-induced state and phasically increase their firing after the elicitation of PGO waves by startle stimuli.
As previously noted, Wu et al. (1989)
reported that during wakefulness and quiet sleep, startle stimuli (clicks) of lower intensities produced EMG suppression, whereas stimuli of higher intensities resulted in a mixed pattern of EMG suppression and excitation. The suppression of the EMG reported by these authors during wakefulness and quiet sleep in response to startle stimuli may be mediated by mechanisms separate from those responsible for the active postsynaptic inhibition of motoneurons during active sleep. The basis for this hypothesis is our finding that before carbachol administration, clicks that did result in activation of CNS alerting mechanisms did not elicit IPSPs in motoneurons. Additional support for this hypothesis is provided by the finding that cats in which the pontine sites implicated in the generation of atonia are lesioned display similar phasic EMG suppression evoked by auditory startle stimuli during active sleep (Wu et al. 1990
).
The findings in this report indicate that only those sensory stimuli that exceed threshold and are capable of exciting components of CNS alerting, or exciting the startle network, are accompanied by an IPSP in motoneurons during activation of the active sleep-specific motor inhibitory system. In accord with this finding, we found that elicitation of IPSPs only occurred in conjunction with the elicitation of PGO waves, indicating that it is not just sensory stimuli that produce an increased inhibition of motoneurons during active sleep, but rather that it is specifically only sensory stimuli that activate the central mechanisms involved in alerting and/or the startle network that are capable of producing the increase in motoneuron inhibition. This finding indicates that a type of selective filtering of sensory information occurs before the excitation of the reticular neurons that are suspected to be involved in the activation of the PGO generators and the active sleep-specific motor inhibitory system. We believe that this selective filtering mechanism acts to prevent sensory stimuli capable of activating the reticular neurons involved in mediating startle responses from generating excitatory motor responses that would, if present, jeopardize the maintenance of atonia and, consequently, continuation of the state of active sleep.
Conclusions
The findings from this study indicate that
-chloralose-anesthesia does not block the responsiveness of the animal to stimuli that produce alerting of the CNS. Further, they suggest that the
-chloralose-anesthetized animal can detect a startle stimulus, and the processing of this sensory input during activation of the active sleep-specific motor inhibitory system involves an increase in the inhibition of motoneurons. We believe that these data provide support for the hypothesis that the spontaneous occurrence of PGO waves during active sleep are the result of the internal activation of mechanisms involved in CNS alerting by highly active reticular neurons. The results of these studies suggest that it is only alerting stimuli (as evidence by the generation of PGO waves) and not just any sensory input that results in elicitation of an inhibitory response in motoneurons. Thus phasic inhibitory events during active sleep may serve to preserve the state of atonia when the organism is exposed to external or internally generated events that would otherwise disrupt the state of active sleep. Phenomenologically, during active sleep, phasic inhibition of motoneurons induced by stimuli that in other behavioral states would elicit excitatory motor responses assures the maintenance of muscle atonia, even in the presence of sensory stimuli.