Department of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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Xi, Ming-Chu,
Francisco R. Morales, and
Michael H. Chase.
The Motor Inhibitory System Operating During Active
Sleep Is Tonically Suppressed by GABAergic Mechanisms During Other
States.
J. Neurophysiol. 86: 1908-1915, 2001.
The present study was
undertaken to explore the neuronal mechanisms responsible for muscle
atonia that occurs after the microinjection of bicuculline into the
nucleus pontis oralis (NPO). Specifically, we wished to test the
hypothesis that motoneurons are postsynaptically inhibited after the
microinjection of bicuculline into the NPO and determine whether the
inhibitory mechanisms are the same as those that are utilized during
naturally occurring active (rapid eye movement) sleep. Accordingly,
intracellular records were obtained from lumbar motoneurons in cats
anesthetized with -chloralose before and during bicuculline-induced
motor inhibition. The microinjection of bicuculline into the NPO
resulted in a sustained reduction in the amplitude of the spinal cord
Ia-monosynaptic reflex. In addition, lumbar motoneurons exhibited
significant changes in their electrophysiological properties [i.e., a
decrease in input resistance and membrane time constant, a reduction in
the amplitude of the action potential's afterhyperpolarization (AHP)
and an increase in rheobase]. Discrete, large-amplitude inhibitory
postsynaptic potentials (IPSPs) were also observed in high-gain
recordings from lumbar motoneurons. These potentials were comparable to
those that are only present during the state of naturally occurring active sleep. Furthermore, stimulation of the medullary nucleus reticularis gigantocellularis evoked a large-amplitude IPSP in lumbar
motoneurons after, but never prior to, the injection of bicuculline;
this reflects the pattern of motor responses that occur in conjunction
with the phenomenon of "reticular response-reversal." The preceding
changes in the electrophysiological properties of motoneurons, as well
as the development of active sleep-specific IPSPs, indicate that lumbar
motoneurons are postsynaptically inhibited following the intrapontine
administration of bicuculline in a manner that is comparable to that
which occurs spontaneously during the atonia of active sleep. The
present results support the conclusion that the brain stem-spinal cord
inhibitory system, which is responsible for motor inhibition during
active sleep, can be activated by the injection of bicuculline into the
NPO. These data suggest that the active sleep-dependent motor
inhibitory system is under constant GABAergic inhibitory control, which
is centered in the NPO. Thus during wakefulness and quiet sleep, the
glycinergically mediated postsynaptic inhibition of motoneurons is
prevented from occurring due to GABAergic mechanisms.
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INTRODUCTION |
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We have recently reported that a behavioral state that resembles
naturally occurring active sleep arises after the microinjection of
bicuculline, a GABAA receptor antagonist, into
the nucleus pontis oralis (NPO) of the pontine reticular formation.
Conversely, microinjections of either GABA or a GABA agonist into the
NPO results in prolonged episodes of wakefulness and increased motor activity (Xi et al. 1999, 2001
). However, it remained to
be determined if the atonia induced by the injection of bicuculline
into the NPO was produced by processes of postsynaptic inhibition or
disfacilitation. If a postsynaptic inhibitory mechanism was
responsible, was this the same system utilized during naturally
occurring active sleep (also referred to as rapid eye movement sleep)
as well as during carbachol-induced active sleep-like state (for
reviews, see Chase and Morales 1990
, 2000
)?
In previous studies we have shown that the neuronal mechanisms of
postsynaptic inhibitions of motoneurons are responsible for motor
inhibition during active sleep. For examples, motoneurons are tonically
hyperpolarized during active sleep (Morales and Chase 1978,
1982
; Morales et al. 1987a
). Superimposed on the
hyperpolarized membrane potential of motoneurons are discrete,
large-amplitude postsynaptic inhibitory potentials (active sleep IPSPs)
(Morales and Chase 1982
; Morales et al.
1987a
; Soja et al. 1991
). In addition, there are
significant changes in the electrophysiological properties of
motoneurons during active sleep such as a decrease in excitability, input resistance and membrane time constant (Morales and Chase 1981
; Soja et al. 1991
). The postsynaptic
inhibition of motoneurons during active sleep is glycinergically
mediated (Chase and Morales 1990
; Chase et al.
1989
; Soja et al. 1991
).
Furthermore, electrical stimulation of brain stem reticular sites
evokes characteristic, large-amplitude IPSPs in motoneurons during
active sleep but not during quiet sleep or wakefulness (Chandler
et al. 1980; Chirwa et al. 1991
; Fung et
al. 1982
). These evoked potentials have been shown to be an
essential aspect of the phenomenon of "reticular
response-reversal," in which responses to a physiological stimulus
are reversed from excitation during wakefulness to inhibition during
active sleep (Chase and Babb 1973
).
We hypothesize that lumbar motoneurons are postsynaptically inhibited
following the microinjection of bicuculline into the NPO, and the
inhibitory mechanisms are the same as those that are utilized during
naturally occurring active sleep as well as during carbachol-induced
active sleep-like state (Chase and Morales 1990, 2000
).
To test this hypothesis, we examined the basic electrophysiological properties and state-dependent synaptic activity impinging on spinal
cord motoneurons after the microinjection of bicuculline into the NPO.
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METHODS |
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Surgical procedures
The present experiments were performed on six adult cats
(3.0-5.0 kg). All experimental procedures were conducted in accord with the "Guide for the Care and Use of Laboratory Animals" (7th edition, National Academy Press, Washington, DC, 1996). Surgical procedures have been described, in detail, in previous papers (Morales et al. 1987b; Xi et al. 1997
).
Briefly, all surgical procedures were carried out under halothane
anesthesia. The left hindlimb nerves of the hamstring (including
posterior biceps and semitendinosus, and anterior biceps and
semimembranosus) and sciatic were excised at their distal ends and
positioned on stimulating electrodes made of silver wire. The
lumbosacral spinal cord was exposed by laminectomy
(L4-S1). The dura was
retracted, and the right L7 dorsal and ventral
roots were cut distally. The left dorsal roots
L5, L6,
L7, S1, and
S2 were excised to eliminate the possibility of
disfacilitation of
motoneurons via the
-loop. Spinal and leg
pools were constructed with skin flaps and filled with warm mineral oil
(37°C).
After completion of all surgical procedures, -chloralose (60 mg/kg
iv) was administered slowly over a period of 1 min while halothane was
discontinued. The chloralose solution was filtered prior to use to
generate a more stable anesthetized preparation (Kohlmeier et
al. 1996
). Supplementary doses of
-chloralose (30 mg/kg, iv)
were administered periodically throughout the remainder of the
experiment, to maintain the animal under a deep anesthesia. Previous
studies from this laboratory (Kohlmeier et al. 1996
; López-Rodríguez et al. 1995
; Xi et
al. 1997
) have shown that the brain stem-spinal cord inhibitory
system that mediates atonia during active sleep can be activated in the
-chloralose-anesthetized preparation following the injection of
carbachol into the NPO. We therefore utilized the
-chloralose-anesthetized preparation to determine if the same
inhibitory system could also be activated following the injection of
bicuculline into the NPO.
During recording sessions, the cats were immobilized with gallamine triethiodide (Flaxedil, 1.0 mg/kg) and artificially ventilated. The level of anesthesia was ensured by checking that the pupils were constricted and that the blood pressure and heart rate were stable and did not alter in response to a paw pinch. The blood pressure and end tidal CO2 were continuously monitored and maintained within the range of normal physiological values (100-140 mmHg for mean blood pressure and 3-5% for end tidal CO2).
Stimulation and recording
To examine the effect of microinjections of bicuculline on the amplitude of the Ia-monosynaptic reflex, both right L7 dorsal and ventral roots were excised at their exit from the dural sac and placed on bipolar silver wire electrodes. The reflex was evoked by the electrical stimulation of the right L7 dorsal root at an intensity just suprathreshold for group I afferents; the reflex response was recorded from the right L7 ventral root.
In experiments in which the phenomenon of reticular response-reversal
(Chandler et al. 1980; Chase and Babb
1973
; Chirwa et al. 1991
; Fung et al.
1982
) was studied, a stainless-steel electrode was lowered into
the medullary nucleus reticularis gigantocellularis (NRGc; P 9, L 1, H
8, Berman 1968
) for monopolar electrical stimulation (4 pulses at 400 Hz; pulse duration, 0.8 ms; intensity: 20-120 µA).
The recording sessions commenced 2 h after the cessation of
halothane administration to ensure the systemic clearance of halothane (Cowles et al. 1968; Yanagida et al.
1975
). Intracellular recordings were obtained from
antidromically identified motoneurons using glass micropipettes filled
with either 2 M K-citrate or 3 M KCl (tip resistances: 10-20 and 5-10
M
, respectively). The electrodes were connected to a high-input
impedance preamplifier (Axoclamp 2A).
High-gain (×100) DC and low-gain (×10) DC intracellular activity as well as extracellular AC records of the Ia-monosynaptic reflex response, recorded from the ventral root, were displayed on an oscilloscope and stored on a video cassette recorder by means of a PCM recording adapter (Vetter, Model 4000). The data were digitized off-line at 20 kHz and analyzed with a microcomputer (Apple Power Macintosh) using specially designed software.
Drug administration
Bicuculline (bicuculline methiodide, a
GABAA antagonist, 0.25 µl, 10 mM in saline),
muscimol (muscimol hydrobromide, a GABAA agonist,
0.25 µl, 10 mM in saline), and carbachol (carbamylcholine chloride,
0.25 µl, 22 mM in saline) were individually injected into the NPO
using a 1.0-µl Hamilton syringe with its tip positioned at the
stereotaxic coordinates P 3.0, L 2.0, H-3.5 (Berman
1968). Motor inhibition following the injection of bicuculline
or carbachol was determined on the basis of a decrease in the amplitude
of the Ia-monosynaptic reflex (Morales at al. 1987b
;
Pereda et al. 1990
).
Data analysis
The following electrophysiological properties of motoneurons were measured: resting membrane potential, amplitude of action potential, input resistance, membrane time constant, rheobase, and the amplitude and time course of the action potential's afterhyperpolarization (AHP).
The methods used to analyze the preceding basic electrophysiological
properties of motoneurons are standard procedures that we have employed
and described in other studies in full detail (Engelhardt et al.
1995; Morales et al. 1987b
; Soja et al.
1991
; Xi et al. 1997
). The following is a brief
summary of these methods:
RESTING MEMBRANE POTENTIAL. The membrane potential was determined by measuring the difference between the DC potential recorded intracellularly and that recorded immediately after withdrawing the microelectrode from its intracellular position.
ACTION POTENTIAL. The amplitude of the antidromically evoked action potentials was determined by measuring the difference between the DC potential recorded at the base and the peak of the action potential.
INPUT RESISTANCE. Input resistance was calculated by the "direct" method using computer-averaged voltage responses (100 trials) to the injection of low-intensity (1-3 nA) depolarizing and/or hyperpolarizing current pulses of 50-ms duration.
MEMBRANE TIME CONSTANT.
A determination of the membrane time constant was based on an analysis
of the decay phase of the averaged cell membrane voltage change
following a 50-ms current pulse. For cells in which the membrane
potential exhibited the nonlinear behavior described by Ito and
Oshima (1965), the raw voltage data were corrected to avoid
underestimating the membrane time constant. This procedure involved
successively "peeling" exponential terms with the longest time
constant from semilog plots of V or
dV/dt versus t.
RHEOBASE. Rheobase was determined as the minimum stimulus intensity of a 50-ms duration intracellular depolarizing current pulse that constantly elicited an action potential.
ACTION POTENTIAL'S AHP. The AHP was examined following action potentials elicited by passing a short (500 µs) suprathreshold current pulse through the intracellular electrode. The duration of the AHP was measured from the beginning of the current pulse to the return of the membrane potential to baseline. The amplitude of the AHP was calculated by subtracting the membrane potential value immediately preceding the initiation of the current pulse from the value at the peak of the AHP. The half-width of the AHP was measured by determining its duration at half its amplitude. The half-decay width was defined as the time between the AHP peak and the data point on its decay phase corresponding to half its amplitude.
Experimental data values are expressed as means ± SE of measurements. The statistical level of significance of the difference between sample means was evaluated using the two-tailed, unpaired or paired Student's t-test (P < 0.05).Histological procedures
At the end of each experiment, the site of drug injection was marked with 0.5 µl of a 2% solution of Chicago sky blue dye in 0.5 M Na-acetate. The animal was then killed with a lethal dose of pentobarbital sodium (Nembutal) and perfused with saline followed by a solution of 10% formaldehyde. Coronal serial sections of brain stem tissue were examined to verify the site of drug injection (Fig. 1A).
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RESULTS |
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Monosynaptic reflex
The microinjection of bicuculline into the NPO resulted in a
sustained reduction in the amplitude of the Ia-monosynaptic reflex within 2-5 min (Fig. 1B). The latency to the decrease in
reflex amplitude was similar to that of muscle atonia observed after the injection of bicuculline in intact, unanesthetized, chronically prepared cats (Xi et al. 1999, 2001
). The suppression of
the reflex usually lasted
2 h; afterward it could be re-induced by
another injection of bicuculline. The mean reflex amplitude was reduced from 0.89 ± 0.09 mV during control conditions (prior to
the injection of bicuculline) to 0.49 ± 0.05 mV after
bicuculline administration. This 44.9% reduction in mean reflex
amplitude, which was statistically significant (7 injections,
P < 0.05), was used as the criterion for the
effectiveness of a bicuculline injection; for ease of communication in
the present manuscript, the suppression of reflex activity will be
referred to as bicuculline-induced motor inhibition.
Basic electrophysiological properties
Data were obtained from 93 lumbar motoneurons with stable resting
membrane potentials and antidromic action potentials 65 mV.
Thirty-four motoneurons were recorded only during control conditions;
37 motoneurons were recorded during bicuculline-induced motor
inhibition; and 3 were recorded both before and during
bicuculline-induced motor inhibition. Another 19 motoneurons were
recorded in the experiments in which a combination of injections of
carbachol into the NPO and the subsequent injection of muscimol into
the same region of the NPO was carried out.
The action potential amplitude of motoneurons ranged from 65.4 to 90.7 mV before and from 65.0 to 90.8 mV during bicuculline-induced motor
inhibition. The mean action potential amplitude of the population of
lumbar motoneurons recorded during bicuculline-induced motor inhibition
was almost identical to that of neurons sampled during control
conditions (Table 1). The mean resting
membrane potential of motoneurons during control conditions was
67.8 ± 1.0 mV, the corresponding value for motoneurons during
bicuculline-induced motor inhibition was
70.7 ± 0.9 mV (Table
1). This hyperpolarization of 2.9 mV in mean membrane potential was
statistically significant (P < 0.05).
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Microinjections of bicuculline resulted in a sustained reduction in input resistance (Table 1). The reduction of 29.4% in the mean input resistance was statistically significant (P < 0.01). There was also a statistically significant decrease of 31.7% in the mean membrane time constant during bicuculline-induced motor inhibition (P < 0.01, Table 1).
The excitability of motoneurons was significantly reduced during bicuculline-induced motor inhibition, which is reflected by an increase in rheobase. There was a statistically significant increase (35.1%) in mean rheobase during bicuculline-induced motor inhibition (P < 0.01, Table 1).
The amplitude, duration, half-width, and half-decay width of the AHP were measured in motoneurons before and during bicuculline-induced motor inhibition. There was a statistically significant decrease of 20.0% in the mean amplitude of the AHP during bicuculline-induced motor inhibition (P < 0.05, Table 1). No statistically significant changes in the mean values of duration, half-width and half-decay width of the AHP were observed during bicuculline-induced motor inhibition (Table 1).
Spontaneous synaptic activity of lumbar motoneurons
During naturally occurring active sleep and carbachol-induced
motor inhibition, large amplitude (>1 mV), repetitively occurring IPSPs bombard somatic motoneurons (Chase et al. 1989;
Morales and Chase 1982
; Morales et al.
1987a
,b
; Soja et al. 1991
; Xi et al.
1997
). These IPSPs are specific to this state, i.e., they are
not observed during any other behavioral state. Therefore high-gain
membrane potential recordings were examined in the present study to
determine whether similar IPSPs were also present during bicuculline-induced motor inhibition.
Prior to the injection of bicuculline, the membrane potential of lumbar motoneurons was characterized by a mixture of small-amplitude, spontaneous depolarizing and hyperpolarizing potentials (Fig. 2A). After the administration of bicuculline, large-amplitude, repetitively occurring hyperpolarizing synaptic potentials (>1 mV) dominated the membrane potential recording (Fig. 2B). The mean frequency of hyperpolarizing synaptic potentials increased from 0.2 ± 0.5 potentials/s (n = 30 cells) to 22.6 ± 1.2 potentials/s (n = 32 cells) after bicuculline administration. The difference between these mean frequencies was statistically significant (P < 0.001).
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The discrete, large-amplitude IPSPs were remarkably similar to those
that appear during active sleep in the chronic cat and during
carbachol-induced motor inhibition (Chase et al. 1989; Morales and Chase 1982
; Morales et al.
1987a
; Soja et al. 1991
). In accord with the
behavior of other IPSPs, the intracellular application of
hyperpolarizing direct current (Fig.
3A) and the injection of
chloride ions (Fig. 3B) reversed the polarity, which exhibited equilibrium potentials between
70 and
80 mV. These results confirm that these hyperpolarizing potentials were
chloride-dependent IPSPs (Coombs et al. 1955
;
Morales and Chase 1982
).
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In two experiments, muscimol, a GABAA agonist, was microinjected into the same area of the NPO ~30 min after the first injection of bicuculline into the NPO. Injections of muscimol completely blocked the discrete, large amplitude IPSPs that had been induced following the injection of bicuculline into the NPO (12 cells).
It is well established that the pontine cholinergic system is
critically involved in the generation of motor inhibition during active
sleep (for review, see Jones 1991; Siegel
2000
; Steriade and McCarley 1990
). We were
therefore interested in determining whether the GABAergic system that
we were manipulating with microinjections of GABA agonists and
antagonists interacts with cholinergic mechanisms in the NPO. For this
purpose, experiments were performed using a combination of injections
of carbachol and muscimol into the NPO at different times, with
muscimol being microinjected into the same area of the NPO at a period
of 25-34 min after the injection of carbachol. Figure
4 shows sample records from a motoneuron in which recordings, which began before the injection of carbachol, were maintained during and after an injection of carbachol and a
subsequent injection of muscimol. Following the injection of carbachol,
discrete, spontaneous IPSPs were observed in this motoneuron (Fig.
4B2) as reported previously (Morales et al.
1987b
; Xi et al. 1997
). These IPSPs were not
presented in motoneurons prior to the injection of carbachol (Fig.
4B1). Twenty-five minutes after the injection of carbachol
into the NPO, muscimol was injected into the same area. High-gain
membrane potential recordings were examined to determine the effect of
muscimol injection on carbachol-induced spontaneous IPSPs. Following
the injection of muscimol, the discrete, large-amplitude IPSPs, which
were present during carbachol-induced motor inhibition, were abolished
and large-amplitude, high-frequency excitatory postsynaptic potentials
(EPSPs) were observed to dominate the membrane potential in 13 of 14 motoneurons (Fig. 4B3).
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Reticular response-reversal
The neural mechanisms underlying the phenomenon of reticular
response-reversal were examined in the present study before and during
bicuculline-induced motor inhibition. Prior to the injection of
bicuculline, electrical stimulation of the NRGc elicited a short-latency EPSPs in a majority of motoneurons (17 of 19). An example
of this excitatory potential is shown in Fig.
5A. Following the injection of
bicuculline, the identical stimulus evoked a longer-latency
large-amplitude IPSP in the same motoneuron and the amplitude of the
EPSP decreased (Fig. 5B). This characteristic large-amplitude IPSP was observed in 16 of 20 motoneurons recorded during bicuculline-induced motor inhibition [peak amplitude: 2.1 ± 0.9 mV; latency to onset (measured from the beginning of the stimulation of the NRGc): 28.9 ± 2.7 ms; latency to peak:
50.1 ± 2.0 ms; half-width: 22.7 ± 2.3 ms; duration:
58.2 ± 2.7 ms]. The waveform parameters of the NRGc-evoked IPSPs
during bicuculline-induced motor inhibition were similar to those
observed during naturally-occurring active sleep and carbachol-induced
motor inhibition (Fung et al. 1982;
López-Rodríguez et al. 1995
; Pereda
et al. 1990
).
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Recordings of NRGc-evoked responses were also made from motoneurons
following an injection of carbachol and a subsequent injection of
muscimol into the NPO. Figure 5C presents records from a
motoneuron demonstrating that electrical stimulation of the NRGc was
capable of evoking a characteristic large-amplitude IPSP during
carbachol-induced motor inhibition, as previous reported
(López-Rodríguez et al. 1995;
Pereda et al. 1990
). In the case of this particular
motoneuron, muscimol was injected into the same area of the NPO, 34 min
following the injection of carbachol. Within 5.4 min of the injection
of muscimol, the amplitude of the IPSP was greatly reduced (Fig. 5D). The mean amplitude of NRGc-evoked IPSPs during
carbachol-induced motor inhibition were significantly reduced following
the injection of muscimol [1.9 ± 0.3 mV (n = 6)
vs. 0.3 ± 0.4 mV (n = 7), before vs. following
the injection of muscimol; P < 0.05].
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DISCUSSION |
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In this discussion, we address the cellular bases of the motor inhibition that is induced by the injection of bicuculline, a GABAA antagonist, into the NPO. We will also compare the electrophysiological properties and synaptic activity of lumbar motoneurons before and during bicuculline-induced motor inhibition. These data will then be examined in relation to comparable data that have been obtained during naturally occurring active sleep and carbachol-induced motor inhibition.
The present results have shown that during motor inhibition produced by
injections of bicuculline into the NPO, the mean resting membrane
potential of the motoneurons was hyperpolarized. In addition, there was
a statistically significant decrease in the mean input resistance and
the mean membrane time constant during bicuculline-induced motor
inhibition. The decrease in both input resistance and membrane time
constant indicates that there was an increase in membrane conductance
of lumbar motoneurons. This increase in membrane conductance was also
associated with an increase in rheobase because more current was
required to bring the reduced input resistance of the motoneurons to
firing threshold. Finally, there was a significant reduction in the AHP
amplitude after the administration of bicuculline. The decrease in the
AHP amplitude can be accounted for by the decrease in input resistance
and the tonic hyperpolarization of the resting membrane potential of
motoneurons during bicuculline-induced motor inhibition. These changes
in the excitability and passive electrophysiological properties of
lumbar motoneurons, both in direction and magnitude, are comparable to
those which occur during naturally occurring active sleep
(Chandler et al. 1980; Glenn and Dement
1981
; Morales and Chase 1981
; Soja et al.
1991
) and during carbachol-induced atonia (Morales et
al. 1987b
; Xi et al. 1997
).
Following the injection of bicuculline into the NPO, discrete
large-amplitude (>1 mV) hyperpolarizing potentials were observed in
lumbar motoneurons. These potentials, which were not present during
control conditions (prior to the injection of bicuculline), were
reversed in polarity both by the intracellular application of
hyperpolarizing DC and by the injection of chloride ions. These potentials exhibited equilibrium potentials in the range of 70 to
80 mV. These findings indicate that these hyperpolarizing potentials
are chloride-dependent IPSPs. The waveforms of these potentials are
remarkably similar to those that occur in lumbar motoneurons during
naturally occurring active sleep and carbachol-induced motor inhibition
(Chase et al. 1989
; Morales and Chase
1981
; Morales et al. 1987a
,b
;
Soja et al. 1991
). These findings support the hypothesis
that the IPSPs that appear during bicuculline-induced motor inhibition
originate from the activation of the same group of interneurons that
are responsible for the postsynaptic inhibition of lumbar motoneurons
during active sleep and during carbachol-induced motor inhibition.
One interesting aspect of the neural control of motoneurons is a
phenomenon called "reticular response-reversal" (Chase and Babb 1973). This phenomenon refers to the difference in motor response to activation of the brain stem reticular formation during different behavioral states. Specifically, electrical stimulation of
the NPO or NRGc during wakefulness and quiet sleep elicits EPSPs in
motoneurons. The identical stimulus during active sleep elicits IPSPs
(Chandler et al. 1980
; Chase and Babb
1973
; Chirwa et al. 1991
; Fung et al.
1982
). In the present study, stimulation of the NRGc evoked a
large-amplitude IPSP in lumbar motoneurons during bicuculline-induced
motor inhibition, whereas the same stimulus evoked an EPSP during
control conditions. An analyses of the waveform parameters of the
NRGc-evoked EPSPs and IPSPs during control conditions and during
bicuculline-induced motor inhibition indicated that these potentials
are similar to those evoked by stimulation of the NRGc during
wakefulness and active sleep, respectively. Based on the present data,
we therefore conclude that the same brain stem-spinal cord inhibitory
system that is responsible for the postsynaptic inhibition of
motoneurons during active sleep is also activated by the injection of
bicuculline into the NPO. In other words, this system is under tonic
inhibitory GABAergic control, which can be released by the
administration of bicuculline.
A number of studies have presented data indicating that the NPO of the
pontine reticular formation is a key region in promoting active sleep
and the accompanying pattern of somatomotor atonia (for review, see
Jones 1991; Siegel 2000
; Steriade
and McCarley 1990
). Anatomical evidence indicates that the NPO
receives cholinergic innervation from both the laterodorsal tegmental
and the pedunculopontine tegmental nuclei of the dorsolateral pons
(Mitani et al. 1988
; Shiromani et al.
1988
). Microinjections of cholinergic agonists into the NPO
reliably induce an active sleep-like state that is indistinguishable
from naturally occurring active sleep (Baghdoyan et al. 1984
,
1987
, 1989
, 1993
; George et al. 1964
;
Yamamato et al. 1990
; Yamuy et al. 1993
).
In addition, a majority of the neurons in the NPO increase their
discharge rate during active sleep (McCarley and Hobson
1971
; McCarley et al. 1995
), although many cells
in this nucleus also increase their discharge rate during waking movements (Siegel et al. 1977
). For these reasons, cells
in the NPO are believed to act as effector neurons that are responsible for active sleep, especially for muscle atonia that is a key
characteristic of this state.
On the basis of the well-established fact that bicuculline blocks synaptic transmission at GABAA receptors, and our experiments that have shown that microinections of bicuculline into the NPO induce a generalized inhibition of motor activity, we suggest that a GABAergic system is a key component of a "gating" mechanism within the NPO that participates in promoting somatomotor atonia during active sleep. This gating mechanism apparently operates in such a way that during wakefulness and quiet sleep the activity of GABAergic synaptic transmission in the NPO is both tonic and dominant; consequently, there is a sustained inhibition of effector neurons in the NPO. Thus motor inhibition occurs when GABAergic synaptic transmission in the NPO is suppressed, which allows the brain stem-spinal cord inhibitory system to become activated. However, additional anatomical and physiological studies are needed to determine the precise nature of GABAergic synaptic transmission in the NPO during the behavioral states of active sleep and wakefulness and to identify the GABAergic system involved.
It is interesting to note that 20-30 min after the state of motor
inhibition was induced by the injection of carbachol into the NPO, the
subsequent injection of muscimol into the same area resulted in the
disappearance of the large-amplitude spontaneous IPSPs and a great
reduction in the amplitude of NRGc-evoked IPSPs. Both types of IPSPs
were present only during carbachol-induced motor inhibition. At the
same time, large-amplitude high-frequency EPSPs dominated the membrane
potential record. The amplitude of the Ia-monosynaptic reflex, which
was reduced due to the effects of carbachol, recovered to the values
that were presented before the injection of carbachol. These results
highlight the importance of the interaction of the GABAergic system
described in the this report with the pontine cholinergic system, which
has shown to be critically involved in the generation of muscle atonia
during active sleep (Baghdoyan et al. 1984, 1987
, 1989
,
1993
; George et al. 1964
; Yamamoto et al.
1990
; Yamuy et al. 1993
).
Why are both GABAergic and cholinergic input present in the NPO? We suggest that activation of NPO neurons, which is responsible for the generation of muscle atonia during active sleep, is modulated by both GABAergic and cholinergic systems. Specifically, we hypothesize that the excitatory cholinergic control of the activity of NPO neurons is gated by a pontine GABAergic inhibitory system. This gate mechanism may exert its effects postysynaptically, by direct GABAergic inhibitory effects on NPO neurons, and/or presynaptically, by GABAergic modulation of the release of acetycholine from synaptic terminals.
The present data demonstrate that the effect of the microinjection of
carbachol into the NPO is suppressed by a subsequent injection of
muscimol into the same region, suggesting that NPO neurons are
postsynaptically inhibited by GABAergic inputs. On the other hand, a
recent in vivo microdialysis study has shown that the microperfusion of
bicuculline into the NPO significantly increases the release of
acetylcholine (Baghdoyan and Vazquez 2000), indicating
that a presynaptic GABAergic mechanism may also be involved in the
control of cholinergic synapses on NPO neurons.
In summary, after the injection of bicuculline into the NPO, the
electrophysiological properties and synaptic activity of lumbar
motoneurons exhibit changes that are comparable to those that are
present during naturally occurring active sleep and carbachol-induced atonia. These data indicate that bicuculline-induced atonia is due to
the postsynaptic inhibition of lumbar motoneurons. Based on the present
study, we conclude that the brain stem-spinal cord inhibitory system,
which is responsible for the postsynaptic inhibition of motoneurons
during active sleep, is inhibited by pontine GABAergic mechanisms
during wakefulness and quiet sleep, and becomes activated during active
sleep resulting in atonia of the somatic musculature during this state.
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ACKNOWLEDGMENTS |
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We thank Dr. J. K. Engelhardt for critical comments regarding the manuscript.
This work was supported by National Institutes of Health Grants NS-23426, NS-09999, MH-43362, and HL-60296.
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FOOTNOTES |
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Address for reprint requests: M. H. Chase, Dept. of Physiology, 53-231 CHS, UCLA School of Medicine, Los Angeles, CA 90095 (E-mail: mchase{at}ucla.edu).
Received 5 March 2001; accepted in final form 22 June 2001.
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REFERENCES |
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