Brain Research, 514 (1990) 175-179 Elsevier
175
BRES 24051
Medullary control of lumbar motoneurons during carbachol-induced motor inhibition Alberto E. Pereda, Francisco R. Morales and Michael H. Chase Department of Physiology and Department of Anatomy and Cell Biology, and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted 2 January 1990) Key words: Carbachol; Motor suppression; Reticular formation; Inhibitory postsynaptic potential; Active sleep
The present study examined the effects of stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) on the Ia-monosynaptic reflex and the membrane potential of lumbar motoneurons. Stimulation of the NRGc was carried out in acute decerebrate cats during m o t o r suppression induced by the intrapontine microinjection of carbachol. During carbachol-induced motor suppression, compared with control conditions (prior to the administration of carbachol), NRGc stimulation resulted in a statistically significant reduction in the Ia-monosynaptic reflex. This effect was maximal at an interval of 45 ms following NRGc stimulation. NRGc stimulation also induced, in lumbar motoneurons, a large amplitude (3.17 mV _+0.36 [S.E.M.]), long duration (54.73 ms + 3.52 [S.E.M.]) inhibitory postsynaptic potential whose peak coincided with the interval of maximum reflex suppression. These results suggest that carbachol activates pontine neurons that excite cells of the medullary NRGc. We believe that these medullary neurons, in addition to those of the nucleus pontis oralis (NPO)7, participate in the modulation of the descending inhibitory pathway that is responsible for the phenomenon of response-reversal 5'15 and generalized atonia during naturally occurring active (i.e. REM) sleep.
Substantial evidence indicates that certain regions of the reticular formation of the pons and medulla participate in the production of motoneuron inhibition during active (i.e. R E M ) sleep (AS) 8A°'14. A number of researchers s'1°'14 have proposed that the critical pontine area is centered in, or in the vicinity of, the nucleus pontis oralis (NPO), whereas others 13 suggest that the region lies in the area of the peri locus coeruleus alpha, which is adjacent to the NPO. There are cholinoceptive neurons in these regions whose activation by the microinjection of carbachol induces a postsynaptic inhibitory drive directed to somatic motoneurons similar to that exercised during naturally occurring episodes of AS 12. These data have led to the supposition that the inhibitory system that produces atonia during AS may also be pharmacologically activated by carbachol administration 1'12. It has also been hypothesized that the N R G c participates in this cholinoceptive inhibitory pathway 7. We explored this possibility by studying the effect of stimulation of the nucleus reticularis gigantocellularis ( N R G c ) on the Ia-monosynaptic reflex and on the lumbar motoneuron membrane potential before and after the induction of atonia by the intrapontine injection of carbachol. In 3 adult cats (2.5-4.6 kg), the lumbosacral spinal cord was exposed by laminectomy ((L 4 to L7) during
anesthesia induced by halothane. The right L 7 ventral root was cut distally and separated into thin filaments, one of which was placed on a bipolar electrode in order to monitor spontaneous motor axon activity before and after the intrapontine microinjection of carbachol. The abolition of this activity was taken to verify the presence of motor inhibition after carbachol injection; E M G activity could not be used for this purpose because the preparation was curarized. The following left hindlimb nerves were excised at their distal ends and positioned on platinum stimulating electrodes: gastrocnemius medialis, gastrocnemius lateralis and soleus, c o m m o n peroneal, tibial (distal to the triceps surae branches and the trunk of the nerves innervating the hamstring muscles), and sciatic (prior to its popliteal division). After craniotomy and cerebral hemispherectomy, the brainstem was transected at the precollicular level. U p o n completion of these surgical procedures, halothane was discontinued; the animals were then immobilized with Flaxedil (1 mg/kg, i.v.) and artificially ventilated. In experiments designed to examine the Ia-monosynaptic reflex, the dorsal roots were left intact and the ventral roots were excised from $2 to L 6 at their exits from the dural sac. The Ia-monosynaptic reflex was evoked by electrical stimulation of the sciatic nerve at
Correspondence: M.H. Chase, Department of Physiology, School of Medicine, UCLA Center for the Health Sciences, Los Angeles, CA 90024-1751, U.S.A.
176 mg/ml solution) was injected into the pontine reticular formation using a 1.0-~1 Hamilton syringe whose tip was positioned at Berman's coordinates P3.9, L2.0, H -5.0 to -7.0 (ref. 2). Low intensity stimulation was delivered to the NRGc (P8 to P10, L1 to L2, H - 6 to H -9) via a movable monopolar stainless steel microelectrode (1-4 pulses of 0.8 ms each, 400 Hz, 10 ms duration, 40-80/xA) before and during carbachol-induced motor suppression. The membrane potential response of lumbar motoneurons to NRGc stimulation was quantified by averaging the evoked activity using a microcomputer (DEC LSI 11/23). At the end of each experiment, the site of carbachol microinjection was marked with an injection of 0.5 ~tl of a 2% solution of Chicago Sky blue dye in 0.5 M Na-acetate. Reference lesions were made in the brainstem by delivering anodal current of 75 /xA for 30 s through the stimulating stainless steel microelectrode. For a more detailed technical description of these methodologies, see Morales et a1.12. The effect of NRGc stimulation on the amplitude of the Ia-monosynaptic reflex was first studied prior to the administration of carbachol and then during the suppres-
intensities just suprathreshold for group I afferents; the response was recorded from the L 7 ventral root that had been placed on silver-chloride electrodes. In experiments in which the motoneuron membrane potential was examined, the ipsilateral dorsal roots L6, L7, S 1 and S2 were excised in order to eliminate gamma loop facilitation of alpha motoneurons. The ipsilaterai ventral roots were left intact, bilaterally, in order to identify lumbar motoneurons by antidromic stimulation. The electrodes used for intracellular recording from motoneurons were bevelled glass micropipettes filled with either 3 M KCI or 2 M K-citrate (tip resistances were 5-10 and 10-20 MI2, respectively). The electrodes were connected to a high-input impedance preamplifier with negative capacitance compensation. Intracellular DC records at 10x and 100x gain and the extracellular AC record of ventral root activity at 1000x gain were recorded on a Vetter Model D instrumentation tape recorder. Blood pressure, end tidal CO 2, and body temperature were monitored continuously; after these physiological parameters had stabilized, carbachol (0.25-1.0/xl of a 16
la MONOSYNAPTIC REFLEX A, Pre-Carbachol
B. Carbachol
1. Control
1. Control
/),veragedReflexe$
C.
~]
4 .
Control NRGc Stimulation
m
3,
I
j
I
>
g "1o
2. NRGc Stimulation
_=_
2. NRGc Stimulation
2,
Q.
E
175
BRES 24051
Medullary control of lumbar motoneurons during carbachol-induced motor inhibition Alberto E. Pereda, Francisco R. Morales and Michael H. Chase Department of Physiology and Department of Anatomy and Cell Biology, and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted 2 January 1990) Key words: Carbachol; Motor suppression; Reticular formation; Inhibitory postsynaptic potential; Active sleep
The present study examined the effects of stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) on the Ia-monosynaptic reflex and the membrane potential of lumbar motoneurons. Stimulation of the NRGc was carried out in acute decerebrate cats during m o t o r suppression induced by the intrapontine microinjection of carbachol. During carbachol-induced motor suppression, compared with control conditions (prior to the administration of carbachol), NRGc stimulation resulted in a statistically significant reduction in the Ia-monosynaptic reflex. This effect was maximal at an interval of 45 ms following NRGc stimulation. NRGc stimulation also induced, in lumbar motoneurons, a large amplitude (3.17 mV _+0.36 [S.E.M.]), long duration (54.73 ms + 3.52 [S.E.M.]) inhibitory postsynaptic potential whose peak coincided with the interval of maximum reflex suppression. These results suggest that carbachol activates pontine neurons that excite cells of the medullary NRGc. We believe that these medullary neurons, in addition to those of the nucleus pontis oralis (NPO)7, participate in the modulation of the descending inhibitory pathway that is responsible for the phenomenon of response-reversal 5'15 and generalized atonia during naturally occurring active (i.e. REM) sleep.
Substantial evidence indicates that certain regions of the reticular formation of the pons and medulla participate in the production of motoneuron inhibition during active (i.e. R E M ) sleep (AS) 8A°'14. A number of researchers s'1°'14 have proposed that the critical pontine area is centered in, or in the vicinity of, the nucleus pontis oralis (NPO), whereas others 13 suggest that the region lies in the area of the peri locus coeruleus alpha, which is adjacent to the NPO. There are cholinoceptive neurons in these regions whose activation by the microinjection of carbachol induces a postsynaptic inhibitory drive directed to somatic motoneurons similar to that exercised during naturally occurring episodes of AS 12. These data have led to the supposition that the inhibitory system that produces atonia during AS may also be pharmacologically activated by carbachol administration 1'12. It has also been hypothesized that the N R G c participates in this cholinoceptive inhibitory pathway 7. We explored this possibility by studying the effect of stimulation of the nucleus reticularis gigantocellularis ( N R G c ) on the Ia-monosynaptic reflex and on the lumbar motoneuron membrane potential before and after the induction of atonia by the intrapontine injection of carbachol. In 3 adult cats (2.5-4.6 kg), the lumbosacral spinal cord was exposed by laminectomy ((L 4 to L7) during
anesthesia induced by halothane. The right L 7 ventral root was cut distally and separated into thin filaments, one of which was placed on a bipolar electrode in order to monitor spontaneous motor axon activity before and after the intrapontine microinjection of carbachol. The abolition of this activity was taken to verify the presence of motor inhibition after carbachol injection; E M G activity could not be used for this purpose because the preparation was curarized. The following left hindlimb nerves were excised at their distal ends and positioned on platinum stimulating electrodes: gastrocnemius medialis, gastrocnemius lateralis and soleus, c o m m o n peroneal, tibial (distal to the triceps surae branches and the trunk of the nerves innervating the hamstring muscles), and sciatic (prior to its popliteal division). After craniotomy and cerebral hemispherectomy, the brainstem was transected at the precollicular level. U p o n completion of these surgical procedures, halothane was discontinued; the animals were then immobilized with Flaxedil (1 mg/kg, i.v.) and artificially ventilated. In experiments designed to examine the Ia-monosynaptic reflex, the dorsal roots were left intact and the ventral roots were excised from $2 to L 6 at their exits from the dural sac. The Ia-monosynaptic reflex was evoked by electrical stimulation of the sciatic nerve at
Correspondence: M.H. Chase, Department of Physiology, School of Medicine, UCLA Center for the Health Sciences, Los Angeles, CA 90024-1751, U.S.A.
176 mg/ml solution) was injected into the pontine reticular formation using a 1.0-~1 Hamilton syringe whose tip was positioned at Berman's coordinates P3.9, L2.0, H -5.0 to -7.0 (ref. 2). Low intensity stimulation was delivered to the NRGc (P8 to P10, L1 to L2, H - 6 to H -9) via a movable monopolar stainless steel microelectrode (1-4 pulses of 0.8 ms each, 400 Hz, 10 ms duration, 40-80/xA) before and during carbachol-induced motor suppression. The membrane potential response of lumbar motoneurons to NRGc stimulation was quantified by averaging the evoked activity using a microcomputer (DEC LSI 11/23). At the end of each experiment, the site of carbachol microinjection was marked with an injection of 0.5 ~tl of a 2% solution of Chicago Sky blue dye in 0.5 M Na-acetate. Reference lesions were made in the brainstem by delivering anodal current of 75 /xA for 30 s through the stimulating stainless steel microelectrode. For a more detailed technical description of these methodologies, see Morales et a1.12. The effect of NRGc stimulation on the amplitude of the Ia-monosynaptic reflex was first studied prior to the administration of carbachol and then during the suppres-
intensities just suprathreshold for group I afferents; the response was recorded from the L 7 ventral root that had been placed on silver-chloride electrodes. In experiments in which the motoneuron membrane potential was examined, the ipsilateral dorsal roots L6, L7, S 1 and S2 were excised in order to eliminate gamma loop facilitation of alpha motoneurons. The ipsilaterai ventral roots were left intact, bilaterally, in order to identify lumbar motoneurons by antidromic stimulation. The electrodes used for intracellular recording from motoneurons were bevelled glass micropipettes filled with either 3 M KCI or 2 M K-citrate (tip resistances were 5-10 and 10-20 MI2, respectively). The electrodes were connected to a high-input impedance preamplifier with negative capacitance compensation. Intracellular DC records at 10x and 100x gain and the extracellular AC record of ventral root activity at 1000x gain were recorded on a Vetter Model D instrumentation tape recorder. Blood pressure, end tidal CO 2, and body temperature were monitored continuously; after these physiological parameters had stabilized, carbachol (0.25-1.0/xl of a 16
la MONOSYNAPTIC REFLEX A, Pre-Carbachol
B. Carbachol
1. Control
1. Control
/),veragedReflexe$
C.
~]
4 .
Control NRGc Stimulation
m
3,
I
j
I
>
g "1o
2. NRGc Stimulation
_=_
2. NRGc Stimulation
2,
Q.
E
