MODULATION ACUTE
OF
SPINAL
SPINAL
RATS
AGONISTS Kazuo
KAWASAKI, Shionogi
REFLEX
Research
Accepted
ANTAGONISTS
TAKESUE Laboratory,
Fukushima-kit,
IN
WITH ƒ¿-ADRENERGIC
AND Hideo
ACTIVITIES
and Akira Shionogi
Osaka September
553,
Co.,
MATSUSHITA Ltd.,
Japan
19, 1977
Although many workers have reported the role of norepinephrine (NE) within various regions of the central nervous system, knowledge of the function of this neurohormone in the brain or spinal cord is far from complete.
Iontophoretically
applied NE produces
inhibition of neuronal activity in various regions including the cerebral cortex, the limbic system, the diencephalon, the cerebellum and the spinal cord, while NE-induced excitation has also been reported in studies of the cerebral cortex or the brain stem (1).
Furthermore,
only a few studies have been systematically designed to investigate the influence of various a-adrenergic blockers on the central action of NE. The purpose of the present study was to investigate the effects of clonidine, a centrally acting a-adrenergic agonist, on the tail-flick reflex in acutely spinalized rats, and to compare differential potentials of several a-adrenergic blockers in modulating the effect of clonidine. Sixty-six male Wistar rats (Shizuoka Laboratory used.
Center), over 14 weeks of age, were
In most experiments, the spinal cord was transected at Th7-Th8 with the rats under
ether anesthesia.
Between spinal surgery and the pharmacological test, 4-5 hours elapsed.
The tail-flick reflex was elicited by pinching the tail tip with an arterial clip of 500 g pressure, calibrated by the method of Takagi et al. (2).
The reflex activities were monitored with an
electromyogram (EMG) of the extensor caudal muscle whose function raises or flicks the tail. To record the EMG, a tungsten bipolar electrode (150 ~n in diameter) was inserted into the muscle unilaterally. a-Adrenergic blockers employed were: phenoxybenzamine HCI (Tokyo Kasei), chlorpromazine HCI (Shionogi), tolazoline HC1(Imidalin, Yamanouchi), yohimbine HCI (Sigma) and phentolamine mesylate (Regitine, CIBA-Geigy). HCl was kindly provided by Drs. Heusner and Heel of Boehringer Ingelheim.
Clonidine All drugs
were injected into the external jugular vein through an inserted polyethylene tube. The EMG activities reflexly induced during a 5-second pinching of the tail were composed of two components, phasic and tonic discharges, as mentioned in a previous study (3).
The electrocardiogram (ECG) was simultaneously monitored in all experiments. As shown in Fig. IA, B, clonidine showed biphasic actions on the tail-flick reflex de
pending on the dosage used; small doses (0.03-0.06 mg/kg) suppressed the tonic discharges, while larger doses (0.5-1 mg/kg) enhanced it and furthermore, prolonged the EMG activities even after the arterial clip had been withdrawn from the tail.
Although drug effects on the
phasic component were difficult to analyze accurately because of the unavoidable artifact in the EMG recording generated by movement of the tail, it seemed likely that the phasic movement of the tail was enhanced by a wide range of clonidine dosages.
This result is
consistent with the experiment of Anden et al. (4) in which an intraperitoneal injection of clonidine invariably produced an increase in the flexor reflex of the hindlimb of acute spinal rats pretreated
with reserpine and a-methyl-p-tyrosine.
We observed that even in rats
prepared according to the method of Anden et al., smaller doses of clonidine showed a marked inhibition of tonic discharges in the caudal extensor muscle. Different methods of provoking reflex activity and evaluating its property and intensity may elucidate this discrepancy. In order to observe the interactions of clonidine with five a-blockers, the animals were pretreated with one of the a-blockers 10 min prior to clonidine injection. The inhibitory effect of clonidine upon the tail-flick reflex was completely antagonized by pretreatment with yohimbine (3 mg/kg), tolazoline (20 mg/ kg), and phentolamine (10 mg/kg).
On the
FIG. 1. Effects of clonidine on EMG activities in the extensor caudal muscle during pinching of the tail of acute spinal rats. A: inhibition with 0.06 mg/kg, B: slight potentiation with 0.5 mg/kg, and C: marked inhibition with 0.5 mg/kg 10 min after chlorpromazine treatment.
other hand, clonidine-induced reflex potentiation was not influenced by pretreatment with higher doses of these three a-blockers; the doses up to 8 mg/kg of yohimbine, 50 mg/kg of tolazoline and 20 mg/kg of phentolamine were all without effect. The interactions of clonidine with phenoxybenzamine or chlorpromazine are complicated.
Pretreatment with
phenoxybenzamine (5 mg/kg) or chlorpromazine (0.5 mg/kg) did not produce any antago nism on clonidine-induced reflex inhibition, but rather potentiated the inhibition, and 0.5 mg/kg of clonidine, which usually enhanced reflex activities in untreated animals, showed a marked inhibition after phenoxybenzamine or chlorpromazine treatment (Fig. 1C). most interesting result was that chlorpromazine
or phenoxybenzamine
The
alone had little
inhibitory effect on the reflex at the dose used here, although it inhibited significantly the tonic component which had been previously elevated by 0.5 mg/kg of clonidine. benzamine or chlorpromazine-treated
In phenoxy
animals, the reflex inhibition by 0.5 mg/kg of clonidine
was completely restored following an injection of yohimbine or tolazoline. Arrhythmic change was always observed in ECG recordings shortly after adminis tration of clonidine, in a dose-dependent manner.
However, there was evidence suggesting
that the reflex changes by clonidine were not due to such a circulatory disturbance: the time course of arrhythmia or bradycardia was inconsistent with that of reflex changes, all dosages of clonidine employed here (0.06-0.5 mg/kg i.v.) invariably resulted in hypertension in acute spinal states, and no arrhythmia
or a very weak bradycardia
was observed in
vagotomized spinal rats where clonidine-induced biphasic reflex changes were still present. Changes in autonomic activities monitored by ECG were partially or completely antagonized by all a-blockers. These
results
rat in two and
that
ways, each
TABLE
1.
suggest one
a-blocker
clonidine
of or
affects
the inhibitory
interacts
Influence
inhibition
that
involving
specifically
treatments
potentiation
with of tail-flick
the tail-flick
reflex
in the acutely
mechanism
and
the other
with
one
or the
some reflex
either
a-antagonists in acute
on spinal
rats
the
other
spinalized
excitatory or both
clonidinc-induced
one, adreno
ceptive
mechanisms.
adrenergic Many
Therefore,
mechanisms further
problems
effects
of clonidine
vestigation
using
action with
the
that
should
be carefully
The present physiological
of the extensor should
chronically
a-blockers
investigated.
remain:
tail reflex and the contraction the site of clonidine
the
are being
caudal
be determined
of NE
should
spinalized
rats.
and
muscle and
be done.
data
pharmacological
should
strict
selected
be more
comparative
These
when
are summarized
problems
central
in Table nature
precisely experiments are now
1.
of the clarified, on the
under
in
REFERENCES 1) BLOOM,F.E.: Amine receptors in CNS. I. Norepinephrine, in Handbook of Psychophar macology, Edited by IVERSEN,L.L., IVERSEN,S.D. AND SNYDER,S.H., Vol. 6, p. 1-22, Plenum Press, New York and London (1975)2) TAKAGI, H., INUKAI, T. AND NAKAMA,M.: A modi fication of Haffner's method for testing analgesics. Japan. J. Pharmacol. 16, 287-294 (1966) 3) MATSUSHITA,A., TAKESUE,H. AND KIDO, R.: Actions of morphine and narcotic antagonist analgesics on the spinal cord of acute and chronic spinal rats. Japan. J. Pharmacol. 21, 134-136 (1971)4) ANDEN, N.-E., GRABOWSKA,M. AND STROMBOM, U.: Different alpha-adrenoreceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents. Arch. Pharmacol. 292, 43-52 (1976)
OF
SPINAL
SPINAL
RATS
AGONISTS Kazuo
KAWASAKI, Shionogi
REFLEX
Research
Accepted
ANTAGONISTS
TAKESUE Laboratory,
Fukushima-kit,
IN
WITH ƒ¿-ADRENERGIC
AND Hideo
ACTIVITIES
and Akira Shionogi
Osaka September
553,
Co.,
MATSUSHITA Ltd.,
Japan
19, 1977
Although many workers have reported the role of norepinephrine (NE) within various regions of the central nervous system, knowledge of the function of this neurohormone in the brain or spinal cord is far from complete.
Iontophoretically
applied NE produces
inhibition of neuronal activity in various regions including the cerebral cortex, the limbic system, the diencephalon, the cerebellum and the spinal cord, while NE-induced excitation has also been reported in studies of the cerebral cortex or the brain stem (1).
Furthermore,
only a few studies have been systematically designed to investigate the influence of various a-adrenergic blockers on the central action of NE. The purpose of the present study was to investigate the effects of clonidine, a centrally acting a-adrenergic agonist, on the tail-flick reflex in acutely spinalized rats, and to compare differential potentials of several a-adrenergic blockers in modulating the effect of clonidine. Sixty-six male Wistar rats (Shizuoka Laboratory used.
Center), over 14 weeks of age, were
In most experiments, the spinal cord was transected at Th7-Th8 with the rats under
ether anesthesia.
Between spinal surgery and the pharmacological test, 4-5 hours elapsed.
The tail-flick reflex was elicited by pinching the tail tip with an arterial clip of 500 g pressure, calibrated by the method of Takagi et al. (2).
The reflex activities were monitored with an
electromyogram (EMG) of the extensor caudal muscle whose function raises or flicks the tail. To record the EMG, a tungsten bipolar electrode (150 ~n in diameter) was inserted into the muscle unilaterally. a-Adrenergic blockers employed were: phenoxybenzamine HCI (Tokyo Kasei), chlorpromazine HCI (Shionogi), tolazoline HC1(Imidalin, Yamanouchi), yohimbine HCI (Sigma) and phentolamine mesylate (Regitine, CIBA-Geigy). HCl was kindly provided by Drs. Heusner and Heel of Boehringer Ingelheim.
Clonidine All drugs
were injected into the external jugular vein through an inserted polyethylene tube. The EMG activities reflexly induced during a 5-second pinching of the tail were composed of two components, phasic and tonic discharges, as mentioned in a previous study (3).
The electrocardiogram (ECG) was simultaneously monitored in all experiments. As shown in Fig. IA, B, clonidine showed biphasic actions on the tail-flick reflex de
pending on the dosage used; small doses (0.03-0.06 mg/kg) suppressed the tonic discharges, while larger doses (0.5-1 mg/kg) enhanced it and furthermore, prolonged the EMG activities even after the arterial clip had been withdrawn from the tail.
Although drug effects on the
phasic component were difficult to analyze accurately because of the unavoidable artifact in the EMG recording generated by movement of the tail, it seemed likely that the phasic movement of the tail was enhanced by a wide range of clonidine dosages.
This result is
consistent with the experiment of Anden et al. (4) in which an intraperitoneal injection of clonidine invariably produced an increase in the flexor reflex of the hindlimb of acute spinal rats pretreated
with reserpine and a-methyl-p-tyrosine.
We observed that even in rats
prepared according to the method of Anden et al., smaller doses of clonidine showed a marked inhibition of tonic discharges in the caudal extensor muscle. Different methods of provoking reflex activity and evaluating its property and intensity may elucidate this discrepancy. In order to observe the interactions of clonidine with five a-blockers, the animals were pretreated with one of the a-blockers 10 min prior to clonidine injection. The inhibitory effect of clonidine upon the tail-flick reflex was completely antagonized by pretreatment with yohimbine (3 mg/kg), tolazoline (20 mg/ kg), and phentolamine (10 mg/kg).
On the
FIG. 1. Effects of clonidine on EMG activities in the extensor caudal muscle during pinching of the tail of acute spinal rats. A: inhibition with 0.06 mg/kg, B: slight potentiation with 0.5 mg/kg, and C: marked inhibition with 0.5 mg/kg 10 min after chlorpromazine treatment.
other hand, clonidine-induced reflex potentiation was not influenced by pretreatment with higher doses of these three a-blockers; the doses up to 8 mg/kg of yohimbine, 50 mg/kg of tolazoline and 20 mg/kg of phentolamine were all without effect. The interactions of clonidine with phenoxybenzamine or chlorpromazine are complicated.
Pretreatment with
phenoxybenzamine (5 mg/kg) or chlorpromazine (0.5 mg/kg) did not produce any antago nism on clonidine-induced reflex inhibition, but rather potentiated the inhibition, and 0.5 mg/kg of clonidine, which usually enhanced reflex activities in untreated animals, showed a marked inhibition after phenoxybenzamine or chlorpromazine treatment (Fig. 1C). most interesting result was that chlorpromazine
or phenoxybenzamine
The
alone had little
inhibitory effect on the reflex at the dose used here, although it inhibited significantly the tonic component which had been previously elevated by 0.5 mg/kg of clonidine. benzamine or chlorpromazine-treated
In phenoxy
animals, the reflex inhibition by 0.5 mg/kg of clonidine
was completely restored following an injection of yohimbine or tolazoline. Arrhythmic change was always observed in ECG recordings shortly after adminis tration of clonidine, in a dose-dependent manner.
However, there was evidence suggesting
that the reflex changes by clonidine were not due to such a circulatory disturbance: the time course of arrhythmia or bradycardia was inconsistent with that of reflex changes, all dosages of clonidine employed here (0.06-0.5 mg/kg i.v.) invariably resulted in hypertension in acute spinal states, and no arrhythmia
or a very weak bradycardia
was observed in
vagotomized spinal rats where clonidine-induced biphasic reflex changes were still present. Changes in autonomic activities monitored by ECG were partially or completely antagonized by all a-blockers. These
results
rat in two and
that
ways, each
TABLE
1.
suggest one
a-blocker
clonidine
of or
affects
the inhibitory
interacts
Influence
inhibition
that
involving
specifically
treatments
potentiation
with of tail-flick
the tail-flick
reflex
in the acutely
mechanism
and
the other
with
one
or the
some reflex
either
a-antagonists in acute
on spinal
rats
the
other
spinalized
excitatory or both
clonidinc-induced
one, adreno
ceptive
mechanisms.
adrenergic Many
Therefore,
mechanisms further
problems
effects
of clonidine
vestigation
using
action with
the
that
should
be carefully
The present physiological
of the extensor should
chronically
a-blockers
investigated.
remain:
tail reflex and the contraction the site of clonidine
the
are being
caudal
be determined
of NE
should
spinalized
rats.
and
muscle and
be done.
data
pharmacological
should
strict
selected
be more
comparative
These
when
are summarized
problems
central
in Table nature
precisely experiments are now
1.
of the clarified, on the
under
in
REFERENCES 1) BLOOM,F.E.: Amine receptors in CNS. I. Norepinephrine, in Handbook of Psychophar macology, Edited by IVERSEN,L.L., IVERSEN,S.D. AND SNYDER,S.H., Vol. 6, p. 1-22, Plenum Press, New York and London (1975)2) TAKAGI, H., INUKAI, T. AND NAKAMA,M.: A modi fication of Haffner's method for testing analgesics. Japan. J. Pharmacol. 16, 287-294 (1966) 3) MATSUSHITA,A., TAKESUE,H. AND KIDO, R.: Actions of morphine and narcotic antagonist analgesics on the spinal cord of acute and chronic spinal rats. Japan. J. Pharmacol. 21, 134-136 (1971)4) ANDEN, N.-E., GRABOWSKA,M. AND STROMBOM, U.: Different alpha-adrenoreceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents. Arch. Pharmacol. 292, 43-52 (1976)
