192
Brain Research, 533 (1990) 192-195 Elsevier
BRES 16042
a-Adrenergic receptor agonists, but not antagonists, alter the tail-flick latency when microinjected into the rostral ventromedial medulla of the lightly anesthetized rat* C.M. Haws, M.M. Heinricher and H.L. Fields Departments of Neurology and Physiology, University of California at San Francisco, San Francisco, CA 94143 (U.S.A.) (Accepted 29 May 1990) Key words: Noradrenaline; Brainstem; Antinociception; Microinjection; Rat
The present experiments, part of an ongoing study designed to characterise the role of norepinephrine (NE) in regulating the activity of putative nociceptive modulatory neurons in the rostral ventromedial medulla (RVM), assessed the effects of a-adrenergic receptor-selective agents on the nociceptive threshold (as measured by the tail-flick withdrawal response on noxious heat). These microinjection studies were carried out in the barbiturate-anesthetized rat, a preparation which is favourable for acute neurophysiological studies. The data obtained demonstrate that, as observed by others in the awake animal, activation of a2-adrenergic receptors in the RVM produces hypoalgesia. However, unlike in the awake animal, when antagonists selective for either the a 1- or a2-adrenergic receptor are microinjected alone into the RVM there is no change in the nociceptive threshold. These data suggest that the a2-adrenergic receptor has a postsynaptic location and that barbiturate anaesthesia suppresses a tonically active or noxious stimulus-activated noradrenergic input to the RVM that is present in the awake animal.
INTRODUCTION Neurons in the rostral ventromedial medulla (RVM), an area that includes the nucleus raphe magnus and adjacent reticular formation, have been demonstrated to play a major role in modulating the transmission of nociceptive information at spinal levels 3"6'7"14'15. The activity of these R V M neurons is, in turn, regulated by inputs from other areas of the brainstem 1"1°, including areas demonstrated to contain norepinephrine (NE) 9" 11.18. Microinjection of a-adrenergic receptor-selective agonists and antagonists into R V M produces significant alterations in the nociceptive threshold of awake rats 11'17. Thus, microinjection of an al-receptor antagonist or of an a2-receptor agonist prolongs the latency of the tail-flick withdrawal response (TF) elicited by noxious heat, whereas microinjection of an a~-receptor agonist or an a2-receptor antagonist shortens T F latency. These data demonstrate that, in the awake animal, activation of a~- and a2-adrenergic receptors in RVM have opposing effects on the nociceptive threshold and, further, indicate that nociceptive modulatory neurons in R V M are u n d e r the influence of a tonic or noxious stimulus-activated noradrenergic input. In contrast with the behavioral studies, investigations
of the cellular mechanisms underlying a 1- and a zreceptor-mediated effects in R V M have been u n d e r t a k e n in the anesthetized animal 12'19'z°. Interpretation of these neurophysiological studies in terms of their behavioral consequences requires that the behavioral and cellular responses be compared u n d e r similar experimental conditions. Thus, in order to characterize the role of N E in regulating the activity of putative nociceptive modulating neurons in the RVM, the present behavioral experiments assessed the effects of microinjected a-adrenergic receptor-selective agents in the barbiturate-anesthetized rat.
MATERIALS AND METHODS Male Sprague-Dawley rats (250-275 g) were initially anesthetized with pentobarbital sodium (55 mg/kg, i.p.), and a cannula inserted into an external jugular vein. The animals were placed in a stereotaxic frame, and a 25-gauge stainless steel guide cannula was lowered through the brainstem to a position 2 mm dorsal to the desired injection site in the RVM. The animals were subsequently maintained in a lightly anesthetized state using a continous infusion of methohexital (15-30 mg/kg/h, i.v.). As described previously, animals show no signs of discomfort at this level of anesthesia. The microinjection protocol was initiated 30 min after starting the methohexital infusion. Body temperature was maintained at approximately 37 °C using a circulating hot-water pad. The TF response was evoked using a projector lamp focussed on the blackened ventral surface of the tail. A thermistor probe placed
* A preliminary report of these findings was presented at the 1988 Annual Meeting of the Society for Neuroscience. Correspondence: C.M. Haws, Department of Pharmacology, University of California, San Francisco, CA 94143, U.S.A. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
193 in contact with the tail surface provided a continuous signal for feedback control of the heat stimulus, which consisted of a linear temperature ramp rising at 1.8 °C/s from a 35 °C holding temperature to a maximum of 53 °C. The heat stimulus was terminated automatically upon occurrence of the TF, or, if no TF occurred, after 10 s had elapsed. TF latency was measured at 5-min intervals. Following determination of baseline latency (mean of 3 consecutive TF trials), an injection was made into the RVM. TF latencies were then determined at 5-min intervals for a minimum of 30 min. In those experiments in which the ability of antagonists to reverse the antinociceptive action of agonist microinjections was tested, the antagonists were administered (by microinjection or systemic administration) 15 min after the agonist microinjection. Drugs were microinjected into RVM through a 31-gauge stainless steel injection cannula inserted through and protruding 2 mm beyond the tip of the guide cannula. The injector was attached to a 1-pl Hamilton syringe by a length of polyethylene tubing (PE-20). A volume of 0.25/d of drug dissolved in 165 mM saline was injected into the RVM over a period of 3 min. The efficacy of the drug injection was monitored by watching the movement of a small air bubble through the tubing. The injection cannula was left in place for approximately 10 min after the injection to minimize flow of the drug solution back up the injector track. Drugs microinjected included: clonidine-HCl (CLON; at-receptor agonist); norepinephrine hydrochloride (NE; a~/az-receptor agonist); yohimbine-HCl (YOH; a2-receptor antagonist); idazoxan (IDA; a2-receptor antagonist) and WB4101 (al-receptor antagonist); phentolamine-HCl (PHEN; cq/a2-receptor antagonist). All drugs were obtained from Sigma or Research Biochemicals Inc., except for IDA, which was a generous gift of Reckitt and Colman. At the completion of the experiment animals were perfused intracardially with 0.9% saline followed by 10% formalin solution. Injection sites were histologically verified and plotted on standardized sections derived from the stereotaxic atlas of Paxinos and Watson ~6.
M e a n baseline T F latency in these experiments was 4.8 + 0.1 s ( m e a n + S.E.M., n = 102). Microinjection of
11111
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(,,) z
,~
In RVM ou~ICM RVI I
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_1
0
(s)
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Brain Research, 533 (1990) 192-195 Elsevier
BRES 16042
a-Adrenergic receptor agonists, but not antagonists, alter the tail-flick latency when microinjected into the rostral ventromedial medulla of the lightly anesthetized rat* C.M. Haws, M.M. Heinricher and H.L. Fields Departments of Neurology and Physiology, University of California at San Francisco, San Francisco, CA 94143 (U.S.A.) (Accepted 29 May 1990) Key words: Noradrenaline; Brainstem; Antinociception; Microinjection; Rat
The present experiments, part of an ongoing study designed to characterise the role of norepinephrine (NE) in regulating the activity of putative nociceptive modulatory neurons in the rostral ventromedial medulla (RVM), assessed the effects of a-adrenergic receptor-selective agents on the nociceptive threshold (as measured by the tail-flick withdrawal response on noxious heat). These microinjection studies were carried out in the barbiturate-anesthetized rat, a preparation which is favourable for acute neurophysiological studies. The data obtained demonstrate that, as observed by others in the awake animal, activation of a2-adrenergic receptors in the RVM produces hypoalgesia. However, unlike in the awake animal, when antagonists selective for either the a 1- or a2-adrenergic receptor are microinjected alone into the RVM there is no change in the nociceptive threshold. These data suggest that the a2-adrenergic receptor has a postsynaptic location and that barbiturate anaesthesia suppresses a tonically active or noxious stimulus-activated noradrenergic input to the RVM that is present in the awake animal.
INTRODUCTION Neurons in the rostral ventromedial medulla (RVM), an area that includes the nucleus raphe magnus and adjacent reticular formation, have been demonstrated to play a major role in modulating the transmission of nociceptive information at spinal levels 3"6'7"14'15. The activity of these R V M neurons is, in turn, regulated by inputs from other areas of the brainstem 1"1°, including areas demonstrated to contain norepinephrine (NE) 9" 11.18. Microinjection of a-adrenergic receptor-selective agonists and antagonists into R V M produces significant alterations in the nociceptive threshold of awake rats 11'17. Thus, microinjection of an al-receptor antagonist or of an a2-receptor agonist prolongs the latency of the tail-flick withdrawal response (TF) elicited by noxious heat, whereas microinjection of an a~-receptor agonist or an a2-receptor antagonist shortens T F latency. These data demonstrate that, in the awake animal, activation of a~- and a2-adrenergic receptors in RVM have opposing effects on the nociceptive threshold and, further, indicate that nociceptive modulatory neurons in R V M are u n d e r the influence of a tonic or noxious stimulus-activated noradrenergic input. In contrast with the behavioral studies, investigations
of the cellular mechanisms underlying a 1- and a zreceptor-mediated effects in R V M have been u n d e r t a k e n in the anesthetized animal 12'19'z°. Interpretation of these neurophysiological studies in terms of their behavioral consequences requires that the behavioral and cellular responses be compared u n d e r similar experimental conditions. Thus, in order to characterize the role of N E in regulating the activity of putative nociceptive modulating neurons in the RVM, the present behavioral experiments assessed the effects of microinjected a-adrenergic receptor-selective agents in the barbiturate-anesthetized rat.
MATERIALS AND METHODS Male Sprague-Dawley rats (250-275 g) were initially anesthetized with pentobarbital sodium (55 mg/kg, i.p.), and a cannula inserted into an external jugular vein. The animals were placed in a stereotaxic frame, and a 25-gauge stainless steel guide cannula was lowered through the brainstem to a position 2 mm dorsal to the desired injection site in the RVM. The animals were subsequently maintained in a lightly anesthetized state using a continous infusion of methohexital (15-30 mg/kg/h, i.v.). As described previously, animals show no signs of discomfort at this level of anesthesia. The microinjection protocol was initiated 30 min after starting the methohexital infusion. Body temperature was maintained at approximately 37 °C using a circulating hot-water pad. The TF response was evoked using a projector lamp focussed on the blackened ventral surface of the tail. A thermistor probe placed
* A preliminary report of these findings was presented at the 1988 Annual Meeting of the Society for Neuroscience. Correspondence: C.M. Haws, Department of Pharmacology, University of California, San Francisco, CA 94143, U.S.A. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
193 in contact with the tail surface provided a continuous signal for feedback control of the heat stimulus, which consisted of a linear temperature ramp rising at 1.8 °C/s from a 35 °C holding temperature to a maximum of 53 °C. The heat stimulus was terminated automatically upon occurrence of the TF, or, if no TF occurred, after 10 s had elapsed. TF latency was measured at 5-min intervals. Following determination of baseline latency (mean of 3 consecutive TF trials), an injection was made into the RVM. TF latencies were then determined at 5-min intervals for a minimum of 30 min. In those experiments in which the ability of antagonists to reverse the antinociceptive action of agonist microinjections was tested, the antagonists were administered (by microinjection or systemic administration) 15 min after the agonist microinjection. Drugs were microinjected into RVM through a 31-gauge stainless steel injection cannula inserted through and protruding 2 mm beyond the tip of the guide cannula. The injector was attached to a 1-pl Hamilton syringe by a length of polyethylene tubing (PE-20). A volume of 0.25/d of drug dissolved in 165 mM saline was injected into the RVM over a period of 3 min. The efficacy of the drug injection was monitored by watching the movement of a small air bubble through the tubing. The injection cannula was left in place for approximately 10 min after the injection to minimize flow of the drug solution back up the injector track. Drugs microinjected included: clonidine-HCl (CLON; at-receptor agonist); norepinephrine hydrochloride (NE; a~/az-receptor agonist); yohimbine-HCl (YOH; a2-receptor antagonist); idazoxan (IDA; a2-receptor antagonist) and WB4101 (al-receptor antagonist); phentolamine-HCl (PHEN; cq/a2-receptor antagonist). All drugs were obtained from Sigma or Research Biochemicals Inc., except for IDA, which was a generous gift of Reckitt and Colman. At the completion of the experiment animals were perfused intracardially with 0.9% saline followed by 10% formalin solution. Injection sites were histologically verified and plotted on standardized sections derived from the stereotaxic atlas of Paxinos and Watson ~6.
M e a n baseline T F latency in these experiments was 4.8 + 0.1 s ( m e a n + S.E.M., n = 102). Microinjection of
11111
_m
(,,) z
,~
In RVM ou~ICM RVI I
(27) [7 /
7s
_1
0
(s)
E z
