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Unit activity from cat area postrema influenced by drugs
Brain Research, 92 (1975) 153-156
153
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Unit activity from cat area postrema influenced by drugs
H. L. BORISON, M. J. H A W K E N , J. !. H U B B A R D AND N A N C Y E. SIRETT
Department of Physiology, University of Otago Medical School, P.O. Box 913, Dunedin (New Zealand) (Accepted March 24th, 1975)
The area postrema (AP) is the most caudal of the 'circumventricular' group of organs in the brain, situated at the obex of the IVth ventricle 1. It has been shown to act as a chemoreceptor for vomitingZ,~, v. The present study was undertaken to more closely examine this function. Firstly, we attempted to record electrical indications of receptor activity from AP and secondly, when such activity was found, we attempted to influence it by drugs known to cause vomiting and by a possible neurochemical mediator. Experiments were performed on 20 cats anesthetized with intraperitoneal pentobarbital sodium. The cat's head was fixed in a stereotaxic frame and the AP was exposed by removing the overlying occipital bone and part of the cerebellum. A retractor, positioned against the remaining cerebellum, served as the reference electrode. Extracellular action potentials (units) were recorded from the medulla oblongata with 4 M NaCl-filled glass mieroelectrodes of 0.5 to 2.0 M~) impedance. Electrodes with this low impedance were selected for an initial survey designed to answer the question 'is there any electrical activity in the AP'? Conventional techniques were used for amplification, display and photography of the units. Electrode placement was oriented by means of an Eccles micromanipulator controlled under direct visualization. Body temperature was maintained near normal and arterial blood pressure was monitored throughout. Temporary support of ventilation was required in a few instances immediately following the cerehellar extirpation. The drugs employed were ouabain octahydrate, adenosine triphosphate disodium (ATP) and apomorphine hydrochloride. The drugs were injected in most cases through a catheter inserted upstream into the right subclavian artery to lie near the origin of the vertebral artery after all other branches had been ligated. In addition, the vertebral-occipital anastomosis on the same side was interrupted at the bifurcation of the common carotid artery, which resulted as well in division of the carotid sinus nerve. Drug access to the AP through the intravertebral route was verified by the injection of trypan blue at the conclusion of the experiment. Eight cats were subjected to right unilateral section of the cranial nerves IX, X, XI and XII plus the cervical sympathetic nerve close to the jugular foramen, in order to eliminate afferent impulses to periventricular structures of the lower medulla oblongata and to prevent
154
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'
Ts
Fig. 1. Loci of drug-reactive units in the vicinity of the area postrema. Upper panel : right side surface view of the medulla oblongata. The fine dashed line represents the point of attachment of the tela chorioidae to the margin of the IVth ventricle. The heavy dashed line shows the level of transection corresponding to the transverse section of the medulla given below. Lower panel: dashed line grid indicates 0.5 mm intervals used for stereotaxic orientation. All of the unit loci in the upper panel are projected onto the single plane of the lower panel. Dots, ouabain activated units, circles, ATP activated units. AP, area postrema; CTZ, chemoreceptor trigger zone; GN, gracile nucleus; FS, fasciculus solitarius; X, dorsal vagal nucleus; XII, hypoglossal nucleus; IV Vent., IVth ventricle.
indirect activation of units in the vicinity of AP by drug actions elsewhere. The drugs were on occasion administered intravenously for comparison with the effects of intravertebral injection. Apomorphine was also applied to the floor of the IVth ventricle. Fig. 1 shows a surface view of the medulla oblongata with the electrode points of penetration corresponding to the drug-reactive unit loci marked below in the transverse plane at the indicated depths derived from m~cromanipulator readings. No attempt was made to localize all spontaneously active units. However, the following criteria apply to those units that were examined for drug reactivity. The field of exploration was restricted to the AP and the structures approximately 1 mm deep and lateral to it. Units in the gracile and cuneate nuclei were identified by stroking the appropriate s kin regions, and were rejected as such. Units firing synchronously with the respiratory or cardiac rhythms were likewise eliminated from consideration. Thus, remaining units were independent of the said somatosensory and homeostatic reflex processes, and only those units are shown that were held sufficiently long for observation of a drug activating effect. It will be noticed that the points are clustered around the margin of AP previously characterized as the emetic chemoreceptor trigger zone (CTZ) 2,3. The initial frequency of such units varied from 1 to 10/sec with amplitudes between 50 and 2(10/~V. Some of the variability may be attributable to a cumulative drug effect, which is suggested by the impression that units became more readily available as the experiment progressed. On the other hand, units were seldom found
155
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.
ii.:!::!:::
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Fig. 2. A: time course of a unit activation by ouabain, 10/~g in 1 ml solution, injected into the vertebral artery over a period of 10 sec (stippled section in graph). B" recorded unit activity at representative times is shown in the panel on the right. Calibration signal is 100 msec (baseline) and 50/zV (upright). Faint action potentials have been retouched. Note large unit recruited during injection of ouabain.
in the body of the AP and these were generally small in amplitude (less than 50 #V) and could be retained for only a few seconds. The effects of ouabain were studied in 11 cats. The drug was given intravertebrally in amounts of 5 or 10/~g in 1 ml of solution injected over a period of 10-30 sec, and was repeated at intervals as short as l 0 min. Eleven units in 6 cats were accelerated within 20-30 sec, lasting less than 1 rain, after the intra-arterial injection of ouabain (e.g., Fig. 2). In 4 instances the effect of ouabain was repeated, but in these units there was a reduction in response with the second dose, both in degree of acceleration and duration, suggesting the development of tachyphylaxis. The magnitude of the acceleration varied over a 3- to 1C0-fold range, depending inversely on the initial frequency. In most trials, additional units were recruited by the drug and the recruited units accelerated in parallel with the original unit. These effects could not be attributed to changes in blood pressure which did not occur until the cumulative dose level reached 60-80 Fg/kg when signs of cardiotoxicity had already become evident. Ouabain failed to produce an effect on 2 units, one of which was tested by intravenous administration with two doses of 10/~g/kg. Four further units lying outside the AP (2 with a respiratory rhythm, lying deep to AP and 2 in the gracile nuclei) were also tested by intravertebral injection without any effect being detected. ATP (I mg/ml per dose injected intravertebrally) was administered as a possible mediator of the action of ouabain 6, and was found to have marked transient acceleratory effects independent of the presence of ouabain. Ten units responded to ATP in 5 cats. Recruitment of units was also observed, as with ouabain. In one experiment
156 a unit was accelerated on 3 successive trials of ATP without evidence of tachyphylaxis. In another case, the effect of ATP was augmented after the administration of ouabain which was ineffective in itself. ATP had no significant influence on the blood pressure. Apomorphine (10 /zg/ml) was tried in 4 cats through the various routes of administration but proved difficult to characterize. An assortment of effects was obtained which did not appear to be adventitious inasmuch as particular responses were repeatable. Apomorphine also caused a fall in blood pressure regardless of the route by which it was given. Our working hypothesis that drug-induced unit activation resulted from chemoreceptor stimulation in AP is supported by the following observations. (1) Units were found mainly in the corridor between AP and the nucleus of the fasciculus solitarius where fibers pass between the CTZ and the vomiting center in the lateral reticular formation. (2) Other known fiber connections of the AP, e.g., to the dorsal vagal nuclei 4, account for those units found ventral to AP. (3) Unit activity was accelerated in the presence of extensive medullary denervation. It seems probable, therefore, that units of the CTZ are spontaneously active and can be accelerated by ouabain, as would be expected i f A P contained the chemoreceptors for vomiting induced by the drug. Furthermore, ouabain could act through the agency of ATP by inhibiting receptor ATPase in a manner analogous to the action of cholinesterase inhibitors in the acetycholine-cholinesterase system. It is also reasonable that the AP should show no unit potentials in performing the role of a chemical transducer that activates the CTZ 5. The effects of apomorphine do not lend themselves to a suitable explanation at this time, but it is apparent that we are dealing with multiple actions of the drug. This work was supported in part by the Medical Research Council of New Zealand and by the United States Public Health Service (Grant NB-04456). Dr. Borison was a Faculty Scholar of the Macy Foundation on sabbatical leave from Dartmouth Medical School in Hanover, N e w Hampshire, U.S.A.
1 BORISON,H. L., Area postrema: chemoreceptor trigger zone for vomiting - - is that all? Life Sci., 14 (1974) 1807-1817. 2 BORISON,H. L., AND BRIZZEE, K. R,, Morphology of emetic chemoreceptor trigger zone in cat medulla oblongata, Proc. Soc. exp. BioL (N.Y.), 77 (1951) 38-42. 3 BORISON,H. L., ANDWANG,S. C., Physiology and pharmacology of vomiting, Pharmacol. Rev., 5 (1953) 193-230. 4 MOREST,D. K., Experimental study of the projections of the tractus solitarius and the area postrema in the cat, J. comp. Neurol., 130 (1967) 277-299. 5 PAINTAL,A. S., Action of drugs on sensory nerve endings, Ann. Rev. PharmacoL, 11 (1971) 231-240. 6 S~:ou, J. C., Enzymatic basis for active transport of Na + and K + across cell membrane, Physiol. Rev., 45 (1965) 596-617. 7 WANG,S. C., AND BORISON,H. L., The vomiting center: a critical experimental analysis, Arch. NeuroL Psychiat., 63 (1950) 928-941.
153
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Unit activity from cat area postrema influenced by drugs
H. L. BORISON, M. J. H A W K E N , J. !. H U B B A R D AND N A N C Y E. SIRETT
Department of Physiology, University of Otago Medical School, P.O. Box 913, Dunedin (New Zealand) (Accepted March 24th, 1975)
The area postrema (AP) is the most caudal of the 'circumventricular' group of organs in the brain, situated at the obex of the IVth ventricle 1. It has been shown to act as a chemoreceptor for vomitingZ,~, v. The present study was undertaken to more closely examine this function. Firstly, we attempted to record electrical indications of receptor activity from AP and secondly, when such activity was found, we attempted to influence it by drugs known to cause vomiting and by a possible neurochemical mediator. Experiments were performed on 20 cats anesthetized with intraperitoneal pentobarbital sodium. The cat's head was fixed in a stereotaxic frame and the AP was exposed by removing the overlying occipital bone and part of the cerebellum. A retractor, positioned against the remaining cerebellum, served as the reference electrode. Extracellular action potentials (units) were recorded from the medulla oblongata with 4 M NaCl-filled glass mieroelectrodes of 0.5 to 2.0 M~) impedance. Electrodes with this low impedance were selected for an initial survey designed to answer the question 'is there any electrical activity in the AP'? Conventional techniques were used for amplification, display and photography of the units. Electrode placement was oriented by means of an Eccles micromanipulator controlled under direct visualization. Body temperature was maintained near normal and arterial blood pressure was monitored throughout. Temporary support of ventilation was required in a few instances immediately following the cerehellar extirpation. The drugs employed were ouabain octahydrate, adenosine triphosphate disodium (ATP) and apomorphine hydrochloride. The drugs were injected in most cases through a catheter inserted upstream into the right subclavian artery to lie near the origin of the vertebral artery after all other branches had been ligated. In addition, the vertebral-occipital anastomosis on the same side was interrupted at the bifurcation of the common carotid artery, which resulted as well in division of the carotid sinus nerve. Drug access to the AP through the intravertebral route was verified by the injection of trypan blue at the conclusion of the experiment. Eight cats were subjected to right unilateral section of the cranial nerves IX, X, XI and XII plus the cervical sympathetic nerve close to the jugular foramen, in order to eliminate afferent impulses to periventricular structures of the lower medulla oblongata and to prevent
154
. /<...j
/ /_
DI 0-Smm~kN i GN! '" .... i
VI_.VT,
'
Ts
Fig. 1. Loci of drug-reactive units in the vicinity of the area postrema. Upper panel : right side surface view of the medulla oblongata. The fine dashed line represents the point of attachment of the tela chorioidae to the margin of the IVth ventricle. The heavy dashed line shows the level of transection corresponding to the transverse section of the medulla given below. Lower panel: dashed line grid indicates 0.5 mm intervals used for stereotaxic orientation. All of the unit loci in the upper panel are projected onto the single plane of the lower panel. Dots, ouabain activated units, circles, ATP activated units. AP, area postrema; CTZ, chemoreceptor trigger zone; GN, gracile nucleus; FS, fasciculus solitarius; X, dorsal vagal nucleus; XII, hypoglossal nucleus; IV Vent., IVth ventricle.
indirect activation of units in the vicinity of AP by drug actions elsewhere. The drugs were on occasion administered intravenously for comparison with the effects of intravertebral injection. Apomorphine was also applied to the floor of the IVth ventricle. Fig. 1 shows a surface view of the medulla oblongata with the electrode points of penetration corresponding to the drug-reactive unit loci marked below in the transverse plane at the indicated depths derived from m~cromanipulator readings. No attempt was made to localize all spontaneously active units. However, the following criteria apply to those units that were examined for drug reactivity. The field of exploration was restricted to the AP and the structures approximately 1 mm deep and lateral to it. Units in the gracile and cuneate nuclei were identified by stroking the appropriate s kin regions, and were rejected as such. Units firing synchronously with the respiratory or cardiac rhythms were likewise eliminated from consideration. Thus, remaining units were independent of the said somatosensory and homeostatic reflex processes, and only those units are shown that were held sufficiently long for observation of a drug activating effect. It will be noticed that the points are clustered around the margin of AP previously characterized as the emetic chemoreceptor trigger zone (CTZ) 2,3. The initial frequency of such units varied from 1 to 10/sec with amplitudes between 50 and 2(10/~V. Some of the variability may be attributable to a cumulative drug effect, which is suggested by the impression that units became more readily available as the experiment progressed. On the other hand, units were seldom found
155
OUABAIN,I.A.
~
BeFo
34 -spikes/sec :.:::.:.:
.
ii.:!::!:::
35see.
::::::..:
"/"
~4 -iiii!ii!ii!i .......
40
sec
BO
70sec.
Fig. 2. A: time course of a unit activation by ouabain, 10/~g in 1 ml solution, injected into the vertebral artery over a period of 10 sec (stippled section in graph). B" recorded unit activity at representative times is shown in the panel on the right. Calibration signal is 100 msec (baseline) and 50/zV (upright). Faint action potentials have been retouched. Note large unit recruited during injection of ouabain.
in the body of the AP and these were generally small in amplitude (less than 50 #V) and could be retained for only a few seconds. The effects of ouabain were studied in 11 cats. The drug was given intravertebrally in amounts of 5 or 10/~g in 1 ml of solution injected over a period of 10-30 sec, and was repeated at intervals as short as l 0 min. Eleven units in 6 cats were accelerated within 20-30 sec, lasting less than 1 rain, after the intra-arterial injection of ouabain (e.g., Fig. 2). In 4 instances the effect of ouabain was repeated, but in these units there was a reduction in response with the second dose, both in degree of acceleration and duration, suggesting the development of tachyphylaxis. The magnitude of the acceleration varied over a 3- to 1C0-fold range, depending inversely on the initial frequency. In most trials, additional units were recruited by the drug and the recruited units accelerated in parallel with the original unit. These effects could not be attributed to changes in blood pressure which did not occur until the cumulative dose level reached 60-80 Fg/kg when signs of cardiotoxicity had already become evident. Ouabain failed to produce an effect on 2 units, one of which was tested by intravenous administration with two doses of 10/~g/kg. Four further units lying outside the AP (2 with a respiratory rhythm, lying deep to AP and 2 in the gracile nuclei) were also tested by intravertebral injection without any effect being detected. ATP (I mg/ml per dose injected intravertebrally) was administered as a possible mediator of the action of ouabain 6, and was found to have marked transient acceleratory effects independent of the presence of ouabain. Ten units responded to ATP in 5 cats. Recruitment of units was also observed, as with ouabain. In one experiment
156 a unit was accelerated on 3 successive trials of ATP without evidence of tachyphylaxis. In another case, the effect of ATP was augmented after the administration of ouabain which was ineffective in itself. ATP had no significant influence on the blood pressure. Apomorphine (10 /zg/ml) was tried in 4 cats through the various routes of administration but proved difficult to characterize. An assortment of effects was obtained which did not appear to be adventitious inasmuch as particular responses were repeatable. Apomorphine also caused a fall in blood pressure regardless of the route by which it was given. Our working hypothesis that drug-induced unit activation resulted from chemoreceptor stimulation in AP is supported by the following observations. (1) Units were found mainly in the corridor between AP and the nucleus of the fasciculus solitarius where fibers pass between the CTZ and the vomiting center in the lateral reticular formation. (2) Other known fiber connections of the AP, e.g., to the dorsal vagal nuclei 4, account for those units found ventral to AP. (3) Unit activity was accelerated in the presence of extensive medullary denervation. It seems probable, therefore, that units of the CTZ are spontaneously active and can be accelerated by ouabain, as would be expected i f A P contained the chemoreceptors for vomiting induced by the drug. Furthermore, ouabain could act through the agency of ATP by inhibiting receptor ATPase in a manner analogous to the action of cholinesterase inhibitors in the acetycholine-cholinesterase system. It is also reasonable that the AP should show no unit potentials in performing the role of a chemical transducer that activates the CTZ 5. The effects of apomorphine do not lend themselves to a suitable explanation at this time, but it is apparent that we are dealing with multiple actions of the drug. This work was supported in part by the Medical Research Council of New Zealand and by the United States Public Health Service (Grant NB-04456). Dr. Borison was a Faculty Scholar of the Macy Foundation on sabbatical leave from Dartmouth Medical School in Hanover, N e w Hampshire, U.S.A.
1 BORISON,H. L., Area postrema: chemoreceptor trigger zone for vomiting - - is that all? Life Sci., 14 (1974) 1807-1817. 2 BORISON,H. L., AND BRIZZEE, K. R,, Morphology of emetic chemoreceptor trigger zone in cat medulla oblongata, Proc. Soc. exp. BioL (N.Y.), 77 (1951) 38-42. 3 BORISON,H. L., ANDWANG,S. C., Physiology and pharmacology of vomiting, Pharmacol. Rev., 5 (1953) 193-230. 4 MOREST,D. K., Experimental study of the projections of the tractus solitarius and the area postrema in the cat, J. comp. Neurol., 130 (1967) 277-299. 5 PAINTAL,A. S., Action of drugs on sensory nerve endings, Ann. Rev. PharmacoL, 11 (1971) 231-240. 6 S~:ou, J. C., Enzymatic basis for active transport of Na + and K + across cell membrane, Physiol. Rev., 45 (1965) 596-617. 7 WANG,S. C., AND BORISON,H. L., The vomiting center: a critical experimental analysis, Arch. NeuroL Psychiat., 63 (1950) 928-941.