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The effect of the potent opiate agonist etorphine on local energy metabolism in the isolated perfused rat brain
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002X-3908
20. pp 901 II> 904. 1981
X1.090901-04$02.00,0 Pergamon Press Ltd
THE EFFECT OF THE POTENT OPIATE AGONIST ETORPHINE ON LOCAL ENERGY METABOLISM IN THE ISOLATED PERFUSED RAT BRAIN
M. Wiister, B. Dirks*, J. Krieglstein" and A. Herz Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatric, Kraepelinstrasse 2, D-8000 Miinchen 40, and Institut fiir Pharmakologie und Toxikologie, Fachbereich Pharmazie und Lebensmittelchemie der Philipps-UniversitZt, Ketzerbach 63, 3550 Marburg/Lahn Federal Republic of Germany
(Accepted
7
J&j
1981)
Summary The isolated perfused rat brain preparation and the 14C-deoxyglucose technique were employed for investigating the effects of acute opiate administration on local energy metabolism in the rat brain. The opiate agonist etorphine decreased glucose utilization in most brain regions: however, in some areas such as the septum, hippocampus and particular thalamic and hypothalamic nuclei, the incorporation of 14C-deoxyglucose was significantly increased.
Introduction Biochemical energy for neuronal activity in the central nervous system is almost exclusively supplied by glucose metabolism. Accordingly, the recently developed 14C-deoxyglucose ( 14C-DOG) technique has proven a useful tool to visualize the functional activity of the CNS under normal and drugaltered conditions (Sokoloff, Reivich, Kennedy, Des Rosiers, Patlak, Pettigrew, Sakurada and Shinohara, 1977). Employing this method, several reports have shown increased or decreased rates of local cerebral glucose utilization (LCGU) induced by different pharmacological agents (e.g. Meibach, Glick, Cox and Maayani, 1979; Wechsler, Savaki and Sokoloff, 1979). In the case of opioids, however, the results of these studies may be invalidated by the severe respiratory depression induced by these drugs; thus, possibly masking the specific opiate effects (Sakurada, Shinohara, Klee, Kennedy and Sokoloff, 1976). The use of the isolated perfused rat brain preparation a peared advantageous over intact animals to study acute opiate effects on l&-DOG incorporation, especially since extensive studies have shown that glucose metabolism in this preparation is not significantly different from that in the intact organism (Krieglstein, Krieglstein and Stock, 1972). For these preliminary experiments, the potent narcotic analgesic etorphine was chosen as a compound with a relatively non-selective action on different types of opiate receptors (Wiister,Schulz and Herz, 1979). Preliminary reports on the visualization of opiate effects in the central nervous system of intact rats by the 14C-DOG technique have revealed effects of intracerebroventricularly administered O-endorphin in certain limbic structures (Henriksen, Morisson and Bloom, 1979).
901
902
Preliminary Notes
Methods
The present investigation employed the isolated rat brain preparation to study the incorporation of 14C-DOG into brain tissue. Preparation followed in general a technique described elsewhere, perfusing the brain via the carotis interna exclusively (Krieglstein et al., 1972). The perfusion medium employed was a synthetic fluorcarbon medium, as described by Dirks, Krieglstein, Lind, Rieger and Schiitz, (1980). Preparation of the brain was performed under urethane anaesthesia and EEG activity and velocity of flow were routinely monitored during the course of the experiment. After preparation, brains were equilibrated by a 30 min perfusion phase with drugfree fluorcarbon medium, during which the EEG returned to normal (B-activity). Thereafter, alternate brains in = 10) were either treated as controls or perfused with a medium containing etorphine (5 nM). This treatment did not alter the velocity of flow. 10 min later, 40 fiCi of 14C-DOG (New England Nuclear) were injected into the supplying catheters, followed by another 30 min perfusion with 14C-DOG-free medium. The brains were then rapidly removed from the skull and frozen on dry ice. Coronal 30 p sections were cut with a cryostat, mounted on glass slides and pressed onto Mamoray-T3-film fhgfa) for 5 days at -6OC. After autoradiography, sections were stained with toluidine blue in order to directly compare their histology with the X-ray images.
Results and Discussion Employing the isolated perfused rat brain preparation proved of critical importance for the significance of the obtained results. Earlier attempts to demonstrate acute opiate effects on LCGU in intact animals have been confronted with the overlying unspecific reduction of energy metabolism due to the respiratory depression induced by narcotics (Sakurada et al., 1976). Extensive studies upon the glucose utilization in the isolated rat brain preparation have proven the similarity with the events in the intact animal (Dirki et al., 1980; Krieglstein et al., 1972), and have subsequently been reaffirmed by the autoradiographs from the present experiments. Comparable to results obtained from intact ayirnal experiments reported elsewhere (Sokoloff et al., 1977), inspection of C-DOG labelling in slices from control preparations revealed an uneven distribution throughout the brain. One major discrepancy was found for4the hippocampus, which reportedly C-DOG in the intact animal displays an intermediate incorporation of (Sokoloff et al., 19771, whereas no significant labelling of this area was found in the control preparations in the present experiments.
Table 1 INCREASED INCORPORATION OF
14 C-DOG UPON ETORPHINE ADMINISTRATION
gyrus dentatus subiculum
++ ++ ++ +t
hippocampus
septum
n.
lateralis
hypothalamus
n. n. n. n.
ventromedialis periventricularis mamilaris anterior
+ + + +
thalamus
n. n.
ventralis medialis reticularis thalam
+ +
+ marked increase as compared to controls, ++ pronounced 14C-DOG incorporation with no detectable incorporation in controls.
Preliminary Notes
control
903
etorphine
Figure 1. Autoradiograms taken from control preparations (left) and etorphine-treated preparations (right). Upper panel: Sections showing lateral septum (sl and caudatoputamen (cp). Note pronounced activity ( 1*C-DOG incorporation) in the lateral septum and decreased activity in caudatoputamen in the etorphine-treated preparation. Lower panel: Sections showing pronounced incorporation of 14C-DOG in hippocampus (hi) upon etorphine treatment. Brain preparations receiving etorphine revealed a dramatic effect of the opiate on LCGU. Whereas most areas displayed a remarkedly lower demand for '*C-DOG incorporated, other areas showed an increased rate of energy metabolism (Table 1). This increase was especially pronounced in the septum and subiculum/hippocampus (Fig. 1). In this latter area, l*C-DOG incorporation appears to be especially concentrated throughout the pyramidal cell layer. Other areas with etorphine-increased energy metabolism were some particular thalamic and hypothalamic nuclei and a circumscribed area of the parietal cortex. Interestingly, these areas can be correlated with the predominant occurrence of g-opiate receptors as revealed by in-vitro autoradio-
904
Preliminary Notes
graphy (Duka, Schubert, Wiister, Stoiber and Herz, 1981). These results demonstrate that the narcotic analgesic etorphine induces sefective effects on LCGU in the isolated rat brain preparation, however, the basic mechanism for the observed changes in opiate-induced energy metabolism remains a matter of speculation. The decreased rate of 14C-DOG incorporation displayed in most brain areas may be associated with the inhibitory impact of opiates on neuronal activity. The peculiarity of an increased activity in some areas appears therefore of special interest and may be consistent with the recent results from electrophysiological experiments which demonstrated an opiate-induced excitation of hippocampal pyramidal cells via an inhibition of neighbouring, tonically inhibiting interneurones (Zieglggnsberger, French, Siggins and Bloom, 1979). Accordingly, similar events may be responsible for the increased LCGU in other areas. Another explanation would be opioidcontrolled connections as e.g. the known projections from the hippocampus to the lateral septum. This latter assumption would fit the recently suggested existence of "opiatergic pathways" connecting the structures of the limbic systems (Herkenham and Pert, 1980). Acknowledgements. We thank Drs. Th. Duka for her helpful advice in this investigation and G.T. Shearman for stylistic revision of the text. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn. References Dirks, B., Krieglstein, J., Lind, H.H., Rieger, H. and Schiitz, H. (1980). Fluorocarbon perfusion medium applied to the isolated rat brain. J. PharmacoL. Methods 4: 95-107. Duka, Th., Schubert, P., Wiister, M., Stoiber, R. and Herz, A. (1381). A selective distribution pattern of different opiate receptors in certain areas of rat brain as revealed by in vitro autoradiography. Neurosci. Lett. 21: 119-124. Henriksen, S.J., Morisson, F. and Bloom, F.E. (1979). B-Endorphin induced epileptiform activity increases local cerebral metabolism in hippocampus, amygdala and septum. Neurosci. Abstr. 528. Herkenham, M. and Pert, C-B., In vitro autoradiography of opiate receptors in rat brain suggests loci of "opiatergic" pathways. Proc. natl. Acad. Sci. USA 77: 4469-4473 (1980). Krieglstein, J., Krieglstein, G. and Stock, R. (1972). Suitability of the isolated perfused rat brain for studying effects on cerebral metabolism. Naunyn-Schmiedeberg's Arch. Pharmacol. 275: 124-134. Meibach, R.C., Glick, S.D., Cox, R. and Maayani, S. (1979). Localization of phencyclidine induced changes in brain energy metabolism. Nature 282: 625-626. Sakurada, O., Shinohara, M., Klee, W.A., Kennedy, C. and Sokoloff, L. (1976). Local cerebral glucose utilization following acute or chronic morphine administration and withdrawal. Neurosci. Abstr. 2: 613. Sokoloff, L., Reivich, M.# Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, 0. and Shinohara, M. (1977). The (14C)deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anaesthetized albino rat. J. Neurochem. 28: 897-916. Wechsler, L.R., Savaki, H.E. and Sokoloff, L. (1979). Effects of d- and l-amphetamine on local cerebral glucose utilization in the conscious rat. J. Neurochem. 32: 15-22. Wiister, M., Schulz, R. and Herz, A. (1979). Specificity of opioids towards the !.I-,6- and s-opiate receptors. Neurosci. Lett. 15: 193-198. ZieglgZnsberger, W., French, E.D., Siggins, G.R. and Bloom, F.E. (1979). Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons. Science 205: 415-417.
002X-3908
20. pp 901 II> 904. 1981
X1.090901-04$02.00,0 Pergamon Press Ltd
THE EFFECT OF THE POTENT OPIATE AGONIST ETORPHINE ON LOCAL ENERGY METABOLISM IN THE ISOLATED PERFUSED RAT BRAIN
M. Wiister, B. Dirks*, J. Krieglstein" and A. Herz Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatric, Kraepelinstrasse 2, D-8000 Miinchen 40, and Institut fiir Pharmakologie und Toxikologie, Fachbereich Pharmazie und Lebensmittelchemie der Philipps-UniversitZt, Ketzerbach 63, 3550 Marburg/Lahn Federal Republic of Germany
(Accepted
7
J&j
1981)
Summary The isolated perfused rat brain preparation and the 14C-deoxyglucose technique were employed for investigating the effects of acute opiate administration on local energy metabolism in the rat brain. The opiate agonist etorphine decreased glucose utilization in most brain regions: however, in some areas such as the septum, hippocampus and particular thalamic and hypothalamic nuclei, the incorporation of 14C-deoxyglucose was significantly increased.
Introduction Biochemical energy for neuronal activity in the central nervous system is almost exclusively supplied by glucose metabolism. Accordingly, the recently developed 14C-deoxyglucose ( 14C-DOG) technique has proven a useful tool to visualize the functional activity of the CNS under normal and drugaltered conditions (Sokoloff, Reivich, Kennedy, Des Rosiers, Patlak, Pettigrew, Sakurada and Shinohara, 1977). Employing this method, several reports have shown increased or decreased rates of local cerebral glucose utilization (LCGU) induced by different pharmacological agents (e.g. Meibach, Glick, Cox and Maayani, 1979; Wechsler, Savaki and Sokoloff, 1979). In the case of opioids, however, the results of these studies may be invalidated by the severe respiratory depression induced by these drugs; thus, possibly masking the specific opiate effects (Sakurada, Shinohara, Klee, Kennedy and Sokoloff, 1976). The use of the isolated perfused rat brain preparation a peared advantageous over intact animals to study acute opiate effects on l&-DOG incorporation, especially since extensive studies have shown that glucose metabolism in this preparation is not significantly different from that in the intact organism (Krieglstein, Krieglstein and Stock, 1972). For these preliminary experiments, the potent narcotic analgesic etorphine was chosen as a compound with a relatively non-selective action on different types of opiate receptors (Wiister,Schulz and Herz, 1979). Preliminary reports on the visualization of opiate effects in the central nervous system of intact rats by the 14C-DOG technique have revealed effects of intracerebroventricularly administered O-endorphin in certain limbic structures (Henriksen, Morisson and Bloom, 1979).
901
902
Preliminary Notes
Methods
The present investigation employed the isolated rat brain preparation to study the incorporation of 14C-DOG into brain tissue. Preparation followed in general a technique described elsewhere, perfusing the brain via the carotis interna exclusively (Krieglstein et al., 1972). The perfusion medium employed was a synthetic fluorcarbon medium, as described by Dirks, Krieglstein, Lind, Rieger and Schiitz, (1980). Preparation of the brain was performed under urethane anaesthesia and EEG activity and velocity of flow were routinely monitored during the course of the experiment. After preparation, brains were equilibrated by a 30 min perfusion phase with drugfree fluorcarbon medium, during which the EEG returned to normal (B-activity). Thereafter, alternate brains in = 10) were either treated as controls or perfused with a medium containing etorphine (5 nM). This treatment did not alter the velocity of flow. 10 min later, 40 fiCi of 14C-DOG (New England Nuclear) were injected into the supplying catheters, followed by another 30 min perfusion with 14C-DOG-free medium. The brains were then rapidly removed from the skull and frozen on dry ice. Coronal 30 p sections were cut with a cryostat, mounted on glass slides and pressed onto Mamoray-T3-film fhgfa) for 5 days at -6OC. After autoradiography, sections were stained with toluidine blue in order to directly compare their histology with the X-ray images.
Results and Discussion Employing the isolated perfused rat brain preparation proved of critical importance for the significance of the obtained results. Earlier attempts to demonstrate acute opiate effects on LCGU in intact animals have been confronted with the overlying unspecific reduction of energy metabolism due to the respiratory depression induced by narcotics (Sakurada et al., 1976). Extensive studies upon the glucose utilization in the isolated rat brain preparation have proven the similarity with the events in the intact animal (Dirki et al., 1980; Krieglstein et al., 1972), and have subsequently been reaffirmed by the autoradiographs from the present experiments. Comparable to results obtained from intact ayirnal experiments reported elsewhere (Sokoloff et al., 1977), inspection of C-DOG labelling in slices from control preparations revealed an uneven distribution throughout the brain. One major discrepancy was found for4the hippocampus, which reportedly C-DOG in the intact animal displays an intermediate incorporation of (Sokoloff et al., 19771, whereas no significant labelling of this area was found in the control preparations in the present experiments.
Table 1 INCREASED INCORPORATION OF
14 C-DOG UPON ETORPHINE ADMINISTRATION
gyrus dentatus subiculum
++ ++ ++ +t
hippocampus
septum
n.
lateralis
hypothalamus
n. n. n. n.
ventromedialis periventricularis mamilaris anterior
+ + + +
thalamus
n. n.
ventralis medialis reticularis thalam
+ +
+ marked increase as compared to controls, ++ pronounced 14C-DOG incorporation with no detectable incorporation in controls.
Preliminary Notes
control
903
etorphine
Figure 1. Autoradiograms taken from control preparations (left) and etorphine-treated preparations (right). Upper panel: Sections showing lateral septum (sl and caudatoputamen (cp). Note pronounced activity ( 1*C-DOG incorporation) in the lateral septum and decreased activity in caudatoputamen in the etorphine-treated preparation. Lower panel: Sections showing pronounced incorporation of 14C-DOG in hippocampus (hi) upon etorphine treatment. Brain preparations receiving etorphine revealed a dramatic effect of the opiate on LCGU. Whereas most areas displayed a remarkedly lower demand for '*C-DOG incorporated, other areas showed an increased rate of energy metabolism (Table 1). This increase was especially pronounced in the septum and subiculum/hippocampus (Fig. 1). In this latter area, l*C-DOG incorporation appears to be especially concentrated throughout the pyramidal cell layer. Other areas with etorphine-increased energy metabolism were some particular thalamic and hypothalamic nuclei and a circumscribed area of the parietal cortex. Interestingly, these areas can be correlated with the predominant occurrence of g-opiate receptors as revealed by in-vitro autoradio-
904
Preliminary Notes
graphy (Duka, Schubert, Wiister, Stoiber and Herz, 1981). These results demonstrate that the narcotic analgesic etorphine induces sefective effects on LCGU in the isolated rat brain preparation, however, the basic mechanism for the observed changes in opiate-induced energy metabolism remains a matter of speculation. The decreased rate of 14C-DOG incorporation displayed in most brain areas may be associated with the inhibitory impact of opiates on neuronal activity. The peculiarity of an increased activity in some areas appears therefore of special interest and may be consistent with the recent results from electrophysiological experiments which demonstrated an opiate-induced excitation of hippocampal pyramidal cells via an inhibition of neighbouring, tonically inhibiting interneurones (Zieglggnsberger, French, Siggins and Bloom, 1979). Accordingly, similar events may be responsible for the increased LCGU in other areas. Another explanation would be opioidcontrolled connections as e.g. the known projections from the hippocampus to the lateral septum. This latter assumption would fit the recently suggested existence of "opiatergic pathways" connecting the structures of the limbic systems (Herkenham and Pert, 1980). Acknowledgements. We thank Drs. Th. Duka for her helpful advice in this investigation and G.T. Shearman for stylistic revision of the text. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn. References Dirks, B., Krieglstein, J., Lind, H.H., Rieger, H. and Schiitz, H. (1980). Fluorocarbon perfusion medium applied to the isolated rat brain. J. PharmacoL. Methods 4: 95-107. Duka, Th., Schubert, P., Wiister, M., Stoiber, R. and Herz, A. (1381). A selective distribution pattern of different opiate receptors in certain areas of rat brain as revealed by in vitro autoradiography. Neurosci. Lett. 21: 119-124. Henriksen, S.J., Morisson, F. and Bloom, F.E. (1979). B-Endorphin induced epileptiform activity increases local cerebral metabolism in hippocampus, amygdala and septum. Neurosci. Abstr. 528. Herkenham, M. and Pert, C-B., In vitro autoradiography of opiate receptors in rat brain suggests loci of "opiatergic" pathways. Proc. natl. Acad. Sci. USA 77: 4469-4473 (1980). Krieglstein, J., Krieglstein, G. and Stock, R. (1972). Suitability of the isolated perfused rat brain for studying effects on cerebral metabolism. Naunyn-Schmiedeberg's Arch. Pharmacol. 275: 124-134. Meibach, R.C., Glick, S.D., Cox, R. and Maayani, S. (1979). Localization of phencyclidine induced changes in brain energy metabolism. Nature 282: 625-626. Sakurada, O., Shinohara, M., Klee, W.A., Kennedy, C. and Sokoloff, L. (1976). Local cerebral glucose utilization following acute or chronic morphine administration and withdrawal. Neurosci. Abstr. 2: 613. Sokoloff, L., Reivich, M.# Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, 0. and Shinohara, M. (1977). The (14C)deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anaesthetized albino rat. J. Neurochem. 28: 897-916. Wechsler, L.R., Savaki, H.E. and Sokoloff, L. (1979). Effects of d- and l-amphetamine on local cerebral glucose utilization in the conscious rat. J. Neurochem. 32: 15-22. Wiister, M., Schulz, R. and Herz, A. (1979). Specificity of opioids towards the !.I-,6- and s-opiate receptors. Neurosci. Lett. 15: 193-198. ZieglgZnsberger, W., French, E.D., Siggins, G.R. and Bloom, F.E. (1979). Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons. Science 205: 415-417.