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Morphine-induced alterations of local cerebral glucose utilization in the basal ganglia of rats
Brain Research, 497 (1989) 205-213
205
Elsevier BRES 14789
Research Reports
Morphine-induced alterations of local cerebral glucose utilization in the basal ganglia of rats T. Beck, J. Wenzel, K. Kuschinsky and J. Krieglstein Institute for Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Marburg (F.R. G.) (Accepted 14 February 1989)
Key words: Morphine; Basal ganglia; 2-Deoxyglucose; Local cerebral glucose utilization The actions of various doses of morphine on the local cerebral glucose utilization (LCGU) were studied by means of the autoradiographic [t4C]2-deoxyglucose technique. Morphine (1-15 mg/kg i.p.) decreased LCGU in most areas of the basal ganglia (caudate nucleus, globus pallidus, nucleus accumbens), but not in the substantia nigra pars compacta. LCGU was also decreased in limbic nuclei, such as sePtum, hippocampus and amygdala, and in most thalamic areas. In most cortical regions, a decrease was found as well. Findings in some efferent nuclei seemed of particular interest, namely in the substantia nigra pars reticulata, anteroventral and lateral nucleus of the thalamus and the subthalamic nucleus, where decreases in LCGU were found after administration of 7.5 mg/kg or sometimes lower doses, but not after 15 mg/kg of morphine. The decreases seem to reflect a general depressory effect of morphine on neuronal activity which is known from electrophysiologicalstudies. Part of these effects might be, in addition, due to an activation of dopaminergic neurons, since dopamine mainly acts as an inhibitory neurotransmitter. This dopaminergic activation leads to characteristic behavioral effects after lower doses of morphine. The largest dose used (15 mg/kg) produces muscular rigidity, probably by a direct action on the striatum. This effect antagonizes and masks the dopaminomimetic effects. The results suggest that it also antagonizes the functional alterations in some efferent nuclei of the basal ganglia manifest after lower doses of morphine. Local injections of morphine (15/~g) led to decreases of LCGU in the various parts of the striatum, but to increases in lateral and anteroventral thalamus. These increases in LCGU in two thalamic areas seem to support the above hypothesis that the rigidity is accompanied, at least in part of the efferent nuclei of the basal ganglia, by increases in LCGU antagonizing the inhibitory effects described above. INTRODUCTION M o r p h i n e produces characteristic alterations in motility when a d m i n i s t e r e d to rats. Lower doses lead to l o c o m o t o r activation, whereas larger doses ( > 1 0 mg/kg) induce l o c o m o t o r depression, catalepsy, and muscular rigidity. It was suggested that lower doses activate d o p a m i n e r g i c neurones, whereas after larger doses, this effect is m a s k e d by additional actions of m o r p h i n e on the striatum, resulting in muscular rigidity, and on the nucleus accumbens, leading to akinesia and catalepsy 7 If this hypothesis is valid, it should be expected that lower doses of m o r p h i n e p r o d u c e certain functional alterations in the basal ganglia and/or their efferent nuclei, whereas the larger doses, in addition, p r o d u c e effects which in part might mask or
antagonize thes¢ functional alterations. O n e biochemical p a r a m e t e r which seems to be useful in this respect is the m e a s u r e m e n t of the local cerebral glucose utilization ( L C G U ) by application of [14C]2deoxyglucose ([14C]DG)19, since the energy requirements of cerebral tissue are almost exclusively derived from glucose catabolism. A l t e r a t i o n s in functional activity, in any brain region, a p p e a r to be associated with changes in energy consumed in that area 17. In the present study, the effects of various low doses of morphine producing l o c o m o t o r activation were c o m p a r e d with those of a large dose (15 mg/kg i.p.) which p r o d u c e s a p r o n o u n c e d catalepsy and muscular rigidity, at least in the rat strain used by us 21. Various findings about the effects of m o r p h i n e on L C G U have already been published 4"6'9"1°, but
Correspondence: K. Kuschinsky, Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Ketzerbach 63, D-3550, Marburg, F.R.G. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 none of these studies dealt with the problem described. For these reasons, the present study was performed. In some additional experiments, morphine was locally injected into the striatum, in a dose which produces muscular rigidity s. MATERIALS AND METHODS Male albino Wistar rats (TNO/W 70 of E Winkelmann, Borchen, E R . G . ) of 180-230 g were used. They were kept in groups of 5 on a light-dark cycle of 12 h (light on at 08.00 h) with food (Altromin) and tap water ad libitum. Metabolic studies: polyethylene catheters were introduced into the tail artery and jugular vein under anesthesia (nitrous oxide:oxygen = 80:20, v/v, in presence of 0.8% halothane). The end of the jugular catheter ran subdermally to the dorsal part of the neck. The tail catheter was fixed subdermally in a similar way and was exteriorized above the tail. The wounds were then treated with lidocaine gel and
closed. This preparation enabled the animals to move freely on a 45 x 60 cm platform after the end of anesthesia. Arterial pC02, pO2, pH (Corning-178, Coming Medical, Giessen, E R . G . ) , blood pressure and pulse rate (Statham P23DB, Statham Medical Instruments, Heto Rey, Puerto Rico; HSE Electromanometer, Hugo Sachs Electronic, Hugstetten, E R . G . ) , plasma glucose (Glucose Analyzer II of Beckman, Munich, E R . G . ) and rectal temperature (Ellab TE-S, Ellab, Copenhagen, Denmark) were checked routinely. Experiments were carried out as described by Sokoioff et al. 19. Morphine hydrochloride (E. Merck, Darmstadt, E R . G . ) was dissolved in saline and injected i.p. Thirty minutes after morphine, []4C]DG, 120 ~Ci/kg (spec. act.: 51.1 mCi/mmol; New England Nuclear, Dreieich, E R . G . ) dissolved in physiological saline was injected within 15 s into the jugular vein. In some experiments, naloxone hydrochloride (1 mg/kg i.p.) (kindly donated by DuPont, Garden City, NY, U.S.A.) was adminis-
[mmHg ]
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Fig. 1. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on p 0 2 , pCO 2 or pH in the blood of rats. Mean values _+ S.D. of n = 6. Significances: *P < 0.05; **P < 0.01, Kruskal-Wallis test, when compared with the controls.
207
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Fig. 2. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg)or saline (controls) on local cerebral glucose utilization (LCGU) (umol/100 g tissue/min) in various parts of the caudate nucleus. Mean values + S.D. of n = 6. Significances: **P < 0.01, ***P < 0.001, Kruskai-Wallis test, when compared with the controls.
tered 5 min before and 25 min after morphine. Sixteen timed arterial blood samples (approximately 80/~l each) were drawn during the succeeding 45 min. They were centrifuged immediately (Microfuge B, Beckman Instruments, Munich, E R . G . ) and aliquots were taken for the determination of glucose and radioactive deoxyglucose (LS 50 Liquid Scintillation Counter, Beckman Instruments, Munich, E R . G . ) . The rats were decapitated 45 min after
injection of 14C, the brain was taken out within 50 s and frozen in 2-methylbutane at -50 °C. The brain was embedded in Tissue-Tek 11 medium (Miles, Naperville, IL, U.S.A.) and sectioned at about -20 °C in a cryostat (Reichert-Jung, Model 2700, Nussloch, F.R.G.) into 20-/~m thick slices, which were thaw-mounted on cover-slips and dried on a hot plate at 60 °C. Additional slices were stained with Cresyl violet (Nissl staining) for histological
208
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Fig. 3. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on local cerebral glucose utilization (LCGU) (~mol/100 g tissue/min) in globus pallidus and nucleus accumbens (A), or in substantia nigra pars compacta, substantia nigra pars reticulata or ventral tegmental area (VTA) (B). Mean values + S.D. of n = 6. Significances: *P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wallis test, when compared with the controls. In the pars reticulata, there is also a difference (P < 0.05, Student's t-test) between the values obtained after 7.5 and 15 mg/kg.
verification of brain structures. F o r autoradiography, the cover-slips were glued to c a r d b o a r d and, together with [laC]methacrylate standards (Amersham-Buchler, Braunschweig, F . R . G . ) exposed to O s r a y M3 film ( A g f a - G e v a e r t , Leverkusen, E R . G . ) for 12 days. The methacrylate standards had previously been calibrated against brain sections according to S a k u r a d a et al. 14. The anatomical structures
were identified by reference to the rat brain atlas of Pellegrino et a1.13 and to the a u t o r a d i o g r a p h i c maps of Schwartz and Sharp 16. Optical densities of the a u t o r a d i o g r a m s were m e a s u r e d with a computerbased d e n s i t o m e t e r u n d e r video control ( D T 1505 Parry Instruments, N e w b u r y , U . K . ; C o m m o d o r e 8032/8050, C o m m o d o r e , F r a n k f u r t , E R . G . ; Panasonic video c a m e r a wv 1550 E, Panasonic video
209
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Fig. 4. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on local cerebral glucose utilization (LCGU) (,umoi/100 g tissue/min) in anteromedial, anteroventral and mediodorsal part of the thalamus (A) or in the lateral thalamus and the subthalamic nucleus (B). Mean values + S.D. of n = 6. Significances: *P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wailis test, when compared with the controls. In both areas, the differences between the values after 7.5 and 15 mg/kg barely missed statistical significance.
m o n i t o r wv 5410, N T S G , Kassel, E R . G . ) . Each brain structure listed was m e a s u r e d 6 times and the average value of the readings was stored on a floppy disk. H i g h e r resolution image analysis was perf o r m e d with an image-processing system ( K o n t r o n Bildanalyse, Eching, F . R . G . ) . Final rates of cerebral glucose utilization were c o m p u t e d from the optical
densities m e a s u r e d and from the 2-deoxyglucose and glucose plasma curves, with the o p e r a t i o n a l equation for changing plasma glucose levels TM. In additional experiments, m o r p h i n e was not injected systemically, but into the h e a d of the caudate nucleus through a previously i m p l a n t e d cannula (for technical details see ref. 22, the coor-
210 TABLE I Effects o f various doses o f morphine (mg/kg i.p.), morphine (15 mg/kg i.p.) + naloxone (1 mg/kg i.p.) or saline on L C G U in various cortical areas
Means + S.D. of 6 experiments ~umol/100g tissue/min).
Occipital Auditory Parietal IV Parietal VI Sensory IV Sensory VI Olfactory Cingulate Frontal
Saline
1.0
87 + 12 135 + 17 118 + 16 88 + 13 115 + 14 82 + 14 114 + 17 100 + 13 93 + 9
78 + 117 + 111 + 68 + 113 + 72 + 96 + 88 + 89 +
3.3
5 5 16 11 15 11 11 13 10
79 + 120 + 113 + 73 + 108 + 67 + 91 + 83 + 82 +
16 24 23 17 17 10 9* 12 13
7.5
15
63 + 7 90 + 7*** 89 + 14" 66 + 12" 91 + 12"* 65 + 14 82 + 6** 74 + 11"* 75 _+7*
67 + 81 + 91 + 72 + 94 + 70 + 68 + 74 + 71 +
15 + naloxone
11 9*** 17 6* 8* 11 10"* 15" 10"*
79 + 116 + 105 + 82 + 108 + 81 + 102 + 88 + 88 +
7 17 40 7 8 7 10 16 10
*P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wallistest, when compared with the saline controls.
dinates of Fifkov~i and Margala 5 were applied: A P : - 1 . 2 5 , V: 4.5, L: 2.6, calculated from the bregma). M o r p h i n e hydrochloride (15/~g/1.5/~1 of physiological saline) was slowly (during 5 rain) injected, starting 10 min before administration of [14]DG. The localization of the injection site was checked histologically. The drug doses are expressed as the free base. RESULTS O b s e r v a t i o n of the gross behavior of the animals showed that the largest dose used (15 mg/kg i.p.) - but not the lower doses - - p r o d u c e d a strong akinesia and palpable signs of rigidity. The motorstimulant effects usually observed after lower doses of m o r p h i n e were not very p r o n o u n c e d in the e n v i r o n m e n t used in the present studies. As shown in Fig. 1, morphine decreased the arterial p O 2 after administration of the largest dose (15 mg/kg i.p.). The doses of 7.5 and 15 mg/kg were effective in increasing arterial p C O 2 and decreasing p H , whereas the lower doses of 1.0 and 3.3 mg/kg did not alter these p a r a m e t e r s in a statistically significant way. These results confirmed the well-known effects of m o r p h i n e leading to a central inhibition in respiration and by this to hypoxia and h y p e r k a p n i a with a respiratory acidosis. In the caudate nucleus (Fig. 2 A ) , 7.5 as well as 15 mg/kg p r o d u c e d statistically significant decreases in L C G U , the dose of 3.3
mg/kg being effective in the medial (Fig. 2B) and dorsomedial part of the striatum (Fig. 2A). Similar effects, with a m o r e p r o n o u n c e d dose d e p e n d e n c y , were found in globus pallidus and nucleus accumbens, where the lowest dose of 1 mg/kg a l r e a d y led to a decrease (Fig. 3A). In many cortical areas, both of the largest doses p r o d u c e d statistically significant decreases in L C G U (Table I). No statistically significant effects (although tendencies to decreases) were found in substantia nigra pars c o m p a c t a and ventral tegmental area (Fig. 3B). In contrast, the dose of 7.5 mg/kg p r o d u c e d a statistically significant decrease in L C G U in the substantia nigra pars reticulata, whereas no reduction at all was found after 15 mg/kg. Similar findings were o b t a i n e d in o t h e r efferent nuclei, namely some thalamic nuclei (anteroventral and lateral nuclei) and in the subthalamic nucleus, where 7.5, but not 15 mg/kg of m o r p h i n e p r o d u c e d statistically significant decreases in L C G U (Fig. 4). In the a n t e r o m e d i a l and m e d i o d o r s a l nuclei of the thalamus, in contrast, both doses p r o d u c e d similar decreases in L C G U (Fig. 4). In the p e r i a q u e d u c t a l gray, the following results were obtained: controls: 73 + 12; 7.5 mg/kg morphine: 48 _+ 5; 15 mg/kg: 55 + 20 (~mol/100 g tissue/min). The results o b t a i n e d after 7.5 mg/kg were significant ( P < 0.01), those after 15 mg/kg of m o r p h i n e not statistically significantly different from the controls. N a l o x o n e (1 mg/kg, given 5 min before and 25 min after mor-
211 TABLE II Effects of morphine (15 Fig) or saline injected into the (left) striatum on L C G U in various brain areas
Means + S.D. of 5-6 experiments ~mol/100 g tissue/min).
Striatum Dorsolateral Dorsomedial Ventrolateral Ventromedial Medial Thalamus Lateral Ventral Mediodorsal Anteromedial Anteroventral Subthalamic nucleus Substantia nigra Parscompacta Pars reticulata
Saline
Morphine
80 + 17 87 + 12 78 + 12 87 + 15 92 + 23
54 _+15" 57 + 10"* 55 + 9** 65 + 11" 53 + 7*
75 + 12 82 + 16 79 + 13 83 + 14 71 + 9 70 + 11
89 + 5* 85 + 5 87 + 11 90 + 6 85 + 6* 72 + 9
49 + 11 54 ± 15
57 + 10 65 + 6
*P < 0.05; **P < 0.01, Student's t-test, when compared with the saline controls. phine) prevented the effects of 15 mg/kg morphine (Figs. 1-4). In another series of experiments, morphine (15 pg) was injected into the striatum (head of the caudate nucleus), and the results were compared with those obtained after intrastriatal saline injection. As shown in Table II, this treatment decreased LCGU in the striatum on the level of the area injected (AP -1.25, calculated from the bregma). In contrast, it was increased in two areas of the thalamus, namely the anteroventral and lateral nuclei, but not ventral, mediodorsal and anteromedial nuclei nor the subthalamic nucleus. There was a tendency to increases, although in a statistically not significant way, in both parts of the substantia nigra. No effects were found in various cortical areas, nucleus accumbens, globus pallidus, ventral tegmentum, corpus geniculatum mediale or laterale, red nucleus, nucleus interpeduncularis, periaqueductal gray, medial or lateral habenula or the CA1 region of the hippocampus (results not shown). DISCUSSION In all brain regions studied, moderate doses of morphine (up to 7.5 mg/kg) decreased the LCGU
significantly or at least showed a tendency to decrease it. These effects were antagonized by naloxone and, therefore, can be regarded as opioidspecific actions. Similar decreases in LCGU were found, after 8.0 mg/kg of morphine s.c., in thalamic nuclei by Fanelli et al. 4. In contrast, these authors did not find any significant alterations in the cortical areas or any of the nuclei of the basal ganglia, whereas they found similar alterations as our group in blood pH, pCO 2 and p O 2. These differences might be due to differences between strains, at least with regard to the effects on the basal ganglia, but also to differences in the route of administration (s.c.) and the time-schedule (15 min before 14C)used by these authors. Furthermore, in our study, but not in that of Fanelli et al., the rats were freely moving. The results of Glick et al. 6 seem to be in apparent discrepancy to our results: these authors found a selective increase in LCGU in the striatum of rats. However, their results might be explained by the completely different experimental protocol: in the experiments described by Glick et al. 6 the rats self-administered morphine i.v., with an average dose of 0.7 mg/kg during the 30-min session. There was no discernible difference between control (drugfree) rats and yoked rats, namely animals who passively obtained the same doses of morphine and simultaneously as the self-administering animals. The increases in LCGU apparently were due to the active self-administration of morphine which was not performed in the present studies. Part of the reductions in L C G U observed in our experiments were observed after doses (1 or 3.3 mg/kg) of morphine which did not significantly alter pO 2, p C O 2 or pH in blood. Accordingly, the inhibitory effects observed cannot be fully explained by secondary effects due to alterations in blood oxygen or carbon dioxide. Since morphine as an agonist predominantly at opioid receptors of the p-type, depresses neuronal firing and neurotransmitter release in many neurons, probably due to a Ca2+-activated increase in K+-conductance (see ref. 11 for review), the general depressory effect of morphine on LCGU might be related to this neuronal effect. Furthermore, morphine, in moderate doses, enhances dopamine release in striatum 1'12 and nucleus accumbens 3"12, probably by a transsynaptic activation of dopaminergic neurons in the mid-
212 brain z°, and dopamine shows more inhibitory than excitatory actions in these nuclei. Therefore, this action of morphine might contribute to the inhibitory actions found in the main target areas of the dopaminergic neurons, such as caudate nucleus and globus pallidus. Whereas the lower doses of morphine enhance apomorphine-induced stereotypies 12, higher doses (10 mg/kg and more) do not do it. In contrast, they produce muscular rigidity21, at least in the rat strain used. This latter effect is likely to be due to an additional action of morphine in the striatum 7 which antagonizes the signs of dopaminergic activation; conversely, the rigidity can be antagonized by dopaminergic stimulants 21. It should be expected, therefore, that either in the central nuclei of the basal ganglia (striatum, nucleus accumbens) or rather in their efferent nuclei (e.g. globus pallidus, substantia nigra pars reticulata, some thalamic nuclei, subthalamic nucleus, periaqueductal gray, see review of Scheel-Kriiger~5), LCGU should be altered in such a way that after administration of a large dose of morphine (e.g. 15 mg/kg i.p.), the effects observed after lower doses are antagonized or even reversed, at least in some of the efferent nuclei. Administration of the large dose did not reverse the reduction of LCGU in such a way that an increase resulted. But in various efferent nuclei of the basal ganglia, it antagonized the reduction in LCGU by morphine, so that no significantly decreased values were observed after 15 mg/kg, although they were manifest after 7.5 mg/kg. Such an antagonism was observed in the substantia nigra pars reticulata, the lateral thalamic and the subthalamic nucleus. Similar effects were observed in the anteroventral thalamic nucleus and the periaqueductal gray. The increase in LCGU for 15 mg/kg of morphine, compared to 7.5 mg/kg may be related to an action of morphine in the striatum which mediates muscular rigidity. In this case, local injections of morphine into the striatum should induce increases in LCGU in at least some of its efferent nuclei as well. The dose injected into the striatum produces muscular rigidity 8. The present results showed that it led to significant increases in LCGU in two thalamic regions, namely the lateral and anteroventral area and to similar tendencies (although statistically not
significant) in most of the other thalamic areas and the substantia nigra pars reticulata. These findings seem to support the assumption that the action of morphine on the striatum which results in muscular rigidity might be related to the (relative) increases in LCGU in some of the efferent nuclei of the basal ganglia. The autoradiographic method used did not enable us to discriminate smaller areas of the thalamus, in particular the ventral thalamic nuclei which might be of particular importance as efferent relay stations from the globus pallidus and the substantia nigra pars reticulata 3,15. Nevertheless, in part of the efferent nuclei of the basal ganglia, the phenomenon predicted above was in fact observed, namely an inhibition of LCGU after 7.5 mg/kg or even lower doses, but not after 15 mg/kg. The globus pallidus did not show this phenomenon, however. Whereas the role of the substantia nigra pars reticulata (as one of the primary efferent relay stations of the striatum and the nucleus accumbens) in mediating and transmitting morphine-induced muscular rigidity in rats was well established 2°, the role of the globus pallidus in this respect remains unsettled as yet. Interestingly, neither the striatum (caudate nucleus + putamen which nuclei cannot be discriminated in the rat) nor the nucleus accumbens showed the above phenomenon: 15 mg/kg of morphine produced a similar decrease in LCGU as 7.5 mg/kg (in the striatum) or even a larger one (in the nucleus accumbens). At first glance, these results seem to contradict our hypothesis. However, the exact localization of the target neurons containing the opioid receptors responsible for opioid-induced akinesia (probably in the nucleus accumbens, see ref. 7) or muscular rigidity (in the striatum) is as yet unknown. It might be possible that the opioid receptors responsible are located on the efferent neurons of these nuclei and therefore do not noticeably contribute to the bulk activity in local cerebral glucose utilization. ACKNOWLEDGEMENTS This work was supported by a Grant (Ku 395/3-1) of the Deutsche Forschungsgemeinschaft.
213 REFERENCES 1 Broderick, P.A., In vivo electrochemical studies of rat striatal dopamine and serotonin release after morphine, Life Sci., 36 (1985) 2269-2276. 2 Di Chiara, G. and Imperato, A., Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats, J. Pharmacol. Exp. Ther., 244 (1988) 10671080. 3 Di Chiara, G., Porceddu, M.L., Morelli, M., Mulas, M.L. and Gessa, G.L., Evidence for a GABAergic projection from the substantia nigra to the ventromedial thalamus and to the superior colliculus of the rat, Brain Research, 176 (1979) 273-284. 4 Fanelli, R.J., Szikszay, M., Jasinski, D.R. and London, D.E., Differential effects of mu and kappa opioid analgesics on cerebral glucose utilization in the rat, Brain Research, 422 (1987) 257-266. 5 Fifkov~i, E. and MarSala, J., Stereotaxic atlases for the cat, rabbit and rat. In J. Bures, M. Petran and J. Zachar (Eds.), Electrophysiological Methods in Biological Research, Academia, Publishing House of the Academy of Sciences, Prague, and Academic Press, New York, 1967, pp. 653-722. 6 Glick, S.D., Cox, R.D. and Meibach, R.C., Selective effect of reinforcing doses of morphine in striatum, Brain Research, 190 (1980) 298-300. 7 Havemann, U. and Kuschinsky, K., Neurochemical aspects of the opioid-induced 'catatonia', Neurochem. Int., 4 (1982) 199-215. 8 Havemann, U., Winkler, M. and Kuschinsky, K., Opioid receptors in the caudate nucleus can mediate EMGrecorded rigidity in rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 313 (1980) 139-144. 9 Ito, M., Suda, S., Namba, H., Sokoioff, L. and Kennedy, C., Effects of acute morphine administration on local cerebral glucose utilization in the rat, J. Cerebral Blood Flow Metab., 3, Suppl. 1 (1983) $73-$74. 10 Levy, R.M., Fields, H.L., Stryker, M.P. and Heinricher, M.H., The effect of analgesic doses of morphine on regional cerebral glucose metabolism in pain-related structures, Brain Research, 368 (1986) 170-173. 11 Miller, R., How do opiates act?, Trends Neurosci., 7 (1984) 184-185. 12 M611er, H.-G. and Kuschinsky, K., Interactions of mor-
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20
21 22
phine with apomorphine: behavioural and biochemical studies, Naunyn-Schmiedeberg's Arch. Pharmacol., 334 (1986) 452-457. Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. Sakurada, O., Kennedy, C., Jehle, J., Brown, J.D., Carbin, G.L. and Sokoioff, L., Measurement of local cerebral blood flow with iodo [14C]antipyrin, Am. J. Physiol., 234 (1978) H59-H66. Scheel-Kriiger, J., Dopamine-GABA interactions: evidence that GABA transmits, modulates and mediates dopaminergic functions in the basal ganglia and the limbic system, Acta Neurol. Scand., 73, Suppl. 107 (1986) 1-54. Schwartz, W.J. and Sharp, ER., Autoradiographic maps of regional brain glucose consumption in resting, awake rats using [14C]2-deoxyglucose, J. Comp. Neurol., 1772 (1978) 335-359. Sokoloff, L., Relation between physiological function and energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. Sokoloff, L., The radioactive deoxyglucose method: theory, procedure, and applications for the measurement of local glucose utilization in the central nervous system. In B.W. Agranoff and M. Aprison (Eds.), Advances in Neurochemistry, Vol. 4, Plenum, New York, 1982, pp. 1-82. Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The [~4C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. Turski, L., Havemann, U. and Kuschinsky, K., Evidence that opioid receptors in the substantia nigra pars reticulata are relevant in regulating the function of striatal efferent pathways, Behav. Brain Res., 5 (1982) 415-422. Wand, P., Kuschinsky, K. and Sontag, K.-H., Morphineinduced muscular ridigity in rats, Eur. J. Pharmacol., 24 (1973) 189-193. Winkler, M., Havemann, U. and Kuschinsky, K., Unilateral injection of morphine into the nucleus accumbens induces akinesia and catalepsy, but no spontaneous muscular rigidity in rats, Naunyn-Schmiedeberg's .Arch. Pharmacol., 318 (1982) 143-147.
205
Elsevier BRES 14789
Research Reports
Morphine-induced alterations of local cerebral glucose utilization in the basal ganglia of rats T. Beck, J. Wenzel, K. Kuschinsky and J. Krieglstein Institute for Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Marburg (F.R. G.) (Accepted 14 February 1989)
Key words: Morphine; Basal ganglia; 2-Deoxyglucose; Local cerebral glucose utilization The actions of various doses of morphine on the local cerebral glucose utilization (LCGU) were studied by means of the autoradiographic [t4C]2-deoxyglucose technique. Morphine (1-15 mg/kg i.p.) decreased LCGU in most areas of the basal ganglia (caudate nucleus, globus pallidus, nucleus accumbens), but not in the substantia nigra pars compacta. LCGU was also decreased in limbic nuclei, such as sePtum, hippocampus and amygdala, and in most thalamic areas. In most cortical regions, a decrease was found as well. Findings in some efferent nuclei seemed of particular interest, namely in the substantia nigra pars reticulata, anteroventral and lateral nucleus of the thalamus and the subthalamic nucleus, where decreases in LCGU were found after administration of 7.5 mg/kg or sometimes lower doses, but not after 15 mg/kg of morphine. The decreases seem to reflect a general depressory effect of morphine on neuronal activity which is known from electrophysiologicalstudies. Part of these effects might be, in addition, due to an activation of dopaminergic neurons, since dopamine mainly acts as an inhibitory neurotransmitter. This dopaminergic activation leads to characteristic behavioral effects after lower doses of morphine. The largest dose used (15 mg/kg) produces muscular rigidity, probably by a direct action on the striatum. This effect antagonizes and masks the dopaminomimetic effects. The results suggest that it also antagonizes the functional alterations in some efferent nuclei of the basal ganglia manifest after lower doses of morphine. Local injections of morphine (15/~g) led to decreases of LCGU in the various parts of the striatum, but to increases in lateral and anteroventral thalamus. These increases in LCGU in two thalamic areas seem to support the above hypothesis that the rigidity is accompanied, at least in part of the efferent nuclei of the basal ganglia, by increases in LCGU antagonizing the inhibitory effects described above. INTRODUCTION M o r p h i n e produces characteristic alterations in motility when a d m i n i s t e r e d to rats. Lower doses lead to l o c o m o t o r activation, whereas larger doses ( > 1 0 mg/kg) induce l o c o m o t o r depression, catalepsy, and muscular rigidity. It was suggested that lower doses activate d o p a m i n e r g i c neurones, whereas after larger doses, this effect is m a s k e d by additional actions of m o r p h i n e on the striatum, resulting in muscular rigidity, and on the nucleus accumbens, leading to akinesia and catalepsy 7 If this hypothesis is valid, it should be expected that lower doses of m o r p h i n e p r o d u c e certain functional alterations in the basal ganglia and/or their efferent nuclei, whereas the larger doses, in addition, p r o d u c e effects which in part might mask or
antagonize thes¢ functional alterations. O n e biochemical p a r a m e t e r which seems to be useful in this respect is the m e a s u r e m e n t of the local cerebral glucose utilization ( L C G U ) by application of [14C]2deoxyglucose ([14C]DG)19, since the energy requirements of cerebral tissue are almost exclusively derived from glucose catabolism. A l t e r a t i o n s in functional activity, in any brain region, a p p e a r to be associated with changes in energy consumed in that area 17. In the present study, the effects of various low doses of morphine producing l o c o m o t o r activation were c o m p a r e d with those of a large dose (15 mg/kg i.p.) which p r o d u c e s a p r o n o u n c e d catalepsy and muscular rigidity, at least in the rat strain used by us 21. Various findings about the effects of m o r p h i n e on L C G U have already been published 4"6'9"1°, but
Correspondence: K. Kuschinsky, Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Ketzerbach 63, D-3550, Marburg, F.R.G. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 none of these studies dealt with the problem described. For these reasons, the present study was performed. In some additional experiments, morphine was locally injected into the striatum, in a dose which produces muscular rigidity s. MATERIALS AND METHODS Male albino Wistar rats (TNO/W 70 of E Winkelmann, Borchen, E R . G . ) of 180-230 g were used. They were kept in groups of 5 on a light-dark cycle of 12 h (light on at 08.00 h) with food (Altromin) and tap water ad libitum. Metabolic studies: polyethylene catheters were introduced into the tail artery and jugular vein under anesthesia (nitrous oxide:oxygen = 80:20, v/v, in presence of 0.8% halothane). The end of the jugular catheter ran subdermally to the dorsal part of the neck. The tail catheter was fixed subdermally in a similar way and was exteriorized above the tail. The wounds were then treated with lidocaine gel and
closed. This preparation enabled the animals to move freely on a 45 x 60 cm platform after the end of anesthesia. Arterial pC02, pO2, pH (Corning-178, Coming Medical, Giessen, E R . G . ) , blood pressure and pulse rate (Statham P23DB, Statham Medical Instruments, Heto Rey, Puerto Rico; HSE Electromanometer, Hugo Sachs Electronic, Hugstetten, E R . G . ) , plasma glucose (Glucose Analyzer II of Beckman, Munich, E R . G . ) and rectal temperature (Ellab TE-S, Ellab, Copenhagen, Denmark) were checked routinely. Experiments were carried out as described by Sokoioff et al. 19. Morphine hydrochloride (E. Merck, Darmstadt, E R . G . ) was dissolved in saline and injected i.p. Thirty minutes after morphine, []4C]DG, 120 ~Ci/kg (spec. act.: 51.1 mCi/mmol; New England Nuclear, Dreieich, E R . G . ) dissolved in physiological saline was injected within 15 s into the jugular vein. In some experiments, naloxone hydrochloride (1 mg/kg i.p.) (kindly donated by DuPont, Garden City, NY, U.S.A.) was adminis-
[mmHg ]
MORPHINE 3 . 3 mg/k9
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NORPHINE/NALOXONE
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T
7.40
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pCO 2
I
i
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Fig. 1. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on p 0 2 , pCO 2 or pH in the blood of rats. Mean values _+ S.D. of n = 6. Significances: *P < 0.05; **P < 0.01, Kruskal-Wallis test, when compared with the controls.
207
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Fig. 2. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg)or saline (controls) on local cerebral glucose utilization (LCGU) (umol/100 g tissue/min) in various parts of the caudate nucleus. Mean values + S.D. of n = 6. Significances: **P < 0.01, ***P < 0.001, Kruskai-Wallis test, when compared with the controls.
tered 5 min before and 25 min after morphine. Sixteen timed arterial blood samples (approximately 80/~l each) were drawn during the succeeding 45 min. They were centrifuged immediately (Microfuge B, Beckman Instruments, Munich, E R . G . ) and aliquots were taken for the determination of glucose and radioactive deoxyglucose (LS 50 Liquid Scintillation Counter, Beckman Instruments, Munich, E R . G . ) . The rats were decapitated 45 min after
injection of 14C, the brain was taken out within 50 s and frozen in 2-methylbutane at -50 °C. The brain was embedded in Tissue-Tek 11 medium (Miles, Naperville, IL, U.S.A.) and sectioned at about -20 °C in a cryostat (Reichert-Jung, Model 2700, Nussloch, F.R.G.) into 20-/~m thick slices, which were thaw-mounted on cover-slips and dried on a hot plate at 60 °C. Additional slices were stained with Cresyl violet (Nissl staining) for histological
208
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Fig. 3. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on local cerebral glucose utilization (LCGU) (~mol/100 g tissue/min) in globus pallidus and nucleus accumbens (A), or in substantia nigra pars compacta, substantia nigra pars reticulata or ventral tegmental area (VTA) (B). Mean values + S.D. of n = 6. Significances: *P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wallis test, when compared with the controls. In the pars reticulata, there is also a difference (P < 0.05, Student's t-test) between the values obtained after 7.5 and 15 mg/kg.
verification of brain structures. F o r autoradiography, the cover-slips were glued to c a r d b o a r d and, together with [laC]methacrylate standards (Amersham-Buchler, Braunschweig, F . R . G . ) exposed to O s r a y M3 film ( A g f a - G e v a e r t , Leverkusen, E R . G . ) for 12 days. The methacrylate standards had previously been calibrated against brain sections according to S a k u r a d a et al. 14. The anatomical structures
were identified by reference to the rat brain atlas of Pellegrino et a1.13 and to the a u t o r a d i o g r a p h i c maps of Schwartz and Sharp 16. Optical densities of the a u t o r a d i o g r a m s were m e a s u r e d with a computerbased d e n s i t o m e t e r u n d e r video control ( D T 1505 Parry Instruments, N e w b u r y , U . K . ; C o m m o d o r e 8032/8050, C o m m o d o r e , F r a n k f u r t , E R . G . ; Panasonic video c a m e r a wv 1550 E, Panasonic video
209
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Fig. 4. Effects of various doses of morphine, morphine (15 mg/kg) + naloxone (1 mg/kg) or saline (controls) on local cerebral glucose utilization (LCGU) (,umoi/100 g tissue/min) in anteromedial, anteroventral and mediodorsal part of the thalamus (A) or in the lateral thalamus and the subthalamic nucleus (B). Mean values + S.D. of n = 6. Significances: *P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wailis test, when compared with the controls. In both areas, the differences between the values after 7.5 and 15 mg/kg barely missed statistical significance.
m o n i t o r wv 5410, N T S G , Kassel, E R . G . ) . Each brain structure listed was m e a s u r e d 6 times and the average value of the readings was stored on a floppy disk. H i g h e r resolution image analysis was perf o r m e d with an image-processing system ( K o n t r o n Bildanalyse, Eching, F . R . G . ) . Final rates of cerebral glucose utilization were c o m p u t e d from the optical
densities m e a s u r e d and from the 2-deoxyglucose and glucose plasma curves, with the o p e r a t i o n a l equation for changing plasma glucose levels TM. In additional experiments, m o r p h i n e was not injected systemically, but into the h e a d of the caudate nucleus through a previously i m p l a n t e d cannula (for technical details see ref. 22, the coor-
210 TABLE I Effects o f various doses o f morphine (mg/kg i.p.), morphine (15 mg/kg i.p.) + naloxone (1 mg/kg i.p.) or saline on L C G U in various cortical areas
Means + S.D. of 6 experiments ~umol/100g tissue/min).
Occipital Auditory Parietal IV Parietal VI Sensory IV Sensory VI Olfactory Cingulate Frontal
Saline
1.0
87 + 12 135 + 17 118 + 16 88 + 13 115 + 14 82 + 14 114 + 17 100 + 13 93 + 9
78 + 117 + 111 + 68 + 113 + 72 + 96 + 88 + 89 +
3.3
5 5 16 11 15 11 11 13 10
79 + 120 + 113 + 73 + 108 + 67 + 91 + 83 + 82 +
16 24 23 17 17 10 9* 12 13
7.5
15
63 + 7 90 + 7*** 89 + 14" 66 + 12" 91 + 12"* 65 + 14 82 + 6** 74 + 11"* 75 _+7*
67 + 81 + 91 + 72 + 94 + 70 + 68 + 74 + 71 +
15 + naloxone
11 9*** 17 6* 8* 11 10"* 15" 10"*
79 + 116 + 105 + 82 + 108 + 81 + 102 + 88 + 88 +
7 17 40 7 8 7 10 16 10
*P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wallistest, when compared with the saline controls.
dinates of Fifkov~i and Margala 5 were applied: A P : - 1 . 2 5 , V: 4.5, L: 2.6, calculated from the bregma). M o r p h i n e hydrochloride (15/~g/1.5/~1 of physiological saline) was slowly (during 5 rain) injected, starting 10 min before administration of [14]DG. The localization of the injection site was checked histologically. The drug doses are expressed as the free base. RESULTS O b s e r v a t i o n of the gross behavior of the animals showed that the largest dose used (15 mg/kg i.p.) - but not the lower doses - - p r o d u c e d a strong akinesia and palpable signs of rigidity. The motorstimulant effects usually observed after lower doses of m o r p h i n e were not very p r o n o u n c e d in the e n v i r o n m e n t used in the present studies. As shown in Fig. 1, morphine decreased the arterial p O 2 after administration of the largest dose (15 mg/kg i.p.). The doses of 7.5 and 15 mg/kg were effective in increasing arterial p C O 2 and decreasing p H , whereas the lower doses of 1.0 and 3.3 mg/kg did not alter these p a r a m e t e r s in a statistically significant way. These results confirmed the well-known effects of m o r p h i n e leading to a central inhibition in respiration and by this to hypoxia and h y p e r k a p n i a with a respiratory acidosis. In the caudate nucleus (Fig. 2 A ) , 7.5 as well as 15 mg/kg p r o d u c e d statistically significant decreases in L C G U , the dose of 3.3
mg/kg being effective in the medial (Fig. 2B) and dorsomedial part of the striatum (Fig. 2A). Similar effects, with a m o r e p r o n o u n c e d dose d e p e n d e n c y , were found in globus pallidus and nucleus accumbens, where the lowest dose of 1 mg/kg a l r e a d y led to a decrease (Fig. 3A). In many cortical areas, both of the largest doses p r o d u c e d statistically significant decreases in L C G U (Table I). No statistically significant effects (although tendencies to decreases) were found in substantia nigra pars c o m p a c t a and ventral tegmental area (Fig. 3B). In contrast, the dose of 7.5 mg/kg p r o d u c e d a statistically significant decrease in L C G U in the substantia nigra pars reticulata, whereas no reduction at all was found after 15 mg/kg. Similar findings were o b t a i n e d in o t h e r efferent nuclei, namely some thalamic nuclei (anteroventral and lateral nuclei) and in the subthalamic nucleus, where 7.5, but not 15 mg/kg of m o r p h i n e p r o d u c e d statistically significant decreases in L C G U (Fig. 4). In the a n t e r o m e d i a l and m e d i o d o r s a l nuclei of the thalamus, in contrast, both doses p r o d u c e d similar decreases in L C G U (Fig. 4). In the p e r i a q u e d u c t a l gray, the following results were obtained: controls: 73 + 12; 7.5 mg/kg morphine: 48 _+ 5; 15 mg/kg: 55 + 20 (~mol/100 g tissue/min). The results o b t a i n e d after 7.5 mg/kg were significant ( P < 0.01), those after 15 mg/kg of m o r p h i n e not statistically significantly different from the controls. N a l o x o n e (1 mg/kg, given 5 min before and 25 min after mor-
211 TABLE II Effects of morphine (15 Fig) or saline injected into the (left) striatum on L C G U in various brain areas
Means + S.D. of 5-6 experiments ~mol/100 g tissue/min).
Striatum Dorsolateral Dorsomedial Ventrolateral Ventromedial Medial Thalamus Lateral Ventral Mediodorsal Anteromedial Anteroventral Subthalamic nucleus Substantia nigra Parscompacta Pars reticulata
Saline
Morphine
80 + 17 87 + 12 78 + 12 87 + 15 92 + 23
54 _+15" 57 + 10"* 55 + 9** 65 + 11" 53 + 7*
75 + 12 82 + 16 79 + 13 83 + 14 71 + 9 70 + 11
89 + 5* 85 + 5 87 + 11 90 + 6 85 + 6* 72 + 9
49 + 11 54 ± 15
57 + 10 65 + 6
*P < 0.05; **P < 0.01, Student's t-test, when compared with the saline controls. phine) prevented the effects of 15 mg/kg morphine (Figs. 1-4). In another series of experiments, morphine (15 pg) was injected into the striatum (head of the caudate nucleus), and the results were compared with those obtained after intrastriatal saline injection. As shown in Table II, this treatment decreased LCGU in the striatum on the level of the area injected (AP -1.25, calculated from the bregma). In contrast, it was increased in two areas of the thalamus, namely the anteroventral and lateral nuclei, but not ventral, mediodorsal and anteromedial nuclei nor the subthalamic nucleus. There was a tendency to increases, although in a statistically not significant way, in both parts of the substantia nigra. No effects were found in various cortical areas, nucleus accumbens, globus pallidus, ventral tegmentum, corpus geniculatum mediale or laterale, red nucleus, nucleus interpeduncularis, periaqueductal gray, medial or lateral habenula or the CA1 region of the hippocampus (results not shown). DISCUSSION In all brain regions studied, moderate doses of morphine (up to 7.5 mg/kg) decreased the LCGU
significantly or at least showed a tendency to decrease it. These effects were antagonized by naloxone and, therefore, can be regarded as opioidspecific actions. Similar decreases in LCGU were found, after 8.0 mg/kg of morphine s.c., in thalamic nuclei by Fanelli et al. 4. In contrast, these authors did not find any significant alterations in the cortical areas or any of the nuclei of the basal ganglia, whereas they found similar alterations as our group in blood pH, pCO 2 and p O 2. These differences might be due to differences between strains, at least with regard to the effects on the basal ganglia, but also to differences in the route of administration (s.c.) and the time-schedule (15 min before 14C)used by these authors. Furthermore, in our study, but not in that of Fanelli et al., the rats were freely moving. The results of Glick et al. 6 seem to be in apparent discrepancy to our results: these authors found a selective increase in LCGU in the striatum of rats. However, their results might be explained by the completely different experimental protocol: in the experiments described by Glick et al. 6 the rats self-administered morphine i.v., with an average dose of 0.7 mg/kg during the 30-min session. There was no discernible difference between control (drugfree) rats and yoked rats, namely animals who passively obtained the same doses of morphine and simultaneously as the self-administering animals. The increases in LCGU apparently were due to the active self-administration of morphine which was not performed in the present studies. Part of the reductions in L C G U observed in our experiments were observed after doses (1 or 3.3 mg/kg) of morphine which did not significantly alter pO 2, p C O 2 or pH in blood. Accordingly, the inhibitory effects observed cannot be fully explained by secondary effects due to alterations in blood oxygen or carbon dioxide. Since morphine as an agonist predominantly at opioid receptors of the p-type, depresses neuronal firing and neurotransmitter release in many neurons, probably due to a Ca2+-activated increase in K+-conductance (see ref. 11 for review), the general depressory effect of morphine on LCGU might be related to this neuronal effect. Furthermore, morphine, in moderate doses, enhances dopamine release in striatum 1'12 and nucleus accumbens 3"12, probably by a transsynaptic activation of dopaminergic neurons in the mid-
212 brain z°, and dopamine shows more inhibitory than excitatory actions in these nuclei. Therefore, this action of morphine might contribute to the inhibitory actions found in the main target areas of the dopaminergic neurons, such as caudate nucleus and globus pallidus. Whereas the lower doses of morphine enhance apomorphine-induced stereotypies 12, higher doses (10 mg/kg and more) do not do it. In contrast, they produce muscular rigidity21, at least in the rat strain used. This latter effect is likely to be due to an additional action of morphine in the striatum 7 which antagonizes the signs of dopaminergic activation; conversely, the rigidity can be antagonized by dopaminergic stimulants 21. It should be expected, therefore, that either in the central nuclei of the basal ganglia (striatum, nucleus accumbens) or rather in their efferent nuclei (e.g. globus pallidus, substantia nigra pars reticulata, some thalamic nuclei, subthalamic nucleus, periaqueductal gray, see review of Scheel-Kriiger~5), LCGU should be altered in such a way that after administration of a large dose of morphine (e.g. 15 mg/kg i.p.), the effects observed after lower doses are antagonized or even reversed, at least in some of the efferent nuclei. Administration of the large dose did not reverse the reduction of LCGU in such a way that an increase resulted. But in various efferent nuclei of the basal ganglia, it antagonized the reduction in LCGU by morphine, so that no significantly decreased values were observed after 15 mg/kg, although they were manifest after 7.5 mg/kg. Such an antagonism was observed in the substantia nigra pars reticulata, the lateral thalamic and the subthalamic nucleus. Similar effects were observed in the anteroventral thalamic nucleus and the periaqueductal gray. The increase in LCGU for 15 mg/kg of morphine, compared to 7.5 mg/kg may be related to an action of morphine in the striatum which mediates muscular rigidity. In this case, local injections of morphine into the striatum should induce increases in LCGU in at least some of its efferent nuclei as well. The dose injected into the striatum produces muscular rigidity 8. The present results showed that it led to significant increases in LCGU in two thalamic regions, namely the lateral and anteroventral area and to similar tendencies (although statistically not
significant) in most of the other thalamic areas and the substantia nigra pars reticulata. These findings seem to support the assumption that the action of morphine on the striatum which results in muscular rigidity might be related to the (relative) increases in LCGU in some of the efferent nuclei of the basal ganglia. The autoradiographic method used did not enable us to discriminate smaller areas of the thalamus, in particular the ventral thalamic nuclei which might be of particular importance as efferent relay stations from the globus pallidus and the substantia nigra pars reticulata 3,15. Nevertheless, in part of the efferent nuclei of the basal ganglia, the phenomenon predicted above was in fact observed, namely an inhibition of LCGU after 7.5 mg/kg or even lower doses, but not after 15 mg/kg. The globus pallidus did not show this phenomenon, however. Whereas the role of the substantia nigra pars reticulata (as one of the primary efferent relay stations of the striatum and the nucleus accumbens) in mediating and transmitting morphine-induced muscular rigidity in rats was well established 2°, the role of the globus pallidus in this respect remains unsettled as yet. Interestingly, neither the striatum (caudate nucleus + putamen which nuclei cannot be discriminated in the rat) nor the nucleus accumbens showed the above phenomenon: 15 mg/kg of morphine produced a similar decrease in LCGU as 7.5 mg/kg (in the striatum) or even a larger one (in the nucleus accumbens). At first glance, these results seem to contradict our hypothesis. However, the exact localization of the target neurons containing the opioid receptors responsible for opioid-induced akinesia (probably in the nucleus accumbens, see ref. 7) or muscular rigidity (in the striatum) is as yet unknown. It might be possible that the opioid receptors responsible are located on the efferent neurons of these nuclei and therefore do not noticeably contribute to the bulk activity in local cerebral glucose utilization. ACKNOWLEDGEMENTS This work was supported by a Grant (Ku 395/3-1) of the Deutsche Forschungsgemeinschaft.
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