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Limbic brain structures are important sites of κ-opioid receptor-mediated actions in the rat: a [14C]-2-deoxyglucose study
Brain Research, 478 (1989) 326-336 Elsevier
326 BRE14203
Limbic brain structures are important sites of r-opioid receptormediated actions in the rat: a [14C]-2-deoxyglucose study A. Ableitner and A. Herz Department of Neuropharmacology, Max-Planck-lnstitutfar Psychiatrie, Planegg-Martinsried(F.R. G. ) (Accepted 19 July 1988)
Key words: r-Agonist; U-50,488H; Glucose utilization; Limbic brain structure; Pituitary; Spinal cord
The [1-t4C]-2-deoxyglucose technique was employed to evaluate the regional pattern of alterations in glucose utilization in the rat brain, pituitary and spinal cord induced by the selective K-opioidagonist U-50,488H (trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl) cyclohexyl]-benzeneacetamide).Within the dose range used (0.5-5 mg/kg), U-50,488H produced a dose-depenc]ent attenuation of nociceptive thresholds and a place aversioh in the place conditioning test, allowingfor a correlation of the regional pattern of changes in glucose utilization with certain behavioral responses. The regional changes in glucose utilization induced by U-50,488H in the brain were most pronounced in components of the limbic forebrain circuit such as the anterior thalamic nuclei, mammillarybody, frontal cortex, lateral septal nucleus, nucleus accumbens and lateral habenular nucleus as well as in the brainstem tegmental nuclei and the dorsal and median raphe nucleus (components of the limbic midbrain area). Glucose utilization was decreased in the frontal cortex and increased in the other regions. An increase in glucose utilization also was observed in the central gray pons. Increases in glucose utilization in the pituitary were restricted to the intermediate lobe. In the lumbar part of the spinal cord, glucose metabolism was enhanced in the region around the central canal and in the ventral horn. The changes in glucose metabolism observed in these structures suggest that the aversive (dysphori¢) effects of U-50,488H may be due to the altered activity of the limbic structures of the forebrain and midbrain which have been implicateo in c.motional and affective processes. The increased activity in the intermediate lobe of the pituitary, furthermore, might reflect a stress component in the effects of this drug. Since the dorsal raphe nucleus and the region of the central gray pons have been implicated in both analgesia and pain processes a supraspinal site of antinociceptive action of U-50,488H, in addition to a spinal site of action, must be considered. INTRODUCTION A n abundance of evidence for the existence of multiple classes of opioid receptors has accumulated in the course of the past decade 14,29,3°. Based upon pharmacological and biochemical studies, iN the central nervous system at least 3 distinct opioid receptor subtypes designated as/~, r and 6 have been cicariy differentiated. Studies seeking to address the localization of t h e r-receptor subtype in the CNS and its functional significance, however, have previously been hampered by the lack of a selective ligand. Thus, for example the benzomorphans, prototypic-agonists at r-receptors, display a high degree of cross-reactivity to both /~ and 6 binding sites 24.2s. Recent studies have shown that U-50,488H (trans-3,4-dichloro-N-methyi-N[2-
(1-pyrolidinyl) cyclohexyi]-benzeneacetamide) and a series of other benzeneacetamides are highly selective agonists at the r-receptor 13.25.42.53.56. These compounds thus provide useful tools for identifying those regions in the CNS affected by selective r-opioid receptor activation and which may be functionally involved in the characteristic dysphoric and analgesic actions of r-agonists 3°,36,43,56. In order to delineate the regions in the brain, spinal cord and pituitary gland involved in the actions of r-agonists, the [1-14C]-2-deoxyglucose (2-DG) technique 5°'51 was employed. This technique is based upon the measurement of glucose utilization in discrete structural and functional components of the CNS. Since a close correlation exists between local rates of glucose utilization and functional activity, the method was used to provide a pictorial repre-
Correspondence: A. Al:leitner, Department of Neuropharmacology, Max-Planck-lnstitut ffir Psychiatric, Am Klopferspitz 18a, D-8033 Planegg-Martinsded, F.R.G. 0006-8993/89/$03.50© lt,~]9Elsevier Science Publishers B.V. (Biomedical Division)
327 sentation of regional changes in neuronal activity upon administration of U-50,488H throughout the CNS. Furthermore, to relate the regional pattern of changes in response to U-50,488H to specific behavioral patterns, both the motivational and antinociceptive effects of this compound were evaluated under similar experimental conditions. MATERIALS AND METHODS
2-Deoxyglucose experiments Preparation of rats Experiments were performed on male SpragueDawley rats (290-340 g). In order to accustom the rats to the plexiglas restrainer, used during the experimental procedure, they were confined to the tubes for 1 h on each of the 3 days preceding the experiments. On the day of experiment catheters were inserted into the left femoral artery and vein under light halothane anesthesia. The incision site was infiltrated with a local anesthetic and closed with metal surgical clips. After surgery, the rats were placed in the plexiglas tubes and allowed to recover from the effects of the anesthesia for 3 h.
exposed to an X-ray film (Mamoray T3, Agfa Gevaert) in X-ray cassettes for 14 days, together with a series of 14C-methylmetacrylate standards of increasing specific activity; the standards were equivalent to 14C-concentrations in dried brain sections of 20/~m thickness (Amersham). To facilitate identification of regions in the autoradiogram, sections adjacent to those used for autoradiography were stained with Toluidine blue and examined with reference to the brain atlases of K6nig and Klippe123 and Paxinos and Watson 41. Local tissue concentrations of 14C were evaluated by quantitative densitometric analysis of the autoradiograms with reference to the standards. For brain regions a Transmission Densitometer DT 1105, P.Y., aperture 0.2 mm diameter (Parry, U.K.) was used and for spinal cord regions and pituitary, a computer-based microscope photometer (Leitz Texture Analyse System) was used. Local glucose utilization was calculated from plasma and brain (or spinal cord and pituitary) radioactivities and plasma glucose concentrations using an equation that takes into consideration a variation in the arterial plasma glucose concentration during the experimental period 4s.
Behavioral experiments ~ algesiometric tests Drug treatment U-50,488H was dissolved in 0.9% (w/v) NaCl and was injected i.v. in a volume of 1 ml/kg b. wt. The same volume of saline was injected in control rats. U-50,488H (0.5, 1, 2 and 5 mg/kg) or saline, were injected 5 rain before the 2-DG.
Determination of regional glucose utilization in the brain, lumbar part of the spinal cord and pituitary Regional glucose utilization was determined as previously described 5°. Briefly, 25 /zCi/rat 2-DG were injected i.v. via a syringe pump within 30 s. Thirteen arterial blood samples were drawn during the following 45 min of the experimental session and assayed for plasma glucose (Beckman Glucose Analyser) and plasma 14C-levels (liquid scintillation counting). The rats were then decapitated, the brain, pituitary and lumbar part of the spinal cord removed and immediately frozen in isopentane chilled (-45 °C) with dry ice. Serial coronal sections (20 ~m) were cut in a cryostat maintained at -20 °C, mounted on coverslips, and dried at 60 °C. The sections were
Preparation of rats A polypropylene catheter filled with a heparin solution (200 U/ml) was inserted into the rat's left jugular vein and externalized in the neck of the animal. Incisions were infiltrated with a local anesthetic and closed with surgical wound clips. These procedures were carried out under halothane anesthesia (1%). After surgery the rats were allowed to recover for 48 h. To accustom the rats to the injection procedure and to avoid blood coagulation in the catheter, the catheter was flushed twice daily with a heparin solution on the two days preceding the experiment.
Experimental procedure Tailflick to heat. The latency for removal of the tail from a beam of noxious light focused on its tip was evaluated. The mean of 3 consecutive readings (separated by intervals of 10 s) was determined. A cut-off of 12 s was enforced in order to preclude tissue damage. Tail withdrawal to pressure. An incremental pres-
328 sure was applied via an accelerating blunt wedgeshaped piston to a point 2 cm from the tip of the tail and the pressure required to elicit withdrawal of the tail was determined. The mean of 3 consecutive values (separated by intervals of 20 s) was determined. A cut-off of 540 g was used. After establishment of baseline thresholds, rats were injected with saline (controls) or U-50,488H (0.5, 1, 2 and 5 mg/kg) and returned to their home cages. Thresholds were re-evaluated 5 min after injection of drug or vehicle.
Behavioral experiments -- place conditioning Experimental procedure Jugular catheters were inserted as described above. Place conditioning was carried out employing apparatus and procedures described previously 36. Briefly, it involved the differential pairing of drug and vehicle with two sets of place cues: a white box with a textured white floor and a black box with a smooth black floor. Each rat was given 3 drug treatments in one box, and 3 vehicle treatments in the other. During training sessions rats were injected with either drug or vehicle, and 5 rain later, placed for 50 min into the training compartment. The time interval between successive exposures to experimental compartments was at least 6 h. Vehicle and drug pairing were conducted on the same day. Testing was generally carried out 1 day following training. Rats were placed on the center platform of the testbox and the amount of time spent on the black or white side during a 15 min period was recorded. The position of the rat was defined by the position of its front paws. Since rats do not show a bias for one or other environmental cue in this apparatus and under this test conditions 35'36, rats were conditioned without pretesting. The order of exposure to the white and black compartments, and the assigning of the drug to a particular color, was counter-balanced for all of the rats.
Statistical analysis Statistical differences between individual means in the 2-DG experiment were assessed by a one-way analysis of variance and the Newman-Keul's multiple range test.
To determine whether an individual drug dose produced significant place conditioning the Wilcoxon test was used. Dose-response data derived from conditioning and algesiometric tests were analysed using a one-way random effects model factorial analysis of variance (ANOVA).
Materials [1-14C]-2-Deoxyglucose (spec. act. 49-55 mCi/ mmol) was obtained from New England Nuclear, Dreieich, FRG; U-50,488H (trans-3,4-dichloro-Nmethyl-N[2-(1-pyrolidinyl) cyclohexyl]-benzeneacetamide) was obtained from the Upjohn Company, Kalamazoo, Michigan, U.S.A. RESULTS
Effects of U-50,488H upon regional glucose utilization in the brain U-50,488H produced dose-dependent changes in glucose utilization, which were pronounced in the forebrain and midbrain limbic regions (Table I, Figs. 1 and 2). A dose of 0.5 mg/kg U-50,488H significantly increased glucose utilization in two of the 45 regions investigated: the lateral habenular nucleus (+20%) and the median raphe nucleus (+13%) (Table I). At a dose of 1 mg/kg further significant increases were observed in the anteromedial thalamic nucleus (+11%) and the central gray pons (+8%). However, this dose caused a significant decrease in glucose utilization in the frontal cortex (+11%) (Table I). In addition to regions affected by 1 mg/kg, a dose of 2 mg/kg increased glucose utilization in the medial mammillary nucleus, medial part (+17%), the posterior mammillary nucleus (+29%) and the •.ntrai tegmental nucleus (+22%) (Table I). A dose of 5 mg/kg increased rates of glucose metabolism in the anterior thalamic nuclei, the mammillary nuclei and the lateral habenular nucleus still greater. In the lateral septal nucleus (+36%), the nucleus accumbens (+26%), the dorsal rapbe (+ 14%) and the dorsal tegmental nucleus further increases in glucose utilization (+ 18%) (Table l) were produced by 5 mg/kg U-50,488H. The ventral tegmental area, the lateral thalamic nucleus, the lateral amygdaloid nucleus, posterior part and the lateral hypothalamus also displayed increased glucose utilization, but this trend
329
TABLE I
Effects of U-50,488H upon local cerebralglucose utilization (l~molllO0g/min) Values shown are means _+ S.E.M. for the number of rats indicated in parentheses.
Brain regions
Vehicle
U-50,48811 0.5 mglkg
1 mglkg
2 mglkg
5 mglkg
Telencephalon Frontal cortex Frontoparietal cortex, motor area Frontoparietal cortex, somatosensory area Visual cortex Auditory cortex Hippocampus Dentate gyrus Medial amygdaloid nucleus Lateral amygdaloid nucleus, posterior part Lateral septal nucleus Caudate nucleus Globus pallidus Nucleus accumbens
(5) (5) (5) (5) (5) (5) (5) (5) (3) (5) (5) (5) (5)
95 + 2 100 + 3 105 + 3 99 + 3 129 + 4 83 + 3 49 + 2 41 + 2 86 + 2 50+1 95 + 3 53 + 2 96 + 3
(6)* (6) (6) (6) (6) (6) (6) (5) (6) (6) (6) (6) (6)
92 + 5 96 + 6 101 + 6 102 + 3 134 + 7 87 + 4 48 + 2 42 + 3 85 + 4 49+3 94 + 4 50 + 3 98 + 5
(5)* (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5)
92 + 8 109 + 5 102 + 4 89 + 5 101 + 3 49 + 2 59 + 1 109 + 3 96 + 4 116_+5 73 _+ 2 105 _+ 4
(5) (5) (5) (5) (5) (5) (4) (5) (5) (5)* (5) (5)
106 + 3 116 + 3 103 + 3 89+2 102 + 3 48+1 68 + 6 112 + 5 108+2 122+5 72 + 2 107 + 3
(6) (6)* (6) (6) (6)
104 +_ 7 118 + 6 107 + 6 91+5 108 + 8
(5) (5)* (5) (5) (5)
52 _+ 5 77 _+ 5 91 _+ 4 56 _+ 2 95 _+ 5 78 _+ 2 96 _+ 8 101 _+ 6 64 _+ 4 79 _+ 2 113 + 8 79 -+ 6
(5) (5) (5) (5) (5)*
51+2 81+2 94 _+ 3 67 + 5 102 + 2 82 + 2 104 + 3 107 _+ 3 68 ± 4 82 + 1 121 + 3 89 + 3
(6)
53+3
(5)
(6) (6)
8 4 + 4 (5) 93 + 4 (5)
106 ± 4 104 + 6 110 + 6 103 + 4 !39 + 7 85 + 3 54 + 2 42 + 2 82 + 3 46 + 2 93 + 4 51 + 2 88 + 4
(6) (6) (6) (4) (6) (6) (6) (6) (6) (6) (6) (6) (6)
100 + 103 + 108 + 94 + !3! + 82 + 48 + 43 + 80 + 48 + 96 + 54 + 93 +
99 + 4 104 + 3 99 + 2 89 + 3 99 + 2 47 + 2 58 + 2 101 + 3 94 + 3 97_+4 72 _+ 3 108 _+ 5
(6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6)
54 _+ 2 87 _+ 4 98 _+ 4 57 _+ 2 84 _+ 4 77 _+ 4 90 _+ 4 99 _+ 4 66 _+ 3 76 _+ 2 126 + 10 88 -+ 4
(6) (6) (6) (6) (6)
3 3 4 6 10 5 4 1 6 3 6 3 4
94 + 1 98 + 2 104 + 4 99 + 1 133 + 5 79 _+ 3 47 + 2 41 + 4 94 + 3 63+1 106 _+ 4 56 _+ 4 111 +_ 4
Diencephalon Anteroventral thalamic nucleus Anteromedial thalamic nucleus Lateral thalamic nucleus Ventral thalamic nucleus Ventromedial thalamic nucleus Hypothalamus, ventromedial nucleus Lateral hypothalamus Medial mammillary nucleus, medial part Posterior mammiilary nucleus Lateral habenular nucleus Lateral geniculate body Medial geniculate body
Mesencephalon + hindbrain Substantia nigra, pars reticulata Superior colliculus, superficial layer lnterpeduncular nucleus Ventral tegmental area Median raphe nucleus Dorsal raphe nucleus Ventral tegmental nucleus Dorsal tegmental nucleus Central gray, dorsal part Central gray, pons Medial vestibular nucleus Nucleus of the lateral lemniscus
(6) (6) (6) (6) (6) (6) (6)
(5) (5) (5) (5) (5) (5) (5)
Cerebellum Cerebellar cortex Cerebellar vermis Dentate nucleus lnterpositus nucleus
50 53 92 95
-+ 3 _+ 3 _+ 4 _+ 4
(6) (6) (6) (6)
54 61 89 92
-+ 6 _+ 5 _+ 5 _+ 6
(5) (5) (5) (5)
White matter G e n u of corpus callosum Internal capsule Cerebellar white
24 _+ 1 (6) 26 _+ 1 (6) 29 _+ 1 (6)
24 _+ 1 (5) 29 _+ 2 (5) 26 _+ 3 (5)
51 57 94 97
_+ 2 + 2 +_ 4 + 3
(6) (6) (6) (6) (6)** (6) (6)
(6) (6)*** (6) (6) (6) (6) (6)* (6) (6) (6) (6) (6) (6)
2 4 + 1 (6) 2 5 + 1 (6) 2 8 + 1 (6)
124 + 2 130 + 3 117 + 3 92+1 115 + 6 5 0 + 2 (5) 51_+4 66 +_ 6 (5) 75 _+ 3 118 + 5 (5)* 131 + 3 i21_+7 (5)** 134_+10 1 4 6 + 8 (5)*** 183_+7 75 + 5 (5) 79 + 2 109 + 5 (5) 110 + 8
57 + 2 105 + 2 85 _+ 2 110 + 3 113 + 3 69 + 3 87 + 2 132 + 6 92 + 5 54 59 96 95
+ 2 + 3 + 4 ___4
(5) (5)*** (5) (5)* (5) (5) (5)** (5) (5) (4) (5) (5) (5)
2 2 + 2 (5) 2 2 + 2 (5) 2 7 + 1 (5)
*P < 0.05; **P < 0.01; ***P < 0.001 significantly different from vehicle (Newman-Keul's multiple range test).
(4) (4) (4) (4) (4) (4) (4) (4) (4) (4)*** (4) (4) (4)*** (4)** (4)*** (4) (4) (4) (4) (3) (4)*** (4)*** (4)*** (4) (4)
(4) (4) 87 + 2 (4) 65 + 3 (4) 106 + 3 (4)** S8 _+ 2 (4)* 111 + 3 (4)* 117 + 5 (4)* 77 + 5 (4) 90 + 2 (4)*** 127 + 6 (4) 92 + 5 (4) 54+3 85-+2
55 63 94 95
+ + + +
3 3 4 4
25+3 28+2 28+1
(4) (4) (4) (4) (4) (4) (4)
A
~
-
•l' ¢'--
, A
Fig. 1. Effects of U-50,488H on autoradiographic grain densities, representing glucose utilization. A - D : grain densities in film exposed to sections from different levels of a control brain. E - H : grain densities in film exposed to section from corresponding brain levels of a rat injected with U-50,488H (5 mg/kg). Abbreviations: acc, nucleu:+accumbens; tav, anteroventral thalamic nucleus; tam, anteromedial thalamic nucleus; Ih, lateral habenular nucleus; mmm, medial mammi!!ary nucleus, medial part; mp, posterior mammillary nucleus.
331
E
0
H
Fig. 2. Effects of U-50,488H on autoradiographic grain densities, representing glucose utilization. A-D: grain densities in film exposed to sections from different levels of a control brain. E-H: grain densities in film exposed to sections from corresponding brain levels of a rat injected with U-50,488H (5 mg/kg). Abbreviations: dr, dorsal raphe nucleus; mr, median raphe nucleus; vtg, ventral tegmental nucleus; dtg, dorsal tegmental nucleus; CGPn, central gray pons.
332 TABLE II
Effects of U-50,488Hupon glucose utifization (l~molllO0glmin) of the neural, intermediateand anteriorlobe of the pituitary Values shown are means _+S.E.M. for the number of rats indicated in parentheses.
Posterior lobe Intermediate lobe Anterior lobe
Vehicle
U-50,488H 0.5 mglkg
1 mglkg
2 mglkg
5 mglkg
41 _+5 (6) 48 + 6 (6) 29 + 3 (6)
57 _+8 (5) 66 + 9 (5)** 34 + 4 (5)
50 _+4 (6) 72 __.5 (6)** 38 + 2 (6)
48 _+2 (5) 81 + 4 (5)** 31 + 4 (5)
54 + 9 (4) 84 + 7 (4)** 39 + 6 (4)
**P < 0.01 significantly different from vehicle (Newman-Keul's multiple range test).
was not statistically significant.
Effects of U-50, 488H upon nociceptive thresholds
Effects of U-50,488H upon local glucose utilization in the spinal cord
U-50,488H produced a dose-dependent attenuation in the response of rats to noxious heat (Fl,14 = 44.92, P = 0.0001) and pressure (Fla 3 = 13.7, P = 0.027) (Fig. 4).
In the lumbar part of the spinal cord, U-50,488H increased glucose utilization in the ventral horn (the m e a s u r e d area comprised Rexed's laminae 8 and 945) a n d the region in the vicinity of the central canal (Rexe d ' s lamina 1045) in a dose-dependent m a n n e r (Table If).
Motivational properties of USO,488H Significant place aversion was p r o d u c e d by all 4 doses of U-50,488H tested (Table IV). No significant differences were found between dose and magnitude of effect (F3,40 = 1.094; P = 0.362).
Effects of U-50, 488H upon local glucose utilization in the pituitary
DISCUSSION
Even the highest dose of U-50,488H (5 mg/kg) did not produce any significant changes in glucose utilization in the anterior and neural lobes of the pituitary (Table III). Glucose utilization in the intermediate lobe, however, was increased significantly at every dose (Table IIl) and in contrast to control animals, the intermediate lobe could be clearly differentiated from the neural and anterior lobes in autoradiograms from rats treated with U-50,488H (Fig. 3).
The 2 - D G method 5° was employed to investigate regions and neural circuits in the rat brain, spinal cord and pituitary involved in the analgesic and aversive (dysphoric) effects of the selective r-opioid receptor agonist U-50,488H. In comparison to recent studies that employed unselective r - r e c e p t o r ligands and evaluated the effects of only single doses on local cerebral glucose utilization aa2 U-50,488H induced a
TABLE III
Effects of U-50,488Hupon glucose utilization (/~mol/lO0g/rain) in the lumbarpart of the spinal cord Values shown are means + S.E.M. for the number of rats indicated in parentheses.
Spinal cord Lumbar Part (L5) Lamina I-III Lamina V-VI LaminaVII-IX Lamina X White matter
Vehicle
36 + 48 + 46 + 48 + 20 +
4 a 5 5 3
U-50,488H
(6) (6) (6) (6) (6)
0.5 mg/kg
1 mg/kg
2 mglkg
5 mglkg
43 + 54 + 52 + 57 + 21 +
43 + 55 + 55 + 58 + 26 +
46 + 56 + 57 + 59 + 22 +
44 + 54 + 61 + 61 + 22 +
2 3 2 2 1
(5) (5) (5) (5)* (5)
3 2 2 2 2
(6) (6) (6) (6)* (6)
*P < 0.05; **P < 0.01 significantly different from vehicle (Newman-Keul's multiple range test).
1 1 1 2 1
(5) (5) (5)* (5)* (5)
2 2 2 2 2
(4) (4) (4)** (4)* (4)
333
A
B
AL
PL
C
IL
Fig. 3. Effects of U-50,488H on autoradiographic grain densities in the pituitary, representing glucose utilization. A: Nissl-stained section. B: corresponding pituitary section of a 2-DG autoradiograph of a control animal. C: 2-DG autoradiograph of an animal treated with U-50,488H (5 mg/kg). Abbreviations: IL, intermediate lobe; AL, anterior lobe; PL, posterior lobe of the pituitary.
more complex pattern of changes. Thus, most pronounced changes in glucose utilization were observed ip limbic forebrain structures and structures that lie within what Nauta 38, has termed the 'limbic midbrain area'. These regions do not entirely correspond with known regions of r-opioid binding sites 29"33. Such discrepancies, however, may be explained by the fact that glucose utilization reflects actions at both the receptor site and the influence of other neuronal circuits. The effects in the lateral habenuiar nucleus, an area apparently devoid of r-opioid binding sites 29'33, therefore, probably reflect an activation of its afferent inputs, which amongst others originate in the nucleus accumbens, and in the median raphe nucleus ~9. These latter regions also displayed changes in glu-
300' ....,. o o II ul Cu
heat
cose utilization and exhibit a dense labeling of r-opiold binding sites 29'33. The increased rates of glucose metabolism in the median and dorsal raphe nucleus, the central gray and the tegmental nuclei, furthermore, might be attributed to modulation by descending projections from the lateral habenular nucleus 1,18.47. In turn, the increased activity of the tegmentai nuclei could induce changes in the mammillary body via the mammiUary peduncle s. Based on known reciprocal connections, however, the activity in the tegmental nuclei vice versa, could be modulated by the mammillary body 1°'18. Besides the mammillary peduncle another important fiber tract originating from the mammillary body is the mammillothalamic tract. This tract projects to the anterior group of the thalamic nuclei 9 which also displayed an increase in glucose utilization. The latter structures and the mammillary body have been described as components of a limbic circuit described by Papez 4°. Further components of this circuit which displayed changed rates of glucose metabolism after U-50,488H were the lateral septal nucleus and the frontal cortex. Taken together, these results indicate that administration of U50,488H selectively activates structures which are
200 ul tO
TABLE IV Place preference of rats trained with different doses of U-50,488H
Values shown are mean differences between time spent on the drug and the vehicle side of the test box (s) + S.E.M. for the number of rats indicated in parentheses.
100
US0~88H (mg/kg) Fig. 4. The influence of acutely applied U-50,488H upon nociceptive thresholds. The dose-response relationship is depicted. Tail heat = triangle; tail pressure = circles. For each dose n = 5; means + S.E.M. are shown.
0.5 mg/kg
I mg/kg
2 mglkg
5 mg/kg
-446 + 74 (12)*
-380 + 62 (12)*
-264 + 60 (10)*
-446 + 100 (10)*
*P < 0.05 significant place aversion (Wilcoxon test).
334 implicated in the circuitry of the limbic system and which are reciprocally interconnected. Functionally these structures have been implicated in the control of various neurobehavioral states. Thus, stimulation and lesions of single components of the limbic forebrain circuits have revealed their involvement in the expression of emotion and mood as well as in the initiation of motivational processes such as avoidance and attack 6"7"t5'32'52. Besides their role in the control of locomotion 5~'5ssleep, analgesia and pain 49 the raphe nuclei w particularly the median raphe nucleus - - have also been implicated in affectire behaviors t7,49. Based upon these data we conclude that the aversive effects produced by the K-agonist U-50,488H in the place conditioning experiments (representing a dysphoric component of this drug) may result from a selective activation of these structures. As regards the analgesic action of U-50,488H, a supraspinal site of pain modulation by cerebral r-receptors has to be considered, since the dorsal raphe nucleus, the region of the central gray ports, and the nucleus accumbens, structures that have been shown to contribute substantially to opiate and stimulus produced analgesia2°,2t,39,46'59, displayed increased activities. Indeed, recent studies examining the antinociceptive effects of i.c.v, administration of U-50,488H indicate a supraspinal site of action of r-opioid ligands n. Glucose utilization at the spinal level was significantly increased in the ventral horn - - the measured area comprised Rexed's lamina 8 and lamina 9 - - and lamina 1045, the gray area surrounding the central canal. Although u-opioid binding sites are mainly concentrated in the superficial layer of the dorsal horn, lower levels are found in other regions with some enrichment in lamina 1 0 1 6 ' 3 4 . In view of evidence indicating a role of the region in the vicinity of the central canal in nociception 27,37, the increased glucose meREFERENCES 1 Aghajanian, G.K. and Wang, R.Y., Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique, Brain Research, 105 (1977) 389-403. 2 Baumgarten, H.G., BjOrklund, A., Holstein, A.F. and Nobin, A., Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary, Z. Zellforsch., 126 (1972) 483-517.
tabolism found in this region in the present experiments may represent a physiological correlate of the dose-dependent increase of nociceptive thresholds demonstrated in the tail-flick and tail-pressure test. Recent studies have suggested a role for r-receptor ligands in the modulation of the stress response 22. Thus, r-agonists potently increased circulating plasma levels of corticosterone and adrenocorticotropic hormone (ACTH). These actions were present in adrenalectomized, but not hypophysectomized, rats. Therefore, a central action or an action at the level of the anterior pituitary is possible e2. In the present experiments, however, no changes in glucose utilization were found in the anterior pituitary. The deoxyglucose method has been validated for neural tissue 5° however, since the anterior pituitary is of non-neural origin, a different lumped constant or a different phosphatase activity in this tissue might result in some inaccuracy of glucose utilization. In contrast to the anterior lobe, the intermediate lobe has an action potential stimulated outflow of a-melanocyte stimulating hormone (a-MSH) and fl-endorphin *~'s4that is modulated by serotoninergic fibers, inputs from dopaminergic neurons in the arcuate area, but also from peripheral sympathetic noradrenergic fibers 2,5. An involvement of a-MSH and fl-endorphin in the organism's response to stress has also been suggested 4'3t'55. Therefore, the increased activity in the intermediate lobe observed in the present experiments may be one reflection of the stress effects of U-50,488H.
ACKNOWLEDGEMENTS We wish to thank K. Metzner for excellent technical assistance; I. Dohle for preparation of the manuscript and Dr. T. Shippenberg and Dr. O.F.X. AImeida for their discussions and comments. 3 Beck, T. and Kriegistein, J., The effects of tifluadom and ketazocine on behaviour, dopamine turnover in the basal ganglia and local cerebral glucose utilization of rats, Brain Research, 381 (1986)327.-335. 4 Berkenbosch, F., Tilders, F.J.H. and Vermes, I., fl-adrenoreceptor activation mediates stress-induced secretion of fl-endorphin-related peptides from intermediate but not anterior pituitary, Nature (Lond.), 305 (1983)237-239. 5 Bj6rklund, A., Moore, R.Y., Nobin, A. and Stenevi, U., The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain,
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336 and Barhsun, C., U-50,488H. A pure kappa receptor agonist with spinal analgesic loci in the mouse, Life Sci., 31 (1982) 1197-1200. 43 Pfeiffer, A., Brantl, V., Herz, A. and Emrich, H., Psychotomimesis mediated by K-opiate receptors, Science, 233 (1986) 774-776. 44 Przewlocki, R., H611t,V., Voigt, K.H. and Herz, A., Modulation of in vitro release of p-endorphin from the separate lobes of the rat pituitary, Life Sci., 24 (1979) 1601-1608. 45 Rexed, B., A cytoarchitectonic atlas of the spinal cord in the cat, J. Comp. Neurol., 100 (1954) 297-380. 46 Richardson, D.E. and Akil, H., Pain reduction by electrical brain stimulation in man. II. Chronic self-administration in the periventricular gray matter, J. Neurosurg., 47 (1977) 184-194. 47 Sakai, K., Salvert, D., Touret, M. and Jouvet, M., Afferent connections of the nucleus raphe dorsalis in the cat as visualized by the horseradish peroxidase technique, Brain Research, 137 (1977) 11-35. 48 Savaki, H.E., Davidsen, L., Smith, C. and Sokoloff, L., Measurement of free glucose turnover in brain, J. Neurochem., 35 (1980) 495-502. 49 Seiden, L.S. and DyKstra, L.A., Serotonin and behaviour. In L.S. Selden and L.A. Dykstra (Eds.), Psychopharmacology: A Biochemical and Behavioral Approach, VanNostrand Reinhold, New York, 1977, pp. 86-116. 50 Sokoloff, L., Reivich, M., Kennedy, C., DesRosier, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The 14C-deoxyglucosemethod for the measurement of local ~rebral glucose utilization: theory, procedure and normal v ~ e s in the conscious and anesthetized albino rat, Y. Neurochem., 288 (1977) 897-916.
51 Sokoloff, L., The 14C-deoxyglucose method: four years later, Acta Neurol. Scand., 72 (Suppl. 60) (1979) 640-649. 52 Sutherland, R.J., The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex, Neurosci. Biobehav. Rev., 6 (1982) 1-13. 53 Szmuszkovicz, J. and VonVoigtlander, P.F., Benzeneacetamide amines: structurally novel non-mu opioids, J. Med. Chem., 25 (1982) 1125-1126. 54 Tomiko, S.A., Taraskovich, P.S. and Douglas, W.W., Potassium-induced secretion of melanocyte-stimulating hormone from isolated pars intermedia cells signals participation of voltage-dependent calcium channels in stimulussecretion coupling, Neuroscience, 6 (1981) 2~9-2267. 55 Vermes, I., Berkenbosch, F., Tilders, F.J.H. and Smelik, P.G., Hypothalamic deaffferentation in the rat appears to discriminate between the anterior lobe and intermediate lobe response to stress, Neurosci. Len., 27 (1981) 89-93. 56 VonVoigtlander, P.F., Lahti, R.A. and Ludens, J.H., U-50,488: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther., 224 (1983) 7-12. 57 Wirtshafter, D. and Asin, K.E., Evidence that electrolytic median raphe lesions increase locomotion but not exploration, Physiol. Behav., 28 (1982) 749-754. 58 Wirtshafter, D. and McWilliams, C., Suppression of locomotor activity produced by acute injections of kainic acid inte the ~edian raphe nucleus, Brain Research, 408 (1987) 349-352. 59 Xuan, J.T., Shi, Y~S., Zhou, Z.F. and Han, J.S., Studies on the mesolimbic loop of antinociception-II. A serotonin-enkephalin interaction in the nucleus accumbens, Neuroscience, 19 (1986) 403-409.
326 BRE14203
Limbic brain structures are important sites of r-opioid receptormediated actions in the rat: a [14C]-2-deoxyglucose study A. Ableitner and A. Herz Department of Neuropharmacology, Max-Planck-lnstitutfar Psychiatrie, Planegg-Martinsried(F.R. G. ) (Accepted 19 July 1988)
Key words: r-Agonist; U-50,488H; Glucose utilization; Limbic brain structure; Pituitary; Spinal cord
The [1-t4C]-2-deoxyglucose technique was employed to evaluate the regional pattern of alterations in glucose utilization in the rat brain, pituitary and spinal cord induced by the selective K-opioidagonist U-50,488H (trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl) cyclohexyl]-benzeneacetamide).Within the dose range used (0.5-5 mg/kg), U-50,488H produced a dose-depenc]ent attenuation of nociceptive thresholds and a place aversioh in the place conditioning test, allowingfor a correlation of the regional pattern of changes in glucose utilization with certain behavioral responses. The regional changes in glucose utilization induced by U-50,488H in the brain were most pronounced in components of the limbic forebrain circuit such as the anterior thalamic nuclei, mammillarybody, frontal cortex, lateral septal nucleus, nucleus accumbens and lateral habenular nucleus as well as in the brainstem tegmental nuclei and the dorsal and median raphe nucleus (components of the limbic midbrain area). Glucose utilization was decreased in the frontal cortex and increased in the other regions. An increase in glucose utilization also was observed in the central gray pons. Increases in glucose utilization in the pituitary were restricted to the intermediate lobe. In the lumbar part of the spinal cord, glucose metabolism was enhanced in the region around the central canal and in the ventral horn. The changes in glucose metabolism observed in these structures suggest that the aversive (dysphori¢) effects of U-50,488H may be due to the altered activity of the limbic structures of the forebrain and midbrain which have been implicateo in c.motional and affective processes. The increased activity in the intermediate lobe of the pituitary, furthermore, might reflect a stress component in the effects of this drug. Since the dorsal raphe nucleus and the region of the central gray pons have been implicated in both analgesia and pain processes a supraspinal site of antinociceptive action of U-50,488H, in addition to a spinal site of action, must be considered. INTRODUCTION A n abundance of evidence for the existence of multiple classes of opioid receptors has accumulated in the course of the past decade 14,29,3°. Based upon pharmacological and biochemical studies, iN the central nervous system at least 3 distinct opioid receptor subtypes designated as/~, r and 6 have been cicariy differentiated. Studies seeking to address the localization of t h e r-receptor subtype in the CNS and its functional significance, however, have previously been hampered by the lack of a selective ligand. Thus, for example the benzomorphans, prototypic-agonists at r-receptors, display a high degree of cross-reactivity to both /~ and 6 binding sites 24.2s. Recent studies have shown that U-50,488H (trans-3,4-dichloro-N-methyi-N[2-
(1-pyrolidinyl) cyclohexyi]-benzeneacetamide) and a series of other benzeneacetamides are highly selective agonists at the r-receptor 13.25.42.53.56. These compounds thus provide useful tools for identifying those regions in the CNS affected by selective r-opioid receptor activation and which may be functionally involved in the characteristic dysphoric and analgesic actions of r-agonists 3°,36,43,56. In order to delineate the regions in the brain, spinal cord and pituitary gland involved in the actions of r-agonists, the [1-14C]-2-deoxyglucose (2-DG) technique 5°'51 was employed. This technique is based upon the measurement of glucose utilization in discrete structural and functional components of the CNS. Since a close correlation exists between local rates of glucose utilization and functional activity, the method was used to provide a pictorial repre-
Correspondence: A. Al:leitner, Department of Neuropharmacology, Max-Planck-lnstitut ffir Psychiatric, Am Klopferspitz 18a, D-8033 Planegg-Martinsded, F.R.G. 0006-8993/89/$03.50© lt,~]9Elsevier Science Publishers B.V. (Biomedical Division)
327 sentation of regional changes in neuronal activity upon administration of U-50,488H throughout the CNS. Furthermore, to relate the regional pattern of changes in response to U-50,488H to specific behavioral patterns, both the motivational and antinociceptive effects of this compound were evaluated under similar experimental conditions. MATERIALS AND METHODS
2-Deoxyglucose experiments Preparation of rats Experiments were performed on male SpragueDawley rats (290-340 g). In order to accustom the rats to the plexiglas restrainer, used during the experimental procedure, they were confined to the tubes for 1 h on each of the 3 days preceding the experiments. On the day of experiment catheters were inserted into the left femoral artery and vein under light halothane anesthesia. The incision site was infiltrated with a local anesthetic and closed with metal surgical clips. After surgery, the rats were placed in the plexiglas tubes and allowed to recover from the effects of the anesthesia for 3 h.
exposed to an X-ray film (Mamoray T3, Agfa Gevaert) in X-ray cassettes for 14 days, together with a series of 14C-methylmetacrylate standards of increasing specific activity; the standards were equivalent to 14C-concentrations in dried brain sections of 20/~m thickness (Amersham). To facilitate identification of regions in the autoradiogram, sections adjacent to those used for autoradiography were stained with Toluidine blue and examined with reference to the brain atlases of K6nig and Klippe123 and Paxinos and Watson 41. Local tissue concentrations of 14C were evaluated by quantitative densitometric analysis of the autoradiograms with reference to the standards. For brain regions a Transmission Densitometer DT 1105, P.Y., aperture 0.2 mm diameter (Parry, U.K.) was used and for spinal cord regions and pituitary, a computer-based microscope photometer (Leitz Texture Analyse System) was used. Local glucose utilization was calculated from plasma and brain (or spinal cord and pituitary) radioactivities and plasma glucose concentrations using an equation that takes into consideration a variation in the arterial plasma glucose concentration during the experimental period 4s.
Behavioral experiments ~ algesiometric tests Drug treatment U-50,488H was dissolved in 0.9% (w/v) NaCl and was injected i.v. in a volume of 1 ml/kg b. wt. The same volume of saline was injected in control rats. U-50,488H (0.5, 1, 2 and 5 mg/kg) or saline, were injected 5 rain before the 2-DG.
Determination of regional glucose utilization in the brain, lumbar part of the spinal cord and pituitary Regional glucose utilization was determined as previously described 5°. Briefly, 25 /zCi/rat 2-DG were injected i.v. via a syringe pump within 30 s. Thirteen arterial blood samples were drawn during the following 45 min of the experimental session and assayed for plasma glucose (Beckman Glucose Analyser) and plasma 14C-levels (liquid scintillation counting). The rats were then decapitated, the brain, pituitary and lumbar part of the spinal cord removed and immediately frozen in isopentane chilled (-45 °C) with dry ice. Serial coronal sections (20 ~m) were cut in a cryostat maintained at -20 °C, mounted on coverslips, and dried at 60 °C. The sections were
Preparation of rats A polypropylene catheter filled with a heparin solution (200 U/ml) was inserted into the rat's left jugular vein and externalized in the neck of the animal. Incisions were infiltrated with a local anesthetic and closed with surgical wound clips. These procedures were carried out under halothane anesthesia (1%). After surgery the rats were allowed to recover for 48 h. To accustom the rats to the injection procedure and to avoid blood coagulation in the catheter, the catheter was flushed twice daily with a heparin solution on the two days preceding the experiment.
Experimental procedure Tailflick to heat. The latency for removal of the tail from a beam of noxious light focused on its tip was evaluated. The mean of 3 consecutive readings (separated by intervals of 10 s) was determined. A cut-off of 12 s was enforced in order to preclude tissue damage. Tail withdrawal to pressure. An incremental pres-
328 sure was applied via an accelerating blunt wedgeshaped piston to a point 2 cm from the tip of the tail and the pressure required to elicit withdrawal of the tail was determined. The mean of 3 consecutive values (separated by intervals of 20 s) was determined. A cut-off of 540 g was used. After establishment of baseline thresholds, rats were injected with saline (controls) or U-50,488H (0.5, 1, 2 and 5 mg/kg) and returned to their home cages. Thresholds were re-evaluated 5 min after injection of drug or vehicle.
Behavioral experiments -- place conditioning Experimental procedure Jugular catheters were inserted as described above. Place conditioning was carried out employing apparatus and procedures described previously 36. Briefly, it involved the differential pairing of drug and vehicle with two sets of place cues: a white box with a textured white floor and a black box with a smooth black floor. Each rat was given 3 drug treatments in one box, and 3 vehicle treatments in the other. During training sessions rats were injected with either drug or vehicle, and 5 rain later, placed for 50 min into the training compartment. The time interval between successive exposures to experimental compartments was at least 6 h. Vehicle and drug pairing were conducted on the same day. Testing was generally carried out 1 day following training. Rats were placed on the center platform of the testbox and the amount of time spent on the black or white side during a 15 min period was recorded. The position of the rat was defined by the position of its front paws. Since rats do not show a bias for one or other environmental cue in this apparatus and under this test conditions 35'36, rats were conditioned without pretesting. The order of exposure to the white and black compartments, and the assigning of the drug to a particular color, was counter-balanced for all of the rats.
Statistical analysis Statistical differences between individual means in the 2-DG experiment were assessed by a one-way analysis of variance and the Newman-Keul's multiple range test.
To determine whether an individual drug dose produced significant place conditioning the Wilcoxon test was used. Dose-response data derived from conditioning and algesiometric tests were analysed using a one-way random effects model factorial analysis of variance (ANOVA).
Materials [1-14C]-2-Deoxyglucose (spec. act. 49-55 mCi/ mmol) was obtained from New England Nuclear, Dreieich, FRG; U-50,488H (trans-3,4-dichloro-Nmethyl-N[2-(1-pyrolidinyl) cyclohexyl]-benzeneacetamide) was obtained from the Upjohn Company, Kalamazoo, Michigan, U.S.A. RESULTS
Effects of U-50,488H upon regional glucose utilization in the brain U-50,488H produced dose-dependent changes in glucose utilization, which were pronounced in the forebrain and midbrain limbic regions (Table I, Figs. 1 and 2). A dose of 0.5 mg/kg U-50,488H significantly increased glucose utilization in two of the 45 regions investigated: the lateral habenular nucleus (+20%) and the median raphe nucleus (+13%) (Table I). At a dose of 1 mg/kg further significant increases were observed in the anteromedial thalamic nucleus (+11%) and the central gray pons (+8%). However, this dose caused a significant decrease in glucose utilization in the frontal cortex (+11%) (Table I). In addition to regions affected by 1 mg/kg, a dose of 2 mg/kg increased glucose utilization in the medial mammillary nucleus, medial part (+17%), the posterior mammillary nucleus (+29%) and the •.ntrai tegmental nucleus (+22%) (Table I). A dose of 5 mg/kg increased rates of glucose metabolism in the anterior thalamic nuclei, the mammillary nuclei and the lateral habenular nucleus still greater. In the lateral septal nucleus (+36%), the nucleus accumbens (+26%), the dorsal rapbe (+ 14%) and the dorsal tegmental nucleus further increases in glucose utilization (+ 18%) (Table l) were produced by 5 mg/kg U-50,488H. The ventral tegmental area, the lateral thalamic nucleus, the lateral amygdaloid nucleus, posterior part and the lateral hypothalamus also displayed increased glucose utilization, but this trend
329
TABLE I
Effects of U-50,488H upon local cerebralglucose utilization (l~molllO0g/min) Values shown are means _+ S.E.M. for the number of rats indicated in parentheses.
Brain regions
Vehicle
U-50,48811 0.5 mglkg
1 mglkg
2 mglkg
5 mglkg
Telencephalon Frontal cortex Frontoparietal cortex, motor area Frontoparietal cortex, somatosensory area Visual cortex Auditory cortex Hippocampus Dentate gyrus Medial amygdaloid nucleus Lateral amygdaloid nucleus, posterior part Lateral septal nucleus Caudate nucleus Globus pallidus Nucleus accumbens
(5) (5) (5) (5) (5) (5) (5) (5) (3) (5) (5) (5) (5)
95 + 2 100 + 3 105 + 3 99 + 3 129 + 4 83 + 3 49 + 2 41 + 2 86 + 2 50+1 95 + 3 53 + 2 96 + 3
(6)* (6) (6) (6) (6) (6) (6) (5) (6) (6) (6) (6) (6)
92 + 5 96 + 6 101 + 6 102 + 3 134 + 7 87 + 4 48 + 2 42 + 3 85 + 4 49+3 94 + 4 50 + 3 98 + 5
(5)* (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5)
92 + 8 109 + 5 102 + 4 89 + 5 101 + 3 49 + 2 59 + 1 109 + 3 96 + 4 116_+5 73 _+ 2 105 _+ 4
(5) (5) (5) (5) (5) (5) (4) (5) (5) (5)* (5) (5)
106 + 3 116 + 3 103 + 3 89+2 102 + 3 48+1 68 + 6 112 + 5 108+2 122+5 72 + 2 107 + 3
(6) (6)* (6) (6) (6)
104 +_ 7 118 + 6 107 + 6 91+5 108 + 8
(5) (5)* (5) (5) (5)
52 _+ 5 77 _+ 5 91 _+ 4 56 _+ 2 95 _+ 5 78 _+ 2 96 _+ 8 101 _+ 6 64 _+ 4 79 _+ 2 113 + 8 79 -+ 6
(5) (5) (5) (5) (5)*
51+2 81+2 94 _+ 3 67 + 5 102 + 2 82 + 2 104 + 3 107 _+ 3 68 ± 4 82 + 1 121 + 3 89 + 3
(6)
53+3
(5)
(6) (6)
8 4 + 4 (5) 93 + 4 (5)
106 ± 4 104 + 6 110 + 6 103 + 4 !39 + 7 85 + 3 54 + 2 42 + 2 82 + 3 46 + 2 93 + 4 51 + 2 88 + 4
(6) (6) (6) (4) (6) (6) (6) (6) (6) (6) (6) (6) (6)
100 + 103 + 108 + 94 + !3! + 82 + 48 + 43 + 80 + 48 + 96 + 54 + 93 +
99 + 4 104 + 3 99 + 2 89 + 3 99 + 2 47 + 2 58 + 2 101 + 3 94 + 3 97_+4 72 _+ 3 108 _+ 5
(6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6)
54 _+ 2 87 _+ 4 98 _+ 4 57 _+ 2 84 _+ 4 77 _+ 4 90 _+ 4 99 _+ 4 66 _+ 3 76 _+ 2 126 + 10 88 -+ 4
(6) (6) (6) (6) (6)
3 3 4 6 10 5 4 1 6 3 6 3 4
94 + 1 98 + 2 104 + 4 99 + 1 133 + 5 79 _+ 3 47 + 2 41 + 4 94 + 3 63+1 106 _+ 4 56 _+ 4 111 +_ 4
Diencephalon Anteroventral thalamic nucleus Anteromedial thalamic nucleus Lateral thalamic nucleus Ventral thalamic nucleus Ventromedial thalamic nucleus Hypothalamus, ventromedial nucleus Lateral hypothalamus Medial mammillary nucleus, medial part Posterior mammiilary nucleus Lateral habenular nucleus Lateral geniculate body Medial geniculate body
Mesencephalon + hindbrain Substantia nigra, pars reticulata Superior colliculus, superficial layer lnterpeduncular nucleus Ventral tegmental area Median raphe nucleus Dorsal raphe nucleus Ventral tegmental nucleus Dorsal tegmental nucleus Central gray, dorsal part Central gray, pons Medial vestibular nucleus Nucleus of the lateral lemniscus
(6) (6) (6) (6) (6) (6) (6)
(5) (5) (5) (5) (5) (5) (5)
Cerebellum Cerebellar cortex Cerebellar vermis Dentate nucleus lnterpositus nucleus
50 53 92 95
-+ 3 _+ 3 _+ 4 _+ 4
(6) (6) (6) (6)
54 61 89 92
-+ 6 _+ 5 _+ 5 _+ 6
(5) (5) (5) (5)
White matter G e n u of corpus callosum Internal capsule Cerebellar white
24 _+ 1 (6) 26 _+ 1 (6) 29 _+ 1 (6)
24 _+ 1 (5) 29 _+ 2 (5) 26 _+ 3 (5)
51 57 94 97
_+ 2 + 2 +_ 4 + 3
(6) (6) (6) (6) (6)** (6) (6)
(6) (6)*** (6) (6) (6) (6) (6)* (6) (6) (6) (6) (6) (6)
2 4 + 1 (6) 2 5 + 1 (6) 2 8 + 1 (6)
124 + 2 130 + 3 117 + 3 92+1 115 + 6 5 0 + 2 (5) 51_+4 66 +_ 6 (5) 75 _+ 3 118 + 5 (5)* 131 + 3 i21_+7 (5)** 134_+10 1 4 6 + 8 (5)*** 183_+7 75 + 5 (5) 79 + 2 109 + 5 (5) 110 + 8
57 + 2 105 + 2 85 _+ 2 110 + 3 113 + 3 69 + 3 87 + 2 132 + 6 92 + 5 54 59 96 95
+ 2 + 3 + 4 ___4
(5) (5)*** (5) (5)* (5) (5) (5)** (5) (5) (4) (5) (5) (5)
2 2 + 2 (5) 2 2 + 2 (5) 2 7 + 1 (5)
*P < 0.05; **P < 0.01; ***P < 0.001 significantly different from vehicle (Newman-Keul's multiple range test).
(4) (4) (4) (4) (4) (4) (4) (4) (4) (4)*** (4) (4) (4)*** (4)** (4)*** (4) (4) (4) (4) (3) (4)*** (4)*** (4)*** (4) (4)
(4) (4) 87 + 2 (4) 65 + 3 (4) 106 + 3 (4)** S8 _+ 2 (4)* 111 + 3 (4)* 117 + 5 (4)* 77 + 5 (4) 90 + 2 (4)*** 127 + 6 (4) 92 + 5 (4) 54+3 85-+2
55 63 94 95
+ + + +
3 3 4 4
25+3 28+2 28+1
(4) (4) (4) (4) (4) (4) (4)
A
~
-
•l' ¢'--
, A
Fig. 1. Effects of U-50,488H on autoradiographic grain densities, representing glucose utilization. A - D : grain densities in film exposed to sections from different levels of a control brain. E - H : grain densities in film exposed to section from corresponding brain levels of a rat injected with U-50,488H (5 mg/kg). Abbreviations: acc, nucleu:+accumbens; tav, anteroventral thalamic nucleus; tam, anteromedial thalamic nucleus; Ih, lateral habenular nucleus; mmm, medial mammi!!ary nucleus, medial part; mp, posterior mammillary nucleus.
331
E
0
H
Fig. 2. Effects of U-50,488H on autoradiographic grain densities, representing glucose utilization. A-D: grain densities in film exposed to sections from different levels of a control brain. E-H: grain densities in film exposed to sections from corresponding brain levels of a rat injected with U-50,488H (5 mg/kg). Abbreviations: dr, dorsal raphe nucleus; mr, median raphe nucleus; vtg, ventral tegmental nucleus; dtg, dorsal tegmental nucleus; CGPn, central gray pons.
332 TABLE II
Effects of U-50,488Hupon glucose utifization (l~molllO0glmin) of the neural, intermediateand anteriorlobe of the pituitary Values shown are means _+S.E.M. for the number of rats indicated in parentheses.
Posterior lobe Intermediate lobe Anterior lobe
Vehicle
U-50,488H 0.5 mglkg
1 mglkg
2 mglkg
5 mglkg
41 _+5 (6) 48 + 6 (6) 29 + 3 (6)
57 _+8 (5) 66 + 9 (5)** 34 + 4 (5)
50 _+4 (6) 72 __.5 (6)** 38 + 2 (6)
48 _+2 (5) 81 + 4 (5)** 31 + 4 (5)
54 + 9 (4) 84 + 7 (4)** 39 + 6 (4)
**P < 0.01 significantly different from vehicle (Newman-Keul's multiple range test).
was not statistically significant.
Effects of U-50, 488H upon nociceptive thresholds
Effects of U-50,488H upon local glucose utilization in the spinal cord
U-50,488H produced a dose-dependent attenuation in the response of rats to noxious heat (Fl,14 = 44.92, P = 0.0001) and pressure (Fla 3 = 13.7, P = 0.027) (Fig. 4).
In the lumbar part of the spinal cord, U-50,488H increased glucose utilization in the ventral horn (the m e a s u r e d area comprised Rexed's laminae 8 and 945) a n d the region in the vicinity of the central canal (Rexe d ' s lamina 1045) in a dose-dependent m a n n e r (Table If).
Motivational properties of USO,488H Significant place aversion was p r o d u c e d by all 4 doses of U-50,488H tested (Table IV). No significant differences were found between dose and magnitude of effect (F3,40 = 1.094; P = 0.362).
Effects of U-50, 488H upon local glucose utilization in the pituitary
DISCUSSION
Even the highest dose of U-50,488H (5 mg/kg) did not produce any significant changes in glucose utilization in the anterior and neural lobes of the pituitary (Table III). Glucose utilization in the intermediate lobe, however, was increased significantly at every dose (Table IIl) and in contrast to control animals, the intermediate lobe could be clearly differentiated from the neural and anterior lobes in autoradiograms from rats treated with U-50,488H (Fig. 3).
The 2 - D G method 5° was employed to investigate regions and neural circuits in the rat brain, spinal cord and pituitary involved in the analgesic and aversive (dysphoric) effects of the selective r-opioid receptor agonist U-50,488H. In comparison to recent studies that employed unselective r - r e c e p t o r ligands and evaluated the effects of only single doses on local cerebral glucose utilization aa2 U-50,488H induced a
TABLE III
Effects of U-50,488Hupon glucose utilization (/~mol/lO0g/rain) in the lumbarpart of the spinal cord Values shown are means + S.E.M. for the number of rats indicated in parentheses.
Spinal cord Lumbar Part (L5) Lamina I-III Lamina V-VI LaminaVII-IX Lamina X White matter
Vehicle
36 + 48 + 46 + 48 + 20 +
4 a 5 5 3
U-50,488H
(6) (6) (6) (6) (6)
0.5 mg/kg
1 mg/kg
2 mglkg
5 mglkg
43 + 54 + 52 + 57 + 21 +
43 + 55 + 55 + 58 + 26 +
46 + 56 + 57 + 59 + 22 +
44 + 54 + 61 + 61 + 22 +
2 3 2 2 1
(5) (5) (5) (5)* (5)
3 2 2 2 2
(6) (6) (6) (6)* (6)
*P < 0.05; **P < 0.01 significantly different from vehicle (Newman-Keul's multiple range test).
1 1 1 2 1
(5) (5) (5)* (5)* (5)
2 2 2 2 2
(4) (4) (4)** (4)* (4)
333
A
B
AL
PL
C
IL
Fig. 3. Effects of U-50,488H on autoradiographic grain densities in the pituitary, representing glucose utilization. A: Nissl-stained section. B: corresponding pituitary section of a 2-DG autoradiograph of a control animal. C: 2-DG autoradiograph of an animal treated with U-50,488H (5 mg/kg). Abbreviations: IL, intermediate lobe; AL, anterior lobe; PL, posterior lobe of the pituitary.
more complex pattern of changes. Thus, most pronounced changes in glucose utilization were observed ip limbic forebrain structures and structures that lie within what Nauta 38, has termed the 'limbic midbrain area'. These regions do not entirely correspond with known regions of r-opioid binding sites 29"33. Such discrepancies, however, may be explained by the fact that glucose utilization reflects actions at both the receptor site and the influence of other neuronal circuits. The effects in the lateral habenuiar nucleus, an area apparently devoid of r-opioid binding sites 29'33, therefore, probably reflect an activation of its afferent inputs, which amongst others originate in the nucleus accumbens, and in the median raphe nucleus ~9. These latter regions also displayed changes in glu-
300' ....,. o o II ul Cu
heat
cose utilization and exhibit a dense labeling of r-opiold binding sites 29'33. The increased rates of glucose metabolism in the median and dorsal raphe nucleus, the central gray and the tegmental nuclei, furthermore, might be attributed to modulation by descending projections from the lateral habenular nucleus 1,18.47. In turn, the increased activity of the tegmentai nuclei could induce changes in the mammillary body via the mammiUary peduncle s. Based on known reciprocal connections, however, the activity in the tegmental nuclei vice versa, could be modulated by the mammillary body 1°'18. Besides the mammillary peduncle another important fiber tract originating from the mammillary body is the mammillothalamic tract. This tract projects to the anterior group of the thalamic nuclei 9 which also displayed an increase in glucose utilization. The latter structures and the mammillary body have been described as components of a limbic circuit described by Papez 4°. Further components of this circuit which displayed changed rates of glucose metabolism after U-50,488H were the lateral septal nucleus and the frontal cortex. Taken together, these results indicate that administration of U50,488H selectively activates structures which are
200 ul tO
TABLE IV Place preference of rats trained with different doses of U-50,488H
Values shown are mean differences between time spent on the drug and the vehicle side of the test box (s) + S.E.M. for the number of rats indicated in parentheses.
100
US0~88H (mg/kg) Fig. 4. The influence of acutely applied U-50,488H upon nociceptive thresholds. The dose-response relationship is depicted. Tail heat = triangle; tail pressure = circles. For each dose n = 5; means + S.E.M. are shown.
0.5 mg/kg
I mg/kg
2 mglkg
5 mg/kg
-446 + 74 (12)*
-380 + 62 (12)*
-264 + 60 (10)*
-446 + 100 (10)*
*P < 0.05 significant place aversion (Wilcoxon test).
334 implicated in the circuitry of the limbic system and which are reciprocally interconnected. Functionally these structures have been implicated in the control of various neurobehavioral states. Thus, stimulation and lesions of single components of the limbic forebrain circuits have revealed their involvement in the expression of emotion and mood as well as in the initiation of motivational processes such as avoidance and attack 6"7"t5'32'52. Besides their role in the control of locomotion 5~'5ssleep, analgesia and pain 49 the raphe nuclei w particularly the median raphe nucleus - - have also been implicated in affectire behaviors t7,49. Based upon these data we conclude that the aversive effects produced by the K-agonist U-50,488H in the place conditioning experiments (representing a dysphoric component of this drug) may result from a selective activation of these structures. As regards the analgesic action of U-50,488H, a supraspinal site of pain modulation by cerebral r-receptors has to be considered, since the dorsal raphe nucleus, the region of the central gray ports, and the nucleus accumbens, structures that have been shown to contribute substantially to opiate and stimulus produced analgesia2°,2t,39,46'59, displayed increased activities. Indeed, recent studies examining the antinociceptive effects of i.c.v, administration of U-50,488H indicate a supraspinal site of action of r-opioid ligands n. Glucose utilization at the spinal level was significantly increased in the ventral horn - - the measured area comprised Rexed's lamina 8 and lamina 9 - - and lamina 1045, the gray area surrounding the central canal. Although u-opioid binding sites are mainly concentrated in the superficial layer of the dorsal horn, lower levels are found in other regions with some enrichment in lamina 1 0 1 6 ' 3 4 . In view of evidence indicating a role of the region in the vicinity of the central canal in nociception 27,37, the increased glucose meREFERENCES 1 Aghajanian, G.K. and Wang, R.Y., Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique, Brain Research, 105 (1977) 389-403. 2 Baumgarten, H.G., BjOrklund, A., Holstein, A.F. and Nobin, A., Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary, Z. Zellforsch., 126 (1972) 483-517.
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