0028-3908/92 $5.00 + 0.00 Copyright 0 1992 PergamonPress Ltd
Neuropharmacology Vol. 31, No. 11, pp. 1193-I 199, 1992 hinted in Great Britain. All rights nscwed
EFFECTS OF CHOLINERGIC AGONISTS ON REGIONAL BRAIN ENERGY METABOLISM IN THE SCOPOLAMINE-TREATED RAT C. A. RAY,’ J. BLIN,~* T. N. CHASESand M. F. Pmacav’t ‘CNS Research, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. and *NINCDS NIH Bldg 10, Rm 5C103, Bethesda, MD, 20892, U.S.A. (Accepted 11 May 1992) Summary-The effects of scopolamine, physostigmine, RS86 and U-80816B on regional energy metabolism were studied in rodents by means of the 2-deoxyglucose autoradiographic technique. Scopolamine depressed metabolism in an area of cerebral cortex, focused around the parietal region. Rats treated with cholinergic direct agonists (U80816B, RS86) as well as with the indirect agonist (physostigmine) all showed decreases in cortical energy metabolism, similar to scopolamine. They also induced an increase in thalamic metabolism. When these drugs were given in conjunction with scopolamine, metabolism tended to change in the opposite direction from the values obtained with the drug alone. These results suggest that there are complex interactions between pre- and post-synaptic muscarinic receptors. Additionally, nicotinic receptors could also be involved in some of the effects of physostigmine. Key words-2-dg
autoradiography,
muscarinic receptors, nicotinic receptors, Alxheimer’s disease.
Degeneration of cholinergic neurons, arising from the medial forebrain is one of the more consistent find-
ings in Alzheimer’s disease (Whitehouse and Au, 1986; McGeer, McGeer, Suzuki, Dolman and Nagai, 1984). One common method for modeling Alzheimer’s disease pharmacologically is treatment with the muscarinic choline@. antagonist, scopolamine (Beatty, Butters and Janowsky, 1986; Smith, 1988). Scopolamine, for example, blocks cholinergic transmission in the cerebral cortex and hippocampus, the major projection areas of the medial forebrain cholinergic system (Frey, Ehrenkaufer and Agranoff, 1985). In addition, like Alzheimer’s disease, scopolamine impairs memory function (Rusted and Warburton, 1988; Sunderland, Tariot, Weingartner, Murphy, Newhouse, Mueller and Cohen, 1986). In a previous study, measuring regional energy metabolism in brain, it was noted that the effects of scopolamine in the parietal cortex of the rat (Piercey, Vogelsang, Franklin and Tang, 1987) seemed to correspond well with the reported depression in the parietal cortex of Alzheimer’s patients (Chase, Foster and Mansi, 1983; Foster, Chase, Mansi, Brooks, Fe&o, Patronas and DiChiro, 1984). This finding has recently been confirmed by a direct collaborative study (Blin, Ray, Chase and Piercey, unpublished). However, the same study indicated that there was no overall correlation between the effects of scopolamine in the brain of either rat or human and the alterations that occur in Alzheimer’s patients.
Nonetheless, it appeared desirable to evaluate further the effects of cholinergic drugs in the scopolamine-treated rat because (1) the metabolic depression in the parietal cortex resembled that of Alzheimer’s disease, (2) metabolic considerations aside, cholinergic denervation and amnesic phenomena are consistent features of Alzheimer’s disease, (3) therapies with cholinergic agonists continue to be evaluated for the symptomatic treatment of Alzheimer’s disease and (4) mapping the functional effects of cholinergic agonists and antagonists could enhance appreciation of the physiology and pharmacology of choline@ systems. 2,8-Diazaspiro(4, S)decane-1,3-dione, 2-ethyl-gmethyl-monohydrobromide (RS86) a direct-acting cholinergic agonist (Bruno, Mohr, Gillespie, Fedio and Chase, 1986) and physostigmine, an indirectly acting choline@ agonist (cholinesterase inhibitor) have previously been used to treat Alzheimer’s disease, with limited success. In the present study, these two compounds were evaluated and compared to U-80816B (1-[4-(5methyl-lH-imisol-1-yl)-2-butynyll2-pyrrolidinone hydrobromide), a partial cholinergic agonist (Moon, Chidester, Heier, Morris, Collins, Russell, Francis, Sage and Sethy, 1991; &thy, Francis, Hyslop, Sage, Oien, Meyer, Collins, Russell, Heier, Hoffmann, Piercey, Nichols, Schreur and Moon, 1991), for their effects on energy metabolism in the brain, using 2-deoxyglucose autoradiography. METHODS
*Present address: Service Hospitalier Frederic Joliot, DRIPP, CEA, Hopital d’orsay, 91401 Orsay, France. tTo whom correspondence should be addressed.
The procedures for 2-deoxyglucose (2-dg) autoradiography have been described in detail previously 1193
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C. A. RAY et al.
(Sokoloff, Reivich, Kennedy, Des Rosiers, Patlak, Pettigrew, Sakurada and Shinohara, 1977; Piercey et al., 1987; Piercey and Ray, 1988a, b; Blin, Ray, Chase and Piercey, 1992). Briefly, Charles River Sprague-Dawley male rats, 275-320 g, were anesthetized with a fluothane-nitrous oxide mix and fitted with arterial and venous cannulae. After cannulation, the animals were lightly restrained in a Brain Tree experimental containment unit. The animals were allowed to recover in the restrainer for a minimum of 30 min, after removal of anesthesia, before any injections of drugs. Following recovery, the animals were given, by intravenous injection, either the drug or an equal volume of saline vehicle. Scopolamine (30 pg/kg) or saline was injected 10 min prior to an intravenous injection of 25 pCi of [i4C]2-dg. Physostigmine (100 or 300 pg/kg), RS86 (2 mg/kg), U-80816B (2 or 7 mg/kg) or saline was injected 5 min prior to the [14C]2-dg. Forty-five minutes after the injection of the label, the rats were sacrificed, their brains rapidly removed and frozen over liquid nitrogen. Utilizing the horizontal autoradiography atlas, designed for rat-human comparative studies (Blin et al., 1992), the brains were mounted dorsal side down and then sectioned horizontally, using a Leitz 1720 motorized cryostat, roughly corresponding to bregma levels:8.6, 7.6, 6.6, 5.6, 5.1, 4.6, 4.1 and 3.1 mm; and at 500, 1000 and 1500 pm after bregma 3.1 mm (Paxinos and Watson, 1986). The 20pm sections were positioned on 25 mm* cover glasses mounted on railroad board, using two-sided tape and exposed to Kodak SB-5 film for 6 days. The resulting autoradiograms were developed and analyzed for local cerebral glucose utilization, using an Amersham RAS 1000 image analysis system. The metabolic activity of regions of the brain were analyzed in polygonal areas of interest, directly from the autoradiograms. Metabolic values from whole brain were obtained by taking, as areas of interest, entire horizontal sections of all levels of the brain and then combining results by weighted averaging, to obtain the mean metabolic rates of all pixels imaged. Average glucose utilizations within each treatment group, were compared statistically using the SAS statistical program (analysis of variance [ANOVA] and Fischer’s least significant difference for multiple comparisons). The 30pg/kg (i.v.) dose of scopolamine was chosen, based on the results of a previous study (unpublished) demonstrating that similar maximum effects were detected between doses of 10-100 pg/kg (i.v.). Two doses of both physostigmine and U-80816B were chosen in order to encompass the range of effects of those compounds. The dose for RS86 was also chosen for its maximum effect in other experiments (V. H. Sethy, personal communication). Copious salivation and lacrimation were observed during the experiments with physostigmine and RS86.
RESULTS
Physostigmine and scopolamine
The effects of physostigmine (100 and 300 pg/kg) on energy metabolism in the brain, compared to control and scopolamine-treated animals are presented in Table 1. Intravenous administration of scopolamine decreased metabolism in the parietal, motor and striate cortices. These results were similar to those previously described using intraperitoneal administration of scopolamine and making measurements from coronal sections (Piercey et al., 1987). At a dose of 100 pg/kg, physostigmine also decreased metabolism in the parietal and striate cortices, to levels similar to those produced by scopolamine. At this dose, physostigmine induced a decrease in activity in the medial septum, while stimulating the anterior thalamic nuclei. When scopolamine and physostigmine, 100 pg/kg, were combined, the values for the cortex were brought back to control levels, while the ventral, anterior and posterior nuclei of the thalamus all showed significant increases in metabolism. The 300 pg/kg dose of physostigmine affected even more regions of the brain. By itself, the larger dose of physostigmine decreased metabolism in the striate, motor, cingulate, parietal and frontal one cortical regions, as well as in the amygdala and accumbens nuclei. It increased metabolism in the anterior and posterior thalamic regions. When 300 pg/kg of physostigmine was administered with scopolamine, energy metabolism in the brain returned to control levels in frontal one and cingulate cortices, areas where physostigmine, but not scopolamine, depressed metabolism. Metabolism in the striate cortex also did not differ from controls in animals, treated with both scopolamine and physostigmine, even though each significantly depressed metabolism when given alone. The depression in metabolic values in the parietal and motor cortices, experienced with 300 pg/kg physostigmine, were not brought back to control levels by scopolamine. However, scopolamine superficially appeared to antagonize, physostigmine given in a large dose in the parietal and motor cortex, since the depression in metabolism was significantly less than that seen with physostigmine alone but did not differ from that observed with scopolamine alone. Just as with small doses, large doses of physostigmine continued to stimulate metabolism in the thalamic nuclei in scopolamine-treated rats. However, the depression in energy metabolism in the amygdala and accumbens were antagonized by administration of scopolamine. Muscarinic agonists and scopolamine
The effects of RS86 and U-80816B, with and without scopolamine, are compared against control and scopolamine-treated animals in Table 2.
Cholinergic agonists and energy metabolism Table
1. Effects of scopolamine
and physostigmine
Name
Control
ScoP 30 ualkg
Brainstem Cerebral peduncle IP Hypothalamus Amygdala Substantia nip Red nucleus Hippocampus Accumbens Caudate Globus DBB Medial septum Medial geniculate Lateral geniculate Ventral tier Ventral thalamus DM thalamus Ant. thalamus Post. thaiamus Centrai grey MHB LHB Inferior colliculus Superior colliculus Colliculi White GCC Cerebraller hemisphere Vermis Frontal cortex one Frontal cortex two Sensory cortex Parietal cortex Auditory cortex Cingolate cortex Temporal cortex Motor cortex Striate cortex Brain
46 * 3.3 25 4 0.7 85 + 4.2 40+ 1.8 49 * 2.7 49 * 4.3 55 f 4.8 56 f 3.6 71 F 4.4 76 + 4.2 49 + 3.4 60+4.1 SO& 3.1 12 f 4.3 53f3.1 61 + 3.2 64+4.1 70 i 3.4 72 * 3.6 65 + 2.9 48 + 2.3 5-l + 2.3 78 f 3.2 91 * 9.4 62 + 4.1 76 ? 6.6 22 & 1.2 252 1.9 40 It. 3.4 56 +_ 5.1 12 + 4.1 74 + 4.4 18 * 5.0 16 f 5.0 84 * 4.9 83 i. 4.3 87 + 5.9 75 * 4.9 83 + 4.6 56 * 3.5
42 f 2.8 22& 1.4 82 f 4.9 38k 1.6 46 k 2.2 47 + 3.0 51 + 3.4 52 + 2.2 66 + 2.0 71 * 3.8 47 f 2.5 58 * 3.3 52 + 3.4 66 rf: 6.2 51 k 4.1 56 & 4.8 57 f 4.5 65 * 4.0 64+4.5 60+4.1 44 f 2.6 54 + 2.9 71 rt 4.1 79 + 5.0 55 + 2.9 66 + 3.7 20% 1.0 25 f 3.9 36 f 2.3 50 f 3.9 64 + 2.4 61 + 2.8 69 f 3.2 67+2.1’ 76 + 5.6 75 * 2.7 72 + 2.8 64 + 2.3” 71 * 1.8’ 51 * 2.5
Physo IM) Ica/kg 47 f 1.4 23 * 1.0 79 f 5.4 39 f 2.5 4b* 1.3 48 4 1.4 52 + 2.0 51 & 1.5 65 f 3.4 70 * 1.4 48* 1.8 53 2 3.2 44* l.OC II f 2.6 S4 + 2.9 64 _L2.7 66 + 2.6 69+ 1.7 79 rf: 2.4’ 68 rt 2.5 47* 1.5 56 k 2.3 14 ~fi3.6 80 f 2.7 62+2.1 68*2.0 22 + 0.9 24rf: 1.5 43 2 2.2 55f 1.9 65 k 2.9 71 * 3.1 75 & 4.2 67 f 3.1’ 79 + 2.3 76 24.1 79 f 4.8 66 f 3.3 13 f 3.4* 53 * 2.0
on regional
1195
energy metabolism
Physo 100 pg/kg +SCOP 30 /&kg 4brfc 1.9 28 + 2.9 16 it 4.3 43ir 1.8 48 rt 1.2 52 & 2.3 59 + 3.0 56f 1.7 61 & 3.3 74 + 2.5 50& 1.4 65 + 2.0 S8& 1.8 78 rt 3.4 58 rf: 1.8 69 rf: 2.9 69 +_ 2.9= 78 * 5.0 87 + 4 2’~~ 72 : 3:3’ 49k2.1 63*4.1 81 f 3.9 81 ri: 1.7 61 rt 2.5 68 F 2.0 25 & 1.0 27 rf: 0.6 43 If: 2.1 56 + 2.7 68 + 0.7 764 1.1 75 + 2.4 71 i 1.9 85 f 2.5 84 + 2.s 78 * 2.4 68 2 0.7 79* 1.8 56+ 1.5
in brainy
Physo 300 pglkg 45 + 22+ 83 * 362 39 + 41 rt 51 * 46 k 58 *
2.5 I.7 7.5 1.2 1.8b” 2.9 3.5 2.4 3.9b 68 rt 4.2 46 5 2.4 51 f 2.2 46 + 3.2 73 + 6.1 56 Y!Z 3.6 64 * 4.4 64 * 3.3 70 + 5.0 89 + 8.3” 71 & 4.4Q 47 f 2.9 53 k 2.9 72 f 6.3 18 * 5.0 63 &-3.7 67 f 4.0 21 + 1.6 21 * 1.7 42 f 2.8 54 k 2.3 58 f 3.4b 64 f 4.9 69 + 4.7 59 5 4.sb 76 +_6.3 70 + 4.48 69 + 5.4 60 5 4.9D 65 + 3.5b _^ __ 50 19.1
Physo 300 &kg +SWP 30rg/kg 48+ 1.6 25 * 1.7 74+ 1.6 425 1.0 47 f 2.0 SO* 1.6 57 + 2.2 S4? 1.6 63% 1.4 70 i. 1.7 49+ 1.3 59% 1.7 525 1.4 74+ 1.6 bO+ 1.7 67? 1.5 68 k 0.9t 12 1 1.5 85 + 2.7”” 70 * 1.1c 47kO.l 61 + 2.7 19 f 3.5 81 &Y2.5 64 + 2.8’ 70 + 2.2 24+ 1.0 28 f 2.4 44 + 0.5 54 + 0.6 70 * 3.0 66 k 2.3 14 + 2.3 66 k 1.5’ 82 i 2.2 75 + 1.9 76 * 2.0 65+ 1.7s 74 + 2.9 55 f 1.2
“P < 0.05, bP < 0.01 compared to control; ‘P c 0.05, dP c 0.01 compared to scopolamine 30 pg/kg tGlucose metabolism &mol glucose/100 g/min). Means + SEM, 4 animals per group.
The drug RS86 caused an increase in metabolism in several subcortical areas, including: brainstem, medial septum, ventral thalamus, anterior thalamus, posterior thalamus, superior colliculus and cerebellar hemisphere. It decreased metabolism in the parietal, motor and striate cortices. When combined with ~opol~ine, RS86 no longer increased metabolism significantly greater than controls, in the anterior thalamus or superior colliculus. However, scopolamine did not antagonize stimulai.ion of the brainstem, ventral or posterior thalamus or cerebellum by RS86. Although both RS86 and scopolamine depressed energy metabolism in the parietal and striate cortices, when either drug was given alone, the metabolic values in these areas were not significantly different from control when the two drugs were administered together. The drug U-80816B (2 mg/kg) caused a decrease in the striate cortex, when compared to control. When combined with scopolamine, the metabolic depression observed with scopolamine in the parietal cortex was abolished, as was the depression observed with each drug when given alone, in the striate cortex. At lmg/kg, U-80816B continued to depress metabolism in the striate cortex but it increased
metabolism in the anterior thalamus, posterior thalamus and brainstem. In the combination experiments, 7 mg/kg U-80816B reversed the depressant effects of scopolamine in the parietal cortex, while scopolamine reversed the stimulant effects of large doses of U80816B in the thalamus. The common depressant effects of scopolamine and U-808 16B were retained in the striate cortex with no additive effects. Treatment with scopolamine did not alter the stimulant effects of U-80816B in the brainstem. DISCUSSION The present experiments, using intravenous administration of scopolamine have confirmed earlier studies demonstrating a depression in metabolism in the parietal cortex with intraperitoneallyadministered scopolamine (Piercey ei al., 1987). Similar depressions in metabolism in the parietal cortex are also seen in Alzheimer’s disease (Chase et al., 1983; Foster et al., 1984; Friedland, Budinger, Koss and Ober, 1985; Hoyer, 1986; Kuhl, Metter and Riege, 1985), although recent experiments have demonstrated that there was no overall correlation between areas showing metabolic effects with
46 + 3.3 25 * 0.7 85 + 4.2 40& I.8 49 + 2.7 49+4.1 55 & 4.8 56 + 3.6 71 + 4.4 76 f 4.2 49 + 3.4 6ok4.1 50+ 3.1 72 _t 4.3 53 & 3.1 61 + 3.2 64 + 4.1 70 + 3.4 72 + 3.6 65 2 2.9 48 + 2.3 57 + 2.3 78 f 3.2 91 + 9.4 62 54.1 76 + 6.6 22 + 1.2 25 + 1.9 40 & 3.4 56 + 5.7 72 + 4.7 74 f 4.4
Brainstem Cerebral peduncle IP Hypothalamus Amygdala Substantia nigra Red nucleus Hippocampus Accumbens Caudate Globus DBB Medial septum Medial geniculate Lateral geniculate Ventral tier Ventral thalamus DM thalamus Ant. thalamus Post. thafamus Central grey MHB LHB Inferior colliculus superior colliculus Colliculi White GCC Cerebcllar hemisphet Vermis Frontal cortex one Frontal cortex two Sensory cortex Parietal cortex Auditory cortex Cingulate cortex Temporal cortex Motor cortex Striate cortex Brain 5.0 4.9 4.3 5.9 4.9 4.6 3.5
42 f 2.8 22+ 1.4 82 ) 4.9 38 5 1.6 46 * 2.2 47 + 3.0 51 f 3.4 52 f 2.2 66 * 2.0 71 + 3.8 47 & 2.5 58 F 3.3 52 f 3.4 66 + 6.2 51 + 4.1 56 * 4.8 57 & 4.5 65 + 4.0 64 + 4.5 6ok4.1 44 + 2.6 54 rt 2.9 71 rt 4.7 79 1 5.0 55 f 2.9 66 f 3.7 20& 1.0 25 + 3.9 36 f 2.3 50 f 3.9 64 + 2.4 67 k 2.8 69 ) 3.2 67 f 2.1” 76 & 5.6 75 + 2.1 72 4 2.8 64 + 2.3” 71 + 1.8” 51 22.5
=JP 30 /%/kg 48 & I .3 25+ I.1 19ir: 5.1 37 & 2.2 43 & 2.4 50+2,1 57k2.1 50 * 1.6 cb+ 1.8 14 f: 1.4 49 & 2.5 58rt3.1 48 & 3.3 70 f 2.1 53 + I.2 63 + 2.6 66 +: 3.0 67k3.l 80 rt 3.8c 69 + 2.8 46i:3‘1 54k2.5 76 -I_2.6 80 + 2.6 59+ I.7 67 If:0.9 23& 1.6 25 & 2.4 43 + 1.9 55+- I,9 72 & 2.6 67+ I.7 76 + I .6 71 + 0.7 79t 1.3 80 ,: 2.9 74+ I.4 70& I.4 70+ 1.7b 54+ 1.3
U-808 I 6B 2 mg/kg 46&2.1 22 * 1.1 87 f 4.8 38 f 1.8 48 f 2.1 51 1: 2.3 57 f 3.2 55 * 1.5 72 + 2.9 82 + 1.5 51 + 2.2 61 f 3.7 54 f 2.0 71 f 2.9 54 * 1.6 62 + 2.2 63 * 2.2’ 73 * 3.3 74 * 2.1 67 i 2.7 50 i 2.5 59 2 3.0 80 + 3.6 82 * 3.7 59 5 2.1 69 k 2.4 23 k 1.5 25 k 0.4 42 5 2.0 59 + 3.2 83 * 1.98” 75 * 2.5 80 + 2.5 77 & 2.3’ 85 * 1.6 92 + 4.1d 85 * 3.0 75 * 2.W 85 + 1.6d 57 I 1.8 53 + 4.7’” 27 * 4.9 84 + 5.2 40 f 2.9 48 4 3.8 55 rt 5.2 62 + 5.5 54 * 5.3 12 + 5.6 78 * 6.3 55 _t 6.2 64i6.3 54 * 6.0 77 * 7.4 57 rt: 4.2 69 rt 6.4 73 + 6.0d 75 4.7.3 86 + 8.2”qd 74 f 6.3”6 SZ-r_Al 58rt4.9 81 + 5.7 80+ 9.3 63 + 4.7’ 70 & 6.8 23 & 3.2 24* 5.1 45 & 5s 59 2 4.5 77 I 6.6” 71 I: 5.2 80 f. 6.2 12 f 5.9 16 * 9.0 85 t 4.6 83 rfr 7.4 73 * 5.7 69 _t 4.6’ 58 f 5.2
U-80816B 7 mg/kg
*P .z 0.05, bP < 0.01 compared to control, 'P-z0.05, dP < 0.01 compared to scopolamine 30 &kg. ~Glucose metabolism @mol glucose/100 gjmin). Means & SEM, 4 animals per group.
76 + 84 f 83 f 87 f 75 + 83 f 56 f
78 + 5.0
Control
Name
Stop 30 i@g + U-80816B 2 mg/kg 53 + 4 784 27 : 314 85 + 6.9 42 + 2.6 49 f 2.7 54 + 2.4 65 + 3.9 54 + 3.4 74 + 3.2 81 + 4.2c 54 * 3.5 67 f 3.5’ 54 + 3.0 70 * 3.9 56 + 4.1 64 + 3.9 69 + 3.8’ 72 + 3.2 74 -r_4.5 70 + 3.v 50+ 3.6 61 & 3.6 87 ) 8.8 86 f 3.3 62 f 3.3 71 + 3.4 24 + 1.8 26 + 2.7 45 + 3.1’ 59 + 2.9 75 it 5.T 69 f 4.4 77 + 4.8 14 + 4.5 84 + 5.5 84 &-5.6 79 + 4.9 71 If:4.7 73 ) 5.0p 57 + 3.6
Stop 30 &kg+ U-80816B 7 mg/kg
Table 2. Effects of scopolamine, U-80816B and RS86 on regional energy metabolism in braint
27I 1.5 85 + 4.4 372 1.4 46It2.2 53 & 0.4 59+ 1.8 57 + 2.3 68 + 2.1 76k I.7 54& I.1 67 f 1.2 60 + 2.1=+ 80 + 3.8 64t 3.5 73 * 3.4 78 + 3 Ob.d 7873:l 91 + 4.4”d 78 + 2.7”d 55 f 2.0 63+ 1.8 a2 * 2.0 90 -+_4.7 74 k 2.6b,d 80 f 3.0 24 + 0.9 26 + 0.6 57 + 2.5b,d 66+ I.1 68 + 3.0 63 * 3.5 72 & 5.5 63 + 3.0b 80 + 4.1 74 & 3.9 71 & 2.3 63 4 4.0b 69 * 4.7b 58 f 2.0
55 + 0.5”
RS86 2 mg/kg
Stop 30
53 4 1.71” 27 F: 2.3 90 * 3.3 44 & 0.4 55 4 l.4*.& 54 rt: 1.0 63 & 0.8 59 2 1.1 76 * l.lc 80 i_ 0.8 561: 1.0 64i: 1.2 56 i: 1.6 8Oc I.8 62+ I.6 73 rt I.7 76 rt 2.2’.* 76+ I.8 82 + O.gd 75 ): 2.2a.c 541: 1.7 6oi: 1.9 81 f 2.8 93 f I#3 68 + l.Od 77 f I.0 27 rt 0.2 30f I.9 49 & l.Wd 59 f 1.4 77 f 1.1’ 74& 1.0 79 & 1.9 73 f 1.2 88 f 2.2 a2 & I.3 84 k 3.2 73 & I.2 81 & I.8 60 F: 0.8
2 mg/kg
B gW&+
Cholinergic agonists and energy metabolism scopolamine in rats and those affected in Alzheimer’s disease (Blin, Ray, Chase and Piercey, ~published). Physostigmine is an indirectly-acting cholinergic agonist (cholinesterase inhibitor), which has had some success in treating Alzheimer’s patients, although with inconsistent results and unwanted peripheral side effects (Davis, Mohs, Davis, Horvath, Greenwald, Rosen, Levy and Johns, 1983; Drachman, Glosser, Fleming and Longenecker, 1982; Thai, Fuld, Masur, Sharpless and Davies, 1983). The potent direct-acting muscarinic agonist RS86, has also been tested in Alzheimer’s patients, although therapeutic effects were minimal and were also limited by unwanted peripheral side effects (Bruno e; al., 1986; Mouradian, Mohr, Williams and Chase, 1’388; Gray, Enz and Spiegel, 1989). Like RS86, U-80816B is also a direct-acting muscarinic agonist but with a much lower intrinsic activity (Moon et al., 1’391; Sethy et ai., 1991). It has been suggested that an agonist with low intrinsic activity might possess a more feasible therapeutic efficacy, because it will be less limited by peripheral side effects (Sethy et al., 1991). However, recent data suggest that the poor symptomatic reversal of Alzheimer’s disease with cholinergic drugs could be due to a significant amount of non-cholinergic pathophysiology in this disease (Blin, Ray, Chase and Piercey, unpublished). All of the cholinergic agonists depressed metabolism in the cerebral cortex, the strongest and most consistent effects being observed in the striate and parietal regions. This result was surprising because scopolamine, a muscarinic antagonist, also depressed energy metabolism in these regions. Furthermore, when any of the cholinergic agonists were combined with scopolamine, there appeared to be a mutual abolition of the depressant effects on the cortex that each of the drugs demonstrated, when administered by themselves. It is well established that there are multiple muscarinic receptors (Ehlert and Tran, 1990; Quiron, 198.5; Quiron, Aubert, Lapchak, Schaum, Teolis, Gauthier and Araujo, 1989). The fact that agonists and antagc,nists produced the same effects in the cerebral cortex suggests that agonists could have their dominant effects on one subtype in the cortex, while antagonists have their dominant effects on another subtype. However, since all the agents can interact at ail muscarinic subtypes, they retain the ability to mutually antagonize each other’s effects. The fact that increasing doses of agonists (e.g. physostigmine, U80816B) resulted in the reappearance of depressant effects in the cortex argues in favor of the suggestion that scopolamine and the agonists can, when combined, interact at common sites. Since the animals were awake, it is possible that cholinergic receptors might be strongly stimulated by the large amount of acetylcholine released in the awake state (Jasper and Tessier, 1971). One hypothetical construct, consistent with the similar effects of scopolamine and the agonists is that, under the
1197
conditions of the ex~~ment, cholinergic activity was near maximum effectiveness and therefore a ready target for depression but with little room for further enhancement. Antagonists like scopolamine could depress cholinergic transmission by blockade of postsynaptic receptors, whereas agonists could depress cholinergic transmission by activating autoreceptors, which depress the release of acetylcholine. ‘In the cerebral cortex, M1 muscarinic receptors function as post-synaptic receptors (Crews, Meyer, Gonzales, Theiss, Otero, Larsen, Raulli and Calderini, 1986), while the M, subtype has been identified as an autoreceptor, depressing release of acetylcholine from cholinergic terminals (Crews et al., 1986; Meyer and Otero, 1985). Acetylcholine is generally an excitatory neurotransmitter in the cerebral cortex (Bradshaw, Sheridan and Szabadi, 1987). Thus, scopolamine could depress energy metabolism in the cortex by blocking this excitatory effect, mediated through the M, postsynaptic receptor. On the other hand, cholinergic agonists could cause a similar functional effect if they depressed cholinergic transmission in the cortex by activating M, autoreceptors, resulting in a decrease in release of acetylcholine. Therefore, it is possible th t, although none of the agents bind selectively to specific muscarinic subtypes, the actions of the drugs may be expressed through specific subtypes, depending upon the state of cholinergic transmission. Moreover, since the agonists and antagonists are all capable of interacting at multiple muscarinic receptors, they could mutually antagonize each other. Although this hypothesis is speculative, it does illustrate one possible explanation for the unusual occurrence of similar but mutually antagoni~ble effects of agonists and antagonists of cholinergic receptors. Although mixed effects were observed, the most consistent effect of the cholinergic agonists in subcortical areas was an increase in metabolism in the thalamus. Scopolamine, by itself, did not alter metabolism in the thalamus. However, scopolamine did antagonize some of the effects of RS86 and U808168. This interaction is readily explained by a simple antagonism of the action of the muscarinic agonist by a muscarinic antagonist. Scopolamine did not antagonize the stimulation of metabolism in the thalamus, evoked by physostigmine, even though its effects were not any greater than those of the muscarinic agonists. Physostigmine, by virtue of its anticholinesterase activity, could increase levels of a~tycholine, non-disc~minately stimulating multiple subtypes of choiinergic receptor, including nicotinic receptors. The resistance of the effects of physostigmine to scopolamine in the thalamus could be due to stimulation of nicotinic receptors, which are known to exist there (Clarke, 1987; Clarke, Schwartz, Paul, Pert and Pert, 1985). in summary, rats treated with direct agonists (U80816B, RS86), as well as with an indirect agonist ~hysosti~ine), all showed decreases in energy
C. A. RAY et al.
1198
metabolism in the cortex, similar to that produced by scopolamine. These drugs increased metabolism in the thalamus. When these cholinergic agonists were given in conjunction with a cholinergic antagonist, the tendency was for metabolism to change in the opposite direction from that occurring when the agonists were administered alone. These results suggest that complex interactions occur between multiple subtypes of cholinergic central nervous receptor in the mammalian system.
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Beatty W. W., Butters N. and Janowsky D. S. (1986)
Patterns of memory failure after scopolamine treatment: implications for choline@ hypotheses of dementia. Behav. Neurol. Biol. 45: 19&211. Blin J., Ray C. A., Chase T. N and Piercey M. F. (1992) Regional cerebral glucose metabolism compared in rodents and humans. Brain Res. 568: 215-222. Bradshaw C. M., Sheridan R. D. and Szabadi E. (1987) Involvement of M,-muscarinic receptors in the excitation of neocortical neurones by acetylcholine. Neuropharmacology 26: 1195-1200. Bruno G., Mohr E., Gillespie M., Fedio P. and Chase T. N. (1986) Muscarinic agonist therapy of Alzheimer’s disease-a clinical trial of RS86. Archs Neurof. 43: 659661.
Chase T. N., Foster N. G. and Mansi L. (1983) Alzheimer’s disease and the parietal lobe. Lancet 2: 225. Clarke P. B. S. (1987) Recent progress in identifying nicotinic cholinoceptors in mammalian brain. Trends Pharmat. Sci. 8: 32-35.
Clarke P. B. S., Schwartz R. D., Paul S. M., Pert C. B. and Pert A. (1985) Nicotinic binding in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine, and [iZSI]-a-bungarotoxin. J. Neurosci. 5: 1307-1315.
Crews F. T., Meyer E. M., Gonzales R. A., Theiss C., Otero D., Larsen K., Raulli R. and Calderini G. (1986) Presynaptic and postsynaptic approaches to enhancing central cholinergic neurotransmission. In: Treatment Development Strategies for Alzheimer’s Disease (Cook T.. Bartus R. T.. Ferris S. and Gershon S., Ed:), pp. 385419. Mark Pdwley, Madison, Connecticut. Davis K. L., Mohs R. C., Davis B. M., Horvath T. B., Greenwald B. S., Rosen W. G., Levy M. I. and Johns C. A. (1983) Oral physostigmine in Alzheimer’s disease. Psychopharmac. Bull. 19: 451-453. Drachman D. A., Glosser G., Fleming P. and Longenecker G. (1982) Memory decline in the aged: treatment with lecithin and physostigmine. Neurology 32: 944-950. Ehlert F. J. and Tran L. L. P. (1990) Regional distribution of M,, M,, and non-M,, non-M, subtypes of muscarinic binding sites in rat brain. J. Pharmac. exp. Ther. 255: 1148-I 157.
Foster N. L., Chase T. N., Mansi L., Brooks R., Fedio P., Patronas N. J. and DiChiro G. (1984) Cortical abnormalities in alzheimer’s disease. Ann. Neural. 16: 649-654. Frev K. A.. Ehrenkaufer R. L. E. and Azranoff B. W. (1985) Quantitative in vivo receptor binding II. Autoradio: graphic imaging of muscarinic cholinergic receptors. J. Neurosci. 5: 2407-2414.
Friedland R. P., Budinger T. F., Koss E. and Ober B. A. (1985) Alzheimer’s disease: anterior-posterior and lateral hemispheric alterations in cortical glucose utilization. Neurosci. Lett. 53: 235-240.
Gray J. A., Enz A. and Spiegel R. (1989) Muscarinic agonists for senile dementia: past experience and future trends. Trena!s Pharmac. Sci. 10: Suppl., 85-88. Hoyer S. (1986) Senile dementia and alzheimer’s disease brain blood flow and metabolism. Prog. Neuropsychopharmac. biol. Psychiat. 10: 447478.
Jasper H. H. and Tessier J. (1971) Acetylcholine liberation from cerebral cortex during paradoxical (REM) sleep. Science 172: 601-602. Kuhl D. E., Metter E. J. and Riege W. H. (1985) Patterns of cerebral glucose utilization in depression, multiple infarct dementia, and alzheimer’s disease. In: Brain Zmaging and Brain Function (Sokoloff L., Ed.), pp. 21 l-226. Raven Press, New York. McGeer P. L., McGeer E. G., Suzuki J., Dolman C. E. and Nagai T. (1984) Aging, alzheimer’s disease, and the choline@ system of the basal forebrain. Neurology 34: 741-745.
Meyer E. M. and Otero D. H. (1985) Pharmacological and ionic characterizations of the muscarinic receptors modulating [‘H]acetylcholine release from rat cortical synaptosomes. J. Neurosci. 5: 1202-1207. Moon M. W., Chidester C. G., Heier R. F., Morris J. K., Collins R. J., Russell R. R., Francis J. W., Sage G. P. and Sethy V. H. (1991) Cholinergic activity of acetylenic imidazoles and related compounds. J. med. Chem. 34: 23142327. Mouradian M. M., Mohr E., Williams J. A. and Chase T. N. (1988) No response to high-dose muscarinic agonist therapy in alzheimer’s disease. Neurology 38: 606608. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates., pp. 87-l 13. Academic Press, Florida. Piercey M. F. and Ray C. A. (1988a) Evidence for 2-DG autoradiography that phencyclidine’s functional effects are mediated by specific PCP rather than sigma receptors. In: Sigma Opioid/Phencyclidine-like Compounds as Melecular Probes in Biology (Domino E. F. and Kamenka J. M., Eds), pp. 285-295. NPP Press, Ann Arbor, Michigan. Piercey M. F. and Ray C. A. (1988b) Dramatic stimulation of limbic and cortical structures via high affinity PCP receptors. Life Sci. 43: 379-385. Piercey M. F., Vogelsang G. D., Franklin S. F. and Tang A. H. (1987) Reversal of scopolamine-induced amnesia and alterations in energy metabolism by the nootropic piracetam: implications regarding identification of brain structures involved in consolidation of memory traces. Brain Res. 424: l-9.
Quiron R. (1985) Comparative localization of putative pre- and postsynaptic markers of muscarinic cholinergic nerve terminals in rat brain. Eur. J. Pharmac. 111: 287-289.
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Smith G. (1988) Animal models of Alzheimer’s disease: experimental cholinergic denervation. Brain Res. Rev. 13: 103-l 18.
Sokoloff L., Reivich M., Kennedy C., Des Rosiers M. H., Patlak C. S., Pettigrew K. D., Sakurada 0. and Shinohara M. (1977) The [i4C]deoxyglucose method for the
Choline@
agonists and energy metabolism
measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetired rat. J. Neurochem. Zs: 897-916. Sunderland T., Tariot P. N., Weingartner H., Murphy D. L., Newhouse P. A., Mueller E. A. and Cohen R. M. (1986) Pharmacologic modelling of alzheimer’s disease. Prog. Neuropsychopharmac.
biol. Psychiar. 10: 599610.
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Neuropharmacology Vol. 31, No. 11, pp. 1193-I 199, 1992 hinted in Great Britain. All rights nscwed
EFFECTS OF CHOLINERGIC AGONISTS ON REGIONAL BRAIN ENERGY METABOLISM IN THE SCOPOLAMINE-TREATED RAT C. A. RAY,’ J. BLIN,~* T. N. CHASESand M. F. Pmacav’t ‘CNS Research, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. and *NINCDS NIH Bldg 10, Rm 5C103, Bethesda, MD, 20892, U.S.A. (Accepted 11 May 1992) Summary-The effects of scopolamine, physostigmine, RS86 and U-80816B on regional energy metabolism were studied in rodents by means of the 2-deoxyglucose autoradiographic technique. Scopolamine depressed metabolism in an area of cerebral cortex, focused around the parietal region. Rats treated with cholinergic direct agonists (U80816B, RS86) as well as with the indirect agonist (physostigmine) all showed decreases in cortical energy metabolism, similar to scopolamine. They also induced an increase in thalamic metabolism. When these drugs were given in conjunction with scopolamine, metabolism tended to change in the opposite direction from the values obtained with the drug alone. These results suggest that there are complex interactions between pre- and post-synaptic muscarinic receptors. Additionally, nicotinic receptors could also be involved in some of the effects of physostigmine. Key words-2-dg
autoradiography,
muscarinic receptors, nicotinic receptors, Alxheimer’s disease.
Degeneration of cholinergic neurons, arising from the medial forebrain is one of the more consistent find-
ings in Alzheimer’s disease (Whitehouse and Au, 1986; McGeer, McGeer, Suzuki, Dolman and Nagai, 1984). One common method for modeling Alzheimer’s disease pharmacologically is treatment with the muscarinic choline@. antagonist, scopolamine (Beatty, Butters and Janowsky, 1986; Smith, 1988). Scopolamine, for example, blocks cholinergic transmission in the cerebral cortex and hippocampus, the major projection areas of the medial forebrain cholinergic system (Frey, Ehrenkaufer and Agranoff, 1985). In addition, like Alzheimer’s disease, scopolamine impairs memory function (Rusted and Warburton, 1988; Sunderland, Tariot, Weingartner, Murphy, Newhouse, Mueller and Cohen, 1986). In a previous study, measuring regional energy metabolism in brain, it was noted that the effects of scopolamine in the parietal cortex of the rat (Piercey, Vogelsang, Franklin and Tang, 1987) seemed to correspond well with the reported depression in the parietal cortex of Alzheimer’s patients (Chase, Foster and Mansi, 1983; Foster, Chase, Mansi, Brooks, Fe&o, Patronas and DiChiro, 1984). This finding has recently been confirmed by a direct collaborative study (Blin, Ray, Chase and Piercey, unpublished). However, the same study indicated that there was no overall correlation between the effects of scopolamine in the brain of either rat or human and the alterations that occur in Alzheimer’s patients.
Nonetheless, it appeared desirable to evaluate further the effects of cholinergic drugs in the scopolamine-treated rat because (1) the metabolic depression in the parietal cortex resembled that of Alzheimer’s disease, (2) metabolic considerations aside, cholinergic denervation and amnesic phenomena are consistent features of Alzheimer’s disease, (3) therapies with cholinergic agonists continue to be evaluated for the symptomatic treatment of Alzheimer’s disease and (4) mapping the functional effects of cholinergic agonists and antagonists could enhance appreciation of the physiology and pharmacology of choline@ systems. 2,8-Diazaspiro(4, S)decane-1,3-dione, 2-ethyl-gmethyl-monohydrobromide (RS86) a direct-acting cholinergic agonist (Bruno, Mohr, Gillespie, Fedio and Chase, 1986) and physostigmine, an indirectly acting choline@ agonist (cholinesterase inhibitor) have previously been used to treat Alzheimer’s disease, with limited success. In the present study, these two compounds were evaluated and compared to U-80816B (1-[4-(5methyl-lH-imisol-1-yl)-2-butynyll2-pyrrolidinone hydrobromide), a partial cholinergic agonist (Moon, Chidester, Heier, Morris, Collins, Russell, Francis, Sage and Sethy, 1991; &thy, Francis, Hyslop, Sage, Oien, Meyer, Collins, Russell, Heier, Hoffmann, Piercey, Nichols, Schreur and Moon, 1991), for their effects on energy metabolism in the brain, using 2-deoxyglucose autoradiography. METHODS
*Present address: Service Hospitalier Frederic Joliot, DRIPP, CEA, Hopital d’orsay, 91401 Orsay, France. tTo whom correspondence should be addressed.
The procedures for 2-deoxyglucose (2-dg) autoradiography have been described in detail previously 1193
1194
C. A. RAY et al.
(Sokoloff, Reivich, Kennedy, Des Rosiers, Patlak, Pettigrew, Sakurada and Shinohara, 1977; Piercey et al., 1987; Piercey and Ray, 1988a, b; Blin, Ray, Chase and Piercey, 1992). Briefly, Charles River Sprague-Dawley male rats, 275-320 g, were anesthetized with a fluothane-nitrous oxide mix and fitted with arterial and venous cannulae. After cannulation, the animals were lightly restrained in a Brain Tree experimental containment unit. The animals were allowed to recover in the restrainer for a minimum of 30 min, after removal of anesthesia, before any injections of drugs. Following recovery, the animals were given, by intravenous injection, either the drug or an equal volume of saline vehicle. Scopolamine (30 pg/kg) or saline was injected 10 min prior to an intravenous injection of 25 pCi of [i4C]2-dg. Physostigmine (100 or 300 pg/kg), RS86 (2 mg/kg), U-80816B (2 or 7 mg/kg) or saline was injected 5 min prior to the [14C]2-dg. Forty-five minutes after the injection of the label, the rats were sacrificed, their brains rapidly removed and frozen over liquid nitrogen. Utilizing the horizontal autoradiography atlas, designed for rat-human comparative studies (Blin et al., 1992), the brains were mounted dorsal side down and then sectioned horizontally, using a Leitz 1720 motorized cryostat, roughly corresponding to bregma levels:8.6, 7.6, 6.6, 5.6, 5.1, 4.6, 4.1 and 3.1 mm; and at 500, 1000 and 1500 pm after bregma 3.1 mm (Paxinos and Watson, 1986). The 20pm sections were positioned on 25 mm* cover glasses mounted on railroad board, using two-sided tape and exposed to Kodak SB-5 film for 6 days. The resulting autoradiograms were developed and analyzed for local cerebral glucose utilization, using an Amersham RAS 1000 image analysis system. The metabolic activity of regions of the brain were analyzed in polygonal areas of interest, directly from the autoradiograms. Metabolic values from whole brain were obtained by taking, as areas of interest, entire horizontal sections of all levels of the brain and then combining results by weighted averaging, to obtain the mean metabolic rates of all pixels imaged. Average glucose utilizations within each treatment group, were compared statistically using the SAS statistical program (analysis of variance [ANOVA] and Fischer’s least significant difference for multiple comparisons). The 30pg/kg (i.v.) dose of scopolamine was chosen, based on the results of a previous study (unpublished) demonstrating that similar maximum effects were detected between doses of 10-100 pg/kg (i.v.). Two doses of both physostigmine and U-80816B were chosen in order to encompass the range of effects of those compounds. The dose for RS86 was also chosen for its maximum effect in other experiments (V. H. Sethy, personal communication). Copious salivation and lacrimation were observed during the experiments with physostigmine and RS86.
RESULTS
Physostigmine and scopolamine
The effects of physostigmine (100 and 300 pg/kg) on energy metabolism in the brain, compared to control and scopolamine-treated animals are presented in Table 1. Intravenous administration of scopolamine decreased metabolism in the parietal, motor and striate cortices. These results were similar to those previously described using intraperitoneal administration of scopolamine and making measurements from coronal sections (Piercey et al., 1987). At a dose of 100 pg/kg, physostigmine also decreased metabolism in the parietal and striate cortices, to levels similar to those produced by scopolamine. At this dose, physostigmine induced a decrease in activity in the medial septum, while stimulating the anterior thalamic nuclei. When scopolamine and physostigmine, 100 pg/kg, were combined, the values for the cortex were brought back to control levels, while the ventral, anterior and posterior nuclei of the thalamus all showed significant increases in metabolism. The 300 pg/kg dose of physostigmine affected even more regions of the brain. By itself, the larger dose of physostigmine decreased metabolism in the striate, motor, cingulate, parietal and frontal one cortical regions, as well as in the amygdala and accumbens nuclei. It increased metabolism in the anterior and posterior thalamic regions. When 300 pg/kg of physostigmine was administered with scopolamine, energy metabolism in the brain returned to control levels in frontal one and cingulate cortices, areas where physostigmine, but not scopolamine, depressed metabolism. Metabolism in the striate cortex also did not differ from controls in animals, treated with both scopolamine and physostigmine, even though each significantly depressed metabolism when given alone. The depression in metabolic values in the parietal and motor cortices, experienced with 300 pg/kg physostigmine, were not brought back to control levels by scopolamine. However, scopolamine superficially appeared to antagonize, physostigmine given in a large dose in the parietal and motor cortex, since the depression in metabolism was significantly less than that seen with physostigmine alone but did not differ from that observed with scopolamine alone. Just as with small doses, large doses of physostigmine continued to stimulate metabolism in the thalamic nuclei in scopolamine-treated rats. However, the depression in energy metabolism in the amygdala and accumbens were antagonized by administration of scopolamine. Muscarinic agonists and scopolamine
The effects of RS86 and U-80816B, with and without scopolamine, are compared against control and scopolamine-treated animals in Table 2.
Cholinergic agonists and energy metabolism Table
1. Effects of scopolamine
and physostigmine
Name
Control
ScoP 30 ualkg
Brainstem Cerebral peduncle IP Hypothalamus Amygdala Substantia nip Red nucleus Hippocampus Accumbens Caudate Globus DBB Medial septum Medial geniculate Lateral geniculate Ventral tier Ventral thalamus DM thalamus Ant. thalamus Post. thaiamus Centrai grey MHB LHB Inferior colliculus Superior colliculus Colliculi White GCC Cerebraller hemisphere Vermis Frontal cortex one Frontal cortex two Sensory cortex Parietal cortex Auditory cortex Cingolate cortex Temporal cortex Motor cortex Striate cortex Brain
46 * 3.3 25 4 0.7 85 + 4.2 40+ 1.8 49 * 2.7 49 * 4.3 55 f 4.8 56 f 3.6 71 F 4.4 76 + 4.2 49 + 3.4 60+4.1 SO& 3.1 12 f 4.3 53f3.1 61 + 3.2 64+4.1 70 i 3.4 72 * 3.6 65 + 2.9 48 + 2.3 5-l + 2.3 78 f 3.2 91 * 9.4 62 + 4.1 76 ? 6.6 22 & 1.2 252 1.9 40 It. 3.4 56 +_ 5.1 12 + 4.1 74 + 4.4 18 * 5.0 16 f 5.0 84 * 4.9 83 i. 4.3 87 + 5.9 75 * 4.9 83 + 4.6 56 * 3.5
42 f 2.8 22& 1.4 82 f 4.9 38k 1.6 46 k 2.2 47 + 3.0 51 + 3.4 52 + 2.2 66 + 2.0 71 * 3.8 47 f 2.5 58 * 3.3 52 + 3.4 66 rf: 6.2 51 k 4.1 56 & 4.8 57 f 4.5 65 * 4.0 64+4.5 60+4.1 44 f 2.6 54 + 2.9 71 rt 4.1 79 + 5.0 55 + 2.9 66 + 3.7 20% 1.0 25 f 3.9 36 f 2.3 50 f 3.9 64 + 2.4 61 + 2.8 69 f 3.2 67+2.1’ 76 + 5.6 75 * 2.7 72 + 2.8 64 + 2.3” 71 * 1.8’ 51 * 2.5
Physo IM) Ica/kg 47 f 1.4 23 * 1.0 79 f 5.4 39 f 2.5 4b* 1.3 48 4 1.4 52 + 2.0 51 & 1.5 65 f 3.4 70 * 1.4 48* 1.8 53 2 3.2 44* l.OC II f 2.6 S4 + 2.9 64 _L2.7 66 + 2.6 69+ 1.7 79 rf: 2.4’ 68 rt 2.5 47* 1.5 56 k 2.3 14 ~fi3.6 80 f 2.7 62+2.1 68*2.0 22 + 0.9 24rf: 1.5 43 2 2.2 55f 1.9 65 k 2.9 71 * 3.1 75 & 4.2 67 f 3.1’ 79 + 2.3 76 24.1 79 f 4.8 66 f 3.3 13 f 3.4* 53 * 2.0
on regional
1195
energy metabolism
Physo 100 pg/kg +SCOP 30 /&kg 4brfc 1.9 28 + 2.9 16 it 4.3 43ir 1.8 48 rt 1.2 52 & 2.3 59 + 3.0 56f 1.7 61 & 3.3 74 + 2.5 50& 1.4 65 + 2.0 S8& 1.8 78 rt 3.4 58 rf: 1.8 69 rf: 2.9 69 +_ 2.9= 78 * 5.0 87 + 4 2’~~ 72 : 3:3’ 49k2.1 63*4.1 81 f 3.9 81 ri: 1.7 61 rt 2.5 68 F 2.0 25 & 1.0 27 rf: 0.6 43 If: 2.1 56 + 2.7 68 + 0.7 764 1.1 75 + 2.4 71 i 1.9 85 f 2.5 84 + 2.s 78 * 2.4 68 2 0.7 79* 1.8 56+ 1.5
in brainy
Physo 300 pglkg 45 + 22+ 83 * 362 39 + 41 rt 51 * 46 k 58 *
2.5 I.7 7.5 1.2 1.8b” 2.9 3.5 2.4 3.9b 68 rt 4.2 46 5 2.4 51 f 2.2 46 + 3.2 73 + 6.1 56 Y!Z 3.6 64 * 4.4 64 * 3.3 70 + 5.0 89 + 8.3” 71 & 4.4Q 47 f 2.9 53 k 2.9 72 f 6.3 18 * 5.0 63 &-3.7 67 f 4.0 21 + 1.6 21 * 1.7 42 f 2.8 54 k 2.3 58 f 3.4b 64 f 4.9 69 + 4.7 59 5 4.sb 76 +_6.3 70 + 4.48 69 + 5.4 60 5 4.9D 65 + 3.5b _^ __ 50 19.1
Physo 300 &kg +SWP 30rg/kg 48+ 1.6 25 * 1.7 74+ 1.6 425 1.0 47 f 2.0 SO* 1.6 57 + 2.2 S4? 1.6 63% 1.4 70 i. 1.7 49+ 1.3 59% 1.7 525 1.4 74+ 1.6 bO+ 1.7 67? 1.5 68 k 0.9t 12 1 1.5 85 + 2.7”” 70 * 1.1c 47kO.l 61 + 2.7 19 f 3.5 81 &Y2.5 64 + 2.8’ 70 + 2.2 24+ 1.0 28 f 2.4 44 + 0.5 54 + 0.6 70 * 3.0 66 k 2.3 14 + 2.3 66 k 1.5’ 82 i 2.2 75 + 1.9 76 * 2.0 65+ 1.7s 74 + 2.9 55 f 1.2
“P < 0.05, bP < 0.01 compared to control; ‘P c 0.05, dP c 0.01 compared to scopolamine 30 pg/kg tGlucose metabolism &mol glucose/100 g/min). Means + SEM, 4 animals per group.
The drug RS86 caused an increase in metabolism in several subcortical areas, including: brainstem, medial septum, ventral thalamus, anterior thalamus, posterior thalamus, superior colliculus and cerebellar hemisphere. It decreased metabolism in the parietal, motor and striate cortices. When combined with ~opol~ine, RS86 no longer increased metabolism significantly greater than controls, in the anterior thalamus or superior colliculus. However, scopolamine did not antagonize stimulai.ion of the brainstem, ventral or posterior thalamus or cerebellum by RS86. Although both RS86 and scopolamine depressed energy metabolism in the parietal and striate cortices, when either drug was given alone, the metabolic values in these areas were not significantly different from control when the two drugs were administered together. The drug U-80816B (2 mg/kg) caused a decrease in the striate cortex, when compared to control. When combined with scopolamine, the metabolic depression observed with scopolamine in the parietal cortex was abolished, as was the depression observed with each drug when given alone, in the striate cortex. At lmg/kg, U-80816B continued to depress metabolism in the striate cortex but it increased
metabolism in the anterior thalamus, posterior thalamus and brainstem. In the combination experiments, 7 mg/kg U-80816B reversed the depressant effects of scopolamine in the parietal cortex, while scopolamine reversed the stimulant effects of large doses of U80816B in the thalamus. The common depressant effects of scopolamine and U-808 16B were retained in the striate cortex with no additive effects. Treatment with scopolamine did not alter the stimulant effects of U-80816B in the brainstem. DISCUSSION The present experiments, using intravenous administration of scopolamine have confirmed earlier studies demonstrating a depression in metabolism in the parietal cortex with intraperitoneallyadministered scopolamine (Piercey ei al., 1987). Similar depressions in metabolism in the parietal cortex are also seen in Alzheimer’s disease (Chase et al., 1983; Foster et al., 1984; Friedland, Budinger, Koss and Ober, 1985; Hoyer, 1986; Kuhl, Metter and Riege, 1985), although recent experiments have demonstrated that there was no overall correlation between areas showing metabolic effects with
46 + 3.3 25 * 0.7 85 + 4.2 40& I.8 49 + 2.7 49+4.1 55 & 4.8 56 + 3.6 71 + 4.4 76 f 4.2 49 + 3.4 6ok4.1 50+ 3.1 72 _t 4.3 53 & 3.1 61 + 3.2 64 + 4.1 70 + 3.4 72 + 3.6 65 2 2.9 48 + 2.3 57 + 2.3 78 f 3.2 91 + 9.4 62 54.1 76 + 6.6 22 + 1.2 25 + 1.9 40 & 3.4 56 + 5.7 72 + 4.7 74 f 4.4
Brainstem Cerebral peduncle IP Hypothalamus Amygdala Substantia nigra Red nucleus Hippocampus Accumbens Caudate Globus DBB Medial septum Medial geniculate Lateral geniculate Ventral tier Ventral thalamus DM thalamus Ant. thalamus Post. thafamus Central grey MHB LHB Inferior colliculus superior colliculus Colliculi White GCC Cerebcllar hemisphet Vermis Frontal cortex one Frontal cortex two Sensory cortex Parietal cortex Auditory cortex Cingulate cortex Temporal cortex Motor cortex Striate cortex Brain 5.0 4.9 4.3 5.9 4.9 4.6 3.5
42 f 2.8 22+ 1.4 82 ) 4.9 38 5 1.6 46 * 2.2 47 + 3.0 51 f 3.4 52 f 2.2 66 * 2.0 71 + 3.8 47 & 2.5 58 F 3.3 52 f 3.4 66 + 6.2 51 + 4.1 56 * 4.8 57 & 4.5 65 + 4.0 64 + 4.5 6ok4.1 44 + 2.6 54 rt 2.9 71 rt 4.7 79 1 5.0 55 f 2.9 66 f 3.7 20& 1.0 25 + 3.9 36 f 2.3 50 f 3.9 64 + 2.4 67 k 2.8 69 ) 3.2 67 f 2.1” 76 & 5.6 75 + 2.1 72 4 2.8 64 + 2.3” 71 + 1.8” 51 22.5
=JP 30 /%/kg 48 & I .3 25+ I.1 19ir: 5.1 37 & 2.2 43 & 2.4 50+2,1 57k2.1 50 * 1.6 cb+ 1.8 14 f: 1.4 49 & 2.5 58rt3.1 48 & 3.3 70 f 2.1 53 + I.2 63 + 2.6 66 +: 3.0 67k3.l 80 rt 3.8c 69 + 2.8 46i:3‘1 54k2.5 76 -I_2.6 80 + 2.6 59+ I.7 67 If:0.9 23& 1.6 25 & 2.4 43 + 1.9 55+- I,9 72 & 2.6 67+ I.7 76 + I .6 71 + 0.7 79t 1.3 80 ,: 2.9 74+ I.4 70& I.4 70+ 1.7b 54+ 1.3
U-808 I 6B 2 mg/kg 46&2.1 22 * 1.1 87 f 4.8 38 f 1.8 48 f 2.1 51 1: 2.3 57 f 3.2 55 * 1.5 72 + 2.9 82 + 1.5 51 + 2.2 61 f 3.7 54 f 2.0 71 f 2.9 54 * 1.6 62 + 2.2 63 * 2.2’ 73 * 3.3 74 * 2.1 67 i 2.7 50 i 2.5 59 2 3.0 80 + 3.6 82 * 3.7 59 5 2.1 69 k 2.4 23 k 1.5 25 k 0.4 42 5 2.0 59 + 3.2 83 * 1.98” 75 * 2.5 80 + 2.5 77 & 2.3’ 85 * 1.6 92 + 4.1d 85 * 3.0 75 * 2.W 85 + 1.6d 57 I 1.8 53 + 4.7’” 27 * 4.9 84 + 5.2 40 f 2.9 48 4 3.8 55 rt 5.2 62 + 5.5 54 * 5.3 12 + 5.6 78 * 6.3 55 _t 6.2 64i6.3 54 * 6.0 77 * 7.4 57 rt: 4.2 69 rt 6.4 73 + 6.0d 75 4.7.3 86 + 8.2”qd 74 f 6.3”6 SZ-r_Al 58rt4.9 81 + 5.7 80+ 9.3 63 + 4.7’ 70 & 6.8 23 & 3.2 24* 5.1 45 & 5s 59 2 4.5 77 I 6.6” 71 I: 5.2 80 f. 6.2 12 f 5.9 16 * 9.0 85 t 4.6 83 rfr 7.4 73 * 5.7 69 _t 4.6’ 58 f 5.2
U-80816B 7 mg/kg
*P .z 0.05, bP < 0.01 compared to control, 'P-z0.05, dP < 0.01 compared to scopolamine 30 &kg. ~Glucose metabolism @mol glucose/100 gjmin). Means & SEM, 4 animals per group.
76 + 84 f 83 f 87 f 75 + 83 f 56 f
78 + 5.0
Control
Name
Stop 30 i@g + U-80816B 2 mg/kg 53 + 4 784 27 : 314 85 + 6.9 42 + 2.6 49 f 2.7 54 + 2.4 65 + 3.9 54 + 3.4 74 + 3.2 81 + 4.2c 54 * 3.5 67 f 3.5’ 54 + 3.0 70 * 3.9 56 + 4.1 64 + 3.9 69 + 3.8’ 72 + 3.2 74 -r_4.5 70 + 3.v 50+ 3.6 61 & 3.6 87 ) 8.8 86 f 3.3 62 f 3.3 71 + 3.4 24 + 1.8 26 + 2.7 45 + 3.1’ 59 + 2.9 75 it 5.T 69 f 4.4 77 + 4.8 14 + 4.5 84 + 5.5 84 &-5.6 79 + 4.9 71 If:4.7 73 ) 5.0p 57 + 3.6
Stop 30 &kg+ U-80816B 7 mg/kg
Table 2. Effects of scopolamine, U-80816B and RS86 on regional energy metabolism in braint
27I 1.5 85 + 4.4 372 1.4 46It2.2 53 & 0.4 59+ 1.8 57 + 2.3 68 + 2.1 76k I.7 54& I.1 67 f 1.2 60 + 2.1=+ 80 + 3.8 64t 3.5 73 * 3.4 78 + 3 Ob.d 7873:l 91 + 4.4”d 78 + 2.7”d 55 f 2.0 63+ 1.8 a2 * 2.0 90 -+_4.7 74 k 2.6b,d 80 f 3.0 24 + 0.9 26 + 0.6 57 + 2.5b,d 66+ I.1 68 + 3.0 63 * 3.5 72 & 5.5 63 + 3.0b 80 + 4.1 74 & 3.9 71 & 2.3 63 4 4.0b 69 * 4.7b 58 f 2.0
55 + 0.5”
RS86 2 mg/kg
Stop 30
53 4 1.71” 27 F: 2.3 90 * 3.3 44 & 0.4 55 4 l.4*.& 54 rt: 1.0 63 & 0.8 59 2 1.1 76 * l.lc 80 i_ 0.8 561: 1.0 64i: 1.2 56 i: 1.6 8Oc I.8 62+ I.6 73 rt I.7 76 rt 2.2’.* 76+ I.8 82 + O.gd 75 ): 2.2a.c 541: 1.7 6oi: 1.9 81 f 2.8 93 f I#3 68 + l.Od 77 f I.0 27 rt 0.2 30f I.9 49 & l.Wd 59 f 1.4 77 f 1.1’ 74& 1.0 79 & 1.9 73 f 1.2 88 f 2.2 a2 & I.3 84 k 3.2 73 & I.2 81 & I.8 60 F: 0.8
2 mg/kg
B gW&+
Cholinergic agonists and energy metabolism scopolamine in rats and those affected in Alzheimer’s disease (Blin, Ray, Chase and Piercey, ~published). Physostigmine is an indirectly-acting cholinergic agonist (cholinesterase inhibitor), which has had some success in treating Alzheimer’s patients, although with inconsistent results and unwanted peripheral side effects (Davis, Mohs, Davis, Horvath, Greenwald, Rosen, Levy and Johns, 1983; Drachman, Glosser, Fleming and Longenecker, 1982; Thai, Fuld, Masur, Sharpless and Davies, 1983). The potent direct-acting muscarinic agonist RS86, has also been tested in Alzheimer’s patients, although therapeutic effects were minimal and were also limited by unwanted peripheral side effects (Bruno e; al., 1986; Mouradian, Mohr, Williams and Chase, 1’388; Gray, Enz and Spiegel, 1989). Like RS86, U-80816B is also a direct-acting muscarinic agonist but with a much lower intrinsic activity (Moon et al., 1’391; Sethy et ai., 1991). It has been suggested that an agonist with low intrinsic activity might possess a more feasible therapeutic efficacy, because it will be less limited by peripheral side effects (Sethy et al., 1991). However, recent data suggest that the poor symptomatic reversal of Alzheimer’s disease with cholinergic drugs could be due to a significant amount of non-cholinergic pathophysiology in this disease (Blin, Ray, Chase and Piercey, unpublished). All of the cholinergic agonists depressed metabolism in the cerebral cortex, the strongest and most consistent effects being observed in the striate and parietal regions. This result was surprising because scopolamine, a muscarinic antagonist, also depressed energy metabolism in these regions. Furthermore, when any of the cholinergic agonists were combined with scopolamine, there appeared to be a mutual abolition of the depressant effects on the cortex that each of the drugs demonstrated, when administered by themselves. It is well established that there are multiple muscarinic receptors (Ehlert and Tran, 1990; Quiron, 198.5; Quiron, Aubert, Lapchak, Schaum, Teolis, Gauthier and Araujo, 1989). The fact that agonists and antagc,nists produced the same effects in the cerebral cortex suggests that agonists could have their dominant effects on one subtype in the cortex, while antagonists have their dominant effects on another subtype. However, since all the agents can interact at ail muscarinic subtypes, they retain the ability to mutually antagonize each other’s effects. The fact that increasing doses of agonists (e.g. physostigmine, U80816B) resulted in the reappearance of depressant effects in the cortex argues in favor of the suggestion that scopolamine and the agonists can, when combined, interact at common sites. Since the animals were awake, it is possible that cholinergic receptors might be strongly stimulated by the large amount of acetylcholine released in the awake state (Jasper and Tessier, 1971). One hypothetical construct, consistent with the similar effects of scopolamine and the agonists is that, under the
1197
conditions of the ex~~ment, cholinergic activity was near maximum effectiveness and therefore a ready target for depression but with little room for further enhancement. Antagonists like scopolamine could depress cholinergic transmission by blockade of postsynaptic receptors, whereas agonists could depress cholinergic transmission by activating autoreceptors, which depress the release of acetylcholine. ‘In the cerebral cortex, M1 muscarinic receptors function as post-synaptic receptors (Crews, Meyer, Gonzales, Theiss, Otero, Larsen, Raulli and Calderini, 1986), while the M, subtype has been identified as an autoreceptor, depressing release of acetylcholine from cholinergic terminals (Crews et al., 1986; Meyer and Otero, 1985). Acetylcholine is generally an excitatory neurotransmitter in the cerebral cortex (Bradshaw, Sheridan and Szabadi, 1987). Thus, scopolamine could depress energy metabolism in the cortex by blocking this excitatory effect, mediated through the M, postsynaptic receptor. On the other hand, cholinergic agonists could cause a similar functional effect if they depressed cholinergic transmission in the cortex by activating M, autoreceptors, resulting in a decrease in release of acetylcholine. Therefore, it is possible th t, although none of the agents bind selectively to specific muscarinic subtypes, the actions of the drugs may be expressed through specific subtypes, depending upon the state of cholinergic transmission. Moreover, since the agonists and antagonists are all capable of interacting at multiple muscarinic receptors, they could mutually antagonize each other. Although this hypothesis is speculative, it does illustrate one possible explanation for the unusual occurrence of similar but mutually antagoni~ble effects of agonists and antagonists of cholinergic receptors. Although mixed effects were observed, the most consistent effect of the cholinergic agonists in subcortical areas was an increase in metabolism in the thalamus. Scopolamine, by itself, did not alter metabolism in the thalamus. However, scopolamine did antagonize some of the effects of RS86 and U808168. This interaction is readily explained by a simple antagonism of the action of the muscarinic agonist by a muscarinic antagonist. Scopolamine did not antagonize the stimulation of metabolism in the thalamus, evoked by physostigmine, even though its effects were not any greater than those of the muscarinic agonists. Physostigmine, by virtue of its anticholinesterase activity, could increase levels of a~tycholine, non-disc~minately stimulating multiple subtypes of choiinergic receptor, including nicotinic receptors. The resistance of the effects of physostigmine to scopolamine in the thalamus could be due to stimulation of nicotinic receptors, which are known to exist there (Clarke, 1987; Clarke, Schwartz, Paul, Pert and Pert, 1985). in summary, rats treated with direct agonists (U80816B, RS86), as well as with an indirect agonist ~hysosti~ine), all showed decreases in energy
C. A. RAY et al.
1198
metabolism in the cortex, similar to that produced by scopolamine. These drugs increased metabolism in the thalamus. When these cholinergic agonists were given in conjunction with a cholinergic antagonist, the tendency was for metabolism to change in the opposite direction from that occurring when the agonists were administered alone. These results suggest that complex interactions occur between multiple subtypes of cholinergic central nervous receptor in the mammalian system.
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