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Mechanism of action of scopolamine as an amnestic
T i P S - M a y 1989 [Vol. 10]
i n v e s t i g a t i o n is r e q u i r e d to determ i n e w h e t h e r this effect is exclusively d u e to a n e l e v a t i o n of c A M P levels a n d s u b s e q u e n t p h o s p h o r y lation of t h e 5-HT3 receptor via a c A M P - d e p e n d e n t p r o t e i n kinase. []
[3
[]
N e u r o n a l clonal cell lines prov i d e a c o n v e n i e n t source of large a m o u n t s of relatively h o m o g e n o u s material: it h a s b e e n e s t i m a t e d t h a t a single cell t h a t p r o d u c e s p r o g e n y that d i v i d e once e v e r y 10-12 h, could y i e l d 4 kg of tissue w i t h i n 12 w e e k s 3°. N o t w i t h s t a n d i n g the s o m e w h a t low d e n s i t y of 5-HT3 r e c o g n i t i o n sites i n the clonal lines 19,2° comj~ared w i t h c e r t a i n b r a i n r e g i o n s z~, t h e y offer a m o s t p r o m i s i n g s t a r t i n g p o i n t for t h e i s o l a t i o n of t h e 5-HT3 receptor a n d e v e n t u a l l y the d e t e r m i n a t i o n of its structure. JOHN A. PETERS A N D J E R E M Y J. L A M B E R T
Neurosciences Research Group, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. References
1 Fozard, J. R. (1987) Trends Pharmacol. Sci. 8, ~A ~5
175 2 Jones, B. J. et al. (1988) Br. ]. Pharmacol. 93, 985-993 3 Cos"ll, B., Domeney, A. M., Kelly,M. E., Naylor, R. J. and Tyers, M. B. (1987) Neurosci. Lett. Suppl. 29, $69 4 Kilpatrick, G. J., Jones, B. J. and Tyers, M. B. (1987) Nature 330, 746-748 5 Watling, K. J. (1988) Trends Pharmacol. Sci. 9, 227-229 6 Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. and Nirenberg, M. (1978) Brain Res. 147, 261-276 7 MacDermot, J. et al. (1979) Proc. Natl Acad. Sci. USA 76, 1135-1139 8 Guharay, F. and Usherwood, P. N. R. (1981) Br. J. Pharmacol. 74 294P 9 Freschi, J. E. and Shain, W. G. (1982) J. Neurosci. 2, 106--112 10 Peters, J. A. and Usnerwood, P. N. R. (1983) Br. J. Pharmacol. 80, 523P 11 Neijt, H. C., Vijverberg, H. P. M. and van den Bercken, J. (1986) Eur. J. Pharmacol. 127, 271-274 12 Neijt, H. C., Te Duits, I. J. and Vijverberg, H. P. M. (1988)Neuropharmacology 27, 301-307 13 Peters, J. A., Hales, T. G. and Lambert, J. J. (1988)Eur. J. Pharmacol. 151,491-495 14 Peters, J. A. and Usherwood, P. N. R. (1984) Br. J. Pharmacol. 83, 419P 15 Kato, E. and Narahashi, T. (1982) J. Physiol. (London) 333, 213-226 16 Hales, T. G., Lambert, J. J. and Peters, J. A. (1988) Br. J. PharmacoL 94, 393P 17 Yakel, J. L. and Jackson, M. B. (1988) Neuron 1, 615-621 18 Lambert, J. J., Peters, J. A., Hales, T. G. and Dempster, J. Br. J. Pharmacol. (in press)
Mechanism of action of scopolamine as an amnestic S c o p o l a m i n e is a well k n o w n a m n e s t i c drug. In fact, scopola m i n e a m n e s i a is w i d e l y cited as a m o d e l for h u m a n d e m e n t i a i n general or for A l z h e i m e r ' s d i s e a s e in p a r t i c u l a r 1. H o w e v e r , in spite of this, t h e r e is still d i s a g r e e m e n t as to w h a t s c o p o l a m i n e actually does to m e m o r y . M e m o r y is n o t a s i m p l e or single e n t i t y that can be altered w h o l e s a l e b y d r u g s or o t h e r variables. M e m o r y is a c o m p o s i t e of v a r i o u s processes ( a c q u i s i t i o n , c o n s o l i d a t i o n , storage, retrieval) w h i c h m a y be i n t e r f e r e d w i t h s e p a r a t e l y (Fig. 1), a n d this m a y lead to v e r y different s y n d r o m e s . W h i c h of these processes d o e s s c o p o l a m i n e affect? C a n a n y of its effects b e e x p l a i n e d b y the i n d u c t i o n of state d e p e n d e n c y ? C a n t h e y be r e v e r s e d b y n o n s p e c i f i c e n h a n cers or o n l y b y c h o l i n o m i m e t i c agents? Four articles in the N o v e m b e r 1988 issue of B e h a v i o r a l a n d N e u r a l B i o l o g y deal w i t h effects of scopol-
a m i n e on m e m o r y in mice 2-5. It w o u l d seem that e v e r y b o d y d e c i d e d at the same time to settle the issue of s c o p o l a m i n e a m n e s i a once a n d for all and, for some reason, m a n y mice s u d d e n l y b e c a m e available w o r l d w i d e ironically, in the year of Mickey's 60th Birthday. R u s h 3 s t u d i e d the effect of pret r a i n i n g , p o s t - t r a i n i n g a n d pretest i.p. s c o p o l a m i n e a d m i n i s t r a t i o n o n r e t e n t i o n of s t e p - t h r o u g h onetrial i n h i b i t o r y a v o i d a n c e in mice. P r e t r a i n i n g injections w o u l d be p r e s u m e d to affect acquisition p r i m a r i l y ; p o s t - t r a i n i n g injections, c o n s o l i d a t i o n ; a n d pretest injections, retrieval. Failure of acquisit i o n or c o n s o l i d a t i o n , or both, w o u l d i m p a i r or p r e v e n t storage 6. R u s h f o u n d that s c o p o l a m i n e did d i s r u p t r e t e n t i o n m e a s u r e d in a test session 24 h later in all cases, b u t that dose--response curves w e r e q u i t e different d e p e n d i n g on w h e n the d r u g was given. M e m o r y was m u c h more sensitive to pre-
1989, Elsevier Science Publishers Ltd. (UK) 0165- 6147/89/$02.00
19 Hoyer, D. and Neijt, H. C. (1988) Mol. Pharmacol. 33, 303-309 20 Neijt H. C., Karpf, A., Schoeffter, P., Engel, G. and Hoyer, D. (1988) NaunynSchmiedebergs Arch. Pharmacol. 337, 493--499 21 Neijt, H. C., Plomp, J. J. and Vijverberg, H. P. M. J. Physiol. (London) (in press) 22 Yakel, J. L., Trussdl, L. O. and Jackson, M. B. (1988) J. Neurosci. 8, 1273-1285 23 Higashi, H. and Nishi, S. (1982) J. Physiol. (London) 323, 543-567 24 Guharay, F., Ramsey, R. L. and Usherwood, P. N. R. (1985) Brain Res. 340, 325-332 25 Cull-Candy, S. G. and Ogden, D. C. (1985) Proc. R. Soc. London Ser. B. 224, 367-373 26 Bevan, S. and Robertson, B. (1987) J. Physiol. (London) 390, 256P 27 Wallis, D. I. and North, R. A. (1978) Neuropharmacology 17, 1023-1028 28 ~dash,H. L. and Wallis, D. I. (1981)Br. J. Pharmacol. 73, 759-772 29 Hoyer, D., Waeber, C., Neijt, H. C. and Palacios, J. M. (1989) Br. J. PharmacoL 96, 7P 30 Paul, J. (1970)Cell and Tissue Culture (4th edn), Livingston GR-38032F: 1,2,3,9-tetrahydro-9-methyl-
3[(2-methyl-lH-imidazol-l-yl) methyl]-Hcarbazol-4-one GR-65630:3-(5-methyl-1H-imidazol-4-yl)1-(1-methyl-lH-indol-3-yl)-l-propanone ICS 205-930: (30~-tropanyl)-lH-indole-3carboxylic acid ester MDL-72222:1~H,30~,50~H-tropan-3-yl-3,5dichlorobenzoate
t r a i n i n g than to p o s t - t r a i n i n g scopolamine: doses of 0.33 . 0 m g k g -1 d i s r u p t e d r e t e n t i o n consistently w h e n given prior to training, whereas at least 30.0 m g kg -1 were n e e d e d to o b t a i n a p o s t - t r a i n i n g amnestic effect. Pretest scopolamine, at a dose of 3.0 m g kg -1, also had a deleterious effect; since this was seen in b o t h a n i m a l s that were and animals that were not treated w i t h this same dose prior to training, state dep e n d e n c y can be ruled out. Therefore, in the h a n d s of Rush 3, scopola m i n e t u r n e d out to be a p o t e n t i n h i b i t o r of acquisition a n d retrieval, a n d a weak i n h i b i t o r of consolidation. As Rush suggested, it is likely that the primary or the most i m p o r t a n t action of scopolamine is on acquisition 3. O n one h a n d , retrieval sessions appear to involve a reacquisition comp o n e n t 4,s. O n the other, pretest scop2olamine is not always effecLive : experiments in h u m a n s , a species in w h i c h scopolamine is well k n o w n to h i n d e r a c q u i s i t i o n (see Ref. 1), have failed to detect an influence of the drug on retrieval
]76
in a test of verbal fluency - a test in which serious deficits are seen in Alzheimer's disease 7. In an experiment on conditioned emotional learning in mice, Brioni and Izquierdo 2 were unable to obtain an impairment of retrieval with 1.0 mg kg -1 of scopolamine, a dose that Rush 3 and many others before him (e.g. Refs 8-10) found to affect acquisition profoundly when given before training. However, Brioni and Izquierdo ~ found that pretest scopolamine did antagonize the stimulant influence of both oxotremorine (0.2 mg kg -1) and [3-endorphin (0.02 mg kg -1) on retrieval. This, by the way, suggests an interaction between (3endorphinergic and central muscarinic processes in retrieval that is of opposite sign to that described by Baratti et al. 11 for the interaction of these systems in consolidation. Unlike Rush 3, Quartermain and Leo 4 were able to detect a profound retrograde amnesia with as little as 1.0mg kg -1 of scopolamine given s.c. to mice immediately after a one-way active avoidance task acquired using an apparatus very similar to that used by Rush. So this task seems to be several times more sensitive to post-training scopolamine than the inhibitory avoidance paradigm, and neither the species nor the shape of the apparatus seems to account for the difference. ~ interesting quirk of the experiment of Quartermain and Leo is that the post-training amnestic effect of scopolamine was detectable only w h e n the mice were tested one, three, 14 or 28 days after training, and not when they were tested seven or ten days after training. In other words, there was a short-lived recovery of memory seven to ten days after training in the scopolaminetreated animals. This suggests that scopolamine effectively weakened the memory trace; a similar 'recovery peak' was observed seven to ten days after training in untreated mice trained to a lower criterion 4. A n u m b e r of data in the literature suggest that an interference with post-training consolidation is one of the most effective ways to weaken a memory6; this seems to be precisely what scopolamine did in the experiment of Quartermain and Leo 4. So, what are we left with? According to 17ush3, scopolamine is a strong inhibitor of acquisition
TiPS - M a y 1989 [Vol. 10]
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retrieval and retrieval and a weak inhibitor of consolidation. In the h a n d s of Quartermain and Leo 4, scopolamine was a strong inhibitor of consolidation. Brioni and Izquierdo 2 found that scopolamine antagonized the effect of other drugs on retrieval but did not inhibit retrieval on its own at a dose that the other authors had found effective to inhibit acquisition 3 or consolidation 4. It would seem that scopolamine really does affect m e m o r y at three different stages - acquisition, consolidation and retrieval- but that it interacts differently with each task, so that in some tasks one of the variables is more sensitive to the drug than others. Very probably, this is because some tasks use more cholii~ergic muscarinic receptors than others. There are, no doubt, major modulatory cholinergic systems in the brain: one based on the nucleus basalis of Meynert, which projects to the amygdala and the cortex; and one based on the medial septum, which projects to the hippocampus 9,12. There is strong evidence that the amygdala is involved in the modulation of consolidation 6'~3, and that the septo-hippocampal system plays a role in the early stages of m e m o r y formation and possibly in retrieval 12"14. It is possible that most, if not all, tasks use these general systems to a similar extent if they are to be acquired, consolidated and retrieved at all. But there are also m a n y other cholinergic synapses in the brain, in sensory, motor and associative pathways 9, and no doubt different tasks may
Fig. 1. Memories are first acquired, then consolidated, stored, and eventually retrieved; they can only be measured by measuring retrieval. Drugs can be given prior to acquisition, consolidation (and storage), or retrieval. The effect can be due to an action upon one or more of the preceding steps or processes, depending on when the drug is given.
use different sets of cholinergic and other synapses here or there in order to be acquired, consolidated or retrieved. The l e a r n i n g _ o r retrieval of verbal material ~, or of an active avoidance task 4 would certainly be presumed to be processed b y other synapses than the learning and retrieval of inhibitory avoidance 3 or of a conditioned emotional response2; m a n y cf those synapses could well be chednergic. It is possible that a generalized blockade of central muscarinic synapses, such as would be expected after a systemic injection of scopolamine 9, might therefore weaken some m e m o r y traces - i.e. those that include more, or more sensitive, muscarinic synapses in their storage processes more than others. This could h a p p e n in addition to and regardless of a specific attenuation of the function of the amygdaloid 6 or septohippocampal systems 12,14involved in acquisition, consolidation or retrieval modulation. To equate learning and memory, with all their inherent complexities, with the functioning of cholinergic modulatory systems seems a little worse than naive. Dementias include amnesia among other cognitive disturbances; and, although it has been proposed that the amnesia m a y be central to all the other cognitive disorders 15, there is no way to be sure of this. In view of the very strong evidence for interactions among brain systems involved in m e m o r y regulation (cholinergic, ~-endorphinergic, noradrenergic, dopaminergic, GABAergic, etc.; -
T i P S - M a y 1989 [Vol. 10]
177
see Refs 2--4,10,11,15,16), it w o u l d also seem u n w i s e to place all bets on any single system as the only one that really counts. Similarly, in view of the m o u n t i n g and also very strong evidence that Alzheimer's disease and other d e m e n t i a s involve multiple brain lesions, i n c l u d i n g lesions in all the brain modulatory systems listed above (see Ref. 16), it also seems u n w i s e to cling to a single-transmitter h y p o t h e s i s of dementia 3"15A6. In particular, scopolamine effects can be reversed not only b y cholinomimetic substances (e.g. Refs 1 a n d 11), but also b y nonspecific enhancers. Stone et al. reported that an injection of glucose can antagonize the a m n e s i a (see Ref. 5) and the hyperactivity 17 produced b y scopolarnine in mice; at least in the latter case, the effect of glucose can be potentiated b y p h y s o s t i g m i n e 5. Inasmuch as previous data f r o m that group have revealed a powerful m e m o r y e n h a n c i n g effect of glucose in rodents TM and h u m a n s ~9, w h i c h led t h e m to postulate a role for
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glucose in m e m o r y modulation 5AsA9, the time may have arrived to wonder whether one m i g h t not look for sweeter ways of treating the amnesia of organic brain disorders - particularly in view of the fact that most drugs tried so far appear to be quite useless 2°. Including cholinergic drugs 21. IVAN IZQUIERDO Centro de Memoria, Departamento de Bioquimica, Instituto de Biociencias, UFRGS (centro), 90049 Porto Alegre, RS, Brazil.
References 1 Bartus, R. T., Dean, R. T., Pontecorvo, M. J. and Flicker, C. (1985) Ann. NYAcad. Sci. 444, 332-358
2 Brioni, J- D. and Izquierdo, I. (1988) Behav. Neural Biol. 50, 251-254 3 Rush, D. K. (1988)Behav. Neural Biol. 50, 255-274 4 Quartermain, D. and Leo, P. (1988) Behav. Neural Biol. 50, 300-310 5 Stone,W. S., Cottrill, K. L., Walker,D. L. and Gold, P. E. (1988)Behav. Neural Biol. 50, 325-334 6 McGaugh,J. L. (1988) in Perspectives on Memory Research (Solomon, P.R., Goethals, G.R., Kelley, C.M. and Stephens, B.R., eds), pp. 33-64, Springer
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Analysis of mercapturic acids as a tool in biotransformation, biomonitoring and toxicological studies Mercapturic acids are S-substituted N-acetyl-g-cysteine derivatives. They are metabolic endproducts of an important p a t h w a y in the biotransformation of xenobiotics, notably the glutathione conjugation pathway. They were first identified as urinary metabolites of halogenated b e n z e n e s more than a century ago 1, and their m e c h a n i s m of formation is n o w well documented. Briefly, substrates possessing an electrophilic center, e.g. a leaving group, react w i t h endogenous glutathione (y-Glu-Cys-Gly; GSH) giving rise to GSH conjugates (Fig. 1). This reaction is usually catalysed b y cytosolic or microsomal GSH transferases present in cells of the liver, blood and to a lesser extent other organs; it can, however, also occur spontaneously, particularly w h e n very reactive
electrophilic substrates are involved. Once the GSH conjugates have been formed in the liver, intestine or kidney, the glutamyl moiety is removed by the action of y-glutamyl transpeptidases. This is followed by removal of glycine by cysteinyl glycinase and N-acetylation of the r e m a i n i n g S-alkylated cysteine conjugate (Fig. 1), giving rise to the mercapturic acids. These are often the major urinary products of GSH conjugation in v i v o , although cysteine conjugates and other sulfur-containing metabolites, such as methylsulfides, methylsulfoxides and methylsulfones (Fig. 1) have also been identified as such 2. The latter metabolic p a t h w a y is initiated b y the action of hepatic, intestinal or renal cysteine-conjugate C-S-lyases or 13-1yases.
7 Beatty, W. W., Butters, N. and Janowsky, D.S. (1986) Behav. Neural Biol. 45, 196--211 8 Bohdanecky, Z. and Jarvik, M. E. (1967) J. Neuropharmacol. 6, 217-222 9 Cheal, M. L. (1981) Behav. Neural Biol. 33, 163-187 10 Flood, J. F. and Cherkin, A. (1986) Behav. Neural Biol. 45, 169--184 11 Baratti, C. M., Introini, I.B. and Huygens, P. (1984) Behav. Neural Biol. 40, 155-169 12 Gray, J. A. (1982) The Neuropsychology of Anxiety: an Enquiry into the Physiology o~ the Septo-Hippocampal System, Clarendon 13 Izquierdo, I. (1988) Trends Pharmacol. Sci. 9, 429--430 14 Izquierdo, I. and Netto, C.A. (1985) Behav. Neural Biol. 44, 249-265 15 Cherkin, A. and Riege, W. H. (1983) in Brain Aging: Neuropathology and Neuropharmacology (Cervos-Navarro, J. and Sarkander, H.I., eds), pp. 415--435, Academic 16 Izquierdo, I. (1987) Trends Pharmacol. Sci. 8, 325-327
17 Stone, W. S., Cottrill, K. L. and Gold, P.E. (1987) Neurosci. Res. Commun. 1, 105-111 18 Gold, P. E. (1986)Behav. Neural Biol. 45, 342-349 19 Gonder-Frederick,L. et al. (1987)Physiol. Behav. 41, 503-504 20 Moos, W. H., Davis, R.E., Schwarz, R. D. and Gamzu, E. R. (1988)Meal. Res. Rev. 8, 353-391 21 Crook, T. (1985)Ann. NY Acad. Sci. 444, 428--436
Until recently GSH conjugation was regarded as a universal detoxification mechanism for potentially (geno)toxic electrophilic xenobiotic chemicals, but in the last few years it has become clear that GSH conjugation may also lead to formation of electrophilic metabolites that are more reactive and have mutagenic, carcinogenic or other toxicological effects 3. Different types of bioactivation via GSH conjugation have been distinguished, including: the formation of sulfur half-mustards from comp o u n d s like 1,2-dibromoethane; and the 13-1yase-mediated generation of generally u n k n o w n 'reactive sulfur intermediates' in the kidney from cysteine conjugates of compounds like hexachlorobutadiene. During the last few years the measurement of mercapturic acids has become easier because of advances in analytical techniques (e.g. HPLC, GLC, MS and NMR) which have allowed the isolation and identification of GSH conjugation-related metabolites in biological fluids. Analysis of urinary mercapturic acids may provide valuable information about the extent and mechanism of
(~ 1989, Elsevier Science Publishers Ltd. (UK)
0165 - 6147/89/$02.00
i n v e s t i g a t i o n is r e q u i r e d to determ i n e w h e t h e r this effect is exclusively d u e to a n e l e v a t i o n of c A M P levels a n d s u b s e q u e n t p h o s p h o r y lation of t h e 5-HT3 receptor via a c A M P - d e p e n d e n t p r o t e i n kinase. []
[3
[]
N e u r o n a l clonal cell lines prov i d e a c o n v e n i e n t source of large a m o u n t s of relatively h o m o g e n o u s material: it h a s b e e n e s t i m a t e d t h a t a single cell t h a t p r o d u c e s p r o g e n y that d i v i d e once e v e r y 10-12 h, could y i e l d 4 kg of tissue w i t h i n 12 w e e k s 3°. N o t w i t h s t a n d i n g the s o m e w h a t low d e n s i t y of 5-HT3 r e c o g n i t i o n sites i n the clonal lines 19,2° comj~ared w i t h c e r t a i n b r a i n r e g i o n s z~, t h e y offer a m o s t p r o m i s i n g s t a r t i n g p o i n t for t h e i s o l a t i o n of t h e 5-HT3 receptor a n d e v e n t u a l l y the d e t e r m i n a t i o n of its structure. JOHN A. PETERS A N D J E R E M Y J. L A M B E R T
Neurosciences Research Group, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. References
1 Fozard, J. R. (1987) Trends Pharmacol. Sci. 8, ~A ~5
175 2 Jones, B. J. et al. (1988) Br. ]. Pharmacol. 93, 985-993 3 Cos"ll, B., Domeney, A. M., Kelly,M. E., Naylor, R. J. and Tyers, M. B. (1987) Neurosci. Lett. Suppl. 29, $69 4 Kilpatrick, G. J., Jones, B. J. and Tyers, M. B. (1987) Nature 330, 746-748 5 Watling, K. J. (1988) Trends Pharmacol. Sci. 9, 227-229 6 Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. and Nirenberg, M. (1978) Brain Res. 147, 261-276 7 MacDermot, J. et al. (1979) Proc. Natl Acad. Sci. USA 76, 1135-1139 8 Guharay, F. and Usherwood, P. N. R. (1981) Br. J. Pharmacol. 74 294P 9 Freschi, J. E. and Shain, W. G. (1982) J. Neurosci. 2, 106--112 10 Peters, J. A. and Usnerwood, P. N. R. (1983) Br. J. Pharmacol. 80, 523P 11 Neijt, H. C., Vijverberg, H. P. M. and van den Bercken, J. (1986) Eur. J. Pharmacol. 127, 271-274 12 Neijt, H. C., Te Duits, I. J. and Vijverberg, H. P. M. (1988)Neuropharmacology 27, 301-307 13 Peters, J. A., Hales, T. G. and Lambert, J. J. (1988)Eur. J. Pharmacol. 151,491-495 14 Peters, J. A. and Usherwood, P. N. R. (1984) Br. J. Pharmacol. 83, 419P 15 Kato, E. and Narahashi, T. (1982) J. Physiol. (London) 333, 213-226 16 Hales, T. G., Lambert, J. J. and Peters, J. A. (1988) Br. J. PharmacoL 94, 393P 17 Yakel, J. L. and Jackson, M. B. (1988) Neuron 1, 615-621 18 Lambert, J. J., Peters, J. A., Hales, T. G. and Dempster, J. Br. J. Pharmacol. (in press)
Mechanism of action of scopolamine as an amnestic S c o p o l a m i n e is a well k n o w n a m n e s t i c drug. In fact, scopola m i n e a m n e s i a is w i d e l y cited as a m o d e l for h u m a n d e m e n t i a i n general or for A l z h e i m e r ' s d i s e a s e in p a r t i c u l a r 1. H o w e v e r , in spite of this, t h e r e is still d i s a g r e e m e n t as to w h a t s c o p o l a m i n e actually does to m e m o r y . M e m o r y is n o t a s i m p l e or single e n t i t y that can be altered w h o l e s a l e b y d r u g s or o t h e r variables. M e m o r y is a c o m p o s i t e of v a r i o u s processes ( a c q u i s i t i o n , c o n s o l i d a t i o n , storage, retrieval) w h i c h m a y be i n t e r f e r e d w i t h s e p a r a t e l y (Fig. 1), a n d this m a y lead to v e r y different s y n d r o m e s . W h i c h of these processes d o e s s c o p o l a m i n e affect? C a n a n y of its effects b e e x p l a i n e d b y the i n d u c t i o n of state d e p e n d e n c y ? C a n t h e y be r e v e r s e d b y n o n s p e c i f i c e n h a n cers or o n l y b y c h o l i n o m i m e t i c agents? Four articles in the N o v e m b e r 1988 issue of B e h a v i o r a l a n d N e u r a l B i o l o g y deal w i t h effects of scopol-
a m i n e on m e m o r y in mice 2-5. It w o u l d seem that e v e r y b o d y d e c i d e d at the same time to settle the issue of s c o p o l a m i n e a m n e s i a once a n d for all and, for some reason, m a n y mice s u d d e n l y b e c a m e available w o r l d w i d e ironically, in the year of Mickey's 60th Birthday. R u s h 3 s t u d i e d the effect of pret r a i n i n g , p o s t - t r a i n i n g a n d pretest i.p. s c o p o l a m i n e a d m i n i s t r a t i o n o n r e t e n t i o n of s t e p - t h r o u g h onetrial i n h i b i t o r y a v o i d a n c e in mice. P r e t r a i n i n g injections w o u l d be p r e s u m e d to affect acquisition p r i m a r i l y ; p o s t - t r a i n i n g injections, c o n s o l i d a t i o n ; a n d pretest injections, retrieval. Failure of acquisit i o n or c o n s o l i d a t i o n , or both, w o u l d i m p a i r or p r e v e n t storage 6. R u s h f o u n d that s c o p o l a m i n e did d i s r u p t r e t e n t i o n m e a s u r e d in a test session 24 h later in all cases, b u t that dose--response curves w e r e q u i t e different d e p e n d i n g on w h e n the d r u g was given. M e m o r y was m u c h more sensitive to pre-
1989, Elsevier Science Publishers Ltd. (UK) 0165- 6147/89/$02.00
19 Hoyer, D. and Neijt, H. C. (1988) Mol. Pharmacol. 33, 303-309 20 Neijt H. C., Karpf, A., Schoeffter, P., Engel, G. and Hoyer, D. (1988) NaunynSchmiedebergs Arch. Pharmacol. 337, 493--499 21 Neijt, H. C., Plomp, J. J. and Vijverberg, H. P. M. J. Physiol. (London) (in press) 22 Yakel, J. L., Trussdl, L. O. and Jackson, M. B. (1988) J. Neurosci. 8, 1273-1285 23 Higashi, H. and Nishi, S. (1982) J. Physiol. (London) 323, 543-567 24 Guharay, F., Ramsey, R. L. and Usherwood, P. N. R. (1985) Brain Res. 340, 325-332 25 Cull-Candy, S. G. and Ogden, D. C. (1985) Proc. R. Soc. London Ser. B. 224, 367-373 26 Bevan, S. and Robertson, B. (1987) J. Physiol. (London) 390, 256P 27 Wallis, D. I. and North, R. A. (1978) Neuropharmacology 17, 1023-1028 28 ~dash,H. L. and Wallis, D. I. (1981)Br. J. Pharmacol. 73, 759-772 29 Hoyer, D., Waeber, C., Neijt, H. C. and Palacios, J. M. (1989) Br. J. PharmacoL 96, 7P 30 Paul, J. (1970)Cell and Tissue Culture (4th edn), Livingston GR-38032F: 1,2,3,9-tetrahydro-9-methyl-
3[(2-methyl-lH-imidazol-l-yl) methyl]-Hcarbazol-4-one GR-65630:3-(5-methyl-1H-imidazol-4-yl)1-(1-methyl-lH-indol-3-yl)-l-propanone ICS 205-930: (30~-tropanyl)-lH-indole-3carboxylic acid ester MDL-72222:1~H,30~,50~H-tropan-3-yl-3,5dichlorobenzoate
t r a i n i n g than to p o s t - t r a i n i n g scopolamine: doses of 0.33 . 0 m g k g -1 d i s r u p t e d r e t e n t i o n consistently w h e n given prior to training, whereas at least 30.0 m g kg -1 were n e e d e d to o b t a i n a p o s t - t r a i n i n g amnestic effect. Pretest scopolamine, at a dose of 3.0 m g kg -1, also had a deleterious effect; since this was seen in b o t h a n i m a l s that were and animals that were not treated w i t h this same dose prior to training, state dep e n d e n c y can be ruled out. Therefore, in the h a n d s of Rush 3, scopola m i n e t u r n e d out to be a p o t e n t i n h i b i t o r of acquisition a n d retrieval, a n d a weak i n h i b i t o r of consolidation. As Rush suggested, it is likely that the primary or the most i m p o r t a n t action of scopolamine is on acquisition 3. O n one h a n d , retrieval sessions appear to involve a reacquisition comp o n e n t 4,s. O n the other, pretest scop2olamine is not always effecLive : experiments in h u m a n s , a species in w h i c h scopolamine is well k n o w n to h i n d e r a c q u i s i t i o n (see Ref. 1), have failed to detect an influence of the drug on retrieval
]76
in a test of verbal fluency - a test in which serious deficits are seen in Alzheimer's disease 7. In an experiment on conditioned emotional learning in mice, Brioni and Izquierdo 2 were unable to obtain an impairment of retrieval with 1.0 mg kg -1 of scopolamine, a dose that Rush 3 and many others before him (e.g. Refs 8-10) found to affect acquisition profoundly when given before training. However, Brioni and Izquierdo ~ found that pretest scopolamine did antagonize the stimulant influence of both oxotremorine (0.2 mg kg -1) and [3-endorphin (0.02 mg kg -1) on retrieval. This, by the way, suggests an interaction between (3endorphinergic and central muscarinic processes in retrieval that is of opposite sign to that described by Baratti et al. 11 for the interaction of these systems in consolidation. Unlike Rush 3, Quartermain and Leo 4 were able to detect a profound retrograde amnesia with as little as 1.0mg kg -1 of scopolamine given s.c. to mice immediately after a one-way active avoidance task acquired using an apparatus very similar to that used by Rush. So this task seems to be several times more sensitive to post-training scopolamine than the inhibitory avoidance paradigm, and neither the species nor the shape of the apparatus seems to account for the difference. ~ interesting quirk of the experiment of Quartermain and Leo is that the post-training amnestic effect of scopolamine was detectable only w h e n the mice were tested one, three, 14 or 28 days after training, and not when they were tested seven or ten days after training. In other words, there was a short-lived recovery of memory seven to ten days after training in the scopolaminetreated animals. This suggests that scopolamine effectively weakened the memory trace; a similar 'recovery peak' was observed seven to ten days after training in untreated mice trained to a lower criterion 4. A n u m b e r of data in the literature suggest that an interference with post-training consolidation is one of the most effective ways to weaken a memory6; this seems to be precisely what scopolamine did in the experiment of Quartermain and Leo 4. So, what are we left with? According to 17ush3, scopolamine is a strong inhibitor of acquisition
TiPS - M a y 1989 [Vol. 10]
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retrieval and retrieval and a weak inhibitor of consolidation. In the h a n d s of Quartermain and Leo 4, scopolamine was a strong inhibitor of consolidation. Brioni and Izquierdo 2 found that scopolamine antagonized the effect of other drugs on retrieval but did not inhibit retrieval on its own at a dose that the other authors had found effective to inhibit acquisition 3 or consolidation 4. It would seem that scopolamine really does affect m e m o r y at three different stages - acquisition, consolidation and retrieval- but that it interacts differently with each task, so that in some tasks one of the variables is more sensitive to the drug than others. Very probably, this is because some tasks use more cholii~ergic muscarinic receptors than others. There are, no doubt, major modulatory cholinergic systems in the brain: one based on the nucleus basalis of Meynert, which projects to the amygdala and the cortex; and one based on the medial septum, which projects to the hippocampus 9,12. There is strong evidence that the amygdala is involved in the modulation of consolidation 6'~3, and that the septo-hippocampal system plays a role in the early stages of m e m o r y formation and possibly in retrieval 12"14. It is possible that most, if not all, tasks use these general systems to a similar extent if they are to be acquired, consolidated and retrieved at all. But there are also m a n y other cholinergic synapses in the brain, in sensory, motor and associative pathways 9, and no doubt different tasks may
Fig. 1. Memories are first acquired, then consolidated, stored, and eventually retrieved; they can only be measured by measuring retrieval. Drugs can be given prior to acquisition, consolidation (and storage), or retrieval. The effect can be due to an action upon one or more of the preceding steps or processes, depending on when the drug is given.
use different sets of cholinergic and other synapses here or there in order to be acquired, consolidated or retrieved. The l e a r n i n g _ o r retrieval of verbal material ~, or of an active avoidance task 4 would certainly be presumed to be processed b y other synapses than the learning and retrieval of inhibitory avoidance 3 or of a conditioned emotional response2; m a n y cf those synapses could well be chednergic. It is possible that a generalized blockade of central muscarinic synapses, such as would be expected after a systemic injection of scopolamine 9, might therefore weaken some m e m o r y traces - i.e. those that include more, or more sensitive, muscarinic synapses in their storage processes more than others. This could h a p p e n in addition to and regardless of a specific attenuation of the function of the amygdaloid 6 or septohippocampal systems 12,14involved in acquisition, consolidation or retrieval modulation. To equate learning and memory, with all their inherent complexities, with the functioning of cholinergic modulatory systems seems a little worse than naive. Dementias include amnesia among other cognitive disturbances; and, although it has been proposed that the amnesia m a y be central to all the other cognitive disorders 15, there is no way to be sure of this. In view of the very strong evidence for interactions among brain systems involved in m e m o r y regulation (cholinergic, ~-endorphinergic, noradrenergic, dopaminergic, GABAergic, etc.; -
T i P S - M a y 1989 [Vol. 10]
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see Refs 2--4,10,11,15,16), it w o u l d also seem u n w i s e to place all bets on any single system as the only one that really counts. Similarly, in view of the m o u n t i n g and also very strong evidence that Alzheimer's disease and other d e m e n t i a s involve multiple brain lesions, i n c l u d i n g lesions in all the brain modulatory systems listed above (see Ref. 16), it also seems u n w i s e to cling to a single-transmitter h y p o t h e s i s of dementia 3"15A6. In particular, scopolamine effects can be reversed not only b y cholinomimetic substances (e.g. Refs 1 a n d 11), but also b y nonspecific enhancers. Stone et al. reported that an injection of glucose can antagonize the a m n e s i a (see Ref. 5) and the hyperactivity 17 produced b y scopolarnine in mice; at least in the latter case, the effect of glucose can be potentiated b y p h y s o s t i g m i n e 5. Inasmuch as previous data f r o m that group have revealed a powerful m e m o r y e n h a n c i n g effect of glucose in rodents TM and h u m a n s ~9, w h i c h led t h e m to postulate a role for
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glucose in m e m o r y modulation 5AsA9, the time may have arrived to wonder whether one m i g h t not look for sweeter ways of treating the amnesia of organic brain disorders - particularly in view of the fact that most drugs tried so far appear to be quite useless 2°. Including cholinergic drugs 21. IVAN IZQUIERDO Centro de Memoria, Departamento de Bioquimica, Instituto de Biociencias, UFRGS (centro), 90049 Porto Alegre, RS, Brazil.
References 1 Bartus, R. T., Dean, R. T., Pontecorvo, M. J. and Flicker, C. (1985) Ann. NYAcad. Sci. 444, 332-358
2 Brioni, J- D. and Izquierdo, I. (1988) Behav. Neural Biol. 50, 251-254 3 Rush, D. K. (1988)Behav. Neural Biol. 50, 255-274 4 Quartermain, D. and Leo, P. (1988) Behav. Neural Biol. 50, 300-310 5 Stone,W. S., Cottrill, K. L., Walker,D. L. and Gold, P. E. (1988)Behav. Neural Biol. 50, 325-334 6 McGaugh,J. L. (1988) in Perspectives on Memory Research (Solomon, P.R., Goethals, G.R., Kelley, C.M. and Stephens, B.R., eds), pp. 33-64, Springer
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Analysis of mercapturic acids as a tool in biotransformation, biomonitoring and toxicological studies Mercapturic acids are S-substituted N-acetyl-g-cysteine derivatives. They are metabolic endproducts of an important p a t h w a y in the biotransformation of xenobiotics, notably the glutathione conjugation pathway. They were first identified as urinary metabolites of halogenated b e n z e n e s more than a century ago 1, and their m e c h a n i s m of formation is n o w well documented. Briefly, substrates possessing an electrophilic center, e.g. a leaving group, react w i t h endogenous glutathione (y-Glu-Cys-Gly; GSH) giving rise to GSH conjugates (Fig. 1). This reaction is usually catalysed b y cytosolic or microsomal GSH transferases present in cells of the liver, blood and to a lesser extent other organs; it can, however, also occur spontaneously, particularly w h e n very reactive
electrophilic substrates are involved. Once the GSH conjugates have been formed in the liver, intestine or kidney, the glutamyl moiety is removed by the action of y-glutamyl transpeptidases. This is followed by removal of glycine by cysteinyl glycinase and N-acetylation of the r e m a i n i n g S-alkylated cysteine conjugate (Fig. 1), giving rise to the mercapturic acids. These are often the major urinary products of GSH conjugation in v i v o , although cysteine conjugates and other sulfur-containing metabolites, such as methylsulfides, methylsulfoxides and methylsulfones (Fig. 1) have also been identified as such 2. The latter metabolic p a t h w a y is initiated b y the action of hepatic, intestinal or renal cysteine-conjugate C-S-lyases or 13-1yases.
7 Beatty, W. W., Butters, N. and Janowsky, D.S. (1986) Behav. Neural Biol. 45, 196--211 8 Bohdanecky, Z. and Jarvik, M. E. (1967) J. Neuropharmacol. 6, 217-222 9 Cheal, M. L. (1981) Behav. Neural Biol. 33, 163-187 10 Flood, J. F. and Cherkin, A. (1986) Behav. Neural Biol. 45, 169--184 11 Baratti, C. M., Introini, I.B. and Huygens, P. (1984) Behav. Neural Biol. 40, 155-169 12 Gray, J. A. (1982) The Neuropsychology of Anxiety: an Enquiry into the Physiology o~ the Septo-Hippocampal System, Clarendon 13 Izquierdo, I. (1988) Trends Pharmacol. Sci. 9, 429--430 14 Izquierdo, I. and Netto, C.A. (1985) Behav. Neural Biol. 44, 249-265 15 Cherkin, A. and Riege, W. H. (1983) in Brain Aging: Neuropathology and Neuropharmacology (Cervos-Navarro, J. and Sarkander, H.I., eds), pp. 415--435, Academic 16 Izquierdo, I. (1987) Trends Pharmacol. Sci. 8, 325-327
17 Stone, W. S., Cottrill, K. L. and Gold, P.E. (1987) Neurosci. Res. Commun. 1, 105-111 18 Gold, P. E. (1986)Behav. Neural Biol. 45, 342-349 19 Gonder-Frederick,L. et al. (1987)Physiol. Behav. 41, 503-504 20 Moos, W. H., Davis, R.E., Schwarz, R. D. and Gamzu, E. R. (1988)Meal. Res. Rev. 8, 353-391 21 Crook, T. (1985)Ann. NY Acad. Sci. 444, 428--436
Until recently GSH conjugation was regarded as a universal detoxification mechanism for potentially (geno)toxic electrophilic xenobiotic chemicals, but in the last few years it has become clear that GSH conjugation may also lead to formation of electrophilic metabolites that are more reactive and have mutagenic, carcinogenic or other toxicological effects 3. Different types of bioactivation via GSH conjugation have been distinguished, including: the formation of sulfur half-mustards from comp o u n d s like 1,2-dibromoethane; and the 13-1yase-mediated generation of generally u n k n o w n 'reactive sulfur intermediates' in the kidney from cysteine conjugates of compounds like hexachlorobutadiene. During the last few years the measurement of mercapturic acids has become easier because of advances in analytical techniques (e.g. HPLC, GLC, MS and NMR) which have allowed the isolation and identification of GSH conjugation-related metabolites in biological fluids. Analysis of urinary mercapturic acids may provide valuable information about the extent and mechanism of
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0165 - 6147/89/$02.00