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Differential response of rat brain polyamines to convulsant agents
Life Sciences, Vol. 48, pp. 77-84 Printed in the U.S.A.
Pergamon Press
DIFFERENTIAL RESPONSE OF RAT BRAIN POLYAMINES TO CONVULSANT AGENTS
E. Mart£nez, N. de Vera* and F.Artigas Department of Neurochemietry and *Department of Pharmacology & Toxicology. Jordl Girona 18-26, E-08034 Barcelona, Spain. (Received in final form November I, 1990) Summarv The polyamlnee putrescine (PUT), spermidine (SD} and spermine (SM} h a v e b e e n s t u d i e d in rat b r a i n a f t e r t r e a t m e n t w i t h s e v e r a l c o n v u l s a n t agents. K a i n l o a c i d (10 m g / k g ) , p i c r o t o x i n i n (1.5 mg/kg}, pentylenetetrazol (60 mg/kg) and lindane ( P - hexachlorocyclohexane) (60 mg/kg) were given to male Wistar rats. Twenty-four hours later, the animals were sacrificed and their brains removed. Cortical polyamines were analyzed by HPLC with fluorimetric detection of their respective daneyl derivatives, using 1,6-diamlnohexane as internal standard for the measurements. Polyamine l e v e l s are not a f f e c t e d by s h o r t p e r i o d s of t i m e (30 mln) of brain exposure to room temperature before freezing the samples, as compared to a quick procedure (less than 40 • f r o m a n i m a l d e a t h ) . K a i n i c acid i n d u c e d a 1 4 - f o l d i n c r e a s e of cortical PUT with respect to control values, leaving unchanged the other polyamlnes. Lindane also increased cortical PUT (4-fold) without affecting S M o r SD. Neither picrotoxinln nor pentylenetetrazol groups were different from controls for any of the polyamines assayed. The results are discussed in relation to the possible mechanism of action of these convulsant agents and the role of the polyamines in cell injury. Brain polyamines (PA) and their rate-limiting enzyme ornlthine decarboxylase (ODC) h a v e b e e n r e l a t e d to d i f f e r e n t k i n d of C N S i n j u r i e s (metabolic, mechanical, thermal or chemlcal), which induce ODC activity in rat brain (8). Ales, an enhanced PA synthesis appears to mediate the blood brain barrier breakdown by cold injury (27). Furthermore, it has been suggested that putrescine, produced during reclrculation following iechemia, contributes to the manifestation of isohemic cell injury (20-22), although a high content of hIppocampal putrescine occurs in certain species (21) without any morphological or functional alteration. On the other hand, some authors have'suggested that increases of PA may be mediating the mechanisms of neuronal repair after axonal injury (11) or partial hemitransection (1). C h a n g e s in b r a i n P A or O D C a c t i v i t y are also p r o d u c e d soon a f t e r convulsions(16,19). The increased PA synthesis following seizures has been related to changes in Ca 2+ fluxes at the cellular level (14), since PA increase the cytoeollc C a 2+ c o n c e n t r a t i o n (10,15,16) and release excitatory aminoacide(3). The present work was aimed at studying the effects of several oonvulsant agents on brain PA. Picrotoxinin (PIC), pentylenetetrazol (PTZ) and kainic acid (KA) are well established convuleant substances. Both PIC and PTZ are GABA A 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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receptor antagonists. Kainlc acid, a structural analog of glutamic acid, has complex a c t i o n s , b e i n g a b l e to b i n d to s p e c i f i c r e c e p t o r s (KA r e c e p t o r s ) localized at both presynaptic and postsynaptlc sites (5), and facilitating the release of glutamate from presynaptic terminals. The insecticide Y-hexachlorocyclohexane (lindane, LIN) is also a strong convulsant agent, probably a c t i n g at the p i c r o t o x i n i n b i n d i n g site associated with the Cl- ionophore of the GABA A receptor (26). Material and Me~hods Animals. Forty eight adult male albino Wistar rats (Interfauna, Sant Feliu de C o d i n e @ , Spain) w e i g h i n g 2 0 0 - 2 5 0 g have b e e n used. R a t s w e r e h o u s e d 3-5 animals per cage, under 12 hr llght-dark cycle with free access to standard chow pellets and water before and during the experiments. Temperature of the room was maintained at 21 ± 2 °C. Rats were received from the supplier and housed for one week before experiments began. All rats were randomly assigned to the different treatment groups. COmpounds. PTZ, PIC and KA obtained from commercial sources were dissolved in saline (NaCl, 0.9 %) before i.p. administration. LIN was dissolved in olive oil and administered "per os". Standard compounds putrescine (PUT), spermidine (SD) and spermine (SM) (hydrochlorides}, 1,6-diaminohexane (1,6-DAH) (free base) and dansylated putrescine (PUT-2DNS) were obtained from Sigma (St. Louis, Mo, USA). Dansyl chloride was also from Sigma and solvents -HPLC gradewere purchased from different conm~ercial sources. HPLC method. A modification of the HPLC method of Desiderio et al. (6) was used. Polyamines were separated on a 15.0 cm long, 4.6 mm i.d., stainless steel column packed with 5 ~m-size reverse-phase ODSl. (Tracer, Barcelona, Spain). The liquid chromatographic system consisted of two Waters solvent delivery pumps (model 590), a U6K injector and an automated gradient controller, also from waters. Column eluents were monitored with a fluorescence spectrophotometer (Hitachi-PE 650 S) equipped with a 20-gl quartz flow cell. F l u o r e s c e n c e c o n d i t i o n s -350 nm and 420 nm for e x c i t a t i o n a n d e m i s s i o n respectively- were adjusted experimentally with a standard solution of PUT-2DNS. The elution was performed with a gradient consisting of solvent A: 1.2 mM Na2HPO 4 and 12 mM NaCl solution (pH: 7.9 - 8.1). Solvent B: Methanol. Initial conditions (40% A, 60% B) were maintained for 2 min and then a 18 min linear gradient from 60% to 90% methanol was run. Final conditions were maintained for 7 min. A short (3 mln) reverse program was run to return to initial conditions. The flow rate was adjusted to 1.2 ml/min. 1,6-DAH was used as internal standard for quantitative purposes. Standard PUT-2DNS was the reference compound to determine the yield of derivatization of PA. Sample preparatioD. Rats were sacrificed by decapitation and brains rapidly removed from the skull (within 40 s). The dissection of frontal cortex was c a r r i e d o u t o n a c o l d p l a t e at 0 °C. S a m p l e s w e r e w e i g h e d and s t o r e d in Eppendorf tubes at -80 °C until analysis. Each sample was homogenized ultrasonically for 10-15 s in 10 vols. of cold perchloric acid (0.4 M) containing 1,6-DAH. After centrifugatlon (10000 x g, 15 min) a l i q u o t s of s u p e r n a t a n t s (400 pl) w e r e d e r i v a t i z e d a c c o r d i n g to Desiderio et al. (6). Briefly, 300 pl Na2CO 3 42.5 M) and 500 gl dansylchloride (5 mg/ml in acetone) were added and samples were kept for 1 hr at 50 °C. The s a m p l e s w e r e t h e n e x t r a c t e d w i t h b e n z e n e (i ml). T h e b e n z e n e p h a s e was evaporated under helium flow and the dry residue was redissolved in methanol
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before HPLC injection. Disposable plastic material was used throughout except for the benzene extraction where i0 - ml glass stoppered tubes were used. -~olvamine levels in frozen vs. ambient temperature samples. Ten rats were sacrificed and frontal cortices were divided in two parts, one immediately frozen in dry ice, the other left for 30 min at room temperature (22 °C). Samples were processed as described before. ExPerimental Daradiam. Five groups of animals received the following treatments PTZ (60 mg/kg), PIC (1.5 mg/kg}, KA (i0 mg/kg) and LIN (60 mg/kg}. The control g r o u p (N-7) w a s a d m i n i s t e r e d i.p. w i t h saline. D o s e s w e r e a d j u s t e d in a previous pilot experiment to obtain a maximum of convulsant rats with a minimum of d e a t h s . C o n v u l s a n t a n i m a l s w e r e kept for 24 hr at s t a n d a r d h o u s i n g c o n d i t i o n s a n d s a c r i f i c e d . The w e i g h t of the a n i m a l s w a s r e c o r d e d b e f o r e administration and Immedlately before sacrifice. Statistical analyses. The effect of temperature on polyamine brain levels was assessed by a paired Student t-Test. The influence of convulsant treatment on brain polyamines was evaluated by non-parametric one-way analysis of variance (Kruskal-Wallis Test} and the post-hoc differences between groups by the non parametric Mann Whitney U-Test. The numerical features of the experimental groups -few animals and different number of animals in each group- recommended the use of non-parametric tests. Internal correlations between different variables were analyzed by the Pearson correlation Test. Tests were carried out with a SPSS (Statistical Package for Social Sciences) program for personal computer. Results HPLC method for the determination of Dolvamines. The chromatographic conditions used enabled the complete separation of PUT, SD, SM and the internal standard 1,6-DAH in a run time of 22 minutes. Retention times for these compounds were, PUTs 11.5 min; 1,6-DAH: 13.1 min; SDz 16.8 min and SM: 20.4 min. Cadaverlne - r e t e n t i o n t i m e of 12.0 m i n - can a l s o be d e t e r m i n e d in t h e s a m e run. The orlginal method from Desidsrlo et al. (61 was modified by increasing the saline strength of the chromatographic eluent. This improved the reproducibility and eliminated some interferences observed when running brain samples. The dansyl derlvatlzatlon and benzene extraction procedures showed the followlng characterlstlcss a) The derlvatlzatlon yield, calculated for PUT vs. standard PUT-2DNS, was 43%, b) Recovery of the benzene extraction calculated for the internal standard in a series of 17 brain samples, was established in 103.7% ± 17.8 (mean ± S.D.), c) Total reproducibility of the method -extraction p l u s d e r l v a t l z a t l o n - for the i n t e r n a l s t a n d a r d was 17.2% ( c o e f f i c i e n t of variation} (N=I7). Using the above procedure, the detection limits (signal/noise ratio of 2.5) for these compounds were, PUTs 55 pg; SDs 70 pg and SMs75 pg of original free bases. The calibration plots of the polyamines/internal standard peak height ratio vs. the polyamines/Internal standard concentration ratio showed a linear response in the range (1s1-lsS0). Slopes of these plots for the three p o l y a m i n e s w e r e v e r y similar, PUTs 0.983; SD= 0 . 8 5 9 a n d SM= 0 . 8 6 5 . T h e regression coefficients for adjusted plots were always higher than r=0.998. Ambient temperature vs. freezinq. Figure 1 shows the level of polyamines in e q u i v a l e n t a l i q u o t s of f r o n t a l c o r t e x a f t e r b o t h h a n d l i n g p r o c e d u r e s . No significant differences in the content of polyamines were found after a fast freezing upon removal of brain from the skull or exposure for 30 min at room temperature. The concentration of cadaverine in these samples was below 40 ng/g in both cases.
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% RELATIVE FROZEN
1
125
100
75
50
25
0
PUT M
8D FROZEN
8M AMBIENT TEMP.
FIG.1 I n f l u e n c e of p o s t - m o r t e m c o n d i t i o n s on t h e c o n t e n t of polyamlnes in rat brain tissue. No difference was observed in the concentration of polyamlnes after a fast freezing of tissue or after leaving it 30 minutes at room temperature. Bars represent Percentage relative to frozen frontal cortex (mean + S.D.) (N=10}. The content of polyamlnes in frozen tissue wae~ (in pg/g, mean; S.D.}, PUT (0.491 0.17}, SD (40.8; 4.4) and SM (54.0; 23.4). P a i r e d S t u d e n t t - T e s t (N.S.). Effect of convulsant aoents. Only those animals showing severe tonlc-clonlc seizures during two hours post-adminlstratlon were included in the experiment. The recorded convulsant activity was as follows= PTZ group (N=7); 7 animals showed tonlc-clonic seizures starting in a time span of 5 to 60 min. One of them died. PIC group (N=7}; 5 animals exhibited tonlc-clonlc convulsions in a span of 15 to 30 min. Two animals did not show seizure activity. KA group (N=10)1 7 animals exhibited wet dog shakes and continuous convulsent activity between 1.5 and 2 hours post-admlnlstration. Three of them died within 24 hours. Three animals did not show abnormal neurobehavloural signs. LIN group (N=7); 5 animals s h o w e d tonic-clonlc seizures in the Period between 10 and 24 mln. One animal died after administration. Another rat did not show convulsant activity. Figure 2 shows the concentration of polyamlnes in the frontal cortex of rats 24 hr after convulsions induced by different neurotoxlc agents. Kruskalwallls analysis of variance indicated a strong influence of the treatment on
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PUT (N=27, 2-18.64; p<0.009). Mann-Whitney U-Test indicated that LIN group (p<0.0074) and KA group (p< 0.0082) had a content of PUT significantly higher than the control group (KA= 1400% of control values and LIN: 400%). The mean concentrations of SD and S M w e r e not influenced by the treatment, although some animals (ca. 25%) of the PIC, PTZ and LIN groups exhibited low values of these t w o p o l y a m l n e s , i n c r e a s i n g t h e d e v i a t i o n of t h e r e s p e c t i v e g r o u p s . This result could be due to a particular sensitivity of some of the animals to the
1800
% OF CONTROLS
1600 1400 1200
6oo .
1000 800
400
2O0 0
CONT ~1
PTZ PUT
PIC ~
8D
KA ~
LIN 8M
FIG.2
Effect of convulsant agents on the content of polyamlnea in rat frontal cortex, 24 hr post-adminlstration. Bars (mean + S.D.) represent percentage of control group (N=7). Pentylenetetrazol (N-6), Picrotoxinln (N-5}, Kainlc acid (N-4) and Lindane (N-5) groups are shown. Concentration of polyamines (gg/g) in control an!male were (mean; S.D.): PUT (0.65; 0.40}, SD (40.26; 15.94) and SM (58.07; 1 9 . 9 7 ) . (*) I)<0.009 from controls (Mann-Whitney U-Test). convulsant agents used. SD and 8M content showed a Positive correlation in all the animals studied (r= 0.9458; N=27; p < 0.0001) that indicates a parallel metabolic regulation after such treatments. However, putrescine did not correlate with any of the other two Polyamine8. Kruakal-Wallls analysis of variance indicated a strong influence of the treatment on the body weight gain for 24 hr post administration (N-27, 2 . 17.86; p<0.0013}. Mann-Whltney U-Test showed that body weight gain in KA group (mean: -42.2 g, S.D.-4.1 g) and LIN group (mean: -5.7 g; S.D.-5.1 g) was significantly different (P <0.05 ) from control group (mean: 0.8 g, S.D.-4.0 g). Figure 3 shows the correlation (r--0.9414; N=27; p<0.0001) between the putrescine level in frontal cortex and body weight gain post administration for a~l the animals included in t h e experiment.
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P U T R E S C I N E L E V E L (pglg) 0
10 8
0 0
0
6
!t -50
,
-40
,
-30
,
,
-20
-10
0
10
20
BODY WEIGHT GAIN (g) o
KA
×
LIN
FIG. 3
Correlation between the cortical content of putrescine and t h e b o d y w e i g h t g a i n 24 hr p o s t - a d m l n i s t r a t i o n . Data r e p r e s e n t all t h e a n i m a l s i n c l u d e d in t h e study. R a t s treated with Kalnic acid and Lindane are represented by special symbols. Body weight gain values in g (mean; S.D.) were8 Control (0.77; 3.99), Pentylenetetrazol (4.26~ 4.46), Picrotoxinin (4.431 2.66), Kainic acid (-42.25; 4.11) and Lindane {-5.73; 5.11). r=-0.9414; p<0.0001 . Discussion
T h e c o n c e n t r a t i o n s of PA f o u n d in rat c o r t e x a r e in l i n e w i t h o t h e r reported values (20), with putrescine in the low gM range. Previous results from our laboratory (data not shown) indicate that the pattern of change of p o l y a m i n e s 24 hr a f t e r KA t r e a t m e n t is s i m i l a r in r a t b r a i n c o r t i c a l a n d hlppocampal areas. This work was aimed to evaluate the posible sensitivity of brain polyamines to neurotoxlc agents with different m e c h a n i s m s of action. For this reason we selected the cortical area b e c a u s e its wide distribution of neurotransmltters and receptors. The lack of changes of PA content after leaving the rat cortices for short times (30 man} at room temperature is consistent with previous results of Shaw et al. (25) showing that different procedures of sacrifice did not modify brain PA. This result is important from a methodological point of view since it enables the dissection of different brain structures within minutes without any artlfactual alteration of the endogenous concentration of PA. The dramatic increase of PUT levels (but not SD and SM) after KA has not been described before. KA has been shown to exert a variety of actions (5) and recent results point to a selectivity of its neurotoxic actions on cholinerglc neurons (24}. This marked effect on PUT levels (1400~ of control values) of convulsant animals is much larger than the effects induced by any of the other convulsant agents employed. The levels of PUT correlate in several brain
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structures with the degree of cell injury in experimental models of ischemla (22) and the neuronal damage produced can be reduced by treatment with calcium antagonists (23). Also, calcium antagonists have been shown to inhibit the increase of ODC activity and PA levels in cold injured rat brain (13}. The profile of change of brain PA found after KA (large increase of PUT a n d no c h a n g e of SM a n d SD} is e n t i r e l y similar to t h a t o b s e r v e d in experimental m o d e l s of c e r e b r a l i s c h e m l a (22). After reclrculatlon, an increase of ODC activity and an inhibition of SAMDC has been reported (8}. In our experimental model this mechanism has not b e e n p r o v e n , and other poslbilities -involving the conversion of SD to PUTcan not be excluded. KA induces dose-related histological lesions in different brain regions at different times post-adminlstration (2,18). The dose and time chosen in the present study have been shown to be effective in yieldlng extensive hippocampal lesions. Furthermore, an increase of PA 24 hr after the lesion has also been observed using the post-ischamic model (20) and in partial hemitransection studies (7). Then, it is likely that the increase of PUT levels observed 24 hr after KA treatment mediates in part the neurotoxicity (cell death) of this agent. It would be interesting in future studies to relate the intensity of brain damage induced by KA with the PUT content in brain. The present results, together with existing literature on this topic, suggest that an increase of putrescine occurs in a variety of injuries to brain tissue. Whether this is a common cause of the injury -perhaps enhancing the release of excitatory aminoacids (3)- or a consequence, in response to cell d a m a g e is n o t clear, a l t h o u g h t h e C a 2 + - m o b i l i z l n g e f f e c t s of P U T a n d t h e partial blockage of damage by Ca2+-antagonists would suggest the former. O u r f i n d i n g t h a t c o n v u l s i o n s p r o d u c e d by c e r t a i n n e u r o t o x l c agents increase putrescine levels agrees with the results of Koenig et al. (16). However, these authors found that both PUT and SD increased in the brain of rats after convulsions induced by electroshock or methionine sulfoximide (MSO), whereas only PUT was increased in KA and LIN-treated rats. Moreover, the extent of the changes observed is very differentt a 20-50 % increase in PUT was produced by electroshock and MSO whereas a 1400% and a 400% have been found after KA and LIN treatment respectively. These differences could be explained by the long *time elapsed (24 hr post-treatment) in our experimental procedure compared with the shorter time (90 seconds for electroshock and 4 hr for MSO) used by those authors. Nevertheless, since normal PA levels were found in PTZ and PIC-treated rats, the whole profile of changes suggests that epileptic seizures "per se" do not induce PA increases, at least 24 hr later. Thus, it is likely that the effects of KA and LIN are due to specific actions exerted by these toxins rather than to a general effect of convulsions. The neurotoxic action of lindane is assumed to be due to interaction with the GABA A receptor complex, probably through the elchannel (26}. Nevertheless, in view of the differential effects induced by PTZ, PIC and LIN, supposedly acting at the same receptor site, LIN may be also acting through another mechanism. LIN increases free I n t r a c e l l u l a r Ca 2+ "in v i t r o " in synaptosomes (4) and in neurohybridoma cells (13). Thus, it may be possible that PUT mediates such increases of free Ca 2÷ induced by LIN. In summary, we have found increased PUT levels after KA and LIN treatments that correlate very strongly with a sign of overall toxicity (body weight loss) (see Fig 3). These PUT increases are not seen after other effective convulsant treatments and suggest that they are related to specific actions of KA and LIN.
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Taking also previous literature into account , the present data indicate that large, non-physiologlcal increases of brain putrescine are involved in the neurotoxic actions of these compounds.
Acknowladoements This work has been supported by a Research Grant from the Panda de Investigaciones Sanitarias (88/1560). The skillful technical assistance of C. Clerie8 is acknowledged. References I. L.F. AGNATI, K. FUXl, M. ZOLI, G. PICCININI
I. ZINI, P. DAVALLI, A. CORTI, L. CRLZA, G. TOFFANO, and M. GOLDSTKIN, Acts Physiol. Stand. 124 499-506
(1985). 2. ¥. BEN-ARI, E. TREMBLA¥, D. RICHE, G. GHILINI and R. NAQUET, Neuroscience 1361-1391 (1981). 3. S.C. BANDY and C.H. WALKER. Brain Res. 371 96-100 (1986). 4. S.C. BANDY and L. HALSALL. Naurotoxlcology ~ 645-652 (1988). 5. J.T. COYLE, J. Neurochem. 41 1-11 (1983). 6. M.A. DESIDERIO, P. DAVALLI and A. PERIN J. of Chromat. 419 285-290 (1987). 7. M.A. DEBIDERIO ,I. ZINI, P. DAVALLI, M. $OLI, A. CORTI, K. FUXE and L.F. AGNATI J. Neurochem. 51 25-31 (1988). 8. G.A. DIENEL and N.F. CRUZ. J. Neurochem. 42 1053-1061 (1984). 9. S. GENEDANI, G. PICCININI and A. BERTOLINI. Life Sci. 34 2407-2412 (1984). i0. S. GENEDANI, S. BERNARDI, S. TAGLIAVINI, A. BOTTICELLI and A. BBRTOLINI. Pharmacol. Toxicol. 61 224-227 (1987). 11. G.M. GILAD and V.H. GILAD. Brain Research 273 191-194 (1983}. 12. Z. IQBAL and H. KOENIG. Blather1. Biophy8. Re8. Commun. 133 563-573 (1985). 13. R.M. JOY and V.W. BURNS. Neurotoxicology 9 637-644 (1988). 14. H. KOENIG, A.D. GOLDSTONE and CH.Y. LU. Prec. Natl. Acad. Sci. USA, ~ 72107214 (1983). 15. H.' KONNIG, A. GOLDSTONE and Ch.Y. LU. Nature 305 530-534 (1983). 16. H. KOENIG, and A. IQBAL. Program and Abstracts, American Neurological Association, 18 155 41985}. 17. H. KOENIG, A.D. GOLDSTONE, and ch.Y. LU. J. Neurochem. 52 101-109 (1989). 18. E.W. LOTHMAN and R.C. COLLINS. Brain Research 218 299-318 41981). 19. A.E.I. PAJUNEN, O.A. HIETALA, E.-L. VIRRANSALO and R.S. PIHA. J. Neurochem. 30 281-283 (1978). 20. W. PASCHEN, R. SCHMIDT-KASTNER, B. DJURICIC, C. MEESE, F. LINN and K.-A. HOSSMAN. J. Neurochem. 49 35-37 (1987). 21. W. PASCHEN, J. HALLMAYER and G. MIES. Neurocham. Pathol. ~ 143-156 (1987). 22. W. PASCHEN, R. SCHMIDT-KASTNER, J. HALLMAYER, and B. DJURICIC. Neurochem. Pathol. 2 1-20 (1988). 23. A. SAUTER , M. RUDIN and K.-H. WIEDERHOLD. Neurochem~ Pathol. ~ 211236 (1988). 24. R. SCHLIEBS, M. ZIVIN, J. STEINBACH and T. RATHE. J. Neurochem. 53 212-218 (1989). 25. G.G. SHAW and A.J. PATERMAN. J. Neurochem. 20 1225-1230 (1973). 26. C. SU~OL, J.M. TUSELL, E. GELPI, and E. RODRIGUEZ-FARRE. Toxicol. Appl. Pharmacol. 100 1-8 41989}. 27. J.J. TROUT, H. KOENIG, A.D. GOLDSTONE, and CH. Y. LU. Lab. Invest. 55 622631 (1986).
Pergamon Press
DIFFERENTIAL RESPONSE OF RAT BRAIN POLYAMINES TO CONVULSANT AGENTS
E. Mart£nez, N. de Vera* and F.Artigas Department of Neurochemietry and *Department of Pharmacology & Toxicology. Jordl Girona 18-26, E-08034 Barcelona, Spain. (Received in final form November I, 1990) Summarv The polyamlnee putrescine (PUT), spermidine (SD} and spermine (SM} h a v e b e e n s t u d i e d in rat b r a i n a f t e r t r e a t m e n t w i t h s e v e r a l c o n v u l s a n t agents. K a i n l o a c i d (10 m g / k g ) , p i c r o t o x i n i n (1.5 mg/kg}, pentylenetetrazol (60 mg/kg) and lindane ( P - hexachlorocyclohexane) (60 mg/kg) were given to male Wistar rats. Twenty-four hours later, the animals were sacrificed and their brains removed. Cortical polyamines were analyzed by HPLC with fluorimetric detection of their respective daneyl derivatives, using 1,6-diamlnohexane as internal standard for the measurements. Polyamine l e v e l s are not a f f e c t e d by s h o r t p e r i o d s of t i m e (30 mln) of brain exposure to room temperature before freezing the samples, as compared to a quick procedure (less than 40 • f r o m a n i m a l d e a t h ) . K a i n i c acid i n d u c e d a 1 4 - f o l d i n c r e a s e of cortical PUT with respect to control values, leaving unchanged the other polyamlnes. Lindane also increased cortical PUT (4-fold) without affecting S M o r SD. Neither picrotoxinln nor pentylenetetrazol groups were different from controls for any of the polyamines assayed. The results are discussed in relation to the possible mechanism of action of these convulsant agents and the role of the polyamines in cell injury. Brain polyamines (PA) and their rate-limiting enzyme ornlthine decarboxylase (ODC) h a v e b e e n r e l a t e d to d i f f e r e n t k i n d of C N S i n j u r i e s (metabolic, mechanical, thermal or chemlcal), which induce ODC activity in rat brain (8). Ales, an enhanced PA synthesis appears to mediate the blood brain barrier breakdown by cold injury (27). Furthermore, it has been suggested that putrescine, produced during reclrculation following iechemia, contributes to the manifestation of isohemic cell injury (20-22), although a high content of hIppocampal putrescine occurs in certain species (21) without any morphological or functional alteration. On the other hand, some authors have'suggested that increases of PA may be mediating the mechanisms of neuronal repair after axonal injury (11) or partial hemitransection (1). C h a n g e s in b r a i n P A or O D C a c t i v i t y are also p r o d u c e d soon a f t e r convulsions(16,19). The increased PA synthesis following seizures has been related to changes in Ca 2+ fluxes at the cellular level (14), since PA increase the cytoeollc C a 2+ c o n c e n t r a t i o n (10,15,16) and release excitatory aminoacide(3). The present work was aimed at studying the effects of several oonvulsant agents on brain PA. Picrotoxinin (PIC), pentylenetetrazol (PTZ) and kainic acid (KA) are well established convuleant substances. Both PIC and PTZ are GABA A 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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receptor antagonists. Kainlc acid, a structural analog of glutamic acid, has complex a c t i o n s , b e i n g a b l e to b i n d to s p e c i f i c r e c e p t o r s (KA r e c e p t o r s ) localized at both presynaptic and postsynaptlc sites (5), and facilitating the release of glutamate from presynaptic terminals. The insecticide Y-hexachlorocyclohexane (lindane, LIN) is also a strong convulsant agent, probably a c t i n g at the p i c r o t o x i n i n b i n d i n g site associated with the Cl- ionophore of the GABA A receptor (26). Material and Me~hods Animals. Forty eight adult male albino Wistar rats (Interfauna, Sant Feliu de C o d i n e @ , Spain) w e i g h i n g 2 0 0 - 2 5 0 g have b e e n used. R a t s w e r e h o u s e d 3-5 animals per cage, under 12 hr llght-dark cycle with free access to standard chow pellets and water before and during the experiments. Temperature of the room was maintained at 21 ± 2 °C. Rats were received from the supplier and housed for one week before experiments began. All rats were randomly assigned to the different treatment groups. COmpounds. PTZ, PIC and KA obtained from commercial sources were dissolved in saline (NaCl, 0.9 %) before i.p. administration. LIN was dissolved in olive oil and administered "per os". Standard compounds putrescine (PUT), spermidine (SD) and spermine (SM) (hydrochlorides}, 1,6-diaminohexane (1,6-DAH) (free base) and dansylated putrescine (PUT-2DNS) were obtained from Sigma (St. Louis, Mo, USA). Dansyl chloride was also from Sigma and solvents -HPLC gradewere purchased from different conm~ercial sources. HPLC method. A modification of the HPLC method of Desiderio et al. (6) was used. Polyamines were separated on a 15.0 cm long, 4.6 mm i.d., stainless steel column packed with 5 ~m-size reverse-phase ODSl. (Tracer, Barcelona, Spain). The liquid chromatographic system consisted of two Waters solvent delivery pumps (model 590), a U6K injector and an automated gradient controller, also from waters. Column eluents were monitored with a fluorescence spectrophotometer (Hitachi-PE 650 S) equipped with a 20-gl quartz flow cell. F l u o r e s c e n c e c o n d i t i o n s -350 nm and 420 nm for e x c i t a t i o n a n d e m i s s i o n respectively- were adjusted experimentally with a standard solution of PUT-2DNS. The elution was performed with a gradient consisting of solvent A: 1.2 mM Na2HPO 4 and 12 mM NaCl solution (pH: 7.9 - 8.1). Solvent B: Methanol. Initial conditions (40% A, 60% B) were maintained for 2 min and then a 18 min linear gradient from 60% to 90% methanol was run. Final conditions were maintained for 7 min. A short (3 mln) reverse program was run to return to initial conditions. The flow rate was adjusted to 1.2 ml/min. 1,6-DAH was used as internal standard for quantitative purposes. Standard PUT-2DNS was the reference compound to determine the yield of derivatization of PA. Sample preparatioD. Rats were sacrificed by decapitation and brains rapidly removed from the skull (within 40 s). The dissection of frontal cortex was c a r r i e d o u t o n a c o l d p l a t e at 0 °C. S a m p l e s w e r e w e i g h e d and s t o r e d in Eppendorf tubes at -80 °C until analysis. Each sample was homogenized ultrasonically for 10-15 s in 10 vols. of cold perchloric acid (0.4 M) containing 1,6-DAH. After centrifugatlon (10000 x g, 15 min) a l i q u o t s of s u p e r n a t a n t s (400 pl) w e r e d e r i v a t i z e d a c c o r d i n g to Desiderio et al. (6). Briefly, 300 pl Na2CO 3 42.5 M) and 500 gl dansylchloride (5 mg/ml in acetone) were added and samples were kept for 1 hr at 50 °C. The s a m p l e s w e r e t h e n e x t r a c t e d w i t h b e n z e n e (i ml). T h e b e n z e n e p h a s e was evaporated under helium flow and the dry residue was redissolved in methanol
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before HPLC injection. Disposable plastic material was used throughout except for the benzene extraction where i0 - ml glass stoppered tubes were used. -~olvamine levels in frozen vs. ambient temperature samples. Ten rats were sacrificed and frontal cortices were divided in two parts, one immediately frozen in dry ice, the other left for 30 min at room temperature (22 °C). Samples were processed as described before. ExPerimental Daradiam. Five groups of animals received the following treatments PTZ (60 mg/kg), PIC (1.5 mg/kg}, KA (i0 mg/kg) and LIN (60 mg/kg}. The control g r o u p (N-7) w a s a d m i n i s t e r e d i.p. w i t h saline. D o s e s w e r e a d j u s t e d in a previous pilot experiment to obtain a maximum of convulsant rats with a minimum of d e a t h s . C o n v u l s a n t a n i m a l s w e r e kept for 24 hr at s t a n d a r d h o u s i n g c o n d i t i o n s a n d s a c r i f i c e d . The w e i g h t of the a n i m a l s w a s r e c o r d e d b e f o r e administration and Immedlately before sacrifice. Statistical analyses. The effect of temperature on polyamine brain levels was assessed by a paired Student t-Test. The influence of convulsant treatment on brain polyamines was evaluated by non-parametric one-way analysis of variance (Kruskal-Wallis Test} and the post-hoc differences between groups by the non parametric Mann Whitney U-Test. The numerical features of the experimental groups -few animals and different number of animals in each group- recommended the use of non-parametric tests. Internal correlations between different variables were analyzed by the Pearson correlation Test. Tests were carried out with a SPSS (Statistical Package for Social Sciences) program for personal computer. Results HPLC method for the determination of Dolvamines. The chromatographic conditions used enabled the complete separation of PUT, SD, SM and the internal standard 1,6-DAH in a run time of 22 minutes. Retention times for these compounds were, PUTs 11.5 min; 1,6-DAH: 13.1 min; SDz 16.8 min and SM: 20.4 min. Cadaverlne - r e t e n t i o n t i m e of 12.0 m i n - can a l s o be d e t e r m i n e d in t h e s a m e run. The orlginal method from Desidsrlo et al. (61 was modified by increasing the saline strength of the chromatographic eluent. This improved the reproducibility and eliminated some interferences observed when running brain samples. The dansyl derlvatlzatlon and benzene extraction procedures showed the followlng characterlstlcss a) The derlvatlzatlon yield, calculated for PUT vs. standard PUT-2DNS, was 43%, b) Recovery of the benzene extraction calculated for the internal standard in a series of 17 brain samples, was established in 103.7% ± 17.8 (mean ± S.D.), c) Total reproducibility of the method -extraction p l u s d e r l v a t l z a t l o n - for the i n t e r n a l s t a n d a r d was 17.2% ( c o e f f i c i e n t of variation} (N=I7). Using the above procedure, the detection limits (signal/noise ratio of 2.5) for these compounds were, PUTs 55 pg; SDs 70 pg and SMs75 pg of original free bases. The calibration plots of the polyamines/internal standard peak height ratio vs. the polyamines/Internal standard concentration ratio showed a linear response in the range (1s1-lsS0). Slopes of these plots for the three p o l y a m i n e s w e r e v e r y similar, PUTs 0.983; SD= 0 . 8 5 9 a n d SM= 0 . 8 6 5 . T h e regression coefficients for adjusted plots were always higher than r=0.998. Ambient temperature vs. freezinq. Figure 1 shows the level of polyamines in e q u i v a l e n t a l i q u o t s of f r o n t a l c o r t e x a f t e r b o t h h a n d l i n g p r o c e d u r e s . No significant differences in the content of polyamines were found after a fast freezing upon removal of brain from the skull or exposure for 30 min at room temperature. The concentration of cadaverine in these samples was below 40 ng/g in both cases.
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% RELATIVE FROZEN
1
125
100
75
50
25
0
PUT M
8D FROZEN
8M AMBIENT TEMP.
FIG.1 I n f l u e n c e of p o s t - m o r t e m c o n d i t i o n s on t h e c o n t e n t of polyamlnes in rat brain tissue. No difference was observed in the concentration of polyamlnes after a fast freezing of tissue or after leaving it 30 minutes at room temperature. Bars represent Percentage relative to frozen frontal cortex (mean + S.D.) (N=10}. The content of polyamlnes in frozen tissue wae~ (in pg/g, mean; S.D.}, PUT (0.491 0.17}, SD (40.8; 4.4) and SM (54.0; 23.4). P a i r e d S t u d e n t t - T e s t (N.S.). Effect of convulsant aoents. Only those animals showing severe tonlc-clonlc seizures during two hours post-adminlstratlon were included in the experiment. The recorded convulsant activity was as follows= PTZ group (N=7); 7 animals showed tonlc-clonic seizures starting in a time span of 5 to 60 min. One of them died. PIC group (N=7}; 5 animals exhibited tonlc-clonlc convulsions in a span of 15 to 30 min. Two animals did not show seizure activity. KA group (N=10)1 7 animals exhibited wet dog shakes and continuous convulsent activity between 1.5 and 2 hours post-admlnlstration. Three of them died within 24 hours. Three animals did not show abnormal neurobehavloural signs. LIN group (N=7); 5 animals s h o w e d tonic-clonlc seizures in the Period between 10 and 24 mln. One animal died after administration. Another rat did not show convulsant activity. Figure 2 shows the concentration of polyamlnes in the frontal cortex of rats 24 hr after convulsions induced by different neurotoxlc agents. Kruskalwallls analysis of variance indicated a strong influence of the treatment on
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PUT (N=27, 2-18.64; p<0.009). Mann-Whitney U-Test indicated that LIN group (p<0.0074) and KA group (p< 0.0082) had a content of PUT significantly higher than the control group (KA= 1400% of control values and LIN: 400%). The mean concentrations of SD and S M w e r e not influenced by the treatment, although some animals (ca. 25%) of the PIC, PTZ and LIN groups exhibited low values of these t w o p o l y a m l n e s , i n c r e a s i n g t h e d e v i a t i o n of t h e r e s p e c t i v e g r o u p s . This result could be due to a particular sensitivity of some of the animals to the
1800
% OF CONTROLS
1600 1400 1200
6oo .
1000 800
400
2O0 0
CONT ~1
PTZ PUT
PIC ~
8D
KA ~
LIN 8M
FIG.2
Effect of convulsant agents on the content of polyamlnea in rat frontal cortex, 24 hr post-adminlstration. Bars (mean + S.D.) represent percentage of control group (N=7). Pentylenetetrazol (N-6), Picrotoxinln (N-5}, Kainlc acid (N-4) and Lindane (N-5) groups are shown. Concentration of polyamines (gg/g) in control an!male were (mean; S.D.): PUT (0.65; 0.40}, SD (40.26; 15.94) and SM (58.07; 1 9 . 9 7 ) . (*) I)<0.009 from controls (Mann-Whitney U-Test). convulsant agents used. SD and 8M content showed a Positive correlation in all the animals studied (r= 0.9458; N=27; p < 0.0001) that indicates a parallel metabolic regulation after such treatments. However, putrescine did not correlate with any of the other two Polyamine8. Kruakal-Wallls analysis of variance indicated a strong influence of the treatment on the body weight gain for 24 hr post administration (N-27, 2 . 17.86; p<0.0013}. Mann-Whltney U-Test showed that body weight gain in KA group (mean: -42.2 g, S.D.-4.1 g) and LIN group (mean: -5.7 g; S.D.-5.1 g) was significantly different (P <0.05 ) from control group (mean: 0.8 g, S.D.-4.0 g). Figure 3 shows the correlation (r--0.9414; N=27; p<0.0001) between the putrescine level in frontal cortex and body weight gain post administration for a~l the animals included in t h e experiment.
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P U T R E S C I N E L E V E L (pglg) 0
10 8
0 0
0
6
!t -50
,
-40
,
-30
,
,
-20
-10
0
10
20
BODY WEIGHT GAIN (g) o
KA
×
LIN
FIG. 3
Correlation between the cortical content of putrescine and t h e b o d y w e i g h t g a i n 24 hr p o s t - a d m l n i s t r a t i o n . Data r e p r e s e n t all t h e a n i m a l s i n c l u d e d in t h e study. R a t s treated with Kalnic acid and Lindane are represented by special symbols. Body weight gain values in g (mean; S.D.) were8 Control (0.77; 3.99), Pentylenetetrazol (4.26~ 4.46), Picrotoxinin (4.431 2.66), Kainic acid (-42.25; 4.11) and Lindane {-5.73; 5.11). r=-0.9414; p<0.0001 . Discussion
T h e c o n c e n t r a t i o n s of PA f o u n d in rat c o r t e x a r e in l i n e w i t h o t h e r reported values (20), with putrescine in the low gM range. Previous results from our laboratory (data not shown) indicate that the pattern of change of p o l y a m i n e s 24 hr a f t e r KA t r e a t m e n t is s i m i l a r in r a t b r a i n c o r t i c a l a n d hlppocampal areas. This work was aimed to evaluate the posible sensitivity of brain polyamines to neurotoxlc agents with different m e c h a n i s m s of action. For this reason we selected the cortical area b e c a u s e its wide distribution of neurotransmltters and receptors. The lack of changes of PA content after leaving the rat cortices for short times (30 man} at room temperature is consistent with previous results of Shaw et al. (25) showing that different procedures of sacrifice did not modify brain PA. This result is important from a methodological point of view since it enables the dissection of different brain structures within minutes without any artlfactual alteration of the endogenous concentration of PA. The dramatic increase of PUT levels (but not SD and SM) after KA has not been described before. KA has been shown to exert a variety of actions (5) and recent results point to a selectivity of its neurotoxic actions on cholinerglc neurons (24}. This marked effect on PUT levels (1400~ of control values) of convulsant animals is much larger than the effects induced by any of the other convulsant agents employed. The levels of PUT correlate in several brain
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structures with the degree of cell injury in experimental models of ischemla (22) and the neuronal damage produced can be reduced by treatment with calcium antagonists (23). Also, calcium antagonists have been shown to inhibit the increase of ODC activity and PA levels in cold injured rat brain (13}. The profile of change of brain PA found after KA (large increase of PUT a n d no c h a n g e of SM a n d SD} is e n t i r e l y similar to t h a t o b s e r v e d in experimental m o d e l s of c e r e b r a l i s c h e m l a (22). After reclrculatlon, an increase of ODC activity and an inhibition of SAMDC has been reported (8}. In our experimental model this mechanism has not b e e n p r o v e n , and other poslbilities -involving the conversion of SD to PUTcan not be excluded. KA induces dose-related histological lesions in different brain regions at different times post-adminlstration (2,18). The dose and time chosen in the present study have been shown to be effective in yieldlng extensive hippocampal lesions. Furthermore, an increase of PA 24 hr after the lesion has also been observed using the post-ischamic model (20) and in partial hemitransection studies (7). Then, it is likely that the increase of PUT levels observed 24 hr after KA treatment mediates in part the neurotoxicity (cell death) of this agent. It would be interesting in future studies to relate the intensity of brain damage induced by KA with the PUT content in brain. The present results, together with existing literature on this topic, suggest that an increase of putrescine occurs in a variety of injuries to brain tissue. Whether this is a common cause of the injury -perhaps enhancing the release of excitatory aminoacids (3)- or a consequence, in response to cell d a m a g e is n o t clear, a l t h o u g h t h e C a 2 + - m o b i l i z l n g e f f e c t s of P U T a n d t h e partial blockage of damage by Ca2+-antagonists would suggest the former. O u r f i n d i n g t h a t c o n v u l s i o n s p r o d u c e d by c e r t a i n n e u r o t o x l c agents increase putrescine levels agrees with the results of Koenig et al. (16). However, these authors found that both PUT and SD increased in the brain of rats after convulsions induced by electroshock or methionine sulfoximide (MSO), whereas only PUT was increased in KA and LIN-treated rats. Moreover, the extent of the changes observed is very differentt a 20-50 % increase in PUT was produced by electroshock and MSO whereas a 1400% and a 400% have been found after KA and LIN treatment respectively. These differences could be explained by the long *time elapsed (24 hr post-treatment) in our experimental procedure compared with the shorter time (90 seconds for electroshock and 4 hr for MSO) used by those authors. Nevertheless, since normal PA levels were found in PTZ and PIC-treated rats, the whole profile of changes suggests that epileptic seizures "per se" do not induce PA increases, at least 24 hr later. Thus, it is likely that the effects of KA and LIN are due to specific actions exerted by these toxins rather than to a general effect of convulsions. The neurotoxic action of lindane is assumed to be due to interaction with the GABA A receptor complex, probably through the elchannel (26}. Nevertheless, in view of the differential effects induced by PTZ, PIC and LIN, supposedly acting at the same receptor site, LIN may be also acting through another mechanism. LIN increases free I n t r a c e l l u l a r Ca 2+ "in v i t r o " in synaptosomes (4) and in neurohybridoma cells (13). Thus, it may be possible that PUT mediates such increases of free Ca 2÷ induced by LIN. In summary, we have found increased PUT levels after KA and LIN treatments that correlate very strongly with a sign of overall toxicity (body weight loss) (see Fig 3). These PUT increases are not seen after other effective convulsant treatments and suggest that they are related to specific actions of KA and LIN.
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Taking also previous literature into account , the present data indicate that large, non-physiologlcal increases of brain putrescine are involved in the neurotoxic actions of these compounds.
Acknowladoements This work has been supported by a Research Grant from the Panda de Investigaciones Sanitarias (88/1560). The skillful technical assistance of C. Clerie8 is acknowledged. References I. L.F. AGNATI, K. FUXl, M. ZOLI, G. PICCININI
I. ZINI, P. DAVALLI, A. CORTI, L. CRLZA, G. TOFFANO, and M. GOLDSTKIN, Acts Physiol. Stand. 124 499-506
(1985). 2. ¥. BEN-ARI, E. TREMBLA¥, D. RICHE, G. GHILINI and R. NAQUET, Neuroscience 1361-1391 (1981). 3. S.C. BANDY and C.H. WALKER. Brain Res. 371 96-100 (1986). 4. S.C. BANDY and L. HALSALL. Naurotoxlcology ~ 645-652 (1988). 5. J.T. COYLE, J. Neurochem. 41 1-11 (1983). 6. M.A. DESIDERIO, P. DAVALLI and A. PERIN J. of Chromat. 419 285-290 (1987). 7. M.A. DEBIDERIO ,I. ZINI, P. DAVALLI, M. $OLI, A. CORTI, K. FUXE and L.F. AGNATI J. Neurochem. 51 25-31 (1988). 8. G.A. DIENEL and N.F. CRUZ. J. Neurochem. 42 1053-1061 (1984). 9. S. GENEDANI, G. PICCININI and A. BERTOLINI. Life Sci. 34 2407-2412 (1984). i0. S. GENEDANI, S. BERNARDI, S. TAGLIAVINI, A. BOTTICELLI and A. BBRTOLINI. Pharmacol. Toxicol. 61 224-227 (1987). 11. G.M. GILAD and V.H. GILAD. Brain Research 273 191-194 (1983}. 12. Z. IQBAL and H. KOENIG. Blather1. Biophy8. Re8. Commun. 133 563-573 (1985). 13. R.M. JOY and V.W. BURNS. Neurotoxicology 9 637-644 (1988). 14. H. KOENIG, A.D. GOLDSTONE and CH.Y. LU. Prec. Natl. Acad. Sci. USA, ~ 72107214 (1983). 15. H.' KONNIG, A. GOLDSTONE and Ch.Y. LU. Nature 305 530-534 (1983). 16. H. KOENIG, and A. IQBAL. Program and Abstracts, American Neurological Association, 18 155 41985}. 17. H. KOENIG, A.D. GOLDSTONE, and ch.Y. LU. J. Neurochem. 52 101-109 (1989). 18. E.W. LOTHMAN and R.C. COLLINS. Brain Research 218 299-318 41981). 19. A.E.I. PAJUNEN, O.A. HIETALA, E.-L. VIRRANSALO and R.S. PIHA. J. Neurochem. 30 281-283 (1978). 20. W. PASCHEN, R. SCHMIDT-KASTNER, B. DJURICIC, C. MEESE, F. LINN and K.-A. HOSSMAN. J. Neurochem. 49 35-37 (1987). 21. W. PASCHEN, J. HALLMAYER and G. MIES. Neurocham. Pathol. ~ 143-156 (1987). 22. W. PASCHEN, R. SCHMIDT-KASTNER, J. HALLMAYER, and B. DJURICIC. Neurochem. Pathol. 2 1-20 (1988). 23. A. SAUTER , M. RUDIN and K.-H. WIEDERHOLD. Neurochem~ Pathol. ~ 211236 (1988). 24. R. SCHLIEBS, M. ZIVIN, J. STEINBACH and T. RATHE. J. Neurochem. 53 212-218 (1989). 25. G.G. SHAW and A.J. PATERMAN. J. Neurochem. 20 1225-1230 (1973). 26. C. SU~OL, J.M. TUSELL, E. GELPI, and E. RODRIGUEZ-FARRE. Toxicol. Appl. Pharmacol. 100 1-8 41989}. 27. J.J. TROUT, H. KOENIG, A.D. GOLDSTONE, and CH. Y. LU. Lab. Invest. 55 622631 (1986).