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Effect of reserpine on sympathetic neuronal RNA
355
SHORT COMMUNICATIONS
Effect of reserpine on sympathetic neuronal RNA Reserpine administration affects sympathetic ganglion cells by causing a depletion of noradrenaline and amine storage granules 1,6. If" the drug is given in a single dose this decrease is followed by an increased synthesis of these compounds 1,s. It appears that the restoration of storage function, i.e. amine storage granules, precedes that of amine levels in the cell 8. Hillarp 5 showed that catecholamines are bound in complex with ATP, proteins and phospholipids. Philippu and Schfimann tl suggested that R N A acts in the binding of catecholamines and ATP to protein. The increased synthesis of amine storage granules (for review see ref. 3) also requires R N A since these granules contain several proteins. Studies by Mytilineo 9 showed that reserpine administration in rats resulted in increased toluidine blue stainability of sympathetic ganglion cells, suggesting an increased R N A content in these cells. In view of the mentioned findings we made a preliminary study on the effect of reserpine on sympathetic neuronal RNA. Reserpine (I0 mg/kg body weight) was injected intraperitoneally in male Sprague-Dawley rats weighing 220-230 g. The rats were killed 30 min, 4 h or 24 h after injection. Controls were injected with saline and killed at the same time intervals. Lumbar sympathetic ganglia were dissected out and fixated in Carnoy's solution. Individual ganglion cells were isolated from deparaffinized sections by micromanipulation 7, R N A was extracted with RNAse in two-cell samples and determined in pg amounts as described by Edstr6m 4. The volume of each cell was calculated prior to the extraction procedure in order to assure that R N A determinations were carried out on cells belonging to the same size population. The results are shown in Table I and reveal that the R N A content did not increase 30 rain after reserpine treatment. After 4 and 24 h the R N A content in the ganglion cells increased significantly over control ~alues. The R N A content in neurons from control rats were almost constant at all time intervals. The results reflect changes in absolute amounts of R N A per cell since the determinations were not TABLE I RNA
CONTENT IN SYMPATHETIC GANGLION CELLS FROM RESERPINIZED RATS AND CONTROLS
Determinations carried out on samples of two cells, n represents the number of determinations. N.S. not significant. Time (h) after reserpine (10 mg/kg body wt., i.p.)
RNA in pg ( ~ S.E.M.)
n
P-value
0.5 Control
363 -~ 14 358 -L 15
17 17
N.S.
4 Control
440 ~ 22 369 :~ 21
19 18
< 0.05
24 Control
457 ± 21 339 ± 11
17 23
< 0.001
Brain Research, 28 (1971) 355-356
356
SHORT COM~,~UNICATIONS
affected by changes in such p a r a m e t e r s as cell v o l u m e or cell mass. Investigation o f R N A base c o m p o s i t i o n showed no changes at any time interval alter reserpine comp a r e d to controls. The q u o t i e n t g u a n i n e ~- cytosine/adenine "- uracil was a r o u n d 1.5 in b o t h e x p e r i m e n t a l and control rats, suggesting that the newly synthetized R N A was o f r i b o s o m a l type. The results s u p p o r t earlier findings, indicating an elevated R N A c o n t e n t m s y m p a t h e t i c ganglion cells after reserpine a d m i n i s t r a t i o n . The time sequence for the R N A increase in the adrenergic p e r i k a r y a c o r r e s p o n d s to w h a t has been shown for n o r a d r e n a l i n e recovery in reserpinized rats. The first sign o f recovery was observed 12 h after reserpine injection in locations indicating b i n d i n g to storage g r a m d e s which were p r e s u m a b l y synthetized before the r e a p p e a r a n c e o f n o r a d r e n a l i n e z.t0. W h e n the n e u r o n s were isolated by m i c r o m a n i p u l a t i o n , it was evident that the p o p u l a t i o n was h e t e r o g e n o u s with respect to cell size. In spite o f the fact that the R N A d e t e r m i n a t i o n s were carried o u t on cells o f the same size, a relatively large v a r i a t i o n in cellular R N A c o n t e n t was found. This suggests that a heterogeneity exists even with respect to R N A c o n c e n t r a t i o n which might be due to different functional states between these cells. Department of Neurobiology, University of GOteborg. G6teborg (Sweden) and Department of Zoology, University of Athens, Athens (Greece)
JAN JARLSTED'I CATHERINE MYTILINEO
1 CARLSSON,A., Drugs which block the storage of 5-hydroxytryptamine and related amines. In O. ErCHLER AND A. FARAH (Eds.), Handbuch der experimentellen Pharmakologie, Springer, Berlin, 1965, p. 529. 2 DAHLSTR6M,A., The effect of reserpine and tetrabenazine on the accumulation of noradrenaline in the rat sciatic nerve after tigation, Acta physiol, scand., 69 (1967) 167-180. 3 DAHLSTR()M,m., ANDH.~GGENDAHL.J.. Axonal transport of amine storage granules in sympathetic adrenergic neurons. In E. COSTA AriD E. GIACOBINItEdsA. Biochemistry of Simple Neuronal Models, Raven, New York, 1970, in press. 4 EDSTR(SM, J.-E., Microextraction and microelectrophoresis for determination and analysis of nucleic acids in isolated cellular units. In D. M. PRESCOTr (Ed.), Methods in Cell Physiology, Academic Press, New York, 1964. pp. 417 444. 5 HILLARP, N. A., Further observations on the state of catecholamines stored in the adrenal medullary granules, Acta physioL scand.. 47 (1959) 271-281. 6 HOLZBAUER,M., AND VOGT. M., Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat, 1 (1956) 8-17. 7 JARLSXEDT,J., Functional localization in the cerebellar cortex studied by quantitative determinations of Purkinje cell RNA. I. RNA changes in rat cerebellar Purkinje cells after proprio- and exteroceptive stimulation, ,4cta physiol, scand.. 67 (1966) 243-252. 8 LUNDBORG,P., Storage function and amine levels of the adrenal medullary granules al various intervals after reserpine treatment, Experientia (Basel), 19 (1963) 479. 9 MVXILINEO, C., Histochemical alterations of sympathetic ganglia of the rat after reserpine administration, Biol. Psychiat., ] (1969) 61-72. 10 NORBERG,K. A., AND HAMBERGER,B., The sympathetic adrenergic neuron. Some characteristics revealed by histochemical studies on the intraneuronal distribution of the transmitter. Acta physiol, scand., 63, Suppl. 238 (1964). 11 PHILIPPU, A., UND SCnOMANN, H. J., Ribonuclease- und Trypsin-wirkung auf isolierte Nebennierenmark-Granula, Experientia /Basel), 19 (1963) 17. (Accepted February 10th, 1971) Brain Research, 28 (1971) 355-356
SHORT COMMUNICATIONS
Effect of reserpine on sympathetic neuronal RNA Reserpine administration affects sympathetic ganglion cells by causing a depletion of noradrenaline and amine storage granules 1,6. If" the drug is given in a single dose this decrease is followed by an increased synthesis of these compounds 1,s. It appears that the restoration of storage function, i.e. amine storage granules, precedes that of amine levels in the cell 8. Hillarp 5 showed that catecholamines are bound in complex with ATP, proteins and phospholipids. Philippu and Schfimann tl suggested that R N A acts in the binding of catecholamines and ATP to protein. The increased synthesis of amine storage granules (for review see ref. 3) also requires R N A since these granules contain several proteins. Studies by Mytilineo 9 showed that reserpine administration in rats resulted in increased toluidine blue stainability of sympathetic ganglion cells, suggesting an increased R N A content in these cells. In view of the mentioned findings we made a preliminary study on the effect of reserpine on sympathetic neuronal RNA. Reserpine (I0 mg/kg body weight) was injected intraperitoneally in male Sprague-Dawley rats weighing 220-230 g. The rats were killed 30 min, 4 h or 24 h after injection. Controls were injected with saline and killed at the same time intervals. Lumbar sympathetic ganglia were dissected out and fixated in Carnoy's solution. Individual ganglion cells were isolated from deparaffinized sections by micromanipulation 7, R N A was extracted with RNAse in two-cell samples and determined in pg amounts as described by Edstr6m 4. The volume of each cell was calculated prior to the extraction procedure in order to assure that R N A determinations were carried out on cells belonging to the same size population. The results are shown in Table I and reveal that the R N A content did not increase 30 rain after reserpine treatment. After 4 and 24 h the R N A content in the ganglion cells increased significantly over control ~alues. The R N A content in neurons from control rats were almost constant at all time intervals. The results reflect changes in absolute amounts of R N A per cell since the determinations were not TABLE I RNA
CONTENT IN SYMPATHETIC GANGLION CELLS FROM RESERPINIZED RATS AND CONTROLS
Determinations carried out on samples of two cells, n represents the number of determinations. N.S. not significant. Time (h) after reserpine (10 mg/kg body wt., i.p.)
RNA in pg ( ~ S.E.M.)
n
P-value
0.5 Control
363 -~ 14 358 -L 15
17 17
N.S.
4 Control
440 ~ 22 369 :~ 21
19 18
< 0.05
24 Control
457 ± 21 339 ± 11
17 23
< 0.001
Brain Research, 28 (1971) 355-356
356
SHORT COM~,~UNICATIONS
affected by changes in such p a r a m e t e r s as cell v o l u m e or cell mass. Investigation o f R N A base c o m p o s i t i o n showed no changes at any time interval alter reserpine comp a r e d to controls. The q u o t i e n t g u a n i n e ~- cytosine/adenine "- uracil was a r o u n d 1.5 in b o t h e x p e r i m e n t a l and control rats, suggesting that the newly synthetized R N A was o f r i b o s o m a l type. The results s u p p o r t earlier findings, indicating an elevated R N A c o n t e n t m s y m p a t h e t i c ganglion cells after reserpine a d m i n i s t r a t i o n . The time sequence for the R N A increase in the adrenergic p e r i k a r y a c o r r e s p o n d s to w h a t has been shown for n o r a d r e n a l i n e recovery in reserpinized rats. The first sign o f recovery was observed 12 h after reserpine injection in locations indicating b i n d i n g to storage g r a m d e s which were p r e s u m a b l y synthetized before the r e a p p e a r a n c e o f n o r a d r e n a l i n e z.t0. W h e n the n e u r o n s were isolated by m i c r o m a n i p u l a t i o n , it was evident that the p o p u l a t i o n was h e t e r o g e n o u s with respect to cell size. In spite o f the fact that the R N A d e t e r m i n a t i o n s were carried o u t on cells o f the same size, a relatively large v a r i a t i o n in cellular R N A c o n t e n t was found. This suggests that a heterogeneity exists even with respect to R N A c o n c e n t r a t i o n which might be due to different functional states between these cells. Department of Neurobiology, University of GOteborg. G6teborg (Sweden) and Department of Zoology, University of Athens, Athens (Greece)
JAN JARLSTED'I CATHERINE MYTILINEO
1 CARLSSON,A., Drugs which block the storage of 5-hydroxytryptamine and related amines. In O. ErCHLER AND A. FARAH (Eds.), Handbuch der experimentellen Pharmakologie, Springer, Berlin, 1965, p. 529. 2 DAHLSTR6M,A., The effect of reserpine and tetrabenazine on the accumulation of noradrenaline in the rat sciatic nerve after tigation, Acta physiol, scand., 69 (1967) 167-180. 3 DAHLSTR()M,m., ANDH.~GGENDAHL.J.. Axonal transport of amine storage granules in sympathetic adrenergic neurons. In E. COSTA AriD E. GIACOBINItEdsA. Biochemistry of Simple Neuronal Models, Raven, New York, 1970, in press. 4 EDSTR(SM, J.-E., Microextraction and microelectrophoresis for determination and analysis of nucleic acids in isolated cellular units. In D. M. PRESCOTr (Ed.), Methods in Cell Physiology, Academic Press, New York, 1964. pp. 417 444. 5 HILLARP, N. A., Further observations on the state of catecholamines stored in the adrenal medullary granules, Acta physioL scand.. 47 (1959) 271-281. 6 HOLZBAUER,M., AND VOGT. M., Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat, 1 (1956) 8-17. 7 JARLSXEDT,J., Functional localization in the cerebellar cortex studied by quantitative determinations of Purkinje cell RNA. I. RNA changes in rat cerebellar Purkinje cells after proprio- and exteroceptive stimulation, ,4cta physiol, scand.. 67 (1966) 243-252. 8 LUNDBORG,P., Storage function and amine levels of the adrenal medullary granules al various intervals after reserpine treatment, Experientia (Basel), 19 (1963) 479. 9 MVXILINEO, C., Histochemical alterations of sympathetic ganglia of the rat after reserpine administration, Biol. Psychiat., ] (1969) 61-72. 10 NORBERG,K. A., AND HAMBERGER,B., The sympathetic adrenergic neuron. Some characteristics revealed by histochemical studies on the intraneuronal distribution of the transmitter. Acta physiol, scand., 63, Suppl. 238 (1964). 11 PHILIPPU, A., UND SCnOMANN, H. J., Ribonuclease- und Trypsin-wirkung auf isolierte Nebennierenmark-Granula, Experientia /Basel), 19 (1963) 17. (Accepted February 10th, 1971) Brain Research, 28 (1971) 355-356