Developmental Brain Research, 51 (1990) 291-294 Elsevier
291
BRESD 60346
Histamine increases ornithine decarboxylase activity in primary cultures of cerebellar granule cells Jose Rodriguez t, Anthony Hunt 2, Isaac Blanco 1 and Ambrish J.
Patel 2
1Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad Aut6noma de Barcelona, Barcelona (Spain) and 2MRC Collaborative Centre and Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, Mill Hill, London (U.K.)
(Accepted 7 November 1989) Key words: Histamine; Ornithine decarboxylase; Cerebellar granule neuron; Development; Neuronal culture
The effect of histamine on the activity of ornithine decarboxylase (ODC) of cerebellar granule neurons was studied using primary cultures grown both in serum-containing medium and in chemically defined medium. In comparison with granule neurons grown in chemically defined medium, the activity of ODC was about twice as great in the neurons grown in serum-containing medium. Treatment of cultured cerebellar neurons with histamine caused a dose-dependent increase in ODC activity. The maximum elevation was observed at 500 nM of histamine, when the increase in ODC activity was about 50% and 120% over controls in granule cells grown in serum-containing medium and in chemically defined medium, respectively. Histamine had no significant effect on the activity of lactate dehydrogenase in these cultures. The present findings provided direct evidence for the involvement of histamine in the regulation of ODC-related non-mitotic growth of granule neurons in the cerebellum. Ornithine decarboxylase ( O D C ) is an important enzyme in the biosynthesis of polyamines. Both O D C and p o l y a m i n e s have been shown to be associated with cellular growth and differentiation in the rat brain (for review see ref. 21). In in vivo and in vitro brain p r e p a r a t i o n s , t r e a t m e n t with several substances increases the activity of O D C 5'7'9'12'17'23. O n e of these c o m p o u n d s is histamine, which acts as a n e u r o t r a n s m i t t e r in the m a m m a l i a n nervous system 19. W h e n administered intracerebroventricularly, this amine was found to elevate O D C activity, suggesting that histamine may also play a trophic role in the brain during d e v e l o p m e n t 15'~7. A t the cellular level, we have recently shown that histamine elicits an increase in O D C activity in cultured astrocytes, derived from either the forebrain or the cerebellum ~8. The effect is associated with an elevation in the incorp o r a t i o n of [3H]thymidine into D N A in both types of astrocyte cultures, indicating that the increase in O D C activity m e d i a t e d by histamine is mainly due to the proliferation of astroglial cells. The c e r e b e l l u m is one of the brain areas in which the relationship between O D C and polyamines with develo p m e n t is relatively clear. In this late maturing brain region, the p e a k in O D C activity coincides with the postnatal d e v e l o p m e n t of granule cells 1'2. M o r e o v e r , the postnatal administration of a - d i f l u o r o m e t h y l o r n i t h i n e ,
an irreversible inhibitor of O D C 13, c a u s e s growth retardation and a reduction in the n u m b e r of granule cells in the rat cerebellum 4. In the p r e s e n t study, to obtain m o r e precise information we have e x a m i n e d the direct effects of histamine on the activity of O D C in cerebellar granule cells cultured both in serum-containing m e d i u m and in chemically defined m e d i u m . Cultures enriched in neurons ( m o r e than 90% granule cells) were o b t a i n e d by plating dissociated cells from the cerebellum of 8-day-old rats, at a density of 265 × 10 3 cells p e r cm 2, in plastic dishes (60-mm d i a m e t e r ) precoated with poly-L-lysine (10/~g/ml; mol. wt. >300,000). The neurons were grown either in a serum-containing or in a chemically defined m e d i u m as described previously 11"14. In the e x p e r i m e n t a l groups, the cultures grown for 4 days in vitro ( D I V ) were t r e a t e d for a further 4 days with different concentrations of histamine ( b e t w e e n 1 nM and 250/~M). A t 8 DIV, the cells were washed twice with p r e w a r m e d p h o s p h a t e - b u f f e r e d saline and once with saline at 37 °C. The cells were then scraped into 10 m M imidazole buffer, p H 7.4 at 0 °C, with a plastic scraper and h o m o g e n i z e d using an all-glass homogenizer. W h o l e h o m o g e n a t e was used for the estimation of O D C (EC 4.1.1.17) and lactate d e h y d r o g e n a s e ( L D H ; E C 1.1.1.27) activities and protein concentration as described previously 16. The data are expressed as enzyme activity per
Correspondence: A.J. Patel, Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
292
mg protein. The results were analyzed by an analysis of variance (ANOVA) followed by Duncan's multiple comparison. The results are given as mean values + S.E.M. The significance of the differences between the mean values of different groups was calculated in the appropriate way. The morphological appearance of cerebellar granule cells grown either in serum-containing medium (supplemented with 20 mM K +) or in chemically defined medium, have already been described in detail tl. The present study confirms these observations by showing that in serum-containing medium, neurons migrate along the fibers to form clumps during development, whereas in chemically defined medium, cell migration and clump formation is more limited (Fig. 1). In comparison with controls, treatment with histamine for 4 days had no visible effect on the morphological appearance of cerebellar granule neurons cultured either in serum-containing or in chemically defined medium (Fig. 1).
In normal 8 DIV cerebellar granule neurons, when they were cultured in serum-containing medium or in chemically defined medium, the specific activity of ODC was about 210 or 75 pmol.h-l-mg -1 protein, respectively (Table I). A possible reason for the lower ODC activity in cells grown in chemically defined medium may be the limited morphological maturation of cerebellar granule neurons normally observed under this culture condition 1~ (Fig. 1). Also, the specific activity of O D C in primary cultures of cerebellar granule cells was much lower (about one tenth) than the activity previously observed in cultured cerebellar astrocytes ~. A plausible explanation of this marked difference may be that the enzyme activity in astrocytes is associated with both proliferation and development of the cells ts, while, as neurons do not divide, the activity of O D C in nerve cells reflects only the non-mitotic growth. A number of previous studies have already described such an association between the activity of ODC and the non-mitotic growth, measured in
Fig. 1. Phase contrast photomicrographs of primary cultures of cerebellar granule neurons grown either in medium containing fetal calf serum (a,b) or in chemically defined medium (c,d). The 8 DIV cerebellar granule cells were controls (a,c) or treated for the last 4 days with 500 nM histamine (b,d). Bar = 100 ~m.
293 TABLE I Effect of histamine on the activity of ornithine decarboxylase (ODC) and lactate dehydrogenase (LDH) in cultured cerebellar granule neurons
Primary cultures of granule cells derived from the cerebellum of 8-day-old rats and grown either in a chemically defined medium or in a basal Eagle's medium containing 10% fetal calf serum. At 4 DIV, cells were treated for a further 4 days with the indicated concentrations of histamine. At 8 DIV, cells were washed, harvested and homogenized in 10 mM imidazole buffer, pH 7.4. Whole homogenate was used for the estimation of ODC and LDH activities. The data were subjected to analysis of variance followed by Duncan's multiple comparison. The values are means _+ S.E.M. of 3 experiments. Histamine concentration
0 (Control) I nM 10nM 500nM 10/~M 250/~M
Chemically defined medium
Serum-containing medium
ODC (pmol.h -r mg -1protein)
ODC (pmol.h-r mg 1pro_ rein)
LDH (pmol.h 1. mg -1prorein)
74+2.2 48+2.5 120+16.1" 47+4.5 163+27.3"* 42_+1.8 153_+12.1"*49_+1.4 -
LDH (pmol.h 1 mg -1 protein)
209+9.4 83_+7.0 256_+11.0" 86_+4.3 315_+14.3"* 77_+12.3 299_+17.3"* 74_+1.7 280_+24.3* 71_+4.6
Significant differences between mean values of the control and experimental groups are indicated: *P < 0.05 and **P < 0.01. terms of axonal regeneration or the survival and differentiation of nerve cells 3'4'6"8'2°. The addition of different amounts of histamine to the culture medium significantly increased the specific activity of O D C in a dose-dependent manner (Table I). The dose of histamine that produced the largest effect was 500 nM, and the increase in concentration up to 250 p M of histamine failed to elevate O D C activity further (Table I). In comparison with controls, the maximal increase in O D C activity was about 50% in granule cells cultured in serum-containing medium, whereas less-differentiated neurons grown in chemically defined medium 11 (Fig. 1) showed an appreciably larger increase (about 120%) in enzyme activity (Table I). These results would suggest that the effect of histamine on O D C activity may depend on the maturation state of the granule neurons. The enhancement in the activity of O D C appeared to be relatively specific, as the treatment with histamine had little effect on the activity of L D H , in both types of cultured neurons (Table I). Recently we have reported that treatment with hista1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions, J. Comp. Neurol., 128 (1966) 431-474.
mine increases the activity of O D C in cultured cerebellar astrocytes 18, and it is possible that these contaminating glial cells present in the neuronal cultures may be responsible for the effect observed in the present study. However, this is highly unlikely due to the following reasons: (1) in the neuronal cell cultures grown either in serum-containing medium supplemented with cytosine arabinoside (10 pM) or in chemically defined medium, the proportion of contaminating astroglial cells is very small, less than 3% of the total cells (see ref. 11); (2) the observed effect of histamine appears to depend on the maturation state of cultured granule neurons (Fig. 1 and Table I); and (3) the neuronal cultures derived from the septum-diagonal band region containing cholinergic and G A B A e r g i c neurons, when grown in a chemically defined medium also contained a similar proportion (less than 3%) of contaminating astrocytes 1°. When these subcortical neuronal cultures were treated with histamine, no significant alteration' was observed in the activity of O D C (unpublished observations). The latter findings not only preclude the possibility that the present effects of histamine are related to small numbers of contaminating astrocytes in the culture, but also emphasize that the increase in the activity of O D C in nerve cells after treatment with histamine must be specific to granule cells of the cerebellum. The histamine effects described in the present study may be mediated by the H1 type of histamine receptor, as the presence of this type of receptor has been described in cultured cerebeUar granule cells 22. On the other hand, earlier studies carried out in situ have indicated that the effect of histamine on O D C activity is mediated by H 2 receptors in the brain ~7. It would, therefore, be interesting to derive further information on the effects of histamine receptor agonists and antagonists on the O D C activity in the cultured cerebellar granule neurons. In conclusion, the present findings may be the first showing an increase mediated by histamine in O D C activity in non-proliferating cells, providing direct evidence for the involvement of this neurotransmitter amine in the O D C - d e p e n d e n t regulation of certain processes related to maturation of granule neurons in the cerebellum. J.R. is a recipient of a P.F.P.I. Fellowship and gratefully acknowledges the award of the British Council grant-in-aid. This study was supported, in part, by Grant DGICYT PB86-0355 and by Beechams Pharmaceuticals. 2 Anderson, T.R. and Schanberg, S.M., Ornithine decarboxylase activity in developing rat brain, J. Neurochem., 19 (1972) 1471-1481. 3 Ando, M., Miwa, M., Kato, K. and Nagata, Y., Effects of denervation and axotomy on nervous system-specific protein, ornithine decarboxylase, and other enzyme activities in the
294
4
5
6
7
8
9
10
11
12
13
superior cervical sympathetic ganglion of the rat, J. Neurochem., 42 (1984) 94-100. Bartolome, J.V., Schweitzer, L., Slotkin, T.A. and Nadler, J.V., Impaired development of cerebellar cortex in rats treated postnatally with a-difluoromethylornithine, Neuroscience, 15 (1985) 203-213. Cousin, M.E., Lando, D. and Moguilewsky, M., Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats, J. Neurochem., 38 (1982) 1296-1304. Davalli, P., Zini, I., Desiderio, M.A. and Zoli, M., Studies on the involvement of polyamines and gangliosides in the regenerative features of the dopamine nigrostriatal pathway after mechanical and neurotoxic lesions. In A. Parin, G. Scalabrino, A. Sessa and M.E. Ferioli (Eds.), Perspectives in Potyamine Research, Wichtig, Milano, 1988, pp. 85-88. Dienel, G.A. and Cruz, N.E, Induction of brain ornithine decarboxylase during recovery from metabolic, mechanical, thermal, or chemical injury, J. Neurochem., 42 (1984) 10531061. Gilad, G.M. and Gilad, V.H., Early rapid and transient increase in ornithine decarboxylase activity within sympathetic neurons after axonal injury, Exp. Neurol., 81 (1983) 158-166. Greene, L.A. and McGuire, J,C., Induction of ornithine decarboxylase by nerve growth factor dissociated from effects on survival and neurite outgrowth, Nature (Lond.), 276 (1978) 191-194. Hayashi, M. and Patel, A.J., An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain, Dev. Brain Res., 36 (1987) 109-120. Kingsbury, A., GaUo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985) 17-25. Lewis, M.E., Lakshmanan, J., Nagaiah, K., MacDonnell, P.C. and Guroff, G., Nerve growth factor increases activity of ornithine decarboxylase in rat brain, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 1021-1023. Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P., Catalytic irreversible inhibition of mammalian
14
15
16
17
18
19
20
21
22
23
ornithine decarboxylase (E.C. 4.1.1.17) by substrate and product analogues, J. Am. Chem, Sot., 100 (1978) 2551-2553. Moran, J. and Patel, A.J., Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development, Dev. Brain Res., 46 (1989) 97-105. Morris, G., Seidler, EJ. and Slotkin, T.A., Stimulation of ornithine decarboxylase by histamine or norepinephrine in brain regions of the developing rat: evidence for biogenic amines as trophic agents in neonatal brain development, Life Sci., 32 (1983) 1565-1571. Patel, A.J., Hunt, A. and Faraji-Shadan, F., Effect of removal of glutamine and addition of dexamethasone on the activities of glutamine synthetase, ornithine decarboxylase and lactate dehydrogenase in primary cultures of forebrain and cerebellar astrocytes, Dev. Brain Res., 26 (1986) 229-238. Rodriguez, J., Toledo, A., Brandner, R., Rodriguez, R., Sabria, J. and Blanco, I., Histamine He-receptor mediated activation of neonatal rat brain ornithine decarboxylase in vivo, Biochem. Pharmaeol., 37 (1988) 551-55& Rodriguez, J., Moran, J., Blanco, I. and Patel, A.J., Effect of histamine on the development of astroglial cells in culture, Neurochem. Res., 14 (1989) 693-700. Schwartz, J.C., Gabarg, M. and Pollard, H., Histaminergic transmission in the brain. In EE. Bloom, V.B. Mountcastle and S.R. Geiger (Eds.), Handbook of Physiology, Section 1, Vol. 4, American Physiological Society, Bethesda, 1986, pp. 257-316. Schwartz, M., Kohsaka, S. and Agranoff, B.W., Ornithine decarboxylase activity in retinal explants of goldfish undergoing optic nerve regeneration, Dev. Brain Res., 1 (1981) 403-413. Slotkin, T.A. and Bartolome, 1., Role of ornithine decarboxylase and the polyamines in nervous system development: a review, Brain Res. Bull., 17 (1986) 307-320. Xu, J. and Chuang, D.-M., Serotonergic, adrenergic and histaminergic receptors coupled to phospholipase C in cultured cerebellar granule cells of rats, Biochem. Pharmacol., 36 (1987) 2353-2358. Yang, J.W., Raizada, M.K. and Fellows, R.E., Effect of insulin on cultured rat brain cells: stimulation of ornithine decarboxylase activity, J. Neurochem.. 36 (1981) 1050-1057.
291
BRESD 60346
Histamine increases ornithine decarboxylase activity in primary cultures of cerebellar granule cells Jose Rodriguez t, Anthony Hunt 2, Isaac Blanco 1 and Ambrish J.
Patel 2
1Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad Aut6noma de Barcelona, Barcelona (Spain) and 2MRC Collaborative Centre and Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, Mill Hill, London (U.K.)
(Accepted 7 November 1989) Key words: Histamine; Ornithine decarboxylase; Cerebellar granule neuron; Development; Neuronal culture
The effect of histamine on the activity of ornithine decarboxylase (ODC) of cerebellar granule neurons was studied using primary cultures grown both in serum-containing medium and in chemically defined medium. In comparison with granule neurons grown in chemically defined medium, the activity of ODC was about twice as great in the neurons grown in serum-containing medium. Treatment of cultured cerebellar neurons with histamine caused a dose-dependent increase in ODC activity. The maximum elevation was observed at 500 nM of histamine, when the increase in ODC activity was about 50% and 120% over controls in granule cells grown in serum-containing medium and in chemically defined medium, respectively. Histamine had no significant effect on the activity of lactate dehydrogenase in these cultures. The present findings provided direct evidence for the involvement of histamine in the regulation of ODC-related non-mitotic growth of granule neurons in the cerebellum. Ornithine decarboxylase ( O D C ) is an important enzyme in the biosynthesis of polyamines. Both O D C and p o l y a m i n e s have been shown to be associated with cellular growth and differentiation in the rat brain (for review see ref. 21). In in vivo and in vitro brain p r e p a r a t i o n s , t r e a t m e n t with several substances increases the activity of O D C 5'7'9'12'17'23. O n e of these c o m p o u n d s is histamine, which acts as a n e u r o t r a n s m i t t e r in the m a m m a l i a n nervous system 19. W h e n administered intracerebroventricularly, this amine was found to elevate O D C activity, suggesting that histamine may also play a trophic role in the brain during d e v e l o p m e n t 15'~7. A t the cellular level, we have recently shown that histamine elicits an increase in O D C activity in cultured astrocytes, derived from either the forebrain or the cerebellum ~8. The effect is associated with an elevation in the incorp o r a t i o n of [3H]thymidine into D N A in both types of astrocyte cultures, indicating that the increase in O D C activity m e d i a t e d by histamine is mainly due to the proliferation of astroglial cells. The c e r e b e l l u m is one of the brain areas in which the relationship between O D C and polyamines with develo p m e n t is relatively clear. In this late maturing brain region, the p e a k in O D C activity coincides with the postnatal d e v e l o p m e n t of granule cells 1'2. M o r e o v e r , the postnatal administration of a - d i f l u o r o m e t h y l o r n i t h i n e ,
an irreversible inhibitor of O D C 13, c a u s e s growth retardation and a reduction in the n u m b e r of granule cells in the rat cerebellum 4. In the p r e s e n t study, to obtain m o r e precise information we have e x a m i n e d the direct effects of histamine on the activity of O D C in cerebellar granule cells cultured both in serum-containing m e d i u m and in chemically defined m e d i u m . Cultures enriched in neurons ( m o r e than 90% granule cells) were o b t a i n e d by plating dissociated cells from the cerebellum of 8-day-old rats, at a density of 265 × 10 3 cells p e r cm 2, in plastic dishes (60-mm d i a m e t e r ) precoated with poly-L-lysine (10/~g/ml; mol. wt. >300,000). The neurons were grown either in a serum-containing or in a chemically defined m e d i u m as described previously 11"14. In the e x p e r i m e n t a l groups, the cultures grown for 4 days in vitro ( D I V ) were t r e a t e d for a further 4 days with different concentrations of histamine ( b e t w e e n 1 nM and 250/~M). A t 8 DIV, the cells were washed twice with p r e w a r m e d p h o s p h a t e - b u f f e r e d saline and once with saline at 37 °C. The cells were then scraped into 10 m M imidazole buffer, p H 7.4 at 0 °C, with a plastic scraper and h o m o g e n i z e d using an all-glass homogenizer. W h o l e h o m o g e n a t e was used for the estimation of O D C (EC 4.1.1.17) and lactate d e h y d r o g e n a s e ( L D H ; E C 1.1.1.27) activities and protein concentration as described previously 16. The data are expressed as enzyme activity per
Correspondence: A.J. Patel, Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
292
mg protein. The results were analyzed by an analysis of variance (ANOVA) followed by Duncan's multiple comparison. The results are given as mean values + S.E.M. The significance of the differences between the mean values of different groups was calculated in the appropriate way. The morphological appearance of cerebellar granule cells grown either in serum-containing medium (supplemented with 20 mM K +) or in chemically defined medium, have already been described in detail tl. The present study confirms these observations by showing that in serum-containing medium, neurons migrate along the fibers to form clumps during development, whereas in chemically defined medium, cell migration and clump formation is more limited (Fig. 1). In comparison with controls, treatment with histamine for 4 days had no visible effect on the morphological appearance of cerebellar granule neurons cultured either in serum-containing or in chemically defined medium (Fig. 1).
In normal 8 DIV cerebellar granule neurons, when they were cultured in serum-containing medium or in chemically defined medium, the specific activity of ODC was about 210 or 75 pmol.h-l-mg -1 protein, respectively (Table I). A possible reason for the lower ODC activity in cells grown in chemically defined medium may be the limited morphological maturation of cerebellar granule neurons normally observed under this culture condition 1~ (Fig. 1). Also, the specific activity of O D C in primary cultures of cerebellar granule cells was much lower (about one tenth) than the activity previously observed in cultured cerebellar astrocytes ~. A plausible explanation of this marked difference may be that the enzyme activity in astrocytes is associated with both proliferation and development of the cells ts, while, as neurons do not divide, the activity of O D C in nerve cells reflects only the non-mitotic growth. A number of previous studies have already described such an association between the activity of ODC and the non-mitotic growth, measured in
Fig. 1. Phase contrast photomicrographs of primary cultures of cerebellar granule neurons grown either in medium containing fetal calf serum (a,b) or in chemically defined medium (c,d). The 8 DIV cerebellar granule cells were controls (a,c) or treated for the last 4 days with 500 nM histamine (b,d). Bar = 100 ~m.
293 TABLE I Effect of histamine on the activity of ornithine decarboxylase (ODC) and lactate dehydrogenase (LDH) in cultured cerebellar granule neurons
Primary cultures of granule cells derived from the cerebellum of 8-day-old rats and grown either in a chemically defined medium or in a basal Eagle's medium containing 10% fetal calf serum. At 4 DIV, cells were treated for a further 4 days with the indicated concentrations of histamine. At 8 DIV, cells were washed, harvested and homogenized in 10 mM imidazole buffer, pH 7.4. Whole homogenate was used for the estimation of ODC and LDH activities. The data were subjected to analysis of variance followed by Duncan's multiple comparison. The values are means _+ S.E.M. of 3 experiments. Histamine concentration
0 (Control) I nM 10nM 500nM 10/~M 250/~M
Chemically defined medium
Serum-containing medium
ODC (pmol.h -r mg -1protein)
ODC (pmol.h-r mg 1pro_ rein)
LDH (pmol.h 1. mg -1prorein)
74+2.2 48+2.5 120+16.1" 47+4.5 163+27.3"* 42_+1.8 153_+12.1"*49_+1.4 -
LDH (pmol.h 1 mg -1 protein)
209+9.4 83_+7.0 256_+11.0" 86_+4.3 315_+14.3"* 77_+12.3 299_+17.3"* 74_+1.7 280_+24.3* 71_+4.6
Significant differences between mean values of the control and experimental groups are indicated: *P < 0.05 and **P < 0.01. terms of axonal regeneration or the survival and differentiation of nerve cells 3'4'6"8'2°. The addition of different amounts of histamine to the culture medium significantly increased the specific activity of O D C in a dose-dependent manner (Table I). The dose of histamine that produced the largest effect was 500 nM, and the increase in concentration up to 250 p M of histamine failed to elevate O D C activity further (Table I). In comparison with controls, the maximal increase in O D C activity was about 50% in granule cells cultured in serum-containing medium, whereas less-differentiated neurons grown in chemically defined medium 11 (Fig. 1) showed an appreciably larger increase (about 120%) in enzyme activity (Table I). These results would suggest that the effect of histamine on O D C activity may depend on the maturation state of the granule neurons. The enhancement in the activity of O D C appeared to be relatively specific, as the treatment with histamine had little effect on the activity of L D H , in both types of cultured neurons (Table I). Recently we have reported that treatment with hista1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions, J. Comp. Neurol., 128 (1966) 431-474.
mine increases the activity of O D C in cultured cerebellar astrocytes 18, and it is possible that these contaminating glial cells present in the neuronal cultures may be responsible for the effect observed in the present study. However, this is highly unlikely due to the following reasons: (1) in the neuronal cell cultures grown either in serum-containing medium supplemented with cytosine arabinoside (10 pM) or in chemically defined medium, the proportion of contaminating astroglial cells is very small, less than 3% of the total cells (see ref. 11); (2) the observed effect of histamine appears to depend on the maturation state of cultured granule neurons (Fig. 1 and Table I); and (3) the neuronal cultures derived from the septum-diagonal band region containing cholinergic and G A B A e r g i c neurons, when grown in a chemically defined medium also contained a similar proportion (less than 3%) of contaminating astrocytes 1°. When these subcortical neuronal cultures were treated with histamine, no significant alteration' was observed in the activity of O D C (unpublished observations). The latter findings not only preclude the possibility that the present effects of histamine are related to small numbers of contaminating astrocytes in the culture, but also emphasize that the increase in the activity of O D C in nerve cells after treatment with histamine must be specific to granule cells of the cerebellum. The histamine effects described in the present study may be mediated by the H1 type of histamine receptor, as the presence of this type of receptor has been described in cultured cerebeUar granule cells 22. On the other hand, earlier studies carried out in situ have indicated that the effect of histamine on O D C activity is mediated by H 2 receptors in the brain ~7. It would, therefore, be interesting to derive further information on the effects of histamine receptor agonists and antagonists on the O D C activity in the cultured cerebellar granule neurons. In conclusion, the present findings may be the first showing an increase mediated by histamine in O D C activity in non-proliferating cells, providing direct evidence for the involvement of this neurotransmitter amine in the O D C - d e p e n d e n t regulation of certain processes related to maturation of granule neurons in the cerebellum. J.R. is a recipient of a P.F.P.I. Fellowship and gratefully acknowledges the award of the British Council grant-in-aid. This study was supported, in part, by Grant DGICYT PB86-0355 and by Beechams Pharmaceuticals. 2 Anderson, T.R. and Schanberg, S.M., Ornithine decarboxylase activity in developing rat brain, J. Neurochem., 19 (1972) 1471-1481. 3 Ando, M., Miwa, M., Kato, K. and Nagata, Y., Effects of denervation and axotomy on nervous system-specific protein, ornithine decarboxylase, and other enzyme activities in the
294
4
5
6
7
8
9
10
11
12
13
superior cervical sympathetic ganglion of the rat, J. Neurochem., 42 (1984) 94-100. Bartolome, J.V., Schweitzer, L., Slotkin, T.A. and Nadler, J.V., Impaired development of cerebellar cortex in rats treated postnatally with a-difluoromethylornithine, Neuroscience, 15 (1985) 203-213. Cousin, M.E., Lando, D. and Moguilewsky, M., Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats, J. Neurochem., 38 (1982) 1296-1304. Davalli, P., Zini, I., Desiderio, M.A. and Zoli, M., Studies on the involvement of polyamines and gangliosides in the regenerative features of the dopamine nigrostriatal pathway after mechanical and neurotoxic lesions. In A. Parin, G. Scalabrino, A. Sessa and M.E. Ferioli (Eds.), Perspectives in Potyamine Research, Wichtig, Milano, 1988, pp. 85-88. Dienel, G.A. and Cruz, N.E, Induction of brain ornithine decarboxylase during recovery from metabolic, mechanical, thermal, or chemical injury, J. Neurochem., 42 (1984) 10531061. Gilad, G.M. and Gilad, V.H., Early rapid and transient increase in ornithine decarboxylase activity within sympathetic neurons after axonal injury, Exp. Neurol., 81 (1983) 158-166. Greene, L.A. and McGuire, J,C., Induction of ornithine decarboxylase by nerve growth factor dissociated from effects on survival and neurite outgrowth, Nature (Lond.), 276 (1978) 191-194. Hayashi, M. and Patel, A.J., An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain, Dev. Brain Res., 36 (1987) 109-120. Kingsbury, A., GaUo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985) 17-25. Lewis, M.E., Lakshmanan, J., Nagaiah, K., MacDonnell, P.C. and Guroff, G., Nerve growth factor increases activity of ornithine decarboxylase in rat brain, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 1021-1023. Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P., Catalytic irreversible inhibition of mammalian
14
15
16
17
18
19
20
21
22
23
ornithine decarboxylase (E.C. 4.1.1.17) by substrate and product analogues, J. Am. Chem, Sot., 100 (1978) 2551-2553. Moran, J. and Patel, A.J., Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development, Dev. Brain Res., 46 (1989) 97-105. Morris, G., Seidler, EJ. and Slotkin, T.A., Stimulation of ornithine decarboxylase by histamine or norepinephrine in brain regions of the developing rat: evidence for biogenic amines as trophic agents in neonatal brain development, Life Sci., 32 (1983) 1565-1571. Patel, A.J., Hunt, A. and Faraji-Shadan, F., Effect of removal of glutamine and addition of dexamethasone on the activities of glutamine synthetase, ornithine decarboxylase and lactate dehydrogenase in primary cultures of forebrain and cerebellar astrocytes, Dev. Brain Res., 26 (1986) 229-238. Rodriguez, J., Toledo, A., Brandner, R., Rodriguez, R., Sabria, J. and Blanco, I., Histamine He-receptor mediated activation of neonatal rat brain ornithine decarboxylase in vivo, Biochem. Pharmaeol., 37 (1988) 551-55& Rodriguez, J., Moran, J., Blanco, I. and Patel, A.J., Effect of histamine on the development of astroglial cells in culture, Neurochem. Res., 14 (1989) 693-700. Schwartz, J.C., Gabarg, M. and Pollard, H., Histaminergic transmission in the brain. In EE. Bloom, V.B. Mountcastle and S.R. Geiger (Eds.), Handbook of Physiology, Section 1, Vol. 4, American Physiological Society, Bethesda, 1986, pp. 257-316. Schwartz, M., Kohsaka, S. and Agranoff, B.W., Ornithine decarboxylase activity in retinal explants of goldfish undergoing optic nerve regeneration, Dev. Brain Res., 1 (1981) 403-413. Slotkin, T.A. and Bartolome, 1., Role of ornithine decarboxylase and the polyamines in nervous system development: a review, Brain Res. Bull., 17 (1986) 307-320. Xu, J. and Chuang, D.-M., Serotonergic, adrenergic and histaminergic receptors coupled to phospholipase C in cultured cerebellar granule cells of rats, Biochem. Pharmacol., 36 (1987) 2353-2358. Yang, J.W., Raizada, M.K. and Fellows, R.E., Effect of insulin on cultured rat brain cells: stimulation of ornithine decarboxylase activity, J. Neurochem.. 36 (1981) 1050-1057.
