Electrically stimulated rapid gene expression in the brain: ornithine decarboxylase and c-fos.

A single electroconvuls1ve shock (ECS) resulted m a maior mductJon of cerebral ormthme decarboxylase (ODC) mRNA and a rapid and transient elevation of ODC enzyme act1v1ty. The proto-oncogene c-fos was also transiently induced under the same conditions. Following a rapid rise in mRNA levels, the messages for these protems take different courses The c-fos mRNA fell to below control levels by 1 h, while the ODC mRNA remained elevated beyond 24 h The ECS-mduced elevation of ODC enzyme act1V1ty was not abolished by adrenalectomy but was attenuated significantly by the anti-convulsant MK-801. These results imply that the mduct10n of cerebral ODC may be neuronal act1V1ty dependent, and suggest that the ODC/polyamme system may be linked to the proposed th1rd messenger cascade, involvmg c-fos, which couples cell stimulation to gene express10n, resulting m long-term adaptive responses


INTRODUCTION
Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine synthetic pathway 23 , and the proto-oncogene c-fos are known to be involved in growth-and division-related phenomena However, a multitude of chemical, thermal and physical stimuli, unrelated to proliferative events, result in the mduction of ODC and c-/os 8 • 11 • 12 • 14 • For every case of c-fos induction, a corollary in ODC stimulation can be found. Therefore, it appears that the polyamines and Fos may have specialized and related functions in the adult central nervous system (CNS).
A cell in the nervous system can react to incoming signals in several ways, including an immediate shortterm response which is mediated by second messenger systems, and a long-term response that requires gene expression 5 . A number of gene products have recently been implicated in coupling cell-surface stimulation to gene transcription. The best studied member of this family, termed 'third messengers' is c-fos, which has been shown to be transiently induced in reaction to neuronal activation 20 . The nuclear localization of Fos 20 , its DNAbmding properties and the ability of Fos to activate the transcription of genes in vitro, is compatible with the role of Fos as a 'third messenger' 3 • 10 • ODC activity has also been reported to be induced by neuronal shmulation 1 • 22 . The polyamines, whose synthe-sis is dependent on ODC, are cationic in nature 26 and have long been shown to exert an effect on DNA replication and stability, RNA expression and protein synthesis. ODC, with both nuclear and cytoplasmic localizations 2 , has one of the fastest turn-over rates of any known protem (<10 min), and can undergo rapid and drastic elevations in activity, primarily through the de novo synthesis of enzyme molecules 23 . The ODC/polyamine system and the nuclear protooncogenes thus share many similar properties. In this study, we demonstrate that electroconvuls1ve shock (ECS) results in the induction of ODC mRNA and enzyme activity as well as c-fos mRNA. We also propose that ODC is an early gene that is activated by neuronal stimulat10n and hence may participate m c-fos and other nuclear proto-oncogene mediated events 21 .

Animals and treatment
Male Fischer CD rats, 6-10 weeks old were used. The ammals were housed at 22 °C with free access to food and water, on a 12 h hght-dark cycle. Electroconvuls1ve shock (ECS) was admm1stered via ear electrodes, with salme-mo1stened contact pads, using a constant·current apparatus Shock was applied to normal, adrenalectomized and sham operated rats at the followmg mtens1t1es· 85 mA for 1 s with 1 ms pulses at a frequency of 50 Hz. Control ammals were treated s1milarly without the apphcat10n of a current. These shock mtens1t1es have been demonstrated to be without detectable morpholog1cal damage to the bram 6 Some ammals were dosed by mterpentoneal miect10ns of MK-801 Correspondence SC Bondy, Dept of C & E Med1cme, Umvers1ty of California, Irvme, CA 92717. US A (1 mg/kg), dissolved in saline Ammals were decapitated and theu corresponding brain reg10ns were dissected out, qmckly placed m dry ice and kept at -70 °C until the time of use Orntthme decarboxylase assay ODC activity was determined by measurement of evolved 14 C0 2 from carboxy- [ 14 C]ormthine (55 9 mCi/nmol, New England Nuclear, Boston, MA) Tissue was homogemzed in 19 volumes of 0 04 M Tris-HCI After centrifugation (26,000 g, 20 mm), 0 9 ml of various tissue preparations was added to 50 µl pyridoxal phosphate solution (1 nM) and 50 µl [ 14 C]ormthine, m the presence of 0.045 M d1th1othre1tol The final ormthine concentrat10n was 2 5 µM Incubations were earned out at 37 °C for 45 min in a sealed tube and terminated by in1ect1on of 1 ml of 2 M acetic acid into the react10n m1xture 25 Evolved 14 C0 2 was trapped on a paper wick containing hyamine suspended above the reaction mixture The decarboxyl-at1on process 1s linear for up to 1 5 h under these conditions Decarboxylation, not attributable to ODC, was determined by runmng a parallel incubation m the presence of 5 mM d1fluoromethylormthine, the specific ODC mh1b1tor 19 _

RNA analysis
Total cellular RNA was isolated from bram regions of control and ECS-treated animals using the phenol-SOS method 16 · 27 , with some modifications The RNA was fractionated according to size m formaldehyde-agarose gels 9 and transferred onto mtrocellulose filter paper The filters were then probed with a 32 P-labelled ODC cDNA plasmid 17 (labelled by mck-translalion), washed under stringent conditions and exposed to X-ray film at -70 °C for 5-7 days 29 S1m1larly, RNA obtained from control and treated ammals was probed with a P-actin cDNA plasmid under the same cond1lions The EcoRIBamHl 2 2 kbp fos insert fragment of pSP65 was also used to generate mck-translated probes mRNA content was estimated by scanning the autorad1ograms with a laser densitometer (LKB, Pharmacia), interfaced with a computer integrator RESULTS ECS-induced seizures were used as an experimental paradigm to stimulate both ODC and c-fos. Followmg the administration of ECS all animals exhibited a 30 s clonic-tonic seizure which resulted in the subsequent 120 ..  Fig. 2 The effect of a smgle ECS on ODC mRNA and P-actin mRNA Total RNA was isolated fractionated on agarose gels and transferred onto mtrocellulose filter paper Filters were then probed with 32 P-mck-translated cDNAs for both ODC and /J-actm The hybridized filters were exposed to X-ray film for 1 week Control (C) ODC and /J-actm mRNA levels are compared to ECS (E)-treated rats, 5 h post-ECS The position of the ribosomal RNA bands 1s shown on the vertical axis as determmed by eth1dmm bromide stammg of the agarose gels elevation of ODC enzyme activity. This elevation reached over 2-fold above controls by 5 h, and was transient, returning to basal values by 10 h (Fig. 1) The induction of ODC enzyme activity in the neocortex was accompanied by a rapid and significant elevation of the ODC mRNA (Figs. 2 and 3). The levels of an mRNA coding for a structural protein, /J-actm, were not changed as a result of electrical stimulation (Fig. 2). In contrast to the transient nse of ODC enzyme actlVlty,   levels of the induced ODC mRNA peaked around the same time as the activity, but remained high for 24 h (Fig. 3). Upon electrical stimulation, there ensued a rapid and transient elevation in the levels of expression for cerebral c-fos mRNA (Figs. 3 and 4). Peak c-fos mRNA induction reached about double that of basal levels by 30 min post-ECS, but fell below control values by 2 h. This sequence is consistent with previously reported find-ings20.
In order to determine whether the induction of ODC was related to neuronal activity or to the altered endocrine state that follows seizures 1 , we administered ECS to adrenalectomized rats. ECS increased ODC enzyme activity of both sham and adrenalectomized rats to the same extent. Thus adrenalectomy did not produce .. 245 any significant effect on the ECS-induced enzyme activity (Fig. 5).
To ascertain the dependence of ODC induction on seizure activity, rats were treated with the anti-convulsant MK-801 prior to the application of ECS. Such treatment, which potently and competitively blocks the NMDA-type of glutamate receptor in the brain 30 , abolished the tonic-clonic seizures produced by electrical shock. There was approximately a 40% attenuation in the ECSinduced enzyme activity following the administration of this agent (Fig. 5). DISCUSSION These data suggest that the previously reported induction of ODC activity in the neocortex in response to ECS 1 • 22 , may be partly due to the activation of the ODC gene (Fig. 2). There is good agreement between the induction of ODC enzyme activity in the neocortex and the elevation of the ODC mRNA. Intracerebral injections of actinomycin D prior to ECS, reduced ECSstimulated increases in enzyme activity (data not presented), further showing that ODC induction is at least partially due to genomic derepression. This derepression is a somewhat selective process because the levels of /3-actin mRNA were not altered by ECS treatment.
The ODC mRNA level, followed a different temporal profile to that of the enzyme activity. The mRNA peaked at similar times as the activity did, but remained elevated for over 24 h (Fig. 3). This phenomenon is not unique to ECS-induced ODC, but has been observed in interleukin-2 stimulation of lymphocytic ODC 15 . The fall in enzyme activity, while the mRNA remains elevated, suggests that the build-up in polyamine levels, as a consequence of heightened enzyme activity, may produce a feed-back inhibition of the translation of the mRNA . The ability of the polyamines, putrescine and spermidine, to mhib1t the translation of the ODC mRNA has been demonstrated in tissue culture systems 13 • 24 . Therefore, the polyammes can exert control over ODC activity by regulating the rate of translation of the ODC mRNA.
The proto-oncogene c-fos was also stimulated by ECS (Figs. 3 and 4). This c-fos induction, recently demonstrated to occur in response to a single electrical shock 7 , was very short lived. In contrast to the ODC mRNA which remained elevated for a longer time, c-fos mRNA rapidly fell to below basal levels (Figs. 3 and 4). This major difference in mRNA stability may have some physiological relevance. The persistent ODC mRNA, if open for translation, might enable ODC levels to respond rapidly to subsequent metabolic demands.
Animals given ECS experience a period of behavioral depression which occurs simultaneously with an increase in cerebral ODC activity. Furthermore, ECS leads to changes in the levels of several humoral factors which can affect ODC activity 28 . In this study, adrenalectomy had no significant effect on ECS-mduced enzyme activity in the neocortex (Fig. 5), indicating that the ODC response to ECS is not modulated by adrenal glucocorticoid action.
The fact that adrenalectomy made no difference to ODC mductlon, suggests that, the elevation of ODC by ECS may be neuronal-activity dependent, and not due to the altered endocrine state that follows such treatment. Mitigation of the response of ODC levels to ECS by MK-801 pretreatment, implies neuronal mediat10n of ODC induction, followmg ECS. MK-801 has been previously reported to block ECS-induced se1zures 18 • We have shown that a stimulus modulating the cellular environment, can result in the parallel activation of the nuclear proto-oncogenes and the ODC/polyamine system. The sequence of activation of these two systems, by ECS, is overlapping, thus it 1s not known 1f either of them 1s responsible for the induction of the other It is more likely that the mteractlons between the ODC/polyamine system and the nuclear proto-oncogenes, is manifested at the level of their target sites.
While both systems can be activated by a wide variety of stimuli, the specificity of the cellular response must depend on the differentiated state of the cell The polyamines may potentiate the effects of the protooncogenes, and may also have their own direct effects on genomic events. These two systems in the brain, may then subserve the long-term adaptive responses following intense or repeated neuronal activation. Such responses may underlie the initiation of repair mechanisms, the onset of maladaptive neural changes, or complex events relatmg to the deposition of memory.