Brain Research, 553 (1991) 117-122 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116750D
117
BRES 16750
Lithium augments pilocarpine-induced
fos gene expression in rat brain
Elyse D. Weiner, Vasundhara D. Kalasapudi, Demitri F. Papolos and Herbert M. Lachman Department of Psychiatry: Program of Behavioral Genetics, Department of Medicine, Albert Einstein Collegeof Medicine, Bronx, NY 10461 (U.S.A.) (Accepted 29 January 1991)
Key words: Muscarinic cholinergic; Pilocarpine; Lithium; Manic-depression; Protein kinase C; .los proto-oncogene
Lithium salts are considered the most effective agents used in treating manic-depression. Previous studies in PC12 pheochromocytoma ceils indicate that lithium has a dramatic augmenting effect on expression of the los proto-oncogene, a component of the AP-1 transcription factor. Although fos expression is activated by agonists that function through different signal transduction pathways, the lithium augmenting effect appears to be specific for receptor and post-receptor stimulators of protein kinase C (PKC). In particular, fos induction mediated by the ml muscarinic receptor linked to PKC activation was found to be exquisitely sensitive to lithium enhancement. We now show that a similar augmenting effect can be demonstrated in rat brain. Following treatment with the muscarinic agonist piloearpine, fos mRNA aeetunulates in the cortex, an effect that is blocked by the ml antagonist pirenzepine. Rats treated with a single intraperitoneal injection of lithium chloride exhibited a substantial increase in pilocarpine-mediated fos expression. In contrast, fos expression induced in several brain regions by a single electroconvulsive shock is not augmented by lithium. The finding that short-term treatment with lithium enhances fos expression in the brain suggests a mechanism for its therapeutic action. INTRODUCTION Despite years of investigation, the mechanism of lithium's unique therapeutic action in manic-depression (bipolar affective disorder) is not well understood. One hypothesis suggests that lithium has an inhibitory effect on phosphoinositide (PI) metabolism resulting from its potent inhibition of myoinositol 1-phosphatase (M1P) is. This enzyme converts myoinositol 1-phosphate, an intermediate in the breakdown of inositol phosphates, into inositol, which is required for synthesis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP 2 plays an important role in signal transduction since it is hydrolyzed into second messengers by ligandactivated phospholipase C (PLC). This leads to the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular Ca 2+, and diacylglycerol (DAG), a potent activator of the serine and threonine kinase protein kinase C (PKC) 4,35. It has been suggested that lithium's inhibition of M1P could lead to a depletion of inositol and PIP2, resulting in reduced PI-mediated neuronal responses; a mechanism referred to as the 'inositol depletion hypothesis '5,25. Other investigators have suggested that lithium may act by inhibiting G-proteins 2. These ubiquitous membranebound proteins play a critical role in signal transduction,
since they are coupled to receptors linked to the regulation of PLC, adenylate cyclase and arachidonic acid 3'14'32. The lithium inhibition of agonist-stimulated cyclic AMP (cAMP) that some investigators have described is thought to be due to G-protein (Gs)- adenylate cyclase uncoupling 2,34. Because of the putative inhibitory influence of lithium on second messenger systems, we have been interested in its potential effect on activation of the los protooncogene 1°. Fos encodes a protein that binds to the product of the jun proto-oncogene to form the AP-1 transcription factor s,3°,37,38. Fo$ gene transcription is rapidly induced by agonists that stimulate PKC and cAMP as a result of binding to the cAMP and phorbol ester DNA-responsive elements located in the gene's 5' regulatory domain 15,29,4°,41. Contrary to the proposed inhibitory action on receptor-mediated PKC and cAMPcoupled events, lithium was found to have a dramatic augmenting effect on los expression in PC12 pheochromocytoma cells23. This effect appears to be specific for receptor and post-receptor activators of PKC since lithium augmented los expression induced by ligands that induce PIP 2 hydrolysis, such as carbachol, bradykinin and nerve growth factor, as well as phorbol esters that directly activate PKC. By contrast, lithium has no effect on los expression induced by receptor or post-receptor
Correspondence: H.M. Lachman, Department of Psychiatry: Program of Behavioral Genetics, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, U.S.A.
118 activation of adenylate cyclase 23 (Divish et al., in press). A l t h o u g h lithium enhances los expression induced by a n u m b e r of P L C / P K C coupled agonists, the m l muscarinic cholinergic r e c e p t o r system was found to be particularly sensitive 23. Similarly, A r e n a n d e r et al. found that lithium a u g m e n t e d los expression in cultured rat astrocytes 1. Since one m o d e l for mania suggests that r e d u c e d cholinergic discharge leads to an imbalance in cholinergic and adrenergic neurotransmission, the augmenting effect of lithium on a muscarinic cholinergic target m a y have therapeutic relevance 22. To further investigate the effect of lithium on muscarinic cholinergic systems, we analyzed los expression in rat cortex induced by t r e a t m e n t with pilocarpine, a centrally active muscarinic agonist. Fos gene expression is induced in specific brain regions by a variety of experimental manipulations including induction of generalized seizures ( E C S ) , exposure to environmental light and sensory stimulation 21'24'31. We now show that los m R N A can also be induced in the rat cortex following treatment with pilocarpine. F u r t h e r m o r e , p r e t r e a t m e n t with lithium chloride enhances this effect, similar to the findings for PC12 cells and astrocytes 1'23. The augmentation of a muscarinic cholinergic response in the CNS, at concentrations of lithium within a therapeutically relevant range, is consistent with its anti-manic action according to the cholinergic m o d e l and supports the hypothesis that lithium's effect on gene expression may be an important e l e m e n t in understanding its therapeutic effectiveness. MATERIALS AND METHODS
Drug administration Male Sprague-Dawley rats (Charles River Laboratories) weighing 200-250 g at the start of the experiments were housed in the animal facility under conditions of uniform light, temperature and humidity (14-h day, 10-h night, 23 °C ambient temperature, 50% relative humidity). Food and water were provided ad libitum. As acute treatment, rats received intraperitoneal (i.p.) injections of lithium chloride (0.75 M solution, 3 meq/kg), or an equivalent amount of sodium chloride as a control. After 7 h, pilocarpine (5 mg/kg in saline) or vehicle was administered i.p. For chronic treatment, rats received 3 meq/kg/day subcutaneously (s.c.), in two divided doses for 5-6 days. Animals treated chronically with lithium were also provided with a water bottle containing 0.9% sodium chloride. Pilocarpine or vehicle were then administered 12 h after the last injection of lithium. The animals were sacrificed by decapitation 75 min after pilocarpine administration. Trunk blood was collected for serum lithium determination and the hippocampus and cortex were removed on a dissecting block that had been stored at -20 °C. The tissue was placed in prelabeled plastic bags and rapidly frozen in liquid nitrogen in order to limit RNA degradation. Samples were stored at -80 *C prior to RNA extraction. Electroconvulsive shock was administered through ear clip electrodes using an ECT generator set at 50 Hz delivering 1 s of electric current. Tonic-clonic seizures lasting 20-30 s were observed. The animals were sacrificed 75 min later and total cellular RNA was extracted from the cortex, hippocampus and cerebellum.
RNA extraction RNA was extracted using guanidine isothiocyanate as a denatur-
ant. Frozen cortical and hippocampal samples were homogenized in 15 and 5 ml respectively of solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl and 0.1 M 2-mercaptoethanol). An equal volume of H20-saturated phenol, one-tenth volume chioroform:isoamyl alcohol and 40 mM sodium acetate, pH 4.0 were added to the homogenate. The samples were shaken vigorously for 10 s, placed on ice for 30 min and the RNA recovered from the aqueous phase by sequential isopropyl alcohol and ethanol precipitations. The final RNA pellet was resuspended in TE buffer (10 mM Tris, pH 7.4 and 1 mM EDTA, pH 8.0). The concentration and purity of the samples were determined by the measuring the absorption of an aliquot at 260 and 280 nm.
RNA hybridization analysis 20-40/~g of total cellular RNA was separated by 0.9% agaroseformaldehyde gel electrophoresis and transferred to nylon filters (Nytran, Schleicter and Schuell) as previously described23. Filters were hybridized with 32P-labelled probes using a nick translation kit (Bethesda Research Labs). The recombinant plasmids used in this experiment included pMMMfos3°, a mouse genomic clone, and pGAPDH 36, a glyceraldehyde phosphate dehydrogenase cDNA. DNA inserts, isolated by electrophoresis through low melting point agarose, were labelled to specific activities of 1-3 x 108 cpm//zg DNA. Following overnight hybridization at 60 °C, the filters were washed free of non-specifically bound counts as previously described23 and exposed to X-ray film with two image-intensifying screens. Autoradiograms were developed after 0.5-7 days. Densitometric analysis was performed on autoradiograms within the linear range of exposure using a Quantimet 920 system. The quantitative data was stored in an Excel microsoft database from which the mean, standard error and Student's t-test were derived.
RESULTS
Induction of los mRNA by pilocarpine The cortex contains a b u n d a n t muscarinic cholinergic receptors of both the m l and m2 subtypes. The m l receptors are coupled to P L C / P K C activation and are found primarily on dendrites and cell bodies, whereas m2 receptors, which inhibit a d e n y l a t e cyclase, are primarily presynaptic 16. Since m l receptors are located on cell bodies and are coupled to P K C , we r e a s o n e d that its activation would result in the induction of los m R N A . A n i m a l s were injected with 5 mg/kg pilocarpine in saline, or with saline alone. A f t e r 75 min, the animals were sacrified by decapitation and total cellular R N A was extracted from dissected cortical tissue. T h e R N A was analyzed by N o r t h e r n filter hybridization as described in Methods. A s seen in Fig. 1, los m R N A is not easily detected in the cortex in the absence of pilocarpine under the conditions of our R N A assay (lane 1). H o w e v e r , following t r e a t m e n t with pilocarpine, the 2.2 kb los m R N A band is d e t e c t e d (lane 2). In o r d e r to d e t e r m i n e whether the induction is due to activation of an m l receptor subtype, the animals were p r e t r e a t e d with pirenzepine (PZ) (100 mg/kg), a muscarinic antagonist that exhibits high affinity for the m l r e c e p t o r 19. A s seen in lane 3, t r e a t m e n t with P Z reduces the pilocarpinem e d i a t e d induction of fos m R N A by 3-fold, suggesting an effect m e d i a t e d primarily by the m l r e c e p t o r subtype.
119
Cort 123
Hipp 123
los--
GAPDH-
)
Fig. 1. Effect of pilocarpine on fos expression. Male SpragueDawley rats were treated with 5 mg/kg pilocarpine i.p. in saline and sacrificed 75 min later. The cortex and hippocampus were dissected and total cellular RNA was extracted. The RNA was analyzed by Northern hybridization analysis using fos and GAPDH specific probes. Lane 1: vehicle; lane 2: pilocarpine; lane 3: pilocarpine following pretreatment for 15 min with pirenzepine (100 mg/kg). Autoradiographic exposure times for fos: cortex-2 days, hippocampus-7 days. Prolonged autoradiographic exposure did not result in the detection of fos mRNA in untreated hippocampal controls. Experiments repeated on 3 sets of animals (control, pilocarpine and pilocarpine plus pirenzepine constitutes a set). Similar results were o b t a i n e d in the hippocampus, except that the level of los m R N A was a p p r o x i m a t e l y onetwentieth that found in the cortex. A s a control for the equal loading and transfer of R N A , the filters were r e p r o b e d with GAPDH, which is expressed constitutively in most cell types 36. A s seen in Fig. 1, the changes in los m R N A that occur u p o n pharmacological manipulation with muscarinic agonists and antagonists are not a c c o m p a n i e d by similar changes in GAPDH expression.
similar e n h a n c e m e n t could be d e t e c t e d in animals. We focussed primarily on cortical los expression, since induction by pilocarpine is a p p r o x i m a t e l y 20-fold greater than in the h i p p o c a m p u s . M a l e S p r a g u e - D a w l e y rats were t r e a t e d with a single i.p. injection of lithium chloride (3 meq/kg) or an equivalent dose of sodium chloride. Seven h later, pilocarpine was injected and the animals were sacrificed after an additional 75 min. The average lithium level o b t a i n e d from trunk blood at the time of sacrifice ranged b e t w e e n 0.45-0.81 meq/1, a concentration similar to the t h e r a p e u t i c dose in humans 6. This strategy was e m p l o y e d because intracellular lithium levels p e a k in a n u m b e r of brain loci after a p p r o x m a t e l y 8 h 33 and fos induction occurs rapidly following agonist stimulation 9"23. A s seen in Fig. 2, p r e t r e a t m e n t with lithium c h l o r i d e ( + ) results in an increase in pilocarpineinduced los m R N A c o m p a r e d to sodium c h l o r i d e ( - ) and pilocarpine-treated controls. D e n s i t o m e t r i c analysis revealed that in this e x p e r i m e n t the e n h a n c e m e n t was a p p r o x i m a t e l y 2-fold. These changes are specific for los since we did n o t detect a similar increase in the expression of the GAPDH m R N A control. F u r t h e r m o r e , t r e a t m e n t with lithium alone does not result in d e t e c t a b l e levels of los m R N A under the conditions of the N o r t h e r n hybridization analysis ( d a t a not shown). In o r d e r to d e t e r m i n e the specificity of the lithium-
Effect of lithium on los induction Since lithium enhances fos expression induced by muscarinic cholinergic activation in cultured neuronal cells 1"23, we were interested in determining whether a
3 .c
PILO
Cort
Cort Hipp
Li: fos - -
.=
ECS
2
Cb
T,
1
mR Na-Acute
GAPDH-Fig. 2. A: effect of lithium on pilocarpine and ECS-induced los expression. Rats were treated with a single i.p. injection of lithium chloride (+) or and equivalent amount of sodium chloride (-) (3 meq/kg). After 7 h, pilocarpine was administered and the animals were sacrificed 75 min later. RNA was extracted from the cortex and analyzed as in Methods and Fig. 1. Alternatively, animals were treated with lithium or sodium chloride and a single electroconvulsive shock (ECS) was induced by a generator through ear clip electrodes. Seventy-five min later, RNA was extracted from the cortex, hippocampus and cerebellum (Cb). The ECS experiments were performed on 3 sets of animals.
U-Acute
Na-Chronic
Li-Chronic
Fig. 3. Accumulative analysis. Rats (n = 7) were treated with pilocarpine and lithium or sodium chloride (control) as in Fig. 1 (acute). In addition, 5 other rats were treated for 5-6 days with lithium or sodium chloride (3 meq/kg/day in 2 divided doses, s.c.), and pilocarpine was administered 12 h after the last dose. Northern filters were analyzed for fos and GAPDH mRNAs. Autoradiograms within the linear range were quantitated by densitometric scanning and thefos/GAPDH ratio determined. The mean of the controls has been arbitrarily established as 1.00 and the relative increase induced by lithium was calculated. Fos/GAPDHratio, acute control: 1.00 _+ 0.13, acute lithium: 2.74 __ 0.59, chronic control: 1.00 _+ 0.19, chronic lithium: 1.73 _+ 0.25. The asterisk denotes statistical significance compared to the controls (P < 0.05, one-tailed Student's t-test).
120 augmenting effect in the brain, we induced los mRNA using another strategy; administration of a single electroconvulsive shock (ECS). Although the mechanism of los induction by ECS has not been clearly elucidated, it is likely due to widespread depolarization and C a 2+ influx which are known to activate los gene expression in PC12 cells 17. As seen in Fig. 2, ECS induces the accumulation of los mRNA in the cortex, hippocampus and cerebellum. In contrast to the effects observed in pilocarpinetreated animals, we do not detect significant changes in the level of ECS stimulated los mRNA in the presence of lithium. Because of potential variation in pilocarpine responsiveness that may occur in individual animals, we analyzed los mRNA in a number of animals. Autoradiograms within the linear range of exposure were quantitated on a Quantimet 920 system and the fos/GAPDH signal ratio was determined to factor out potential loading and transfer artifacts. As seen in the cumulative analysis (Fig. 3), acute lithium treatment increased pilocarpine-induced cortical los mRNA by approximately 2.7 fold (n = 7, P < 0.05). Since the therapeutic action of lithium in patients with manic-depression takes approximately one week 6, we also examined the effect of chronic lithium treatment on los expression. According to the 'inositol depletion hypothesis', an inhibitory effect of lithium on the phosphoinisitide (PI) pathway, caused by inositol and PIP E depletion, would not be observed until pools of PIP E synthesized prior to lithium treatment are exhausted. This would not be expected to occur in acutely treated animals. Consequently, rats were treated for 5-6 days with 3 meq/kg/day lithium chloride or sodium chloride administered in 2 divided doses s.c. Twelve h after the final dose, animals were treated with pilocarpine for 75 min, cortical tissue was dissected and analyzed for fos mRNA. As seen in Fig. 3, chronic lithium treatment augments los expression by 1.7-fold, similar to the results obtained in acutely treated animals (n = 5, P < 0.05). DISCUSSION Although a number of models have been proposed, the molecular and genetic factors that result in the vulnerability to manic-depression are still not well understood. These models are based, in part, on the behavioral effects of centrally active drugs, and the putative molecular basis lithium's action. For example, Janowsky et al. 22 and Dilsaver 1~ developed the cholinergic hypothesis which postulates that reduced cholinergic drive, resulting in an imbalance of cholinergic and adrenergic neurotransmission, plays a role in the development of mania. The enhancing effect of lithium on
cholinergic pathways, which we and other investigators have described, is consistent with its anti-manic action according to this model 1"23"26. It has also been suggested that manic-depression could be caused by an overactive neurotransmitter or neuropeptide system linked to a PI pathway 5'25. Consequently, reduced inositol phosphate recycling, resulting from lithium's inhibition of myoinositol 1-phosphatase (MIP), would lead to PIP E depletion and attenuation of overactive, PI-mediated neuronal activity. The enhancing effect on los gene expression, described in this and other reports, may represent a novel mechanism of lithium's therapeutic action. In PC12 cells, lithium augmentation is specific for agonists that stimulate los by activating PKC. By contrast, no enhancing effect is detected upon induction by agonists that stimulate cAMP synthesis 23 (Divish et al., in press). Since los expression induced by phorbol esters is also augmented by lithium, the findings suggested that lithium may act on the pathway leading from PKC activation to gene expression, rather than at the level of receptor-PKC coupling. However, the molecular basis of this effect has not been clearly elucidated. Using concentrations of lithium within the therapeutic range, the enhancing effect on los mRNA in PC12 cells has only been observed following muscarinic cholinergic stimulation. Other PKC stimulating agonists require 5- to 10-fold higher levels of lithium 23. We now find that therapeutic concentrations of lithium also augments pilocarpine-induced cortical los expression. This effect appears to be mediated by an ml receptor subtype linked to PIP 2 hydrolysis and PKC activation since it is blocked by pirenzepine (PZ). While PZ has been shown to inhibit ml and m2 receptors, it has a higher affinity for the ml subtype 19. Although the cortex contains both subtypes, m2 is primarily a presynaptic autoreceptor linked to the inhibition of cAMP. Stimulation of this system would not be expected to induce fos expression since cAMP actually activates the gene 15'23'4°'41. Also, autoreceptors in the synaptic terminal that reduce transynaptic signalling would be an unlikely target for agonists that stimulate gene expression in the nucleus. Regardless of the mechanism, the enhancement of los expression by therapeutic concentrations of lithium supports the hypothesis that gene expression may be an important target of its effect in mood disorders. The augmentation of cholinergic-mediated los expression is consistent with the in vivo enhancing effect of lithium observed by other investigators26"39. However, it contrasts with the inhibitory effect described by Worley et al. in ex vivo hippocampal slices45. One explanation consistent with these observations is that lithium exhibits both inhibitory and excitatory effects on PI-mediated
121 signal transduction. Perhaps under conditions that favor inositol depletion, such as repetitive agonist stimulation in the presence of lithium, as used in the Worley experiment, inhibition of events mediated by PIP 2 hydrolysis would occur. However, in the absence of inositol depletion, one would observe augmentation of a PIP 2mediated target (such as fos) through lithium's putative effect on PKC-mediated gene expression. Alternatively, an inhibitory effect of lithium on the PI pathway could be due to an action at the G-protein level, as suggested by several investigators 2'34, since subtypes of this family of proteins regulate PLC. In this model, the intracellular concentration of lithium would determine whether inhibition of a G-protein linked to PLC, or augmentation of the C-kinase/fos pathway occurs. Although we have used fos expression as a convenient experimental marker to study the effect of lithium on PI-mediated responses in the brain, it is interesting to speculate on the potential role of gene expression in its therapeutic action. A number of genes expressed in the CNS, such as those encoding some neuropeptides, contain AP-1 and phorbol ester-responsive domains that could serve as targets for lithium's effect on PKC-induced fos expression 43. The observation by D o b n e r et al., that lithium enhances the expression of neurotensin/neurome-
REFERENCES 1 Arenander, A.T., De Vellis, J. and Herschman, H.R., Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters, J. Neurosci. Res., 24 (1989) 107-114. 2 Avissar, S., Schreiber, G., Danon, A. and Belmaker, R.H., Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex, Nature, 331 (1988) 440-442. 3 Axelrod, J., Burch, R.M. and Jelserra, C.L., Receptor mediated activation of phospholiplase A 2 via GTP binding protein: arachidonic acid and its metabolites as second messengers, Trends Neurosci., 11 (1988) 117-127. 4 Berridge, M. and Irvine, R.E, Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature, 312 (1984) 315-321. 5 Berridge, M.J. and Irvine, R.E, Inositol phophates and cell signalling, Nature, 341 (1989) 197-205. 6 Braastroup, EC., Poulsen, J.C., Schou, M., Thomsen, K. and Amidsen, A., Prophylactic lithium: double-blind discontinuation in manic-depression and recurrent-depressive illness, Lancet, 2 (1970) 326-330. 7 Buckley, N.J., Bonner, T.I. and Brann, M.R., Localization of a family of muscarinic receptor mRNAs in rat brain, J. Neurosci., 8(12) (1988) 4646-4652. 8 Chiu, R., Boyle, W.J., Meek, J., Smeal, T., Hunter, T. and Karin, M., The c-los proto-oncogene interacts with c-]un/AP-1 to stimulate transcription from AP-l-sensitive genes, Cell, 54 (1988) 541-552. 9 Curran, T. and Morgan, J.I., Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines, Science, 229 (1985) 1265-1268. 10 Curran, T. and Teich, N.M., Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000 dalton phosphoprotein, J. Virol., 42 (1982) 114-122. 11 Davis, J.L., Nakajima, T., Gieiter, C.H., Post, R.M. and
din N, a phorbol ester responsive gene, could be explained by an effect at the P K C and/or los level 13. Preliminary results from our lab, indicating that acute treatment with lithium increases the expression neuropeptide Y m R N A , and the findings by several groups that lithium increases the expression of other neuropeptide encoding genes 28'42, are also consistent with this hypothesis. Some of the behavioral and neuroendocrine abnormalities found in m o o d disorders are thought to be due to abnormal expression of neuropeptides 2°. Furthermore, neuroendocrine stressors that function by influencing gene expression, such as glucocorticoids 46, are capable of causing mood disturbances in vulnerable individuals 44. Consequently, the effect of lithium on gene expression appears to be a particularly relevant target for further experimental investigation.
Acknowledgements. The Program in Behavioral Genetics is supported by the Ruane Family Fund and the G. Harold and Leila Y. Mathers charitable foundation. H.M.L. is a fellow of the Irma T. Hirschl and Monique Weill-Caulier charitable trust. D.EP is a NARSAD young investigator. The authors wish to thank Thelma ParAs for performing serum lithium determinations. We would also like to acknowledge Dr. Herman van Praag for his encouragement and support in estabhshing the Program of Behavioral Genetics.
Marangos, e.J., Mouse brain c-fos mRNA distribution following
a single electroconvulsive shock, J. Neurochem., 52 (1989) 1954-1957. 12 Dilsaver, S.C., Cholinergic mechanisms in depression, Brain Res. Rev., 11 (1986) 285-316. 13 Dobner, P.R., Tischler, A.S., Lee, Y.C., Bloom, S.R. and Donahue, S.R., Lithium dramatically potentiates neurotensin/ neuromedin N gene expression, J. Biol. Chem., 263 (1988) 13983-13986. 14 Gilman, A.G., G-proteins and regulation of adenylate cyclas¢, JAMA, 262 (1989) 1819-1825. 15 Gilman, M.Z., Wilson, R.N. and Weinberg, R.A., Multiple protein binding sites in the 5' flanking region regulate c-fos expression, Mol. Cell. Biol., 6 (1986) 4305-4314. 16 Goyal, R.K., Muscarinic receptor subtypes, New Engl. J. Med., 321 (1989) 1022-1029. 17 Greenberg, M.E., Ziff, E.B. and Greene, L.A., Stimulation of neuronal acetylreceptors induces rapid gene transcription, Science, 234 (1986) 80-83. 18 Halicher, L. and Sherman, W.R., The effects of lithium ion and other agents on the activity of myo-inositol-l-phosphatase from bovine brain, J. Biol. Chem., 261 (1980) 8100-8130. 19 Hammer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C., Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature, 283 (1980) 90-92. 20 Heilig, M., Soderpalm, B., Engel, J.A. and Widerlov, E., Centrally administered neuropeptide Y produces anxiolytic-like effects in animal anxiety models, Psychopharmacology, 98 (1989) 524-529. 21 Hunt, S.P., Pini, A. and Evan, G., Induced activation of c-los like protein in spinal neurons following sensory stimulation, Nature, 328 (1987) 632-634. 22 Janowsky, D.S., Risch, S.C., Parker, D., Huey, L.Y. and Judd, L., Increased vulnerability to cholinergic stimulation in affective disorder patients, Psychopharmacol. Bull., 16 (1980) 29-31.16.
122 23 Kalasapudi, V.D., Sheftei, G., Divish, M.M., Papoios, D.E and Lachman, H.M., Lithium augments los proto-oncogene expression in PC12 pheochromocytoma cells: implications for therapeutic action of lithium, Brain Research, 521 (1990) 47-54. 24 Kornhauser, J.M., Nelson, D., Mayo, K.E. and Takahashi, J.S., Photic and circadian regulation of c-los gene expression in the hamster superehiasmatic nucleus, Neuron, 5 (1990) 127-134. 25 Lachman, H.M. and Papolos, D.E, Abnormal signal transduction: a hypothetical model for bipolar affective disorder, Life Sci., 45 (1989) 1413-1426. 26 Lerer, B. and Stanley, M., Effect of lithium on cholinergically mediated responses and [3H]QNB binding in rat brain, Brain Research, 344 (1985) 211-219. 27 Levy, A., Zohar, J. and Belmaker, R.H., The effect of chronic lithium pretreatment on rat brain muscarinic receptor regulation, Neuropsychopharmacology, 21 (1983) 1199-1201. 28 Mathe, A.A., Jousisto-Hanson, J., Stenfors, C. and Theodorsson, E., Effect of lithium on tachykinins, calcitonin gene-related peptide, and neuropeptide Y in rat brain, J. Neurosci. Res., 26 (1990) 233-237. 29 Mellon, EL., Clegg, C., Correll, L.A. and McKnight, G.S., Regulation of transcription by cAMP-dependent protein kinase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 488-491. 30 Miller, A.D., Curran, T. and Verma, I.M., C-los protooncogene can induce transformation: a novel mechanism of activation of a cellular oneogene, Cell, 36 (1984) 51-60. 31 Morgan, J.I. Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping pattern of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 32 Moriarty, T.M., Giilo, B., Carty, B.J., Premont, R.T., Landau, E.M. and Iyengar R., Beta-gamma subunit of GTP binding proteins inhibits muscarinic receptor stimulation of phospholipase C, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 8865-8869. 33 Mukherjee, B.P., Bailey, P.T. and Pradhan, S.N., Temporal and regional differences in brain concentrations of lithium in rats, Psychopharmacology, 48 (1976) 119-121. 34 Newman, M.E. and Belmaker, R.H., Effects of lithium in vitro and ex vivo on components of the adenylcydase system in membranes from cerbral cortex of the rat, Neuropharmacology, 26 (1987) 211-217. 35 Nishizuka, Y., Studies and perspectives of protein kinase C, 233
(1986) 305-312. 36 Piechaczyk, M., Blanchard, J.M., Marty, L., Dani, C., Panatieres, E, EI-Sabouty, S., Fort, P. and Jeanteur, P., Posttranscriptional regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression in rat tissue, Nucleic Acids Res., 12 (1984) 6951-6963. 37 Rauscher, F.J., Cohen, D.R., Curran, T., Bos, T.J., Vogt, P.K., Bohmann, D., Tijan, R. and Franza, R., Fos-associated protein p39 is the product of the jun proto-oncogene, Science, 240 (1988) 1010-1016. 38 Rauscher, EJ., Sambucetti, L.C., Curran, T., Distel, R.J. and Spiegalman, B.M., Common DNA binding site for los protein complexes and transcription factor AP-1, Cell, 52 (1988) 471-480. 39 Roth, P., Hamburder-Bar, R. and Lerer, B., Peripheral vs central manifestations in toxic interaction of lithium and pilocarpine, Biol. Psychiatry, 25 (1989) 153-158. 40 Sassone-Corsi, P., Visvader, J., Ferland, L., Mellon, P.L. and Verma, I.M., Induction of proto-oncogene los transcription through the adenylate cyclase pathway. Characterization of a cAMP responsive element, Genes Dev., 2 (1988) 1529-1538. 41 Sheng, M. and Greenberg, M., The regulation and function of c-los and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 42 Sivam, S.P., Krause, J.E., Takeuchi, K., Li, S., McGinyy, J.F. and Hong, J.-S., Lithium increases rat striatal beta and gamma preprotachykinin messenger RNA's, 1. Pharm. Exp. Ther., 248 (1989) 1297-1301. 43 Sonnenberg, J.L., Rauscher, F.J., Morgan, J.I. and Curran, T., Regulation of proenkephalin by fos and jun, Science, 246 (1989) 1622-1625. 44 Wolkowitz, O.M., Rubinow, D., Doran, A.R., Breier, A., Berrettini, W.H., Kling, M.A. and Pickar, D., Prednisone effects on neurochemistry and behavior, Arch. Gen. Psychiatry, 47 (1990) 963-968. 45 Worley, P.F., Heller, W.A., Snyder, S.H. and Baraban, J.M., Lithium blocks a phosphoinositide-mediated cholinergic response in hippocampal slices, Science, 239 (1988) 1428--1429. 46 Yamamato, K.R., Steroid receptor-regulated transcription of specific genes and gene networks, Ann. Rev. Genet., 19 (1985) 209-252.
117
BRES 16750
Lithium augments pilocarpine-induced
fos gene expression in rat brain
Elyse D. Weiner, Vasundhara D. Kalasapudi, Demitri F. Papolos and Herbert M. Lachman Department of Psychiatry: Program of Behavioral Genetics, Department of Medicine, Albert Einstein Collegeof Medicine, Bronx, NY 10461 (U.S.A.) (Accepted 29 January 1991)
Key words: Muscarinic cholinergic; Pilocarpine; Lithium; Manic-depression; Protein kinase C; .los proto-oncogene
Lithium salts are considered the most effective agents used in treating manic-depression. Previous studies in PC12 pheochromocytoma ceils indicate that lithium has a dramatic augmenting effect on expression of the los proto-oncogene, a component of the AP-1 transcription factor. Although fos expression is activated by agonists that function through different signal transduction pathways, the lithium augmenting effect appears to be specific for receptor and post-receptor stimulators of protein kinase C (PKC). In particular, fos induction mediated by the ml muscarinic receptor linked to PKC activation was found to be exquisitely sensitive to lithium enhancement. We now show that a similar augmenting effect can be demonstrated in rat brain. Following treatment with the muscarinic agonist piloearpine, fos mRNA aeetunulates in the cortex, an effect that is blocked by the ml antagonist pirenzepine. Rats treated with a single intraperitoneal injection of lithium chloride exhibited a substantial increase in pilocarpine-mediated fos expression. In contrast, fos expression induced in several brain regions by a single electroconvulsive shock is not augmented by lithium. The finding that short-term treatment with lithium enhances fos expression in the brain suggests a mechanism for its therapeutic action. INTRODUCTION Despite years of investigation, the mechanism of lithium's unique therapeutic action in manic-depression (bipolar affective disorder) is not well understood. One hypothesis suggests that lithium has an inhibitory effect on phosphoinositide (PI) metabolism resulting from its potent inhibition of myoinositol 1-phosphatase (M1P) is. This enzyme converts myoinositol 1-phosphate, an intermediate in the breakdown of inositol phosphates, into inositol, which is required for synthesis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP 2 plays an important role in signal transduction since it is hydrolyzed into second messengers by ligandactivated phospholipase C (PLC). This leads to the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular Ca 2+, and diacylglycerol (DAG), a potent activator of the serine and threonine kinase protein kinase C (PKC) 4,35. It has been suggested that lithium's inhibition of M1P could lead to a depletion of inositol and PIP2, resulting in reduced PI-mediated neuronal responses; a mechanism referred to as the 'inositol depletion hypothesis '5,25. Other investigators have suggested that lithium may act by inhibiting G-proteins 2. These ubiquitous membranebound proteins play a critical role in signal transduction,
since they are coupled to receptors linked to the regulation of PLC, adenylate cyclase and arachidonic acid 3'14'32. The lithium inhibition of agonist-stimulated cyclic AMP (cAMP) that some investigators have described is thought to be due to G-protein (Gs)- adenylate cyclase uncoupling 2,34. Because of the putative inhibitory influence of lithium on second messenger systems, we have been interested in its potential effect on activation of the los protooncogene 1°. Fos encodes a protein that binds to the product of the jun proto-oncogene to form the AP-1 transcription factor s,3°,37,38. Fo$ gene transcription is rapidly induced by agonists that stimulate PKC and cAMP as a result of binding to the cAMP and phorbol ester DNA-responsive elements located in the gene's 5' regulatory domain 15,29,4°,41. Contrary to the proposed inhibitory action on receptor-mediated PKC and cAMPcoupled events, lithium was found to have a dramatic augmenting effect on los expression in PC12 pheochromocytoma cells23. This effect appears to be specific for receptor and post-receptor activators of PKC since lithium augmented los expression induced by ligands that induce PIP 2 hydrolysis, such as carbachol, bradykinin and nerve growth factor, as well as phorbol esters that directly activate PKC. By contrast, lithium has no effect on los expression induced by receptor or post-receptor
Correspondence: H.M. Lachman, Department of Psychiatry: Program of Behavioral Genetics, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, U.S.A.
118 activation of adenylate cyclase 23 (Divish et al., in press). A l t h o u g h lithium enhances los expression induced by a n u m b e r of P L C / P K C coupled agonists, the m l muscarinic cholinergic r e c e p t o r system was found to be particularly sensitive 23. Similarly, A r e n a n d e r et al. found that lithium a u g m e n t e d los expression in cultured rat astrocytes 1. Since one m o d e l for mania suggests that r e d u c e d cholinergic discharge leads to an imbalance in cholinergic and adrenergic neurotransmission, the augmenting effect of lithium on a muscarinic cholinergic target m a y have therapeutic relevance 22. To further investigate the effect of lithium on muscarinic cholinergic systems, we analyzed los expression in rat cortex induced by t r e a t m e n t with pilocarpine, a centrally active muscarinic agonist. Fos gene expression is induced in specific brain regions by a variety of experimental manipulations including induction of generalized seizures ( E C S ) , exposure to environmental light and sensory stimulation 21'24'31. We now show that los m R N A can also be induced in the rat cortex following treatment with pilocarpine. F u r t h e r m o r e , p r e t r e a t m e n t with lithium chloride enhances this effect, similar to the findings for PC12 cells and astrocytes 1'23. The augmentation of a muscarinic cholinergic response in the CNS, at concentrations of lithium within a therapeutically relevant range, is consistent with its anti-manic action according to the cholinergic m o d e l and supports the hypothesis that lithium's effect on gene expression may be an important e l e m e n t in understanding its therapeutic effectiveness. MATERIALS AND METHODS
Drug administration Male Sprague-Dawley rats (Charles River Laboratories) weighing 200-250 g at the start of the experiments were housed in the animal facility under conditions of uniform light, temperature and humidity (14-h day, 10-h night, 23 °C ambient temperature, 50% relative humidity). Food and water were provided ad libitum. As acute treatment, rats received intraperitoneal (i.p.) injections of lithium chloride (0.75 M solution, 3 meq/kg), or an equivalent amount of sodium chloride as a control. After 7 h, pilocarpine (5 mg/kg in saline) or vehicle was administered i.p. For chronic treatment, rats received 3 meq/kg/day subcutaneously (s.c.), in two divided doses for 5-6 days. Animals treated chronically with lithium were also provided with a water bottle containing 0.9% sodium chloride. Pilocarpine or vehicle were then administered 12 h after the last injection of lithium. The animals were sacrificed by decapitation 75 min after pilocarpine administration. Trunk blood was collected for serum lithium determination and the hippocampus and cortex were removed on a dissecting block that had been stored at -20 °C. The tissue was placed in prelabeled plastic bags and rapidly frozen in liquid nitrogen in order to limit RNA degradation. Samples were stored at -80 *C prior to RNA extraction. Electroconvulsive shock was administered through ear clip electrodes using an ECT generator set at 50 Hz delivering 1 s of electric current. Tonic-clonic seizures lasting 20-30 s were observed. The animals were sacrificed 75 min later and total cellular RNA was extracted from the cortex, hippocampus and cerebellum.
RNA extraction RNA was extracted using guanidine isothiocyanate as a denatur-
ant. Frozen cortical and hippocampal samples were homogenized in 15 and 5 ml respectively of solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl and 0.1 M 2-mercaptoethanol). An equal volume of H20-saturated phenol, one-tenth volume chioroform:isoamyl alcohol and 40 mM sodium acetate, pH 4.0 were added to the homogenate. The samples were shaken vigorously for 10 s, placed on ice for 30 min and the RNA recovered from the aqueous phase by sequential isopropyl alcohol and ethanol precipitations. The final RNA pellet was resuspended in TE buffer (10 mM Tris, pH 7.4 and 1 mM EDTA, pH 8.0). The concentration and purity of the samples were determined by the measuring the absorption of an aliquot at 260 and 280 nm.
RNA hybridization analysis 20-40/~g of total cellular RNA was separated by 0.9% agaroseformaldehyde gel electrophoresis and transferred to nylon filters (Nytran, Schleicter and Schuell) as previously described23. Filters were hybridized with 32P-labelled probes using a nick translation kit (Bethesda Research Labs). The recombinant plasmids used in this experiment included pMMMfos3°, a mouse genomic clone, and pGAPDH 36, a glyceraldehyde phosphate dehydrogenase cDNA. DNA inserts, isolated by electrophoresis through low melting point agarose, were labelled to specific activities of 1-3 x 108 cpm//zg DNA. Following overnight hybridization at 60 °C, the filters were washed free of non-specifically bound counts as previously described23 and exposed to X-ray film with two image-intensifying screens. Autoradiograms were developed after 0.5-7 days. Densitometric analysis was performed on autoradiograms within the linear range of exposure using a Quantimet 920 system. The quantitative data was stored in an Excel microsoft database from which the mean, standard error and Student's t-test were derived.
RESULTS
Induction of los mRNA by pilocarpine The cortex contains a b u n d a n t muscarinic cholinergic receptors of both the m l and m2 subtypes. The m l receptors are coupled to P L C / P K C activation and are found primarily on dendrites and cell bodies, whereas m2 receptors, which inhibit a d e n y l a t e cyclase, are primarily presynaptic 16. Since m l receptors are located on cell bodies and are coupled to P K C , we r e a s o n e d that its activation would result in the induction of los m R N A . A n i m a l s were injected with 5 mg/kg pilocarpine in saline, or with saline alone. A f t e r 75 min, the animals were sacrified by decapitation and total cellular R N A was extracted from dissected cortical tissue. T h e R N A was analyzed by N o r t h e r n filter hybridization as described in Methods. A s seen in Fig. 1, los m R N A is not easily detected in the cortex in the absence of pilocarpine under the conditions of our R N A assay (lane 1). H o w e v e r , following t r e a t m e n t with pilocarpine, the 2.2 kb los m R N A band is d e t e c t e d (lane 2). In o r d e r to d e t e r m i n e whether the induction is due to activation of an m l receptor subtype, the animals were p r e t r e a t e d with pirenzepine (PZ) (100 mg/kg), a muscarinic antagonist that exhibits high affinity for the m l r e c e p t o r 19. A s seen in lane 3, t r e a t m e n t with P Z reduces the pilocarpinem e d i a t e d induction of fos m R N A by 3-fold, suggesting an effect m e d i a t e d primarily by the m l r e c e p t o r subtype.
119
Cort 123
Hipp 123
los--
GAPDH-
)
Fig. 1. Effect of pilocarpine on fos expression. Male SpragueDawley rats were treated with 5 mg/kg pilocarpine i.p. in saline and sacrificed 75 min later. The cortex and hippocampus were dissected and total cellular RNA was extracted. The RNA was analyzed by Northern hybridization analysis using fos and GAPDH specific probes. Lane 1: vehicle; lane 2: pilocarpine; lane 3: pilocarpine following pretreatment for 15 min with pirenzepine (100 mg/kg). Autoradiographic exposure times for fos: cortex-2 days, hippocampus-7 days. Prolonged autoradiographic exposure did not result in the detection of fos mRNA in untreated hippocampal controls. Experiments repeated on 3 sets of animals (control, pilocarpine and pilocarpine plus pirenzepine constitutes a set). Similar results were o b t a i n e d in the hippocampus, except that the level of los m R N A was a p p r o x i m a t e l y onetwentieth that found in the cortex. A s a control for the equal loading and transfer of R N A , the filters were r e p r o b e d with GAPDH, which is expressed constitutively in most cell types 36. A s seen in Fig. 1, the changes in los m R N A that occur u p o n pharmacological manipulation with muscarinic agonists and antagonists are not a c c o m p a n i e d by similar changes in GAPDH expression.
similar e n h a n c e m e n t could be d e t e c t e d in animals. We focussed primarily on cortical los expression, since induction by pilocarpine is a p p r o x i m a t e l y 20-fold greater than in the h i p p o c a m p u s . M a l e S p r a g u e - D a w l e y rats were t r e a t e d with a single i.p. injection of lithium chloride (3 meq/kg) or an equivalent dose of sodium chloride. Seven h later, pilocarpine was injected and the animals were sacrificed after an additional 75 min. The average lithium level o b t a i n e d from trunk blood at the time of sacrifice ranged b e t w e e n 0.45-0.81 meq/1, a concentration similar to the t h e r a p e u t i c dose in humans 6. This strategy was e m p l o y e d because intracellular lithium levels p e a k in a n u m b e r of brain loci after a p p r o x m a t e l y 8 h 33 and fos induction occurs rapidly following agonist stimulation 9"23. A s seen in Fig. 2, p r e t r e a t m e n t with lithium c h l o r i d e ( + ) results in an increase in pilocarpineinduced los m R N A c o m p a r e d to sodium c h l o r i d e ( - ) and pilocarpine-treated controls. D e n s i t o m e t r i c analysis revealed that in this e x p e r i m e n t the e n h a n c e m e n t was a p p r o x i m a t e l y 2-fold. These changes are specific for los since we did n o t detect a similar increase in the expression of the GAPDH m R N A control. F u r t h e r m o r e , t r e a t m e n t with lithium alone does not result in d e t e c t a b l e levels of los m R N A under the conditions of the N o r t h e r n hybridization analysis ( d a t a not shown). In o r d e r to d e t e r m i n e the specificity of the lithium-
Effect of lithium on los induction Since lithium enhances fos expression induced by muscarinic cholinergic activation in cultured neuronal cells 1"23, we were interested in determining whether a
3 .c
PILO
Cort
Cort Hipp
Li: fos - -
.=
ECS
2
Cb
T,
1
mR Na-Acute
GAPDH-Fig. 2. A: effect of lithium on pilocarpine and ECS-induced los expression. Rats were treated with a single i.p. injection of lithium chloride (+) or and equivalent amount of sodium chloride (-) (3 meq/kg). After 7 h, pilocarpine was administered and the animals were sacrificed 75 min later. RNA was extracted from the cortex and analyzed as in Methods and Fig. 1. Alternatively, animals were treated with lithium or sodium chloride and a single electroconvulsive shock (ECS) was induced by a generator through ear clip electrodes. Seventy-five min later, RNA was extracted from the cortex, hippocampus and cerebellum (Cb). The ECS experiments were performed on 3 sets of animals.
U-Acute
Na-Chronic
Li-Chronic
Fig. 3. Accumulative analysis. Rats (n = 7) were treated with pilocarpine and lithium or sodium chloride (control) as in Fig. 1 (acute). In addition, 5 other rats were treated for 5-6 days with lithium or sodium chloride (3 meq/kg/day in 2 divided doses, s.c.), and pilocarpine was administered 12 h after the last dose. Northern filters were analyzed for fos and GAPDH mRNAs. Autoradiograms within the linear range were quantitated by densitometric scanning and thefos/GAPDH ratio determined. The mean of the controls has been arbitrarily established as 1.00 and the relative increase induced by lithium was calculated. Fos/GAPDHratio, acute control: 1.00 _+ 0.13, acute lithium: 2.74 __ 0.59, chronic control: 1.00 _+ 0.19, chronic lithium: 1.73 _+ 0.25. The asterisk denotes statistical significance compared to the controls (P < 0.05, one-tailed Student's t-test).
120 augmenting effect in the brain, we induced los mRNA using another strategy; administration of a single electroconvulsive shock (ECS). Although the mechanism of los induction by ECS has not been clearly elucidated, it is likely due to widespread depolarization and C a 2+ influx which are known to activate los gene expression in PC12 cells 17. As seen in Fig. 2, ECS induces the accumulation of los mRNA in the cortex, hippocampus and cerebellum. In contrast to the effects observed in pilocarpinetreated animals, we do not detect significant changes in the level of ECS stimulated los mRNA in the presence of lithium. Because of potential variation in pilocarpine responsiveness that may occur in individual animals, we analyzed los mRNA in a number of animals. Autoradiograms within the linear range of exposure were quantitated on a Quantimet 920 system and the fos/GAPDH signal ratio was determined to factor out potential loading and transfer artifacts. As seen in the cumulative analysis (Fig. 3), acute lithium treatment increased pilocarpine-induced cortical los mRNA by approximately 2.7 fold (n = 7, P < 0.05). Since the therapeutic action of lithium in patients with manic-depression takes approximately one week 6, we also examined the effect of chronic lithium treatment on los expression. According to the 'inositol depletion hypothesis', an inhibitory effect of lithium on the phosphoinisitide (PI) pathway, caused by inositol and PIP E depletion, would not be observed until pools of PIP E synthesized prior to lithium treatment are exhausted. This would not be expected to occur in acutely treated animals. Consequently, rats were treated for 5-6 days with 3 meq/kg/day lithium chloride or sodium chloride administered in 2 divided doses s.c. Twelve h after the final dose, animals were treated with pilocarpine for 75 min, cortical tissue was dissected and analyzed for fos mRNA. As seen in Fig. 3, chronic lithium treatment augments los expression by 1.7-fold, similar to the results obtained in acutely treated animals (n = 5, P < 0.05). DISCUSSION Although a number of models have been proposed, the molecular and genetic factors that result in the vulnerability to manic-depression are still not well understood. These models are based, in part, on the behavioral effects of centrally active drugs, and the putative molecular basis lithium's action. For example, Janowsky et al. 22 and Dilsaver 1~ developed the cholinergic hypothesis which postulates that reduced cholinergic drive, resulting in an imbalance of cholinergic and adrenergic neurotransmission, plays a role in the development of mania. The enhancing effect of lithium on
cholinergic pathways, which we and other investigators have described, is consistent with its anti-manic action according to this model 1"23"26. It has also been suggested that manic-depression could be caused by an overactive neurotransmitter or neuropeptide system linked to a PI pathway 5'25. Consequently, reduced inositol phosphate recycling, resulting from lithium's inhibition of myoinositol 1-phosphatase (MIP), would lead to PIP E depletion and attenuation of overactive, PI-mediated neuronal activity. The enhancing effect on los gene expression, described in this and other reports, may represent a novel mechanism of lithium's therapeutic action. In PC12 cells, lithium augmentation is specific for agonists that stimulate los by activating PKC. By contrast, no enhancing effect is detected upon induction by agonists that stimulate cAMP synthesis 23 (Divish et al., in press). Since los expression induced by phorbol esters is also augmented by lithium, the findings suggested that lithium may act on the pathway leading from PKC activation to gene expression, rather than at the level of receptor-PKC coupling. However, the molecular basis of this effect has not been clearly elucidated. Using concentrations of lithium within the therapeutic range, the enhancing effect on los mRNA in PC12 cells has only been observed following muscarinic cholinergic stimulation. Other PKC stimulating agonists require 5- to 10-fold higher levels of lithium 23. We now find that therapeutic concentrations of lithium also augments pilocarpine-induced cortical los expression. This effect appears to be mediated by an ml receptor subtype linked to PIP 2 hydrolysis and PKC activation since it is blocked by pirenzepine (PZ). While PZ has been shown to inhibit ml and m2 receptors, it has a higher affinity for the ml subtype 19. Although the cortex contains both subtypes, m2 is primarily a presynaptic autoreceptor linked to the inhibition of cAMP. Stimulation of this system would not be expected to induce fos expression since cAMP actually activates the gene 15'23'4°'41. Also, autoreceptors in the synaptic terminal that reduce transynaptic signalling would be an unlikely target for agonists that stimulate gene expression in the nucleus. Regardless of the mechanism, the enhancement of los expression by therapeutic concentrations of lithium supports the hypothesis that gene expression may be an important target of its effect in mood disorders. The augmentation of cholinergic-mediated los expression is consistent with the in vivo enhancing effect of lithium observed by other investigators26"39. However, it contrasts with the inhibitory effect described by Worley et al. in ex vivo hippocampal slices45. One explanation consistent with these observations is that lithium exhibits both inhibitory and excitatory effects on PI-mediated
121 signal transduction. Perhaps under conditions that favor inositol depletion, such as repetitive agonist stimulation in the presence of lithium, as used in the Worley experiment, inhibition of events mediated by PIP 2 hydrolysis would occur. However, in the absence of inositol depletion, one would observe augmentation of a PIP 2mediated target (such as fos) through lithium's putative effect on PKC-mediated gene expression. Alternatively, an inhibitory effect of lithium on the PI pathway could be due to an action at the G-protein level, as suggested by several investigators 2'34, since subtypes of this family of proteins regulate PLC. In this model, the intracellular concentration of lithium would determine whether inhibition of a G-protein linked to PLC, or augmentation of the C-kinase/fos pathway occurs. Although we have used fos expression as a convenient experimental marker to study the effect of lithium on PI-mediated responses in the brain, it is interesting to speculate on the potential role of gene expression in its therapeutic action. A number of genes expressed in the CNS, such as those encoding some neuropeptides, contain AP-1 and phorbol ester-responsive domains that could serve as targets for lithium's effect on PKC-induced fos expression 43. The observation by D o b n e r et al., that lithium enhances the expression of neurotensin/neurome-
REFERENCES 1 Arenander, A.T., De Vellis, J. and Herschman, H.R., Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters, J. Neurosci. Res., 24 (1989) 107-114. 2 Avissar, S., Schreiber, G., Danon, A. and Belmaker, R.H., Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex, Nature, 331 (1988) 440-442. 3 Axelrod, J., Burch, R.M. and Jelserra, C.L., Receptor mediated activation of phospholiplase A 2 via GTP binding protein: arachidonic acid and its metabolites as second messengers, Trends Neurosci., 11 (1988) 117-127. 4 Berridge, M. and Irvine, R.E, Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature, 312 (1984) 315-321. 5 Berridge, M.J. and Irvine, R.E, Inositol phophates and cell signalling, Nature, 341 (1989) 197-205. 6 Braastroup, EC., Poulsen, J.C., Schou, M., Thomsen, K. and Amidsen, A., Prophylactic lithium: double-blind discontinuation in manic-depression and recurrent-depressive illness, Lancet, 2 (1970) 326-330. 7 Buckley, N.J., Bonner, T.I. and Brann, M.R., Localization of a family of muscarinic receptor mRNAs in rat brain, J. Neurosci., 8(12) (1988) 4646-4652. 8 Chiu, R., Boyle, W.J., Meek, J., Smeal, T., Hunter, T. and Karin, M., The c-los proto-oncogene interacts with c-]un/AP-1 to stimulate transcription from AP-l-sensitive genes, Cell, 54 (1988) 541-552. 9 Curran, T. and Morgan, J.I., Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines, Science, 229 (1985) 1265-1268. 10 Curran, T. and Teich, N.M., Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000 dalton phosphoprotein, J. Virol., 42 (1982) 114-122. 11 Davis, J.L., Nakajima, T., Gieiter, C.H., Post, R.M. and
din N, a phorbol ester responsive gene, could be explained by an effect at the P K C and/or los level 13. Preliminary results from our lab, indicating that acute treatment with lithium increases the expression neuropeptide Y m R N A , and the findings by several groups that lithium increases the expression of other neuropeptide encoding genes 28'42, are also consistent with this hypothesis. Some of the behavioral and neuroendocrine abnormalities found in m o o d disorders are thought to be due to abnormal expression of neuropeptides 2°. Furthermore, neuroendocrine stressors that function by influencing gene expression, such as glucocorticoids 46, are capable of causing mood disturbances in vulnerable individuals 44. Consequently, the effect of lithium on gene expression appears to be a particularly relevant target for further experimental investigation.
Acknowledgements. The Program in Behavioral Genetics is supported by the Ruane Family Fund and the G. Harold and Leila Y. Mathers charitable foundation. H.M.L. is a fellow of the Irma T. Hirschl and Monique Weill-Caulier charitable trust. D.EP is a NARSAD young investigator. The authors wish to thank Thelma ParAs for performing serum lithium determinations. We would also like to acknowledge Dr. Herman van Praag for his encouragement and support in estabhshing the Program of Behavioral Genetics.
Marangos, e.J., Mouse brain c-fos mRNA distribution following
a single electroconvulsive shock, J. Neurochem., 52 (1989) 1954-1957. 12 Dilsaver, S.C., Cholinergic mechanisms in depression, Brain Res. Rev., 11 (1986) 285-316. 13 Dobner, P.R., Tischler, A.S., Lee, Y.C., Bloom, S.R. and Donahue, S.R., Lithium dramatically potentiates neurotensin/ neuromedin N gene expression, J. Biol. Chem., 263 (1988) 13983-13986. 14 Gilman, A.G., G-proteins and regulation of adenylate cyclas¢, JAMA, 262 (1989) 1819-1825. 15 Gilman, M.Z., Wilson, R.N. and Weinberg, R.A., Multiple protein binding sites in the 5' flanking region regulate c-fos expression, Mol. Cell. Biol., 6 (1986) 4305-4314. 16 Goyal, R.K., Muscarinic receptor subtypes, New Engl. J. Med., 321 (1989) 1022-1029. 17 Greenberg, M.E., Ziff, E.B. and Greene, L.A., Stimulation of neuronal acetylreceptors induces rapid gene transcription, Science, 234 (1986) 80-83. 18 Halicher, L. and Sherman, W.R., The effects of lithium ion and other agents on the activity of myo-inositol-l-phosphatase from bovine brain, J. Biol. Chem., 261 (1980) 8100-8130. 19 Hammer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C., Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature, 283 (1980) 90-92. 20 Heilig, M., Soderpalm, B., Engel, J.A. and Widerlov, E., Centrally administered neuropeptide Y produces anxiolytic-like effects in animal anxiety models, Psychopharmacology, 98 (1989) 524-529. 21 Hunt, S.P., Pini, A. and Evan, G., Induced activation of c-los like protein in spinal neurons following sensory stimulation, Nature, 328 (1987) 632-634. 22 Janowsky, D.S., Risch, S.C., Parker, D., Huey, L.Y. and Judd, L., Increased vulnerability to cholinergic stimulation in affective disorder patients, Psychopharmacol. Bull., 16 (1980) 29-31.16.
122 23 Kalasapudi, V.D., Sheftei, G., Divish, M.M., Papoios, D.E and Lachman, H.M., Lithium augments los proto-oncogene expression in PC12 pheochromocytoma cells: implications for therapeutic action of lithium, Brain Research, 521 (1990) 47-54. 24 Kornhauser, J.M., Nelson, D., Mayo, K.E. and Takahashi, J.S., Photic and circadian regulation of c-los gene expression in the hamster superehiasmatic nucleus, Neuron, 5 (1990) 127-134. 25 Lachman, H.M. and Papolos, D.E, Abnormal signal transduction: a hypothetical model for bipolar affective disorder, Life Sci., 45 (1989) 1413-1426. 26 Lerer, B. and Stanley, M., Effect of lithium on cholinergically mediated responses and [3H]QNB binding in rat brain, Brain Research, 344 (1985) 211-219. 27 Levy, A., Zohar, J. and Belmaker, R.H., The effect of chronic lithium pretreatment on rat brain muscarinic receptor regulation, Neuropsychopharmacology, 21 (1983) 1199-1201. 28 Mathe, A.A., Jousisto-Hanson, J., Stenfors, C. and Theodorsson, E., Effect of lithium on tachykinins, calcitonin gene-related peptide, and neuropeptide Y in rat brain, J. Neurosci. Res., 26 (1990) 233-237. 29 Mellon, EL., Clegg, C., Correll, L.A. and McKnight, G.S., Regulation of transcription by cAMP-dependent protein kinase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 488-491. 30 Miller, A.D., Curran, T. and Verma, I.M., C-los protooncogene can induce transformation: a novel mechanism of activation of a cellular oneogene, Cell, 36 (1984) 51-60. 31 Morgan, J.I. Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping pattern of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 32 Moriarty, T.M., Giilo, B., Carty, B.J., Premont, R.T., Landau, E.M. and Iyengar R., Beta-gamma subunit of GTP binding proteins inhibits muscarinic receptor stimulation of phospholipase C, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 8865-8869. 33 Mukherjee, B.P., Bailey, P.T. and Pradhan, S.N., Temporal and regional differences in brain concentrations of lithium in rats, Psychopharmacology, 48 (1976) 119-121. 34 Newman, M.E. and Belmaker, R.H., Effects of lithium in vitro and ex vivo on components of the adenylcydase system in membranes from cerbral cortex of the rat, Neuropharmacology, 26 (1987) 211-217. 35 Nishizuka, Y., Studies and perspectives of protein kinase C, 233
(1986) 305-312. 36 Piechaczyk, M., Blanchard, J.M., Marty, L., Dani, C., Panatieres, E, EI-Sabouty, S., Fort, P. and Jeanteur, P., Posttranscriptional regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression in rat tissue, Nucleic Acids Res., 12 (1984) 6951-6963. 37 Rauscher, F.J., Cohen, D.R., Curran, T., Bos, T.J., Vogt, P.K., Bohmann, D., Tijan, R. and Franza, R., Fos-associated protein p39 is the product of the jun proto-oncogene, Science, 240 (1988) 1010-1016. 38 Rauscher, EJ., Sambucetti, L.C., Curran, T., Distel, R.J. and Spiegalman, B.M., Common DNA binding site for los protein complexes and transcription factor AP-1, Cell, 52 (1988) 471-480. 39 Roth, P., Hamburder-Bar, R. and Lerer, B., Peripheral vs central manifestations in toxic interaction of lithium and pilocarpine, Biol. Psychiatry, 25 (1989) 153-158. 40 Sassone-Corsi, P., Visvader, J., Ferland, L., Mellon, P.L. and Verma, I.M., Induction of proto-oncogene los transcription through the adenylate cyclase pathway. Characterization of a cAMP responsive element, Genes Dev., 2 (1988) 1529-1538. 41 Sheng, M. and Greenberg, M., The regulation and function of c-los and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 42 Sivam, S.P., Krause, J.E., Takeuchi, K., Li, S., McGinyy, J.F. and Hong, J.-S., Lithium increases rat striatal beta and gamma preprotachykinin messenger RNA's, 1. Pharm. Exp. Ther., 248 (1989) 1297-1301. 43 Sonnenberg, J.L., Rauscher, F.J., Morgan, J.I. and Curran, T., Regulation of proenkephalin by fos and jun, Science, 246 (1989) 1622-1625. 44 Wolkowitz, O.M., Rubinow, D., Doran, A.R., Breier, A., Berrettini, W.H., Kling, M.A. and Pickar, D., Prednisone effects on neurochemistry and behavior, Arch. Gen. Psychiatry, 47 (1990) 963-968. 45 Worley, P.F., Heller, W.A., Snyder, S.H. and Baraban, J.M., Lithium blocks a phosphoinositide-mediated cholinergic response in hippocampal slices, Science, 239 (1988) 1428--1429. 46 Yamamato, K.R., Steroid receptor-regulated transcription of specific genes and gene networks, Ann. Rev. Genet., 19 (1985) 209-252.
