Abstract
The mood-stabilizing effects of lithium are well documented, although its mechanism of action remains unknown. Increases in gray matter volume detected in patients with bipolar disorder who were treated with lithium suggest that changes in the number of synapses might underlie its therapeutic effects. We investigated the effects of lithium on the number of synaptic connections between hippocampal neurons in culture. Confocal imaging of neurons expressing postsynaptic density protein 95 fused to green fluorescent protein (PSD95-GFP) enabled visualization of synaptic sites. PSD95-GFP fluorescent puncta represented functional synapses, and lithium (4 h, 5 mM) increased their number by 150 ± 12%. The increase was time- and concentration-dependent (EC50 = 1.0 ± 0.6 mM). Lithium induced a parallel increase in the presynaptic marker synaptophysin-GFP. Valproic acid, another mood stabilizer, also increased the number of fluorescent puncta at a clinically relevant concentration. Inhibition of postsynaptic glutamate receptors or presynaptic inhibition of neurotransmitter release significantly reduced lithium-induced synapse formation, indicating that glutamatergic synaptic transmission was required. Pretreatment with exogenous myo-inositol inhibited synapse formation, demonstrating that depletion of inositol was necessary to increase synaptic connections. In contrast, inhibition of glycogen synthase kinase 3β did not mimic lithium-induced synapse formation. Pharmacological and lipid reconstitution experiments showed that new synapses formed as a result of depletion of phosphatidylinositol-4-phosphate rather than a build-up of polyphosphoinositides or changes in the activity of phospholipase C, protein kinase C, or phosphatidylinositol-3-kinase. Increased synaptic connections may underlie the mood-stabilizing effects of lithium in patients with bipolar disorder and could contribute to the convulsions produced by excessive doses of this drug.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA007304, DA024428] and the National Science Foundation [Grant IOS0814549].
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ABBREVIATIONS: IMPase, inositol monophosphatase; IPPase, inositol polyphosphate 1-phosphatase; PSD95, postsynaptic density protein 95; GFP, green fluorescent protein; PSD95-GFP, postsynaptic density protein 95 fused to green fluorescent protein; PtdIns(4)P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-phosphate; GSK-3β, glycogen synthase kinase-3β; NMDA, N-methyl-d-aspartate; MK801, dizocilpine; DMEM, Dulbecco's modified Eagle's medium; HHSS, HEPES-buffered Hanks' salt solution; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; FAK, focal adhesion kinase; FM4-64FX, fixable version of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide; PKC, protein kinase C; ANOVA, analysis of variance; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; L803-mts, N-myristol-GKEAPPAPPQS(p)P; SB216763, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; SB415286, 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione; U73122, 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; Ro-31-8220, 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX); Win 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received September 29, 2008.
- Accepted February 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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