Research on Expression and Function of Phosphorylated DARPP-32 on Pentylenetetrazol-induced Epilepsy Model of Rat_Journal of Biomedical Engineering

Authors：

WANGWeiwen ¹ ,  LIAOXiaoyang ² , YANGZhenghui ¹ , LINHang ¹ , WANGQingsong ¹ , WUYuxian ¹ , LIUYu ¹

1. Department of Neurology, Chengdu Military General Hospital, Chengdu 610083, China;
2. General Practice Unit, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

LIAOXiaoyang, Email: liaoxiaoyang@sohu.com

Keywords：

phosphorylated DARPP-32; epilepsy; pentylenetetrazol; rat

DOI：

10.7507/1001-5515.20140119

Video：

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The present study is to explore the change process and distribution of phosphorylated DARPP-32 (p-DARPP-32) in rat brain including cortex, hippocampus and striatum and to further deduce whether p-DARPP-32 was possibly involved in epilepsy induced by repetitive low doses of pentylenetetrazol (PTZ). PTZ-induced epilepsy model in rat was established with 30 male SD rats randomly divided into 6 groups, control group and five trial groups [PTZ 1 h,PTZ 6 h,PTZ 24 h,PTZ 48 h and PTZ 72 h respectively, after onset of status epilepticus (SE)]. Immunohistochemistry and immunofluorescence double-labeling were used to detect the temporal time change and distribution of p-DARPP-32 expression and to analyze the coexpression of DARPP-32 and p-DARPP-32 in rat brain after the onset of PTZ-induced generalized SE. The results showed that there was a temporal time change of p-DARPP-32 expression in rat brain after the onset of SE. The number of p-DARPP-32-positive cells increased significantly and reached the peaks at the ends of 1 hour and 6 hours after the onset of SE, but decreased at the end of 24 hours. The moderate to strong p-DARPP-32-immunopositive neurons were observed in cortex, hippocampus and striatum, and located in cell cytoplasm and cell nucleus. Further immunofluorescence double-labeling revealed that denser colocalization of p-DARPP-32 and DARPP-32 in the neurons existed in the area mentioned above. Therefore, PTZ-induced SE may cause phosphorylation of DARPP-32 in rat brain. The temporal time change and distribution of p-DARPP-32 suggest that phosphorylation of DARPP-32 may be involved in PTZ-induced epilepsy in rat brain including cortex, hippocampus and striatum, and p-DARPP-32 may play a central role in the onset of SE.

Citation： WANGWeiwen, LIAOXiaoyang, YANGZhenghui, LINHang, WANGQingsong, WUYuxian, LIUYu. Research on Expression and Function of Phosphorylated DARPP-32 on Pentylenetetrazol-induced Epilepsy Model of Rat. Journal of Biomedical Engineering, 2014, 31(3): 637-641. doi: 10.7507/1001-5515.20140119 Copy

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2.	SHAROPOV S, MOSER J, CHEN Rongqing, et al. Dopaminergic modulation of low-Mg²⁺-induced epileptiform activity in the intact hippocampus of the newborn mouse in vitro[J]. J Neurosci Res, 2012, 90(10):2020-2033.
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6.	FLORES-HERNANDEZ J, HERNANDEZ S, SNYDER G L, et al. D(1) dopamine receptor activation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/DARPP-32/PP1 signaling cascade[J]. J Neurophysiol, 2000, 83(5):2996-3004.
7.	NEHLIG A, PEREIRA DE VASCONCELOS A. The model of pentylenetetrazol-induced status epilepticus in the immature rat:short- and long-term effects[J]. Epilepsy Res, 1996, 26(1):93-103.
8.	GUBITS R M, HAZELTON J L, SIMANTOV R. Variations in c-fos gene expression during rat brain development[J]. Brain Res, 1988, 427(2):197-201.
9.	WANG W W, CAO R, RAO Z R, et al. Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats[J]. Brain Res, 2004, 998(2):174-183.
10.	DAZZI L, SERRA M, PORCEDDU M L, et al. Enhancement of basal and pentylenetetrazol (PTZ)-stimulated dopamine release in the brain of freely moving rats by PTZ-induced kindling[J]. Synapse, 1997, 26(4):351-358.
11.	BOURNE J A, FOSBRAEY P, HALLIDAY J. Changes in striatal electroencephalography and neurochemistry induced by kainic acid seizures are modified by dopamine receptor antagonists[J]. Eur J Pharmacol, 2001, 413(2-3):189-198.
12.	HEMMINGS H C Jr, GREENGARD P, TUNG H Y, et al. DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1[J]. Nature, 1984, 310(5977):503-505.
13.	CEPEDA C, COLWELL C S, ITRI J N, et al. Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons[J]. Eur J Neurosci, 1998, 10(11):3491-3497.
14.	SNYDER G L, FIENBERG A A, HUGANIR R L, et al. A dopamine/D1 receptor/protein kinase A/dopamine- and cAMP-regulated phosphoprotein (Mr 32 kDa)/protein phosphatase-1 pathway regulates dephosphorylation of the NMDA receptor[J]. J Neurosci, 1998, 18(24):10297-10303.
15.	KOÓS T, TEPPER J M. Inhibitory control of neostriatal projection neurons by GABAergic interneurons[J]. Nat Neurosci, 1999, 2(5):467-472.

1. REKTOR I, KUBA R, BRÁZDIL M, et al. Do the basal ganglia inhibit seizure activity in temporal lobe epilepsy?[J]. Epilepsy Behav, 2012, 25(1):56-59.
2. SHAROPOV S, MOSER J, CHEN Rongqing, et al. Dopaminergic modulation of low-Mg²⁺-induced epileptiform activity in the intact hippocampus of the newborn mouse in vitro[J]. J Neurosci Res, 2012, 90(10):2020-2033.
3. ROCHA L, ALONSO-VANEGAS M, VILLEDA-HERNÁNDEZ J, et al. Dopamine abnormalities in the neocortex of patients with temporal lobe epilepsy[J]. Neurobiol Dis, 2012, 45(1):499-507.
4. HAUT S R, ALBIN R L. Dopamine and epilepsy:hints of complex subcortical roles[J]. Neurology, 2008, 71(11):784-785.
5. GREENGARD P, NAIRN A C, GIRAULT J A, et al. The DARPP-32/protein phosphatase-1 cascade:a model for signal integration[J]. Brain Res Rev, 1998, 26(2-3):274-284.
6. FLORES-HERNANDEZ J, HERNANDEZ S, SNYDER G L, et al. D(1) dopamine receptor activation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/DARPP-32/PP1 signaling cascade[J]. J Neurophysiol, 2000, 83(5):2996-3004.
7. NEHLIG A, PEREIRA DE VASCONCELOS A. The model of pentylenetetrazol-induced status epilepticus in the immature rat:short- and long-term effects[J]. Epilepsy Res, 1996, 26(1):93-103.
8. GUBITS R M, HAZELTON J L, SIMANTOV R. Variations in c-fos gene expression during rat brain development[J]. Brain Res, 1988, 427(2):197-201.
9. WANG W W, CAO R, RAO Z R, et al. Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats[J]. Brain Res, 2004, 998(2):174-183.
10. DAZZI L, SERRA M, PORCEDDU M L, et al. Enhancement of basal and pentylenetetrazol (PTZ)-stimulated dopamine release in the brain of freely moving rats by PTZ-induced kindling[J]. Synapse, 1997, 26(4):351-358.
11. BOURNE J A, FOSBRAEY P, HALLIDAY J. Changes in striatal electroencephalography and neurochemistry induced by kainic acid seizures are modified by dopamine receptor antagonists[J]. Eur J Pharmacol, 2001, 413(2-3):189-198.
12. HEMMINGS H C Jr, GREENGARD P, TUNG H Y, et al. DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1[J]. Nature, 1984, 310(5977):503-505.
13. CEPEDA C, COLWELL C S, ITRI J N, et al. Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons[J]. Eur J Neurosci, 1998, 10(11):3491-3497.
14. SNYDER G L, FIENBERG A A, HUGANIR R L, et al. A dopamine/D1 receptor/protein kinase A/dopamine- and cAMP-regulated phosphoprotein (Mr 32 kDa)/protein phosphatase-1 pathway regulates dephosphorylation of the NMDA receptor[J]. J Neurosci, 1998, 18(24):10297-10303.
15. KOÓS T, TEPPER J M. Inhibitory control of neostriatal projection neurons by GABAergic interneurons[J]. Nat Neurosci, 1999, 2(5):467-472.

Journal of Biomedical Engineering

Research on Expression and Function of Phosphorylated DARPP-32 on Pentylenetetrazol-induced Epilepsy Model of Rat

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