Effect of neuronal excitotoxicity on Munc18-1 distribution in nuclei of rat hippocampal neuron and primary cultured neuron

Abstract

Objective Munc18-1 has an important role in neurotransmitter release, and controls every step in the exocytotic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Munc18 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid (KA) solution into hippocampus of Sprague-Dawley (SD) rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immunochemistry and immunoelectron microscopy with anti-Munc18-1 antibody were used to determine the nuclear localization of Munc18-1. Immunoblotting was used to detect the protein level of Munc18-1. Results The localization of Munc18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunoblotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Munc18-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expression level of Munc18-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relationship between the change of Munc18-1 expression in neuronal nuclei and neuronal over-activation was also tested in primary cultured neurons. After treatment with 50 μmol/L glutamate acid for 3 h, Munc18-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excitatory stimulation can induce the distribution change of Munc18-1 in neuron, which may subsequently modulate neuronal functions in brain.

Neuronal hibernation following hippocampal demyelination

“…Six transcripts were decreased by demyelination, and reductions in five of these have been associated with decreased neuronal activity (Htr3A [ 60 ], CNIH2 [ 61 ], loss of LTP (Drd5 [ 62 ] and Efnb3 [ 63 ], and altered cognitive function (Htr3A [ 60 ] Drd5 [ 62 ], Efnb3 [ 63 ], CNIH2 [ 61 ], and GABARa5 [ 64 ]). The thirteen transcripts increased by demyelination are associated with a variety of neuronal functions including gene transcription (ETV5 [ 65 ] and MEF2c [ 66 ], neurotransmitter receptors (NTRK2 [ 67 ], GRIK3 [ 68 ], and HTR2C [ 69 ]), synaptic vesicle docking (Stxbp1 [ 70 ], RIMS4 and RIMS3 [71;72]), G protein signaling (GNAI2 [ 73 ] and CRK1 [ 74 ]) and dendritic development (ETV5 [ 65 ] and CRK1 [ 74 ]).…”

Local regulation and function of importin-β1 in hippocampal neurons during transcription-dependent plasticity

“…On the similar vein, Stxbp1, one of the 83 shortlisted candidate protein, has also been indirectly reported to exit the nucleus to the cytoplasm following glutamatergic stimulation (Fig. S7) (Zhang et al, 2011).…”