Use of calcium imaging for analysis of neuronal gap junction coupling.
We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36. The results indicate that Ca2+ imaging is a useful tool to quantify the amount of neuronal gap junction coupling in cultures.
Arumugam
Harsha
H
Department of Cell and Molecular Biology, Tulane University, New Orleans, USA.
Denisova
Janna V
JV
Neve
Rachael L
RL
Corriveau
Roderick A
RA
Belousov
Andrei B
AB
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000704:Analysis of Variance; D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D050882:CREB-Binding Protein / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D017630:Connexins / Q000172:deficiency; D003952:Diagnostic Imaging / Q000379:methods*; D004622:Embryo, Mammalian; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D017629:Gap Junctions / Q000378:metabolism*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D006221:Halothane / Q000494:pharmacology; D007033:Hypothalamus, Middle / Q000166:cytology; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000172:deficiency; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology; D000097004:Gap Junction delta-2 Protein
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D000777:Anesthetics; D001640:Bicuculline; D050882:CREB-Binding Protein; D002118:Calcium; D017630:Connexins; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D006034:Glycyrrhetinic Acid; D006221:Halothane; D016194:Receptors, N-Methyl-D-Aspartate; D014633:Valine; D000097004:Gap Junction delta-2 Protein; C056639:2-amino-5-phosphopentanoic acid
J Neurophysiol. 2007 Nov;98(5):2878-86
17855590
Nat Neurosci. 2005 Jan;8(1):61-6
15580271
Science. 1996 May 24;272(5265):1182-7
8638165
Science. 1998 Dec 18;282(5397):2272-5
9856954
J Pharmacol Exp Ther. 1988 Sep;246(3):1104-7
3418512
Biochem Biophys Res Commun. 1986 Jan 14;134(1):29-36
3947327
Trends Neurosci. 2001 Jun;24(6):353-60
11356508
J Neurophysiol. 2002 Sep;88(3):1352-62
12205156
Nat Neurosci. 2005 Dec;8(12):1720-6
16299502
J Neurosci. 1999 Nov 15;19(22):9848-55
10559394
Brain Res. 2000 May 19;865(1):121-38
10814742
J Physiol. 2003 Dec 15;553(Pt 3):729-45
14514879
Neuroreport. 2004 Jan 19;15(1):113-7
15106842
J Neurophysiol. 2004 Aug;92(2):1252-6
15028741
Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2693-8
9122258
J Neurosci. 1997 Feb 15;17(4):1435-46
9006985
J Neurosci. 2001 Mar 15;21(6):2015-27
11245685
Cell. 1994 Feb 11;76(3):427-37
8313466
Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7573-8
10861019
J Neurosci. 1999 Apr 15;19(8):3007-22
10191318
Neuron. 2002 Nov 14;36(4):703-12
12441058
Nat Rev Mol Cell Biol. 2003 Apr;4(4):285-94
12671651
Neuron. 2004 Feb 19;41(4):495-511
14980200