Glutamate is the principal excitatory transmitter within the CNS and is involved in normal brain function as well as long term adaptive responses such as synaptic plasticity and mediates its effects through both ionotropic and metabotropic receptorsGroup 1 metabotropic glutamate receptors (I-mGluRs) are proposed to mediate their effects via activation of phospholipase Cβ, thereby catalysing the formationof inositol-1,4,5 -trisphosphate (IP3) and diacylglycerol. IP3 activates IP3Rs on the neuronal endoplasmic reticulum (ER) membrane, mobilising intracellular calcium from the ER to the cytosol.
Recently however, it has been suggested that I-mGluR-mediated intracellular calcium signals are largely dependent on cyclic ADP ribose (cADPR)-mediated activation of ER-bound ryanodine receptors (RYRs) in acutely dissociated rat hippocampal neurons1. The current study therefore sought to determine if I-mGluR-mediated calcium signals in cultured rat hippocampal neurons were also dependent on cADPR activation of RYRs.
Experiments were carried out utilising cultured primary hippocampal neurons obtained from 3-4 day old Sprague Dawley rat pups as previously described2. Conventional calcium imaging was used to record intracellular somatic calcium signals from cells loaded with the calcium sensitive dye, fluo-2 AM (150μM). Experiments were carried out at room temperature with neurons continuously perfused (2ml/min) with a standard HEPES-buffered saline solution (HBSS). Under control conditions the specific I-mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 50 μM; 2 min) was applied following depolarisation of the neurons with an elevated K+ (10mM) - containing HBSS solution. This protocol was then repeated in the presence of various antagonists of the cADPR-dependent signalling pathway. Data are expressed as means ± S.E.M.
Initial experiments demonstrated that a minimum washout time for DHPG between additions of 50mins was required in order to prevent desensitisation of I-mGluR-mediated responses(n=15). Application of the cADPR antagonist nicotinamide (5mM) significantly reduced the amplitude of DHPG-evoked [Ca2+]i signals elicited in 10mM K+-HBSS by 28 ± 7 % (n= 40; P < 0.05). Similarly, application of the RYR antagonist dantrolene (10µM) significantly reduced the amplitude of DHPG-evoked [Ca2+]i signals elicited in 10mM K+-HBSS by 18 ± 4% (n= 61; P < 0.01). In conclusion these results suggest I-mGluR-evoked [Ca2+]i signals may be mediated, at least in part, via cADPR-dependent activation of RYRs in cultured rat hippocampal neurons.
References
1. Sohn et al.(2011) PMID 22028929
2. Rae & Irving (2000) PMID 11102467