Group 1 metabotropic glutamate receptors (I-mGluRs) are important neuromodulators of synaptic plasticity and also play a role in aetiology of Alzheimer’s disease. For many years I-mGluRs have been thought to mediate their effects primarily by evoking the mobilisation of intracellular calcium from the neuronal endoplasmic reticulum (ER) via the production of inositol 1,4,5-trisphosphate (IP3), catalysed by the activation of phospholipase C (PLC). However, recent work suggests that in acutely dissociated rat hippocampal neurons I-mGluR-mediated intracellular Ca2+ mobilisation is at least partly dependent on cADP ribose (cADPR)-dependent activation of ryanodine receptors (RyRs)1. It was the aim, therefore, of the current study to determine if I-mGluR –mediated Ca2+ signals within cultured rat hippocampal neurons, previously ascribed to activation of the PLC/IP3 signalling pathway 2, may be mediated via the generation of cADPR.
Experiments were carried out utilising cultured hippocampal neurones obtained from 3-4 day old Sprague Dawley rat pups as previously described 2. Conventional calcium imaging was used to record intracellular somatic calcium measurements from cells loaded with the calcium sensitive dye, fluo -2 AM (150M). Experiments were carried out at room temperature with neurons continuously perfused (2ml/min) with a standard HEPES-buffered saline solution (HBSS). All drugs were added to the perfusate. Data are expressed as means ± S.E.M.
Stimulation of I-mGluRs, using the specific agonist, (S)-3, 5 -dihydroxyphenylglycine (DHPG; 50 µM; 2 min) under control conditions evoked somatic [Ca2+]i signals of 4678 ± 503 units (n = 60) (as determined by measuring area under the curve). Following depolarisation with elevated extracellular K+ (15 mM)-containing HBSS solution, DHPG- evoked [Ca2+]i signals were significantly enhanced relative to controls by 26.1 ± 9.5% (P<0.05; n=60) in line with previous studies on rat hippocampal neurones. Application of the cADPR antagonist nicotinamide (5mM) significantly reduced the amplitude of DHPG- evoked [Ca2+]i signals elicited in 15mM K+-HBSS by 23.8 ± 14.7 (n= 34; P< 0.01).
Application of the IP3R antagonist, 2-APB (250M), did not significantly affect DHPG- evoked [Ca2+]i (n = 17; P = 0.97). However, the PLC inhibitor, U73122 (5M), did significantly inhibit the DHPG- evoked [Ca2+]i signals by 56.5 ± 16.3% (n = 9; P<0.05).
The nicotinamide and 2-APB experiments support the suggestion that I-mGluR-evoked [Ca2+]i signals may be mediated, at least in part, via cADPR-dependent signalling. However, the majority of the DHPG-evoked response is still mediated primarily by generation of IP3 as evidenced by the effect of the PLC inhibitor U73122.
1. Sohn et al., (2011) Plos One, 6 (10), e26625
2. Rae et al., (2000) J. Neurosci., 20, 8628-8636