Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB258

Poster Communications

Glutamate and acetylcholine receptor-dependence of optogenetically-induced hippocampal synaptic LTD in vivo

K. J. O'Riordan1, N. Hu1, P. Monahan1, M. J. Rowan1

1. Pharmacology & Therapeutics, Trinity College Dublin, Dublin, Dublin, Ireland.

Recently, we reported that the induction of long-term depression (LTD) by electrical low frequency stimulation (LFS) in the rat hippocampus was blocked by an antagonist of muscarinic acetylcholine (mAChRs), but not NMDA, receptors (Hu et al, Nat Comms 2014). In order to study LTD induction with minimal direct activation of non-glutamatergic inputs, we applied optical excitation of channelrhodopsin selectively expressed in excitatory neurons of male Wistar and Hooded Lister rats (300-330g) in accordance with EU animal legislation. Following transfection with AAV5-CaMKIIa-hChR2(H134R)-EYFP (K. Diesseroth) in the CA3 area of the dorsal hippocampus under recovery anaesthesia (1mL cocktail i.p. injection of ketamine 80 mg/kg and xylazine 8 mg/kg), optically evoked field EPSPs were recorded under urethane anaesthesia (1.5 g/kg i.p.). We compared the ability of optical LFS (900 high intensity pulses, 1 Hz) to induce LTD of optical responses (oLTD) with the ability of similar electrical LFS to induce LTD of electrical responses (eLTD). Values are mean±SEM % baseline at 1 h post-LFS and compared statistically with an unpaired t-test. We examined the possible role of NMDA receptors in LTD induction using CPP (10 mg/kg, i.p.) which strongly inhibited oLTD (88.7 ± 3.7%, n=5, p<0.05 Vs 57.4 ± 6.9% in controls, n=8). The GluN2B selective antagonist, Ro 25-6981 (6 mg/kg, i.p.) also blocked oLTD (98.9 ± 7.3%, n=5, p<0.05 Vs 66.9 ± 5.3% in controls, n=5). Consistent with our previous report, the induction of eLTD was not affected by CPP (64.4 ± 10.1%, n = 10, p>0.05 Vs 63.5 ± 4.2% in controls, n=19). Next, we tested the involvement of metabotropic glutamate 5 (mGlu5) receptors using the antagonist MTEP (3 mg/kg, i.p.). Neither oLTD (67.9 ± 10.4%, n=5, p>0.05 Vs 69.3± 4.7% in controls, n=8) nor eLTD (74.5 ± 8.7%, n=5, p>0.05 Vs 64.3 ± 7.8% in controls, n=9) were significantly affected by pretreatment with MTEP. In order to determine if NMDA and mGlu5 receptors both have the capacity to independently mediate eLTD induction, MTEP was administered with CPP. Consistent with this hypothesis, eLTD was now strongly inhibited (96.7 ± 5.1%, n = 8, p<0.05 Vs 63.5 ± 4.2% in controls, n = 19). In contrast, co-administration of Ro 25-6981 with MTEP, had no effect on eLTD (68.7± 5.9%, n = 7, p>0.05 Vs controls, 63.7 ± 2.2%, n = 5). Finally, similar to our previous findings, pretreatment with the mAChR antagonist scopolamine (0.2 mg/kg, i.p.) also inhibited oLTD (83.2± 5.4%, n=14, p<0.05 Vs controls, 68.7± 3.9%, n=14). These findings indicate that LFS of glutamatergic neurons is sufficient to induce a NMDA receptor-dependent synaptic LTD that also at least partly requires cholinergic activation, most likely due to background tonic cholinergic neuron drive in vivo.

Where applicable, experiments conform with Society ethical requirements