In the dendritic cilia of olfactory receptor neurons (ORNs), cyclic nucleotides link stimulation of G protein-coupled odorant receptors to membrane depolarization by activating cyclic nucleotide-gated channels (CNGCs). CNGCs and odorant receptors may also be expressed in the nerve terminals of ORNs, where they have been proposed to contribute to the remarkably precise targeting of ORN axons to specific olfactory bulb glomeruli. Here we asked whether CNGCs in ORN nerve terminals modulate synaptic transmission.
Olfactory bulb slices (~300 µm) were prepared from 2- to 4-week-old Sprague-Dawley rats or c57bl6 mice in accordance with USA and institutional guidelines. Periglomerular (PG) neurons, which receive glutamatergic input from olfactory nerve (ON) fibres, were visualized via IR-DIC microscopy and patch clamped with electrodes containing a Cs-based internal solution. Data are presented as means ± S.E.M.
Bath application of forskolin (25-50 µM), an activator of adenylyl cyclase, or 8-Br-cGMP (500 µM), a membrane-permeable cGMP analogue, depressed the amplitude of ON-evoked EPSCs in PG neurons by 91.3 ± 1.9 % (n = 5) and 75.2 ± 1.3 % (n = 5), respectively. Forskolin and 8-Br-cGMP altered the frequency but not amplitude of mEPSCs, suggesting that cyclic nucleotides modulate transmission presynapticallly. To further address the specificity with which cyclic nucleotides altered synaptic transmission, we recorded glomerular field EPSPs evoked by ON and mitral cell stimulation. 8-Br-cGMP and forskolin inhibited ON- but not mitral cell-evoked fEPSPs, indicating that the effects of cyclic nucleotides were specific to ON-mediated transmission.
Curiously, the inhibition of ON-evoked fEPSPs by cyclic nucleotides occurred in parallel with an increase in presynaptic Ca2+. Cyclic nucleotides largely abolished action potentials in ON fibres, an effect that was mimicked by applying an elevated (~7.5 mM) K+ solution. These results suggest that cyclic nucleotides inhibit transmitter release by modulating the excitability of ON fibres.
To directly test whether the actions of cyclic nucleotides were due to CNGC activation, we examined the effects of cyclic nucleotides on synaptic transmission and ON excitability in mice lacking olfactory CNGCs. Cyclic nucleotides had no effect on transmission or ON excitability in CNGC mutant mice, indicating that the effects of 8-Br-cGMP and forskolin were attributable to CNGC activation. These results identify a novel role for CNGCs in neurotransmission.
G.J.M. is supported by an NRSA predocotoral fellowship from the NIH (NIDCD). J.S.I. is supported by a Burroughs-Welcome Fund Career Award, a McKnight Scholar award, and the NIH (NIDCD).