Evidence for Cholinergic Collateral Projections between Sympathetic Neurons in the Murine Stellate Ganglia.

Postganglionic neurons of the stellate ganglia (SG) are an integral component of sympathetic regulation of the heart. SG neurons receive preganglionic cholinergic inputs via T-2 and send noradrenergic projections to the heart via the inferior cardiac nerve (iCN) to influence cardiac activity. SG neurons are thought to receive simple monosynaptic inputs from preganglionic neurons, but early studies have hinted that cholinergic collateral projections may be present in the SG. In culture, SG neurons form noradrenergic and cholinergic synapses between one another, suggesting that SG neurons express dual neurochemical phenotypes. This study tests the hypothesis that cholinergic collateral projections are present between sympathetic neurons of the intact SG in mice. Whole-cell patch clamp recordings were made of intact SG neurons from male (N=8) and female (N=6) ChAT mice with and without tamoxifen treatment (N=7 ChAT KO mice and N=7 control mice, respectively). Tamoxifen treatment in ChAT mice induced the deletion of choline acetyltransferase (ChAT) in tyrosine hydroxylase positive (i.e. noradrenergic) cells. This strategy blocks putative acetylcholine synthesis in SG neurons while leaving preganglionic cholinergic synapses intact. To search for collaterals, the preganglionic nerve trunk, T-2, was electrically stimulated to evoke excitatory postsynaptic currents (eEPSCs). The amplitude and jitter (i.e. variability in latency) of eEPSCs were examined. Additionally, the postganglionic nerve trunk, the iCN, was electrically stimulated to evoke retrograde action currents (rAC) and the presence of evoked synaptic currents was assessed. The amplitude and frequency of spontaneous EPSCs (sEPSCs) were also examined. ChAT KO significantly reduced the frequency of sEPSCs compared to control mice (1.21±0.559events/s vs. 0.13±0.048events/s, P<0.05 from Student's unpaired t-test, N=5-6 cells) while the amplitude was unaffected (74.4±26.10pA vs. 39.4±10.46pA, P>0.05 from Student's unpaired t-test, N=5-6 cells). This suggests that ChAT KO reduces the presynaptic activity of postganglionic noradrenergic neurons within the SG. In control mice, stimulation of T-2 evoked high jitter eEPSCs, which was significantly reduced in ChAT KO mice (327.1±92.14μs vs. 39.2±15.69μs, P<0.05 from Student's unpaired t-test, N=5-6 cells) Additionally, the proportion of neurons that exhibited evoked synaptic currents following stimulation of the iCN was significantly reduced in ChAT KO mice (3/4 cells vs. 1/9 cells, P<0.05 from χ² test). Chemical transmission at additional synapses accounts for the high jitter events observed in disynaptic pathways. High jitter responses following T-2 stimulation and evoked synaptic responses after iCN stimulation in control neurons are both consistent with the collateral hypothesis. The loss of high jitter responses and evoked synaptic currents in ChAT KO mice also suggests these collateral projections are cholinergic. This suggests that collateral synaptic activity contributes to the increased frequency of sEPSCs in control mice. Understanding SG neurocircuitry is critical in deciphering changes in sympathetic activity in many pathophysiological conditions such as myocardial infarction and heart failure and may identify novel therapeutic targets for the treatment of autonomic imbalance.