Cortical nNOS/NK1 Neurons Are Regulated by Cholinergic Inputs

Abstract

Cortical GABAergic interneurons shown to co-express neuronal nitric oxide synthase (nNOS) and the neurokinin-1 (NK1) receptor are activated during sleep (Dittrich et al., 2012, Front. Neural Cir). These neurons have been proposed to play a role in sleep homeostasis (Gerashchenko et al., 2008, PNAS)as the proportion of this population expressing cFOS correlates with homeostatic sleep drive (Morairty et al 2013, PNAS). As these cells appear to “”sense”” sleep need, we investigated whether cholinergic inputs affect the electrophysiological properties of cortical nNOS/NK1 neurons. We prepared coronal mouse brain slices (250µm thick) for whole-cell patch-clamp recording in both voltage clamp and current clamp modes. After a brief application of the fluorescent NK1 agonist tetramethylrhodamine (TMR), cortical nNOS/NK1 neurons were readily identifiable. Following a 60min wash-out period to eliminate any residual TMR-mediated response, carbachol (CCh, a cholinergic mimetic) was applied. All cells tested responded to CCh (50µM; n=34). CCh had biphasic effects: an initial membrane hyperpolarization (-2.24±0.57mV, n=6) and outward current (+3.74±0.66pA, n=12) was followed by a large membrane depolarization (+9.18±1.87mV, n=8) and inward current (-5.59±2.22pA, n=12). In addition, CCh predominantly increased spontaneous excitatory postsynaptic currents (sEPSCs; +805.8±62.68%, n=15). In the presence of tetrodotoxin (TTX), both outward (+3.18±0.74pA, n=9), and inward (-11.96±1.88pA, n=15) currents remained, indicating responses mediated by postsynaptic receptors. We tested whether the excitatory response mediated by CCh was attributable to activation of the Gq-coupled muscarinic type 1 receptors (M1R). Application of CCh in the presence of the M1R antagonist VU0235535 (5μM) reduced the membrane depolarization by 47.9±16.9% and inward current by 32.0±17.7% in 3/5 cells tested. The 2 remaining cells showed no change in their excitatory response but an increase in the outward current (+5.76±2.11pA, n=2). In contrast to sEPSCs, CCh decreased miniature EPSCs (mEPSCs) by -45.6±2.2% (n=6). The presence of VU0235535 did little to alter the reduction in mEPSCs seen following CCh application (after vs baseline: -53.1±2.8%, n=4), suggesting that M1Rs are largely postsynaptic. In summary, CCh has a network effect on excitability within the cortex, as both presynaptic and postsynaptic effects result in an oscillating pattern of nNOS/NK1 neuron excitability. When all inputs are blocked, the predominant response is excitation. We are currently attempting to determine the source of cholinergic inputs to cortical nNOS/NK1 neurons.

Keywords: acetylcholine, arousal, cortex, muscarinic, nicotinic, nitric oxide, sleep, wakefulness.