Puncta of neuronal nitric oxide synthase (nNOS) mediate NMDA-receptor signalling in the auditory midbrain.

Nitric oxide (NO) is a neurotransmitter synthesized in the brain by neuronal nitric oxide synthase (nNOS). Using immunohistochemistry and confocal imaging in the inferior colliculus (IC, auditory midbrain) of the guinea pig (Cavia porcellus, male and female), we show that nNOS occurs in two distinct cellular distributions. We confirm that, in the cortices of the IC, a subset of neurons show cytoplasmic labeling for nNOS, whereas in the central nucleus (ICC), such neurons are not present. However, we demonstrate that all neurons in the ICC do in fact express nNOS in the form of discrete puncta found at the cell membrane. Our multi-labeling studies reveal that nNOS puncta form multiprotein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals, which is indicative of synaptic function. Interestingly, these glutamatergic terminals express both vesicular glutamate transporters 1 and 2 denoting a specific source of brainstem inputs. With in vivo electrophysiological recordings of multiunit activity in the ICC, we found that local application of NMDA enhances sound-driven activity in a concentration-dependent and reversible fashion. This response is abolished by blockade of nNOS or sGC, indicating that the NMD Aeffect is mediated solely via the NO and cGMP signaling pathway. This discovery of a ubiquitous, but highly localized, expression of nNOS throughout the ICC and demonstration of the dramatic influence of the NMDA activated NO pathway on sound-driven neuronal activity imply a key role for NO signaling in auditory processing.