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

Nitric oxide (NO), is a neurotransmitter synthesised 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 labelling for nNOS, while 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-labelling studies reveal that nNOS puncta form multi-protein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals 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 NMDA effect is mediated solely via the NO and cGMP signalling pathway. This discovery of a ubiquitous, but highly localised 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 signalling in auditory processing. SIGNIFICANCE STATEMENT We show that nNOS, the enzyme that synthesises nitric oxide (NO), occurs as puncta in apparently all neurons in the central nucleus of the inferior colliculus (ICc) in the auditory midbrain. Punctate nNOS appears at glutamatergic synapses in a complex with glutamate NMDA receptors, soluble guanylyl cyclase (the NO receptor), and PSD-95 (a protein that anchors receptors and enzymes at the postsynaptic density). We show that NMDA-receptor modulation of sound-driven activity in the ICc is solely mediated by activation of nNOS and soluble guanylyl cyclase. The presence of nNOS throughout this sensory nucleus, argues for a major role of NO in hearing. Furthermore, this punctate form of nNOS expression may exist and have gone unnoticed in other brain regions.