Type-I nNOS neurons orchestrate cortical neural activity and vasomotion

Reviewer #1 (Public review):

Turner et al. present an original approach to investigate the role of Type-1 nNOS interneurons in driving neuronal network activity and in controlling vascular network dynamics in awake head-fixed mice. Selective activation or suppression of Type-1 nNOS interneurons has previously been achieved using either chemogenetic, optogenetic, or local pharmacology. Here, the authors took advantage of the fact that Type-1 nNOS interneurons are the only cortical cells that express the tachykinin receptor 1 to ablate them with a local injection of saporin conjugated to substance P (SP-SAP). SP-SAP causes cell death in 90 % of type1 nNOS interneurons without affecting microglia, astrocytes, and neurons. The authors report that the ablation has no major effects on sleep or behavior. Refining the analysis by scoring neural and hemodynamic signals with electrode recordings, calcium signal imaging, and wide-field optical imaging, the authors observe that Type-1 nNOS interneuron ablation does not change the various phases of the sleep/wake cycle. However, it does reduce low-frequency neural activity, irrespective of the classification of arousal state. Analyzing neurovascular coupling using multiple approaches, they report small changes in resting-state neural-hemodynamic correlations across arousal states, primarily mediated by changes in neural activity. Finally, they show that nNOS type 1 interneurons play a role in controlling interhemispheric coherence and vasomotion.

In conclusion, these results are interesting, use state-of-the-art methods, and are well supported by the data and their analysis. I have only a few comments on the stimulus-evoked haemodynamic responses, and these can be easily addressed.

Reviewer #2 (Public review):

Summary:

This important study by Turner et al. examines the functional role of a sparse but unique population of neurons in the cortex that express Nitric oxide synthase (Nos1). To do this, they pharmacologically ablate these neurons in the focal region of whisker-related primary somatosensory (S1) cortex using a saponin-substance P conjugate. Using widefield and 2-photon microscopy, as well as field recordings, they examine the impact of this cell-specific lesion on blood flow dynamics and neuronal population activity. Locally within the S1 cortex, they find changes in neural activity patterns, decreased delta band power, and reduced sensory-evoked changes in blood flow (specifically eliminating the sustained blood flow change after stimulation). Surprisingly, given the tiny fraction of cortical neurons removed by the lesion, they also find far-reaching effects on neural activity patterns and blood volume oscillations between the cerebral hemispheres.

Strengths:

This was a technically challenging study and the experiments were executed in an expert manner. The manuscript was well written and I appreciated the cartoon summary diagrams included in each figure. The analysis was rigorous and appropriate. Their discovery that Nos1 neurons can have far-reaching effects on blood flow dynamics and neural activity is quite novel and surprising (to me at least) and should seed many follow-up, mechanistic experiments to explain this phenomenon. The conclusions were justified by the convincing data presented.

Weaknesses:

I did not find any major flaws in the study. I have noted some potential issues with the authors' characterization of the lesion and its extent. The authors may want to re-analyse some of their data to further strengthen their conclusions. Lastly, some methodological information was missing, which should be addressed.

Reviewer #3 (Public review):

The role of type-I nNOS neurons is not fully understood. The data presented in this paper addresses this gap through optical and electrophysiological recordings in adult mice (awake and asleep).

This manuscript reports on a study on type-I nNOS neurons in the somatosensory cortex of adult mice, from 3 to 9 months of age. Most data were acquired using a combination of IOS and electrophysiological recordings in awake and asleep mice. Pharmacological ablation of the type-I nNOS populations of cells led to decreased coherence in gamma band coupling between left and right hemispheres; decreased ultra-low frequency coupling between blood volume in each hemisphere; decreased (superficial) vascular responses to sustained sensory stimulus and abolishment of the post-stimulus CBV undershoot. While the findings shed new light on the role of type-I nNOS neurons, the etiology of the discrepancies between current observations and literature observations is not clear and many potential explanations are put forth in the discussion.