Inhibitory interneurons can evoke vasodilation and vasoconstriction, making them potential cellular drivers of neurovascular coupling. However, the specific regulatory roles played by particular interneuron subpopulations remain unclear. Our purpose was therefore to adopt a cell-specific optogenetic approach to investigate how somatostatin (SST) and neuronal nitric oxide synthase (nNOS)-expressing interneurons might influence the neurovascular relationship. In mice, specific activation of SST- or nNOS-interneurons was sufficient to evoke hemodynamic changes. In the case of nNOS-interneurons, robust hemodynamic changes occurred with minimal changes in neural activity, suggesting that the ability of blood oxygen level dependent functional magnetic resonance imaging (BOLD fMRI) to reliably reflect changes in neuronal activity may be dependent on type of neuron recruited. Conversely, activation of SST-interneurons produced robust changes in evoked neural activity with shallow cortical excitation and pronounced deep layer cortical inhibition. Prolonged activation of SST-interneurons often resulted in an increase in blood volume in the centrally activated area with an accompanying decrease in blood volume in the surrounding brain regions, analogous to the negative BOLD signal. These results demonstrate the role of specific populations of cortical interneurons in the active control of neurovascular function.

20Inhibitory interneurons can evoke vasodilation and vasoconstriction, making them 21 potential cellular drivers of neurovascular coupling. However, the specific regulatory 22 roles played by particular interneuron subpopulations remain unclear. Our purpose 23 was therefore to adopt a cell-specific optogenetic approach to investigate how 24 somatostatin (SST) and neuronal nitric oxide synthase (NOS1)-expressing 25 interneurons might influence neurovascular relationships. In mice, specific activation 26 of SST-or NOS1-interneurons was sufficient to evoke haemodynamic changes similar 27 to those evoked by physiological whisker stimulation. In the case of NOS1-28 interneurons, robust haemodynamic changes occurred with minimal changes in neural 29 activity. Conversely, activation of SST-interneurons produced robust changes in 30 evoked neural activity with shallow cortical excitation and pronounced deep layer 31 cortical inhibition. This often resulted in a central increase in blood volume with 32 corresponding surround decrease, analogous to the negative BOLD signal. These 33 results demonstrate the role of specific populations of cortical interneurons in the 34 active control of neurovascular function.35 36 80 of cortical GABAergic interneurons have specific roles in NVC. Also, that the ability of 81 BOLD signals to act as a surrogate measure of local neural activation may in part be 82 dependent upon which subpopulation of neurons are being activated.83 84 4 Results 85 Short duration optogenetic stimulation of specific interneurons evokes a 86 localised haemodynamic response 87 Genetically modified mice expressing channelrhodopsin-2 (ChR2) in either SST-or 88 NOS1-expressing interneurons (referred to as SST-ChR2 or NOS1-ChR2 mice, 89 respectively) were used to investigate how light induced activity of these inhibitory 90 interneurons may alter cortical haemodynamics. Using an anaesthetised mouse 91 (Figure 1), we assessed whether short duration optogenetic stimulation of specific 92 subtypes of interneuron evoked a localised haemodynamic response, comparable to 93 that evoked by a mild physiological stimulus (mechanical whisker stimulation). 2-94 dimensional optical imaging spectroscopy (2D-OIS) was used to record high-95 resolution 2D maps of the changes in blood volume (Hbt), oxygenated haemoglobin 96 (HbO2) and reduced haemoglobin (Hbr) evoked by stimulation. Each animal initially 97 received a mechanical whisker stimulation (2s, 5Hz), evoking changes in Hbt, HbO2 98 and Hbr which were localised to the whisker barrel cortex (Figure 2A). These 99 haemodynamic changes allowed us to map the whisker barrel cortex and, in turn, 100 guide the placement of the optical fibre used for photostimulation (Figure 1). The time 101 series of the haemodynamic response to whisker stimulation shows an increase in Hbt 102and HbO2 during the stimulation with a corresponding washout of Hbr (Figure 2A). 103 5 104 A fibre-coupled blue (470nm) LED, placed directly above the whisker barrel cortex, 105 was used to apply photostimulat...

Wolfram syndrome is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years old, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after Tunicamycin treatment. Interestingly, both mutants lead to a decrease of their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for Wolfram syndrome.

Neurovascular coupling ensures that changes in neural activity are accompanied by localised changes in cerebral blood flow. While much is known about the involvement of excitatory neurons in neurovascular coupling, the role of inhibitory interneurons is unresolved. While nNOS-expressing interneurons have been shown to be capable of eliciting vasodilation, the role of nitric oxide in functional hyperemia remains a matter of debate. Therefore in the present study we applied a combination of optogenetic and pharmacological approaches, 2-dimensional optical imaging spectroscopy, and electrophysiology to investigate the role of nitric oxide in neurovascular coupling responses evoked by nNOS-expressing interneurons and whisker stimulation in mouse sensory cortex. The haemodynamic response evoked by nNOS-expressing interneurons was significantly altered in the presence of the NOS inhibitor LNAME, revealing a large initial 20-HETE-dependent vasoconstriction. In contrast, the haemodynamic response induced by sensory stimulation was largely unchanged by LNAME. Our results suggest that while nitric oxide plays a key role in neurovascular responses evoked by nNOS-expressing interneurons it does not mediate the initial sensory-induced neurovascular coupling response in mouse cortex. Thus, our results call into question the involvement of nNOS-expressing interneurons and nitric oxide in sensory-evoked functional hyperemia.

This is a repository copy of Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear.