# Bidirectional control of neurovascular coupling by cortical somatostatin interneurons

Boubacar Mohamed, Julie O’Reilly, Sandrine Picaud, Benjamin Le Gac, Esther Belzic, Liliana Castro, Hédi Soula, Isabelle Dusart, Dongdong Li, Bruno Cauli

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Boubacar Mohamed, Julie O’Reilly, Sandrine Picaud, Benjamin Le Gac, Esther Belzic, et al., Bidirectional control of neurovascular coupling by cortical somatostatin interneurons. 2025. hal-05349150

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Bidirectional control of neurovascular coupling by cortical somatostatin
interneurons

Boubacar Mohamed¹, Julie O’Reilly¹, Sandrine Picaud¹, Benjamin Le Gac¹, Esther Belzic¹,
Liliana R V Castro¹, Hédi Soula², Isabelle Dusart¹, Dongdong Li¹, Bruno Cauli¹*

1. Sorbonne Université, CNRS, INSERM, Neurosciences Paris Seine - Institut de Biologie Paris Seine (NPS-IBPS), 9 quai Saint Bernard, 75005 Paris, France.

2. Sorbonne Université, INSERM, Nutrition and Obesities: Systemic Approaches, NutriOmics, research unit, 91 boulevard de l'hôpital, 75013 Paris, France.

*Correspondence: Bruno CAULI

9 Quai Saint Bernard, 75 005 Paris, France

Phone number: + 33 1 44 27 25 01

e-mail: bruno.cauli@upmc.fr

**Running title:** Control of neurovascular coupling by Sst neurons

**Keywords:** Neurovascular coupling, somatostatin interneurons, nitric oxide signaling, neuropeptide Y, optogenetics
1 ABSTRACT

2 Neurovascular coupling, linking neuronal activity to cerebral blood flow, is altered early in
3 neurological disorders and underlies functional brain imaging. This process involves
4 numerous cellular players. Among them inhibitory interneurons receive increasing attention,
5 but how they control blood flow remains elusive. This study elucidates the mechanisms by
6 which somatostatin interneurons bidirectionally control neurovascular coupling. Patch clamp
7 recordings in, ex vivo, cortical slices from mice expressing channelrhodopsin-2 in
8 somatostatin interneurons, revealed that these neurons are supralinearly activated at low-
9 frequencies (< 5 Hz) and efficiently photostimulated at frequencies up to 20 Hz. Ex vivo
10 vascular imaging showed that low-frequency (2 Hz) photostimulation triggered vasodilation
11 whereas high-frequency (20 Hz) photostimulation induced vasoconstriction. Histochemistry
12 revealed that subpopulations of cortical somatostatin interneurons expressed the neuronal
13 nitric oxide synthase, and/or neuropeptide Y to a greater extent. Consistently,
14 pharmacological investigations showed that vasodilation induced by low-frequency
15 photostimulation involves nitric oxide release and activation of soluble guanylate cyclase. In
16 contrast, the vasoconstriction induced at high-frequency photostimulation involves
17 neuropeptide Y release and activation of the Y1 vascular receptor. These findings provide
18 valuable insights into neurovascular coupling and help to understand the cellular mechanism
19 underlying the functional brain imaging signals.

20

21
22 INTRODUCTION

23 Brain function and integrity are critically dependent on a permanent blood supply provided
24 by a dense vascular network (Schaeffer and Iadecola, 2021). Cerebral blood flow (CBF) is also
25 tightly controlled by neuronal activity, an essential process called neurovascular coupling
26 (NVC) that is impaired in neurological disorders (Iadecola, 2017). The NVC is thought to
27 ensure the optimal supply of metabolic needs to, and removal of waste products and heat
28 from the activated brain regions, although its precise motive remains to be understood
29 (Drew, 2022). It allows CBF to surge within seconds of increased neuronal activity and serves
30 as the physiological basis for functional brain imaging techniques to map brain function
31 (Iadecola, 2017). In the cerebral cortex, this hyperemic response is supported by dynamically
32 controlled vasodilation that is spatially and temporally confined by vasoconstriction (Devor
33 et al., 2007; Boorman et al., 2010; Uhlirova et al., 2016).

34 CBF is critically orchestrated by pial and penetrating arterioles, which have a higher density
35 of contractile mural cells and control their diameter more rapidly than capillaries (Hill et al.,
36 2015; Rungta et al., 2018; Hartmann et al., 2021). NVC is mediated by multiple vasoactive
37 agents produced by excitatory neurons, astrocytes, GABAergic interneurons, and/or
38 endothelial cells (Cauli and Hamel, 2018; Kaplan et al., 2020; Schaeffer and Iadecola, 2021)
39 that control vessel diameter and CBF along the vascular tree (Rungta et al., 2018). These
40 agents may be vasodilatory [e.g. arachidonic acid derivatives, nitric oxide (NO))],
41 vasoconstrictive [e.g. Neuropeptide Y (NPY) (Uhlirova et al., 2016)], or both [e.g. glutamate,
42 K⁺ (Girouard et al., 2010; Zhang et al., 2024)].

43 The cellular diversity that characterizes cortical GABAergic interneurons (Ascoli et al., 2008),
44 has made it challenging to study their contribution to NVC, and the conditions under which
45 they release specific vasoactive agents have long remained elusive (Cauli et al., 2004). A
46 major breakthrough in understanding their role came with the advent of transgenic mice
47 and optogenetics, which allowed confirmation of their ability to induce vasodilation and/or
48 vasoconstriction in vivo (Anenberg et al., 2015; Uhlirova et al., 2016). More recently the use
49 of different Cre-line drivers has made it possible to investigate the contribution of different
50 subpopulations of GABAergic interneurons (Krawchuk et al., 2019; Lee et al., 2020;
51 Echagarruga et al., 2020; Krogsgaard et al., 2023; Ruff et al., 2024). Among these subclasses,
52 somatostatin (Sst) interneurons have been found to either increase and/or decrease CBF
depending on the stimulation paradigm (Krawchuk et al., 2019; Lee et al., 2020), but the
underlying mechanisms of this bimodal response have not been determined.

To address this unresolved issue, we used *ex vivo* approaches in combination with
optogenetics to precisely control Sst interneurons firing in the mouse barrel cortex while
monitoring the resulting arteriolar response. We found that Sst interneurons are
supralinearly activated by low-frequency (< 5 Hz) photostimulation and are efficiently and
reliably activated up to 20 Hz. Low frequency stimulation (2 Hz) induced predominantly
vasodilation, whereas 20 Hz photostimulation resulted in vasoconstriction. Histochemical
analyses showed that subpopulations of Sst interneurons expressed the neuronal isoform of
NO synthase, NOS-1, and/or NPY at much greater extents. Pharmacological investigations
revealed that both types of vascular responses were dependent on action potential (AP)
firing. The vasodilation required NO synthesis and activation of the soluble guanylate cyclase
(sGC), whereas the vasoconstriction involved NPY and activation of its Y1 receptor. Thus, our
study elucidates the mechanisms by which Sst interneurons can induce vasodilation or
vasoconstriction depending on their firing frequency.

68

## MATERIALS AND METHODS

### Animal models

Homozygous Sst-IRES-Cre mice [Jackson Laboratory, B6J.Cg-Ssttm2.1(cre)Zjh/MwarJ, stock# 028864, (Taniguchi et al., 2011)] were crossed with homozygous Ai32 mice [Jackson Laboratory, B6.Cg-Gt(ROSA)26Sor tm32(CAG-COP4*H134R/EYFP)Hze/J stock #024109, (Madisen et al., 2012)] or with homozygous Ai95d mice [Jackson Laboratory, B6J.Cg-Gt(ROSA)26Sor tm95.1(CAG-GCaMP6f)Hze/MwarJ, stock# 028865 (Madisen et al., 2015)], to obtain heterozygous SstCre/WT::Ai32ChR2/WT (Sst-ChR2) for optogenetic stimulations or SstCre/Wt::Ai95dGCaMP6f/Wt (Sst-GCaMP6f) mice for immunohistochemistry. C57BL/6RJ mice were used for control optogenetic experiments and exogenous NPY applications. 16-21 days postnatal old females and males were used for all experiments. All experimental procedures involving animals were carried out in strict compliance with French regulations (Code Rural R214/87 to R214/130) and in accordance with the ethical guidelines of the Directive of the Council of the European Communities of September 22, 2010 (2010/63/EU).

83
84 **Ex vivo slice preparation**

85 Mice were deeply anesthetized by isoflurane (IsoVet Piramal Healthcare UK or IsoFlo, 86 Axience) evaporation in an induction box then euthanized by decapitation. The brain was 87 quickly removed and immersed in cold (~4°C) oxygenated artificial cerebrospinal fluid (aCSF) 88 containing in (mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, 26 NaHCO₃, 10 89 glucose, 15 sucrose and 1 kynurenic acid (Sigma-Aldrich). 300 µm-thick coronal slices 90 containing the barrel cortex were cut with a vibratome (VT1000s; Leica) and were allowed to 91 recover at room temperature for at least 45 min with oxygenated aCSF (95% O₂/5% CO₂) 92 (Devienne et al., 2018). The slices were transferred to a recording chamber and continuously 93 perfused at a rate of ~2 ml/min with oxygenated aCSF lacking kynurenic acid. 94

95 **Patch-clamp recording**

96 Borosilicate glass patch pipettes (5.0 ± 0.4 MΩ) were filled with an internal solution 97 containing (in mM): 144 K-gluconate, 3 MgCl₂, 0.5 EGTA, 10 HEPES, pH 7.2 (285/295 mOsm). 98 Sst-ChR2 interneurons were first visually identified by their EYFP fluorescence. Whole-cell 99 recordings were performed at room temperature (20-25 °C) using a patch clamp amplifier 100 (Axopatch 200B, Molecular Devices) at a wavelength of 780 nm, which does not activate 101 ChR2 (Lin et al., 2009). Data were filtered at 5-10 kHz and digitized at 50 kHz using an 102 acquisition board (Digidata 1440, MDS) attached to a personal computer running pCLAMP 103 10.7 software package (MDS). Electrophysiological properties were determined using the 104 fast I-clamp mode of the amplifier and were measured as previously described (Karagiannis 105 et al., 2021, 2009). Membrane potential values were not corrected for liquid junction 106 potential. Only neurons with a resting membrane potential more hyperpolarized than -55 107 mV were further analyzed. 108

109 **Optogenetic stimulation**

110 Optogenetic stimulation performed through the objective using a 470 nm light-emitting 111 diode (LED) device (CoolLED brand, Precise Excite model) attached to the epifluorescence 112 port of a BX51WI microscope (Olympus) and a set of multiband filters consisting of an 113 excitation filter (HC 392/474/554/635, Semrock), a dichroic mirror (BS 409/493/573/652, 114 Semrock), and an emission filter (HC 432/515/595/730, Semrock). Photostimulation
consisted of a 10-s train of 5 ms light pulses at an intensity of 38 mW/mm² and delivered at six different frequencies (1, 2, 5, 10, 20 and 40 Hz).

## Vascular imaging

Blood vessels and cells were observed in slices under infrared illumination with Dodt gradient contrast optics (IR-DGC Luigs and Neumann; (Dodt and Zieglgansberger, 1998)) using a collimated LED (780 nm; ThorLabs) as the transmitted light source, a 40X (LUMPlanF/IR, 40X/0.80 W, Olympus) and a digital camera (OrcaFlash 4.0LT+, Hamamatsu) attached to the microscope. Diving arterioles were selected by IR-DGC based on their well-defined luminal diameter (16.7 ± 0.5 µm, n = 144 arterioles), their length remaining in the focal plane of at least 50 µm, and the thickness of their wall (5.5 ± 0.1 µm, n = 144 arterioles). A resting period of at least 30 min (Lovick et al., 1999; Zonta et al., 2003) was observed after slice transfer. IR-DGC images were acquired at 0.1 Hz for pharmacological applications and at 1 Hz for optogenetic experiments using Imaging Workbench 6.1 software (Indec Biosystems). The focal plane was continuously maintained on-line using IR-DGC images of cells as anatomical landmark (Lacroix et al., 2015). After light-induced responses, arteriolar identity and reactivity were systematically verified by assessing their contractility (Hartmann et al., 2021) using the thromboxane A2 analog, U46619 (100 nM) (Cauli et al., 2004; Rancillac et al., 2006). Blood vessels that failed to constrict after this pharmacological application were discarded. Only one arteriole was monitored per slice.

## Double fluorescence immunolabeling

Mice were euthanized with pentobarbital (150 mg/kg, i.p., Euthasol Vet) and their brains were perfusion fixed (50 ml of ice-cold 4 % paraformaldehyde (PFA), in 0.1M phosphate buffer, pH 7.4), and post-fixed by immersion in 4 % PFA (2 h, 4°C). Brains were then cut in 50 µm-thick coronal sections with a VT-1000S vibratome (Leica). After washing in PBS, free-floating sections were blocked for 2 h at room temperature in a PBS 1X/0.25% Triton X-100/0,2% gelatin solution before being incubated overnight at 4°C with primary antibodies. Sections from 3 to 4 different mouse brains were simultaneously incubated overnight with the primary antibodies against GFP and specific markers. Antibodies included chicken anti-GFP (1:1000; Aves Labs GFP-1020, (Tricoire et al., 2010)), rabbit anti-NOS-1 (1:1000, Millipore AB5380, (Tricoire et al., 2010)), sheep anti-NPY (1:2000, Abcam ab6173, (Lee et al., 2010)). The respective immunoreactions were visualized with the following secondary
antibodies: goat anti-chicken Alexafluor 488 (1:1000, Invitrogen A11039), donkey anti-rabbit
Alexafluor 555 (1:1000, Invitrogen A31572) donkey anti-sheep Alexafluor 555 (1:1000,
Invitrogen A21436). Nuclear counterstaining was performed with 100 ng/ml DAPI (4',6-
diamidino-2-phenylindole; Invitrogen) solution in PBS for 20 min. Sections were mounted
with Fluoromount G (SouthernBiotech) on gelatin-coated slides for visualization. Images of
immunostained material were acquired and processed in Airyscan 2 mode using a LSM 980
confocal microscope with a 20x objective (N Plan-Apochromat 20x) and ZEN Blue 3 software
(Zeiss). Cell counting was achieved using the cell counter plug-in of FIJI software.

155

156 **Drugs**

All pharmacological compounds were bath-applied after a 5-minute control period (aCSF)
and vascular dynamics were recorded during bath application. The following drugs were
dissolved in water: NPY (Enzo, ALX-163-003), Nω-Nitro-L-arginine methyl ester hydrochloride
(L-NAME, Sigma-Aldrich, N5751), BIBP3226 (1 µM, Tocris, 2707). Tetrodotoxin (TTX, 1 µM,
Latoxan, L8503) was dissolved in 90% acetic acid. 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-
one (ODQ, Sigma-Aldrich, O3636) was dissolved in DMSO and 9,11-dideoxy-9α,11α-
methanoepoxy prostaglandin F2α (U-46619, 100 nM, Enzo, BML-PG023) in ethanol. Acetic
acid, DMSO and ethanol doses were always used below 0.1% to avoid vascular interactions
(Sun et al., 2010). NO pathway inhibitors, BIBP3226 and TTX were applied at least 30 min
before optogenetic stimulation or exogenous NPY application.

167

168 **Vascular reactivity analysis**

To compensate for potential x-y drifts, all images were realigned off-line using the
"StackReg" plug-in (Thevenaz et al., 1998) of the ImageJ 1.54f software. Luminal diameter
was measured on registered images using custom analysis software developed in MATLAB
(MathWorks) (Lacroix et al., 2015). Only arterioles with a stable luminal diameter were
analyzed further. Arterioles were considered stable if the relative standard deviation (RSD)
of their luminal diameter during the baseline period was less than 5% (Lacroix et al., 2015).
The impact of blockers, inhibitors, and antagonists on arterioles was evaluated by comparing
the mean luminal diameter during the initial 5 minutes of the baseline period and the final 5
minutes of the treatment period prior to optogenetic stimulation or NPY application.
Diameter changes (ΔD/D₀) were expressed according to: (Dt – D₀)/D₀ where Dt is the luminal

170

171

172

173

174

175

176

177

178
179 diameter at the time t and D₀ the average diameter during the 5 minutes baseline period.
180 Vasomotor responses and their parameters were determined from the diameter change
181 traces [ΔD/D₀ = f(t)] plotted using Clampfit 10.7 (MDS). To determine whether arterioles
182 dilated or constricted, a Z-score was calculated from the diameter change traces using the
183 formula: Z = (x - μ)/ σ, where both the mean μ and standard deviation σ were calculated
184 using the values before photostimulation or NPY application. Vascular responses were
185 classified as vasodilation or vasoconstriction, respectively, if the Z-score exceeded a value of
186 1.96 (95% criteria) or fell below -1.96 for 10 seconds and 1 minute for photostimulation and
187 NPY application, respectively. For each vascular response, the time of onset and peak
188 (relative to the beginning of stimulation), duration, maximal amplitude and magnitude (area
189 under the curve [AUC]) were determined (Supplementary Fig. 2). When multiple events of
190 vasodilation or vasoconstriction occurred, the earliest onset was used for global onset, the
191 time and amplitude of the largest maximum amplitude were used for global time to peak
192 and global maximum amplitude, and the sum of durations and magnitudes was used for
193 global duration and magnitude.

194

195 Statistical analyses

196 Statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software,
197 La Jolla, California, USA). All values are expressed as means ± SEM. Normality of distribution
198 was assessed using the Shapiro-Wilk test. Parametric tests were only used if this criterion
199 was met. The mean spike success rate per light pulse between photostimulation frequencies
200 was compared using the Q Friedman test. Comparisons of vascular responses were
201 determined using Brown-Forsythe ANOVA or Kruskal-Wallis tests. Given p-values were
202 corrected for multiple comparisons using the original False Discovery Rate method of
203 Benjamini and Hochberg (Benjamini and Hochberg, 1995). Statistical significance on all
204 figures uses the following convention: *p < 0.05, **p < 0.01 and ***p < 0.001.

205
206 **RESULTS**

207 Sst interneurons are efficiently activated by optogenetic stimulation over a wide range of
208 frequencies.

209 To evaluate the frequency dependence of the vascular response driven by Sst interneurons,
210 we optogenetically elicited their AP firing while visualizing the evoked vascular response in
211 cortical brain slices from Sst<sup>Cre/WT</sup>::Ai32<sup>ChR2/WT</sup> mice (Madisen et al., 2012; Taniguchi et al.,
212 2011) conditionally expressing the H134R variant of channelrhodopsin-2 (ChR2) in Sst
213 interneurons. We first determined the electrophysiological phenotype of Sst-ChR2 cortical
214 neurons using whole-cell patch clamp recordings. Cortical neurons expressing EYFP/ChR2
215 were selected based on their EYFP fluorescence (Fig. 1a and Supplementary Fig. 1) in layers
216 II-III (n=13 neurons) and V (n=3 neurons). Most neurons exhibited electrophysiological
217 features typical of adapting-Sst interneurons (Karagiannis et al., 2009, 2021; Perrenoud et
218 al., 2012a), including a relatively high membrane resistance (608 ± 231 MΩ) and a
219 pronounced voltage sag (21.2 ± 10.8 %) in response to hyperpolarizing current pulses (Fig.
220 1a, middle traces). They also exhibited a low rheobase current (8 ± 14 pA, Fig. 1a) and a
221 slowly developing early frequency adaptation (adaptation time constant: 60.5 ± 23.8 ms)
222 when depolarized by high stimulation intensities (Fig. 1a). Consistently, the
223 hyperpolarization (AHP) phase of their action potentials consisted of a fast AHP, followed by
224 an afterdepolarization (ADP) and a medium AHP (Fig. 1a and Supplementary Table 1).
225 However, as previously reported in Sst-Cre cortical neurons (Hu et al., 2013) and expected
226 from the occasional expression of Sst transcripts in parvalbumin neurons (Cauli et al., 1997;
227 Tasic et al., 2016), one Sst-ChR2 neuron exhibited the electrophysiological features of a fast
228 spiking cell with a low input resistance (129 MΩ), a fast time constant (11.7 ms), a high
229 rheobase (185 pA), short spikes (0.6 ms) with a sharp fast AHP (-26.5 mV), and the ability to
230 fire high firing rates (maximal frequency: 99 Hz) with little frequency adaptation
231 (Supplementary Fig. 1 and Table 1).

232 Next, we evaluated the efficiency of optogenetic stimulation by recording the voltage
233 response of the patched Sst-ChR2 neurons. Wide-field photostimulation was achieved using
234 10-second trains of 5 ms light pulses (see Materials and Methods) delivered at six different
235 frequencies (1, 2, 5, 10, 20 and 40 Hz, Fig. 1b, lower traces). Surprisingly, at the beginning of
236 the light pulse trains, Sst-ChR2 neurons fired up to five APs per light pulse (Fig. 1b, upper
237 traces). Such bursting response declined within seconds before reaching a steady state level
(Fig. 1b, middle traces). The instantaneous spike success rate remained above 100 % at stimulation frequencies below 10 Hz, while it decreased below 100% at higher frequencies (Fig. 1b, middle traces). Consistent with the kinetic properties of the H134R ChR2 variant (Lin et al., 2009), Sst-ChR2 neurons were not able to sustain the firing of action potentials when photostimulated at 40 Hz (Fig. 1b6). The mean spike success rate over the 10 s train differed between photostimulation frequencies (Fig. 1c, $Q_{(6,9)}= 41.89$, p= 6.203 $10^{-8}$, 1 Hz: 176 ± 13%; 2 Hz: 197 ± 15%; 5 Hz: 118 ± 11%; 10 Hz: 103 ± 10%; 20 Hz: 97 ± 5%; 40 Hz: 25 ± 7%, n=9 Sst-ChR2 neurons). Indeed, photostimulation at 1 Hz and 2 Hz showed a higher number of APs per light pulse than that observed at frequencies above 5 Hz (Fig. 1c, 1Hz vs. 10 Hz, p= 0.0374; 1Hz vs. 20 Hz, p= 0.0063; 1 Hz vs. 40 Hz, p= 7.000 $10^{-6}$; 2 Hz vs. 10 Hz, p= 0.0245; 2Hz vs. 20 Hz, p= 0.0039, 2 Hz vs. 40 Hz, p=3.597 $10^{-6}$). Taken together, these observations indicate that optogenetic stimulation efficiently and reliably activates Sst-ChR2 neurons, mostly of the adapting Sst subtype, at frequencies up to 20 Hz. They also reveal that these neurons over-respond to low frequencies of photostimulation.

**The polarity of the vascular response induced by the optogenetic stimulation of Sst-ChR2 neurons is frequency dependent.**

Next, we visualized the optogenetically induced response of diving arterioles in cortical slices (Fig. 2). We focused on the photostimulations at 2 Hz and 20 Hz, which correspond respectively to the highest AP number per light pulse and to the frequency that produced the highest total AP number, respectively (Fig. 1b,c). When photostimulation was delivered at 2 Hz in Sst-ChR2 slices, we observed that the vast majority of arterioles exhibited vasodilatory events (n= 13 of 14 arterioles, Fig. 2a,b), which could be accompanied by vasoconstrictive events (n=6 of 14). On average, this resulted in a slight vasodilation (Fig. 2c). High light intensity can cause dilation (Rungta et al., 2017) or constriction (Choi et al., 2010) independent of ChR2, so we did control experiments by repeating 2 Hz photostimulation in slices of C57Bl6 mice that do not express ChR2 (Fig. 2c). Although the overall vascular response, as estimated by the area under the curve (AUC) of the response following the optogenetic stimulation did not differ between Sst-ChR2 (55.8 ± 24.1 %.min, n= 14 arterioles) and C57Bl6 arterioles (37.4 ± 15.3 %.min, n= 6 arterioles, p= 0.15146, Fig. 2g1), an evident tendency was observed toward vasodilation in Sst-ChR2 arterioles (Fig. 2c). Because both vasodilation and vasoconstriction can occur in the same arteriole (Fig. 2b), we refined
270 the analysis of vascular responses by separating vasodilatory events from vasoconstrictive
271 events based on a Z-score test (supplementary Fig. 2a-b, see Methods). The magnitude of
272 vasodilation was greater in Sst-ChR2 arterioles (69.6 ± 19.9 %.min, Fig. 2g2) than in control
273 arterioles (8.5 ± 4.5 %.min, p=0.03415), although not the maximal amplitude (Sst-ChR2: 6.1 ±
274 1.3 % vs. C57BI6: 2.2 ± 0.7 %, p= 0.12763, Fig. 2g3). Vasodilation also occurred earlier and
275 lasted longer in Sst-ChR2 arterioles (onset: 221 ± 130 s, duration: 1121 ± 195 s) than in
276 control arterioles (onset: 1013 ± 314 s, p= 0.02501, Fig. 2g4, duration: 304 ± 135 s, p=
277 0.04865, Fig. 2g6) but did not differ in their time to peak (Sst-ChR2: 669 ± 155 s vs. C57BI6:
278 1014 ± 314 s, p= 0.32174, Fig. 2g5). In contrast, the amplitude, magnitude, and kinetic
279 properties of vasoconstriction induced by photostimulation at 2 Hz did not differ between
280 Sst-ChR2 and C57BI6 arterioles (supplementary Fig. 2c1-5).

281 We next evaluated whether a stronger optogenetic stimulation could induce a similar
282 vascular response. Surprisingly, photostimulation at 20 Hz of Sst-ChR2 slices (n= 16) induced
283 vasoconstrictive events in the vast majority of arterioles (n= 15 of 16), but also vasodilatory
284 events to a lesser extent (n=9 of 16, Fig. 2d,e). This resulted on average in a progressive
285 vasoconstriction that was not observed in control arterioles (n= 4, Fig. 2f). Although the
286 overall vascular response was not statistically different between Sst-ChR2 and C57BI6
287 arterioles (p=0.13458, Fig. 2h1), similarly to 2 Hz photostimulation it was more
288 heterogeneous in Sst-ChR2 arterioles (-83.3 ± 46.7 %.min) than in C57BI6 arterioles (0.0 ±
289 25.3 %.min) and may be associated with both vasoconstriction and vasodilation (Fig. 2d,e
290 and supplemental Fig. 2 a,b). Both the magnitude of vasoconstriction and the maximal
291 vasoconstriction were greater in Sst-ChR2 arterioles (AUC: -121.7 ± 35.4 %.min, Fig. 2h2;
292 Maximal constriction: -12.2 ± 2.7 %, Fig. 2h3) than in control C57BI6 (AUC: -17.8 ± 17.8
293 %.min, p=0.01810; Maximal constriction: -1.6 ± 1.6 %, p=0.00505). The onset and time to
294 peak of vasoconstriction were also earlier in Sst-ChR2 arterioles (onset: 267 ± 120 s, Fig. 2h4;
295 time to peak: 874 ± 181 s, Fig. 2h5) compared to control C57BI6 arterioles (onset: 1361 ± 439
296 s; p= 0.01993; time to peak: 1680 ± 121 s, p= 0.04397). The contraction duration did not
297 differ between Sst-ChR2 arterioles (981 ± 152 s) and C57BI6 (424 ± 424 s, p= 0.13849, Fig.
298 2h6). In contrast, the amplitude, magnitude, and kinetic properties of vasodilatory events
299 induced by photostimulation at 20 Hz did not differ between Sst-ChR2 and C57BI6 arterioles
300 (supplementary Fig. 2d1-5). Taken together, these observations indicate that, depending on
301 the frequency of optogenetic stimulation Sst-ChR2 neurons elicit a bidirectional vascular
302 response consisting mainly of vasodilation and vasoconstriction at 2 Hz and at 20 Hz,
303 respectively.

304 NOS-1 and NPY are expressed by subpopulations of Sst cortical interneurons

305 Subpopulation of Sst-expressing cortical interneurons have been reported to produce NO
306 and/or NPY (Hendry et al., 1984; Kubota et al., 1994, 2011; Perrenoud et al., 2012a). These
307 mediators may explain the bidirectional vascular responses induced by the photostimulation
308 of Sst-ChR2 interneurons at 2 and 20 Hz, respectively, as they have been shown to induce
309 vasodilation and vasoconstriction, respectively (Cauli et al., 2004; Rancillac et al., 2006). We
310 therefore used double immunohistofluorescence to determine the extent to which Sst
311 interneurons have the ability to produce NO and NPY. Consistent with previous reports
312 (Kubota et al., 1994, 2011; Perrenoud et al., 2012a), NOS-1 immunolabeling revealed that
313 strongly immunoreactive somata appeared denser in deep than in superficial cortical layers
314 (Fig. 3a2-3 and supplementary Fig. 3). Some of these neurons were clearly identified as Sst-
315 ChR2 interneurons as shown by immunolabelling of the ChR2-EYFP fusion transgene using an
316 anti-GFP antibody (supplementary Fig. 3). However, consistent with the membrane
317 localization of ChR2-EYFP (Fig. 1a and supplementary Fig. 1), GFP immunofluorescence was
318 present at the periphery of the somata and in a dense fiber plexus (supplementary Fig. 3)
319 making it difficult to evaluate the degree of co-expression. Therefore, we thus used Sst-
320 GCamp6 mice (n= 3 mice) to obtain somatic Cre-dependent expression of the circularly
321 permuted GFP of GCamp6 (Madisen et al., 2015). As previously reported in Sst-IRES-Cre mice
322 (Taniguchi et al., 2011), Cre-dependent GFP expression was observed in the somata of
323 neurons distributed throughout all cortical layers except layer I (Fig. 3a1,b1), which
324 nevertheless showed diffuse GFP labelling presumably originating from the ascending axon
325 of Martinotti cells branching in this layer (DeFelipe et al., 2013). The average density of GFP-
326 positive cells was comparable between sections immunostained for NOS-1 (49.9 ± 2.7
cells/mm², n=3 mice, Fig. 3a1) and NPY (41.0 ± 7.1 cells/mm², n= 3 mice, Fig. 3b1,
327 t(2.55619)= 1.16763, p= 0.3403). A similar density of neurons immunolabeled for NOS-1 was
328 observed in GFP positive (2.0 ± 0.1 cells/mm²) and negative cells (1.9 ± 0.4 cells/mm²,
329 t(2.28582)= 0.215387, p= 0.8472, Fig. 3a3-4). However, the intensity of NOS-1
330 immunostaining was stronger in GFP-positive cells than in negative cells (Fig. 3a1,2),
331 indicating that they likely correspond to type 1 and type 2 NO-producing interneurons,
332 respectively. Consistent with previous reports in Sst interneurons, (Kubota et al., 1994, 2011;
333
Karagiannis et al., 2009; Perrenoud et al., 2012a; Tricoire et al., 2013), putative type 1 NO interneurons represented only a minority of GFP-positive cells (4.0 ± 0.1%). NPY immunostaining showed a relatively high density of positive cells (29.4 ± 6.0 cells/mm²) and fiber plexus in superficial and deep layers (Fig. 3b2) as reported in other mammals (Hendry et al., 1984). The vast majority of NPY immunoreactive neurons were also GFP positive (81.4 ± 2.1%, Fig. 3b3-4) with the remaining NPY interneurons likely corresponding to neurogliaform cells (Cauli et al., 2004; Price et al., 2005; Karagiannis et al., 2009; Kubota et al., 2011; Perrenoud et al., 2012a), as suggested by their round immunolabeled somata (Fig. 3b3-4). More precisely, the proportion of GFP-positive cells immunostained for NPY was much higher (58.9 + 8.2%, t(2.00020)=6.70695, p= 0.0215) than those immunolabeled for NOS-1, which is known to represent a subpopulation of NPY interneurons (Estrada and DeFelipe, 1998; Karagiannis et al., 2009; Tasic et al., 2016). These observations collectively indicate that the Cre-dependent expression of the transgenes in Sst-IRES-Cre mice occurs in at least two subpopulations of interneurons that express NOS-1 and/or NPY to a greater extent.

349

Vasodilation induced by 2 Hz photostimulation of Sst-ChR2 interneurons requires AP firing and NO signaling

Since a subpopulation of Sst interneurons (Fig. 3a3-4 and supplementary 3) can produce the vasodilator NO (Cauli et al., 2004), we next investigated the mechanisms by which they induce vasodilation when photostimulated at 2 Hz. We first determined whether spiking activity was required by blocking APs with the voltage-activated sodium channel blocker tetrodotoxin (TTX 1 µM (Zonta et al., 2003), n= 10 arterioles). Bath application of TTX did not change arteriolar diameter (diameter before: 17.0 ± 1.8 µm vs. after TTX: 16.9 ± 1.7 µm, t(9)= 1.0031, p=0.3420, Supplementary Fig. 4a,b), indicating that basal network activity does not influence the resting vascular tone. By contrast, it dramatically reduced the overall vascular response (-19.6 ± 10.5 %.min, p= 0.04966, Fig. 4a and d1) and the magnitude of evoked vasodilation (6.8 ± 2.4 %.min, p= 0.00844, Fig. 4d2) without altering the maximal dilation (2.2 ± 0.6%, p=0.11114, Fig. 4d3) or its time to peak (1256 ± 192 s, p= 0.07716, Fig. 4e2). In addition, the onset and the duration of vasodilation were respectively delayed (1142 ± 210 s, p= 0.00152, Fig. 4e1) and shortened (306 ± 121 s, p= 0.01623, Fig. 4e3). Of note, the vasoconstrictive events were not altered except for their onset and time to peak

352

353

354

355

356

357

358

359

360

361

362

363

364

365
366 (Supplementary Fig. 4g1-5). These observations indicate that AP firing is mandatory for the vasodilation induced by 2 Hz photostimulation.
367

368 We next asked whether NO signaling could be responsible for the vasodilation induced by 2 Hz photostimulation. Inhibiting nitric oxide synthases with L-NAME (100 µM (Zonta et al., 2003), n = 9 arterioles) did not alter the arteriolar diameter (diameter before: 15.3 ± 2.1 µm vs. after L-NAME: 15.4 ± 2.3 µm, W(9) = -2, p=0.9453, Supplementary Fig. 4c,d) indicating that basal NO synthase activity does not influence resting vascular tone. By contrast L-NAME treatment completely abolished the evoked vasodilation as evidenced by its dramatic impact on the overall vascular response (AUC: -71.0 ± 20.4 %.min, p= 0.00042, Fig. 4b and 4d1), as well as on the magnitude (4.9 ± 2.9 %.min, p = 0.00096, Fig. 4d2) and maximal amplitude of vasodilation (1.5 ± 0.9%, p= 0.00172, Fig. 4d3). It also strongly delayed the apparent onset of vasodilation (1407 ± 210 s, p= 0.0004, Fig. 4e1) and reduced its duration (169 ± 100 s, p=0.00059, Fig. 4e3), but not the time to peak (1410 ± 210 s, H(4,54)= 8.299, p= 0.08123, Fig. 4e2). The overall negative responses observed under NOS inhibition suggest that vasoconstriction was uncovered. Indeed, L-NAME increased the magnitude (-70.1 ± 19.7 %.min, p= 0.00234, Supplementary Fig. 4g1) and the maximal amplitude (-8.6 ± 1.9 %, p= 0.00089, Supplementary Fig. 4g2) of vasoconstrictive events induced by 2 Hz photostimulation. It also shortened their onset (475 ± 207s, p= 0.01292, Supplementary Fig. 4g3) without affecting their time to peak (1056 ± 185 s, p= 0.06502, Supplementary Fig. 4g4) and duration (926 ± 155 s, H(4,54)=9.278, p= 0.05452, Supplementary Fig. 4g5). These observations suggest that the vasodilation evoked by 2 Hz photostimulation involves NO synthesis, and that its blockade unmasks vasoconstriction. To confirm that NO signaling is required, we next used ODQ (10 µM (Hepp et al., 2007), n= 12 arterioles) to inhibit the sGC, the intracellular receptor responsible for NO-mediated vasodilation. Similar to NOS inhibition, blockade of NO-signaling did not alter the basal diameter (diameter before: 19.3 ± 2.9 µm vs. after ODQ: 19.2 ± 2.9 µm, W(12)= -14, p=0.6055, Supplementary Fig. 4c,d) confirming that NO signaling does not influence resting vascular tone. Similar to NOS inhibition by L-NAME, ODQ dramatically altered the overall vascular response (-27.6 ± 10.2 %.min, p= 0.02253, Fig. 4c and d1) and reduced the magnitude (3.0 ± 1.2%, p=0.00093, Fig. 4d2) and maximal amplitude of vasodilation (0.9 ± 0.3 %, p= 0.00172, Fig. 4d3). It also strongly delayed the vasodilation onset (1119 ± 216 s, p= 0.00130, Fig. 4e1), and reduced its duration (147 ± 58 s, p= 0.00123, Fig. 4e3) without impacting the time to peak of
vasodilation (1119 ± 216 s, Fig. 4e2). In contrast to L-NAME treatment, ODQ, did not alter the magnitude, amplitude, or kinetic properties of vasoconstrictive events (Supplementary Fig. 4g1-5). Taken together, these results indicate that AP firing is mandatory for the vasodilation evoked by 2 Hz photostimulation, which is mediated by NO synthesis and signaling. Furthermore, they show that 2 Hz optogenetic stimulation induces vasoconstriction that is masked by NO-dependent vasodilation.

**NPY-induced vasoconstriction involves the Y1 receptor independently of spiking activity.**

Since the majority of Sst interneurons are positive for NPY (Fig. 3b1,2) and virtually all cortical type 1 NO-producing interneurons express this neuropeptide (Dawson et al., 1991; Estrada and DeFelipe, 1998; Kubota et al., 1994, 2011; Perrenoud et al., 2012a), NPY could be responsible for the vasoconstriction observed with 20 Hz photostimulation, but also with 2 Hz photostimulation under NOS inhibition. We first verified that NPY induced vasoconstriction in a dose-dependent manner (Supplementary Fig. 5). 10 nM NPY (n= 11 arterioles) evoked very small vasoconstriction (Supplementary Fig. 5b e1-3), that only differed from control condition (n= 3 arterioles) for their onset (10 nM: 548 ± 194 s, p= 0.04430, Supplementary Fig. 5f1). 100 nM (n= 10 arterioles) and 1 µM NPY (n= 9 arterioles) evoked much stronger vasoconstriction, as evidenced by their magnitude (100 nM: -154.9 ± 40.7 %.min, p= 0.01154; 1 µM: -162.5 ± 23.0 %.min, p= 0.00474, Supplementary Fig. 5e1), maximal amplitude (100 nM: -13.1 ± 2.2 %, p= 0.02023; 1 µM: -25.1 ± 3.4 %, p= 0.00062, Supplementary Fig. 5e2). Vasoconstriction evoked by 100 nM and 1 µM occurred also much faster (100 nM: 282 ± 43 s, p= 0.04430; 1µM: 147 ± 18 s, p= 0.00281, Supplementary Fig. 5e3) and time to peak (100 nM: 545 ± 116 s, p= 0.02912; 1µM: 311 ± 38 s, p= 0.00395, Supplementary Fig. 5e4). They also lasted longer (998 ± 124 s, p= 0.01731; 1µM: 1234 ± 104s, p= 0.00113, Supplementary Fig. 5e5). Since the vasoconstriction induced by 20 Hz optogenetic stimulation of Sst-ChR2 and 100 nM NPY were of similar magnitude and maximal amplitude (Fig. 2f2,3 and supplementary Fig. 5e1,2) we next evaluated whether the NPY Y1 antagonist BIBP3226 could abolish the vasoconstriction induced by 100 nM. Pretreatment with BIBP3226 (1µM (Perrenoud et al., 2012b), n= 10 arterioles) dramatically reduced the vasoconstriction induced by 100 nM NPY (Fig. 5a), as evidenced by its effect on the magnitude (-8.0 ± 7.6 %.min, p= 0.00181, Fig. 5c1) and maximum amplitude (-0.8 ± 0.7 %, p= 0.00144, Fig. 5c2). BIBP3226 also dramatically delayed the onset (1486 ± 212 s, p=
430 0.01467, Fig. 5c3) and time to peak (1622 ± 137 s, p= 0.00207, Fig. 5c4) of NPY-induced
431 vasoconstriction and reduced its duration (175 ± 148 s, p= 0.01261, Fig. 5c5). These
432 observations confirm that NPY induces strong arteriolar vasoconstriction via Y1 receptors.
433 Since Y1 receptor is expressed by smooth muscle cells (Vanlandewijck et al., 2018; Zhang et
434 al., 2024) but also by neurons (Smith et al., 2019; Tasic et al., 2016; Zeisel et al., 2015) it has
435 been proposed that NPY-induced vasoconstriction could be mediated by intermediary
436 neuronal types (Kaplan et al., 2020). To rule out this possibility we investigated the impact of
437 AP firing inhibition on NPY-induced vasoconstriction. We found that TTX treatment (1 µM,
438 n= 9 arterioles) had no effect on the magnitude (-173.7 ± 58.8 %.min, p= 0.62911, Fig. 5c1),
439 maximum amplitude (-14.3 ± 4.2 %, p= 0.64910, Fig. 5c2), onset (554 ± 239s, p= 0.88537, Fig.
440 5c3), time to peak (752 ± 211 s, p= 0.45002, Fig. 5c4) and duration (903 ± 199s, p= 0.80606,
441 Fig. 2c5) of vasoconstriction induced by 100 nM NPY. Collectively these observations indicate
442 that NPY induces vasoconstriction via the Y1 receptor independently of neuronal activity.
443

444 Vasoconstriction induced by 20 Hz photostimulation of Sst-ChR2 interneurons requires AP
445 firing and NPY Y1 signaling

446 To investigate the mechanisms responsible for the vasoconstriction induced by 20 Hz
447 optogenetic stimulation of Sst-ChR2 interneurons, we first evaluated the requirement of AP
448 firing. TTX treatment (1µM, n= 10 arterioles) dramatically reduced the magnitude (-14.1 ±
449 5.8, %.min, p= 0.01810, Fig. 6a, c2), maximum amplitude (-1.9 ± 0.5 %, p= 0.00092, Fig. 6c3)
450 of vasoconstriction induced by 20 Hz photostimulation. It also delayed the vasoconstriction
451 onset (990 ± 236 s, p= 0.01993, Fig. 6d1) without significantly altering its apparent time to
452 peak (1163 ± 187 s, Fig. 6d2) and duration (571 ± 196 s, Fig. 6d3). TTX had also no impact on
453 vasodilatory events evoked by 20 Hz photostimulation (Supplementary Fig. 6c1-5). These
454 observations indicate that AP firing is mandatory for vasoconstriction induced by 20 Hz
455 optogenetic stimulation of Sst-ChR2 interneurons. We next investigated whether this
456 vascular response could be mediated by NPY. Antagonizing Y1 receptor in Sst-ChR2 slices
457 with BIBP3226 (1µM, n=10 arterioles) did not alter the arteriolar diameter (diameter before:
458 17.1 ± 2.0 µm vs. after BIBP3226: 17.0 ± 2.0 µm, t(9)= 0.82945, p= 0.4283, Supplementary Fig.
459 6a,b) indicating that tonic NPY level does not influence resting vascular tone. In contrast,
460 BIBP3226 dramatically reduced the magnitude (-21.8 ± 8.6 %.min, p= 0.01810, Fig. 6a,c2)
461 and the maximum amplitude (-2.0 ± 0.6 %, p= 0.00092, Fig. 6c3) of vasoconstriction induced
462 by 20 Hz optogenetic stimulation. It also delayed the onset of vasoconstriction (934 ± 244 s,

463 p= 0.01993, Fig 6d1) without altering its time to peak (1148 ± 201s, Fig. 6d2) and duration

464 (643 ± 224s, Fig. 6d3). Similar to TTX, BIBP3226 had no impact on the vasodilatory events

465 induced by 20 Hz Photostimulation (Supplementary Fig. 6c1-5). Taken together, these

466 observations indicate that vasoconstriction induced by 20 Hz photostimulation of Sst-ChR2

467 neurons requires both AP firing and NPY signaling through the Y1 receptor.

468

469

470
471 **DISCUSSION**

472 Here we used optogenetics in mouse cortical slices to show that Sst interneurons exert a
473 bimodal vascular response that requires AP firing and whose polarity depends on the
474 stimulation frequency. The vasodilation induced by 2 Hz photostimulation involves NO
475 synthesis and signaling via the sGC, whereas the vasoconstriction induced by 20 Hz
476 photostimulation implies NPY and activation of its Y1 receptor. Consistently, histochemical
477 investigations revealed that subpopulations of photostimulated Sst interneurons express the
478 NOS-1 and/or NPY (Fig. 7).
479

479

480 **Frequency dependence of the neuronal and vascular responses**

481 The observation that Sst interneurons respond supralinearly to low-frequency optogenetic
482 stimulation is reminiscent of the higher impedance observed at low frequency in
483 hippocampal Sst-positive oriens lacunosum-moleculare cells (Zemankovics et al., 2010),
484 which depends on a high capacitance and a pronounced hyperpolarisation-activated cation
485 current Ih, two features shared by cortical Sst interneurons (Halabisky et al., 2006;
486 Karagiannis et al., 2009, 2021; Perrenoud et al., 2012a). Indeed, according to Ohm's law, a
487 higher impedance at low frequency would have resulted in a larger voltage response
488 induced by the ChR2 photocurrents, resulting in a higher number of spikes per light pulse.
489 We found that depending on the AP firing frequency, Sst interneurons can induce
490 vasodilation or vasoconstriction. These results confirm and extend our previous observations
491 in rat cortical slices showing that the evoked firing of single Sst interneurons at a frequency
492 of at least 8 Hz results in vasoconstriction (Cauli et al., 2004). Similarly, the in vivo
493 optogenetic stimulation of GABAergic interneurons was found to induce
494 hyperemia/vasodilation, which can be followed by vasoconstriction (Anenberg et al., 2015;
495 Uhlirova et al., 2016). In addition, photostimulation of Sst interneurons also induced a
496 hyperemic response, followed by a decrease in blood flow that became more pronounced
497 with increasing duration of stimulation (Krawchuk et al., 2019; Lee et al., 2020). Our findings
498 are further supported by the observations showing that the selective optogenetic,
499 chemogenetic or pharmacological stimulation of NOS-1 expressing Sst interneurons induced
500 hyperemia/vasodilation (Echagarruga et al., 2020; Ruff et al., 2024) also support our
501 findings. Nevertheless compared to those in vivo, the responses we observed in slices were
502 notably much slower and longer lasting (Krawchuk et al., 2019; Lee et al., 2020). This is likely
503 due to the lower temperature in ex vivo experiments which has been reported to slowdown
504 the synthesis of vasoactive messenger (Rancillac et al., 2006) and subsequent reactions. In
505 contrast to in vivo observations (Uhlirova et al., 2016; Krawchuk et al., 2019; Lee et al.,
506 2020), the ex vivo magnitude of vasoconstriction was greater than that of vasodilation. Here,
507 the absence of vascular tone in slices, which favors vasoconstriction (Blanco et al., 2008),
508 probably led to an underestimation of vasodilation. On the other hand the absence of blood
509 flow in the slices may have reduced the scavenging of NO by hemoglobin (Rancillac et al.,
510 2006), whose in vivo concentration locally increases during functional hyperemia (Devor et
511 al., 2007; Lee et al., 2020), thereby prolonging ex vivo vasodilation.

512

513 Cellular and molecular mechanisms of vasodilation and vasoconstriction.

514 We found that the vasodilation induced by the 2 Hz optogenetic stimulation was dependent
515 on AP firing, NO synthesis and activation of soluble guanylate cyclase (sGC). The mandatory
516 role of APs indicates that Ca²⁺ entry via ChR2 alone is insufficient to drive vasodilation. This
517 likely reflects the essential role of voltage-gated Ca²⁺ channels in somatic Ca²⁺ elevation
518 (Mao et al., 2001) and the subsequent Ca²⁺-dependent activation of NOS-1, NO release and
519 arteriolar vasodilation (Rancillac et al., 2006; Attwell et al., 2010; Cauli and Hamel, 2010;
520 Mishra et al., 2016; Iadecola, 2017; Cauli and Hamel, 2018). Consistently, some
521 supragranular NOS-1 neurons showed an increase in somatic Ca²⁺ prior to vasodilation
522 evoked by whisker stimulation and/or spontaneous locomotion in awake head-fixed mice
523 whereas others showed a gradual and delayed activation (Ahn et al., 2023). Although type 1
524 NOS-1 interneurons are preferentially located in deep layers (Perrenoud et al., 2012a), our
525 histochemical observations revealed the presence of NOS-1 positive Sst-Cre interneurons in
526 superficial layers. Together with the high excitability of Sst interneurons (Karagiannis et al.,
527 2009, 2021; Perrenoud et al., 2012a), these observations suggest that some of these
528 interneurons correspond to the fast responder NOS-1 neurons (Ahn et al., 2023). The
529 abolition of the optogenetically induced vasodilation evoked by 2 Hz photostimulation by
530 both NOS and sGC inhibition strongly suggests that it is mediated by NO synthesis, release
531 and direct action on smooth muscle cells, leading to their relaxation (Fig. 7). However, NOS
532 inhibition revealed a vasoconstriction, most likely mediated by NPY (Uhlirova et al., 2016),
533 that was not observed when sGC or AP firing was blocked. These observations suggest that a
534 NO-sensitive, but soluble guanylate cyclase-independent, mechanism of vasodilation
counterbalanced the vasoconstriction disclosed by L-NAME. Active astrocytes, which have been shown to produce vasodilatory epoxyeicosatrienoic acids via the P450 epoxygenase (Alkayed et al., 1997), may be responsible for such a vasodilation. Indeed, astrocytes have been reported to be robustly activated by the repetitive firing of Sst interneurons (Mariotti et al., 2018), and the P450 epoxygenase, which is expressed in arteriolar but not cappillary endfeet (Mishra et al., 2016), is inhibited by NO (Roman, 2002; Metea and Newman, 2006; Attwell et al., 2010).

We found that vasoconstriction predominates over vasodilation when Sst-ChR2 interneurons are photostimulated at 20 Hz. This reflects the much higher number of NPY-positive Sst-ChR2 interneurons compared to type 1 NOS-1 interneurons (Karagiannis et al., 2009; Perrenoud et al., 2012a) and the dense network of NPY positive terminals on blood vessels (Abounader and Hamel, 1997). Furthermore, the release of NPY from large dense-core vesicles requires a higher firing frequency than the release of neurotransmitters from synaptic vesicles (Baraban and Tallent, 2004). It is also possible that the release of NO requires a lower firing frequency than that of NPY. We found that optogenetically induced vasoconstriction was almost completely abolished by the antagonism of Y1 receptors, suggesting that this effect was essentially mediated by NPY, as reported for the optogenetic stimulation of GABAergic interneurons (Uhlirova et al., 2016). We cannot exclude the indirect contribution of Sst and GABA which can exert vasoconstrictive effects (Long et al., 1992; Fergus and Lee, 1997). Indeed, the repetitive firing of Sst interneurons robustly activates astrocytes via Sst and GABA<sub>B</sub> receptors (Mariotti et al., 2018), which could in turn induce vasoconstriction via 20-hydroxyeicosatetraenoic acid (Mulligan and MacVicar, 2004) and/or K+ (Girouard et al., 2010). However, Sst is not as efficient as NPY in inducing vasoconstriction in cortical slices (Cauli et al., 2004), and the amount of Sst in the cerebral cortex is reduced in Sst-Cre mice (Viollet et al., 2017), but not that of NPY. On the other hand, the vasoconstrictor effect of the GABA<sub>B</sub> receptor activation requires AP firing (Fergus and Lee, 1997), suggesting that it is mediated by neurons and not by astrocytes. Thus, it is more likely that the vasoconstriction induced by 20 Hz optogenetic stimulation of Sst-ChR2 interneurons is essentially mediated by NPY and the Y1 receptors.

**Physiological relevance of the bidirectional control of neurovascular coupling by Sst-interneurons**
The ability of Sst interneurons to drive a bimodal vascular response ex vivo depending on firing frequency is consistent with in vivo observations showing that increasing the duration and/or the frequency of photostimulation of Sst-ChR2 interneurons favors the observation of decreased blood flow, in addition to hyperemia (Krawchuk et al., 2019; Lee et al., 2020). However, the evoked decrease in blood flow appears to be more pronounced at a site different from the site of hyperemia (Lee et al., 2020). This is reminiscent of the center/surround pattern of vasodilation/vasoconstriction observed in vivo in response to sensory stimulation, with the vasodilation in the activated area preceding the peripheral vasoconstriction (Devor et al., 2007; Boorman et al., 2010; Uhlirova et al., 2016). Because fast-responding NOS-1 neurons are activated before the onset of vasodilation (Ahn et al., 2023), they are most likely involved in the initiation of vasodilation. Consistently, local NO production precedes functional hyperemia but disappears before the end of stimulation (Buerk et al., 2003), presumably because of the short half-life of NO (Wood and Garthwaite, 1994) and its increased scavenging induced by functional hyperemia. The delayed and/or peripheral vasoconstriction (Devor et al., 2007; Uhlirova et al., 2016; Krawchuk et al., 2019; Lee et al., 2020) could be due to its blunting by early occurring vasodilation, a higher level of activity required for NPY release (Baraban and Tallent, 2004), as suggested by our observations, and/or a different site of action of NO and NPY (Estrada and DeFelipe, 1998). Sst/NOS-1/NPY neurons largely correspond to long-range GABAergic neurons (Tomioka et al., 2005) and have been shown to locally induce vasodilation (Echagarruga et al., 2020; Ruff et al., 2024). It remains to determine whether their interhemispheric long-range projection (Ruff et al., 2024) could explain the vasoconstriction observed contralaterally to the activated area (Devor et al., 2008) when their activity is elevated. Sst/NOS-1/NPY also correspond to cortical sleep active neurons (Gerashchenko et al., 2008; Kilduff et al., 2011). Furthermore, Sst and Sst/NOS-1 interneurons are involved in slow and delta wave activities, respectively (Morairty et al., 2013; Funk et al., 2017; Zielinski et al., 2019), a frequency range associated with sleep that we showed was the most efficient at activating them. Interestingly, oscillating vasodilation of large amplitude and a vasoconstriction are observed during non REM sleep and upon awakening, respectively (Turner et al., 2020; Bojarskaite et al., 2023). Whether the coordinated action of NO and NPY could participate in these hemodynamic changes in a frequency-dependent manner remains also to be determined.
599 **Bidirectional control of neurovascular coupling by Sst interneurons in pathological**
600 **conditions**

601 Several neurological disorders, such as epilepsy (Sada et al., 2015; Gaxiola-Valdez et al., 2017) or Alzheimer's disease (Palop et al., 2007; Kimbrough et al., 2015; Le Douce J. et al., 2020), associate hyperactivity with gliovascular and metabolic deficits that are likely to alter the control of neurovascular coupling by Sst interneurons. For example, cerebral lactate, which is produced by astrocytes from glucose during excitatory transmission (Pellerin and Magistretti, 1994; Cauli et al., 2023), is dramatically increased in epilepsy (Slais et al., 2008).

607 Drugs that interfere with lactate metabolism by neurons have emerged as a promising strategy for treating epilepsy (Sada et al., 2015), an effect that involves the neuronal uptake of lactate, its oxidative metabolism and the subsequent closure of ATP-sensitive potassium channels. Since Sst interneurons functionally express these channels (Karagiannis et al., 2021) and are among the neurons showing the highest mitochondrial content (Gulyas et al., 2006), their activity is likely to be efficiently enhanced by the presence of lactate and its oxidative metabolism. This could favor the GABAergic inhibition of pyramidal cells (Silberberg and Markram, 2007), but also decreased glutamatergic transmission (Bacci et al., 2002), and Y1-mediated vasoconstriction (Uhlirova et al., 2016), thereby mitigating lactate-induced hyperactivity (Sada et al., 2015).

617 Hyperactivity has also been reported in several models of Alzheimer's disease (Palop et al., 2007; Bezzina et al., 2015; Zott et al., 2019), and loss of Sst interneurons has been observed in patients (Gaspar et al., 1989) and in the early stages of a mouse model of the disease (Ramos et al., 2006). Furthermore, Sst/NOS-1/NPY interneurons play a key role in non-REM sleep (Gerashchenko et al., 2008; Morairty et al., 2013; Zielinski et al., 2019), during which the fluctuations in the perivascular space favor the clearance of β-amyloid peptides (Iliff et al., 2013; Xie et al., 2013; Bojarskaite et al., 2023). These observations suggest that the control of NVC by Sst interneurons may be altered early, accelerating amyloid plaque accumulation and disease progression. However, NVC has been shown to be impaired (Niwa et al., 2002; Kimbrough et al., 2015; Lourenco et al., 2017), unchanged (Sharp et al., 2020) or even enhanced (Shabir et al., 2020; Kim et al., 2024), making it difficult to assess its alterations. Whether these differences depend on the mouse model, the stage of disease progression, or the experimental condition remains to be investigated.
630 **Conclusion**

631 Here, using a combination of approaches, we describe the mechanisms by which Sst
632 interneurons bidirectionally control NVC as a function of their firing frequency. Vasodilation
633 was preferentially evoked at low-frequency photostimulation and primarily involves NO
634 release and activation of its vascular receptor sGC. In contrast, vasoconstriction was more
635 reliably induced at high frequency and involves NPY release and activation of the vascular
636 receptor Y1. This finding will help to update the interpretation of the functional brain
637 imaging signals used to map network activity in health and disease (ladecola, 2017; Zhang
638 and Raichle, 2010). This bimodal control of neurovascular coupling may also help to define
639 therapeutic targets for epilepsy and Alzheimer's disease in which hyperactivity, gliovascular
640 and metabolic deficits overlap (Palop and Mucke, 2010; Sada et al., 2015; Gaxiola-Valdez et
641 al., 2017).

642

643 **Data availability statement**

644 The data that support the results of this study are available from the corresponding author
645 upon reasonable request.

646

647 **Competing interests**

648 The authors declare no competing interests.

649

650 **Acknowledgements**

651 We acknowledge the invaluable support of the animal (RongIBPS) and imaging facilities of
652 the IBPS. Financial support was provided by grants from the Agence Nationale de la
653 Recherche (ANR-17-CE37-0010-03, B.C.; ANR-20-CE14-0025, D.L.; ANR-23-CE14-0038-01,
654 B.C.), and the i-Bio initiative of Sorbonne University (B.C.). B.M was supported by a
655 fellowship from Centre National de la Recherche Scientifique. B.L.G. and E.B. were supported
656 by fellowships from Fondation pour la Recherche sur Alzheimer.

657

658 **Author contributions**

659 B.M., J.O.R, S.P. B.L.G., M.T., and E.B. designed experiments, acquired and analyzed the data,
660 and edited the manuscript. B.M. and B.C. drafted the manuscript. L.R.V.C. edited the
661 manuscript. I.D. designed experiments and edited the manuscript. H.S. analyzed the data.
662 D.L. provided resources and edited the manuscript. B.C. designed experiments, analyzed the data and edited the manuscript.

664

665

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983 **FIGURE LEGEND**
<figure><img src="image_2.png" /><figcaption>Figure 1: Electrophysiological characterization of cortical Sst-ChR2 neurons. (a) Representative recording showing the voltage responses induced by 800 ms current injections (bottom traces) of -100 pA, -80 pA, -60 pA, -40 pA, -20 pA, 0 pA, +15 and +150 pA in a cortical EYFP/ChR2-expressing neuron from a Sst-Cre:Ai32 mouse. Note the pronounced voltage sag after the initial peak response (arrow) to hyperpolarizing current injections (middle traces). Just-above threshold current pulse (+15 pA) triggered a discharge of three action potentials (middle traces). Note the complex AHP consisting of a fast AHP, a small ADP, and a medium AHP. Near saturation, a strong depolarizing current (+150 pA) elicited a discharge of action potentials with a marked frequency adaptation and a monotonous amplitude accommodation (upper trace). Top inset: IR videomicroscopy picture of the recorded neuron, the pial surface is upward (scale bar, 20 µm). Bottom insets, corresponding field of view showing the EYFP fluorescence of the recorded neuron. (b1-6) Voltage responses (upper traces) of the Sst-ChR2 neuron shown in (a) induced by photostimulations (470 nm, 10 s train, 5 ms pulses) delivered at 1, 2, 5, 10, 20 and 40 Hz (cyan lower traces) and instantaneous spike success rate (middle trace). The red boxes highlight the first second of the zoomed recording in the right insets</figcaption></figure>
(scaled bars 200 ms, 40 mV). The red lines represent a spike success rate of 100%. Note that more
than one action potential is elicited per light pulse delivered at 1 or 2 Hz whereas at frequencies
above 5 Hz this success rate rapidly decreases to a maximum of one spike per light pulse. (c)
Histogram showing the average spike success rate induced by photostimulations at 1, 2, 5, 10, 20, or
40 Hz (n = 9 Sst-ChR2 cortical neurons from 6 SST-Cre:Ai32 mice). Note the average spike success rate
exceeding 100 % at photostimulation frequencies lower than 10 Hz. Data are presented as means ±
SEM. The SEMs envelope the mean traces Statistically different with *p< 0.05, **p< 0.01 and ***p<
0.001; n.s.: not statistically significant.

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<figure><img src="image_3.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>g1</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>g2</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>g3</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>h1</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>h2</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>h3</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>g4</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>g5</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>g6</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>h4</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>h5</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>h6</figcaption></figure>

<figure><img src="image_20.png" /></figure>

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<caption>Figure 2: Bidirectional frequency-dependent vascular response induced by optogenetic stimulation of cortical Sst-ChR2 neurons. (a, d) Representative examples of infrared pictures of diving arterioles and diameter measurements before (0 min) and after (6, 11, 22 min) photostimulation at 2 Hz (a) and 20 Hz (d) in Sst-Cre:Ai32 cortical slices. The pial surface is upward. Scale bars 20 µm. (a, d) White, red and blue calipers correspond to baseline, dilated, and contracted arteriolar diameters, respectively. The dashed yellow lines indicate the initial diameters. Note the slight increase (a) and decrease followed by an increase (d) in diameter induced by photostimulation delivered at 2 Hz (a)</caption>
and 20 Hz (d), respectively. (b, e) Waterfall plots showing the kinetics of changes in diameter of single arterioles induced by photostimulation at 2 Hz (b) and 20 Hz (e). The inset shows the color code used for constriction (blue) and dilation (red) used in both panels. Arterioles are sorted by increasing values of total vascular changes. The cyan planes and arrows indicate the onset of photostimulation. Black arrows indicate changes in diameter of the arterioles shown in (a) and (b). (c, f) Kinetics of mean diameter changes induced by 2 Hz (c) or 20 Hz (f) photostimulation. The dashed yellow lines correspond to the initial diameters. (c) Note the average small diameter increase induced by 2 Hz photostimulation in Sst-Cre:Ai32 (red, n= 14 arterioles from 8 mice) but not in C57Bl6 arterioles (black, n= 6 arterioles from 5 mice) and (f) the average progressive diameter decrease induced by 20 Hz photostimulation in Sst-Cre:Ai32 (blue, n= 16 arterioles from 10 mice) but not in WT arterioles (black, n= 4 arterioles from 3 mice). (g1-6, h1-6) Violin plots summarizing the comparison of overall vascular response (g1, h1), magnitude of dilation (g2) and contraction (h2), maximum dilation (g3) and contraction (h3), onset (g4,h4) time to peak (g5,h5) and duration (g6,h6) of dilation (g2-6) and contraction (h2-6) between arterioles from slices of Sst-Cre:Ai32 and C57Bl6 mice stimulated at 2 (g1-6) and 20 Hz (h1-6). Note the larger (g2), earlier (g4) and longer (g6) dilation in Sst-ChR2 slices stimulated at 2 Hz and the larger (h2,3), and earlier onset (h4) and time to peak (h5) contraction in Sst-ChR2 slices stimulated at 20 Hz Data are presented as individual values. Solid and dashed black bars correspond to median and quartile values. * and ** statistically different from C57Bl6 mice with p< 0.05 and 0.01, respectively. n.s.: not statistically significant.
<figure><img src="image_21.png" /></figure>

a1

GFP

<figure><img src="image_22.png" /></figure>

b1

GFP

<figure><img src="image_23.png" /></figure>

a2

NOS-1

<figure><img src="image_24.png" /></figure>

b2

NPY

<figure><img src="image_25.png" /></figure>

a3

Merge

<figure><img src="image_26.png" /></figure>

b3

Merge

<figure><img src="image_27.png" /></figure>

a4

<figure><img src="image_28.png" /></figure>

b4
Figure 3: Expression of NOS-1 and NPY in Sst-Cre neurons. (a1,b1) Representative maximum intensity projections of 30 µm thick stacked confocal images showing GFP (green) expression in cortical slices from Sst-GCaMP6f mice. (a2) NOS-1 immunolabeling (red) is present in rare neurons. (b2) Numerous GFP-positive neurons are immunolabeled for NPY (red). (a3,b3) Double fluorescence images showing the co-expression of NOS-1 in rare GFP-positive neurons (a3) and NPY in numerous GFP positive neurons (b3). The white rectangles in a3 and b3 correspond to the zoomed regions shown in a4 and b4, respectively. Arrowheads indicate GPF-positive neurons immunolabelled for NOS-1 (a4) or NPY (b4). Asterisks denote GFP-negative neurons immunostained for NOS-1 (a4) or NPY (b4). Scale bars: 200 µm (a3, b3) and 40 µm (a4,b4).

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<figure><img src="image_29.png" /></figure>

Figure 4: Vasodilation induced by 2 Hz photostimulation of Sst-ChR2 neurons requires spiking activity and nitric oxide signaling. (a, b, c) Effects of tetrodotoxin (TTX, 1 µM, brown, n= 10 arterioles from 6 mice), NOS inhibitor (L-NAME, orange, 100 µM, n= 9 arterioles from 6 mice) and ODQ (10 µM, purple, n= 12 arterioles from 7 mice), on the kinetics of the vascular response induced by 2 Hz photostimulation of Sst-ChR2 neurons. The red traces correspond to the control condition shown in Fig. 2b. The SEMs envelope the mean traces. Vasodilation is abolished by blocking action potentials with TTX (a), inhibiting nitric oxide synthase with L-NAME (b) or sGC with ODQ (c). The dashed black lines indicate the normalized initial diameters. Violin plots summarizing the effects of TTX (brown), L-NAME (orange) and ODQ (purple) on the overall vascular response (d1, H₅,₅₁= 16.105, p= 0.00288), magnitude (d2, H₅,₅₁= 18.625, p= 0.00093), maximum (d3, H₅,₅₁= 14.834, p=0.00506), onset (e1, H₅,₅₁= 20.592, p= 0.00038), time to peak (e2, H₅,₅₁= 8.299, p= 0.08123), and duration (e3, H₅,₅₁= 16.867, p= 0.00205) of vasodilation induced by 2 Hz photostimulation (red). Data are presented as individual values. Solid and dashed black bars correspond to median and quartile values. * and ** statistically different from control condition with p<0.05 and 0.01 respectively.
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<figure><img src="image_30.png" /><figcaption>Figure 5: NPY-induced vasoconstriction involves the Y1 receptor independently of spiking activity. (a,b) Kinetics of diameter changes induced by 5 minutes exogenous application of 100 nM NPY (blue bars) under control condition (a, blue trace) and in presence of 1 µM BIBP3226 (a, green trace, n= 10 arterioles from 6 mice) or 1 µM TTX (b, brown trace, n= 9 arterioles from 4 mice). The SEMs envelope the mean traces. Violin plots summarizing the effects of BIBP 3226 (green) and TTX (brown) on the on the magnitude (c1, H<sub>(4,32)</sub>= 17.244, p= 0.00063), maximum (c2, H<sub>(4,32)</sub>= 17.859, p= 0.00047), onset (c3, H<sub>(4,32)</sub>= 13.529, p= 0.00362), time to peak (c4, H<sub>(4,32)</sub>= 16.209, p= 0.00103), and duration (c5, H<sub>(4,32)</sub>= 13.557, p= 0.00357) of vasoconstriction induced by NPY. The selective NPY Y1 receptor antagonist BIBP3226 but not TTX abolished the NPY-induced vasoconstriction. Data are presented as individual values. Solid and dashed black bars correspond to median and quartile values. * and ** statistically different from the 100 nM control condition with p< 0.05 and 0.01, respectively.</figcaption></figure>

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<figure><img src="image_31.png" /><figcaption>Figure 6: Vasoconstriction induced by 20 Hz photostimulation of Sst-ChR2 neurons requires spiking activity and NPY signaling. (a, b) Effects of tetrodotoxin (TTX, 1 µM, brown, n= 10 arterioles from 5 mice) and the selective Y1 receptor antagonist (BIBP 3226, 1 µM, green, n= 11 arterioles from 9 mice)</figcaption></figure>

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1084 on the kinetics of the vascular response induced by 20 Hz photostimulation of Sst-ChR2 neurons. The
1085 blue trace corresponds to the control condition shown in Fig. 2e. The SEMs envelope the mean
1086 traces. Vasoconstriction is abolished by blocking action potentials with TTX (a) or by the selective
1087 antagonist of Y1 receptor, BIBP 3226 (b). Violin plots summarizing the effects of TTX (brown) and
1088 BIBP 3226 (green) on the overall vascular response (c1, F*(3,22.04)= 2.557, p= 0.08117), magnitude (c2,
1089 H(4,41)= 10.774, p= 0.01301), maximum (c3, H(4,41)= 19.152, p= 0.00025), onset (d1, H(4,41)= 10.671, p=
1090 0.01364), time to peak (d2, H(4,41)= 6.373, p= 0.09481), and duration (d3, H(4,41)= 4.726, p= 0.19300) of
1091 vasoconstriction induced by 20 Hz photostimulation (cyan). Data are presented as individual values.
1092 Solid and dashed black bars correspond to median and quartile values. *, ** and *** statistically
1093 different from control condition with p<0.05, p<0.01 and 0.001 respectively.
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<figure><img src="image_32.png" /></figure>

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<caption>**Figure 7:** Schematic representation of the mechanisms of bidirectional control of neurovascular coupling by Sst-ChR2 interneurons. Bidirectional responses are shown on both sides of the Sst-NPY-NOS-1 interneuron. **Left:** 2 Hz stimulation of Sst-ChR2 interneurons activates NOS-1 and releases NO which in turn activates the sGC of smooth muscle cells leading to their relaxation and vasodilation. This effect is abolished by TTX (brown) and by inhibition of NOS with L-NAME (orange) and sGC with ODQ (purple). **Right:** 20 Hz photostimulation of Sst-ChR2 interneurons induces NPY release and activation of vascular receptor Y1 leading to smooth muscle cell contraction and vasoconstriction. This effect is suppressed by the TTX (brown) and the Y1 receptor antagonist, BIBP3226 (green).</caption>
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