neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide

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Author's Accepted Manuscript Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide Cátia F. Lourenço, Ricardo M. Santos, Rui M. Barbosa, Enrique Cadenas, Rafael Radi, João Laranjinha PII: S0891-5849(14)00237-8 DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021 Reference: FRB12030 To appear in: Free Radical Biology and Medicine Received date: 12 February 2014 Revised date: 19 May 2014 Accepted date: 23 May 2014 Cite this article as: Cátia F. Lourenço, Ricardo M. Santos, Rui M. Barbosa, Enrique Cadenas, Rafael Radi, João Laranjinha, Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. www.elsevier.com/locate/freerad- biomed

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Author's Accepted Manuscript

Neurovascular coupling in hippocampus ismediated via diffusion by neuronal-derivednitric oxide

Cátia F. Lourenço, Ricardo M. Santos, Rui M.Barbosa, Enrique Cadenas, Rafael Radi, JoãoLaranjinha

PII: S0891-5849(14)00237-8DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021Reference: FRB12030

To appear in: Free Radical Biology and Medicine

Received date: 12 February 2014Revised date: 19 May 2014Accepted date: 23 May 2014

Cite this article as: Cátia F. Lourenço, Ricardo M. Santos, Rui M. Barbosa,Enrique Cadenas, Rafael Radi, João Laranjinha, Neurovascular coupling inhippocampus is mediated via diffusion by neuronal-derived nitric oxide, FreeRadical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021

This is a PDF file of an unedited manuscript that has been accepted forpublication. As a service to our customers we are providing this early version ofthe manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting galley proof before it is published in its final citable form.Please note that during the production process errors may be discovered whichcould affect the content, and all legal disclaimers that apply to the journalpertain.

www.elsevier.com/locate/freerad-

biomed

� �

1�

Neurovascular�coupling�in�hippocampus�is�mediated�via�diffusion�by�neuronal�1�

derived�nitric�oxide�2�

Cátia�F.�Lourenço1,�Ricardo�M.�Santos1,�Rui�M.�Barbosa1,�Enrique�Cadenas2,�Rafael�Radi3,�João�3�

Laranjinha1*�4�

�5�1Faculty� of� Pharmacy� and� Center� for� Neurosciences� and� Cell� Biology,� University� of� Coimbra,� Health�6�Sciences�Campus,�Azinhaga�de�Santa�Comba,�3000�548�Coimbra,�Portugal2�7�2Department�of�Pharmacology�and�Pharmaceutical�Sciences,�School�of�Pharmacy,�University�of�Southern�8�California,�Los�Angeles,�CA�90089,�USA.�9�3Department� of� Biochemistry� and� Center� for� Free� Radical� and� Biomedical� Research,� Facultad� de�10�Medicina,�Universidad�de�la�Republica,�Montevideo,�Uruguay.�11�

�12�

*To�whom�correspondence�should�be�addressed:�João�Laranjinha,�Center�for�Neurosciences�13�and�Cell�Biology�and�Faculty�of�Pharmacy,�University�of�Coimbra,�Health�Sciences�Campus,�14�Azinhaga�de�Santa�Comba,�3000�548�Coimbra,�Portugal.�E�mail:�[email protected]�15�

�16��17�Abbreviated�title:�Nitric�oxide�mediates�the�neurovascular�coupling�18��19�Conflict�of�Interest:��The�authors�declare�no�competing�financial�interests�20��21�Abstract:��22�

�23�

The� coupling� between� neuronal� activity� and� cerebral� blood� flow� (CBF)� is� essential� for� normal� brain�24�

function.� The� mechanisms� behind� such� neurovascular� coupling� process� remain� elusive,� mainly� due� to�25�

difficulties� to�probe�dynamically� the� functional�and�coordinated� interaction�between�neurons�and� the�26�

vasculature� in� vivo.� Direct� and� simultaneous� measurements� of� nitric� oxide� (�NO)� dynamics� and� CBF�27�

changes�in�hippocampus�in�vivo�support�that�during�glutamatergic�activation�nNOS�derived��NO�induces�28�

a�time,�spatially,�and�amplitude�coupled�increase�in�the�local�CBF,�later�followed�by�a�transient�increase�29�

in� local� O2� tension.� These� events� are� dependent� on� the� activation� of� NMDA�glutamate� receptor� and�30�

2�

nNOS,� without� a� significant� contribution� of� endothelial�derived� �NO� and� astrocyte�neuron� signaling�1�

pathways.�Upon�diffusion�of��NO�from�active�neurons,�the�vascular�response�encompasses�the�activation�2�

of�soluble�guanylate�cyclase.�Hence,�in�the�hippocampus,�neurovascular�coupling�is�mediated�by�nNOS�3�

derived��NO�via�a�diffusional�connection�between�active�glutamatergic�neurons�and�blood�vessels.�4�

�5�

Introduction�6�

�7�

Neuronal�activity�imposes�a�need�for�blood�flow�carrying�substrates�in�order�for�the�brain�to�maintain�its�8�

functional�and�structural�integrity[1].�Intensive�research�during�the�last�decades�aimed�to�untangle�the�9�

underlying�mechanisms�that�couple�the�neuronal�activity�to�cerebral�blood�flow�(CBF)�–�neurovascular�10�

coupling[2].�Strong�evidences� indicate� that�astrocytes�are�critical�contributors� to� the�process,�bridging�11�

neurons� and� blood� vessels,� although� several� questions� still� persist[3]� and� disparate� temporal� events�12�

demand� reconciliation[4].� Other,� non�astrocytic� physiological� mechanisms� have� been� proposed� to�13�

underlie�the�coupling�between�neuronal�activity�and�the�changes�in�CBF[5�7].�In�this�regard,�the�concept�14�

that��NO,�produced�upon�neuronal�activation,�reaches�blood�vessels�by�diffusion�from�neurons,�inducing�15�

vasodilation,� has� been� a� tempting� suggestion[8�12].� In� neurons,� �NO� is� produced� upon� glutamatergic�16�

activation� by� the� neuronal� isoform� of� nitric� oxide� synthase� (nNOS),� an� enzyme� physically� and�17�

functionally� coupled� to� the� NMDA� glutamate� receptors� [13,� 14].� Because� �NO� is� highly� diffusible� and�18�

overcomes� specific� ligand�receptor� interaction,� its� biological� action� is� critically� determined� by� its�19�

concentration�and�temporal�dynamics,�as�well�as�by�the�distribution�of�the�potential�targets;�by�this�way,�20�

the�profile�of��NO�change�in�time�and�space�is�translated�into�a�biological�action�[15].�These�unorthodox�21�

properties� of� �NO� have� raised� difficulties� to� directly� substantiate� its� role� as� a� diffusible� messenger� in�22�

neurovascular�coupling,�as�well�as�in�other�signaling�pathways.��23�

3�

Pharmacological� approaches� have� provided� insights� on� the� involvement� of� �NO� in� neurovascular�1�

coupling�[10,�12,�16,�17],�but�also�opposite�evidences�[18�20].�It�has�also�been�suggested�that,�although�2�

�NO�is�required,�it�does�not�directly�mediate�the�neuron�to�vessels�signaling,�at�least�in�somatosensory�3�

cortex[21].� A� step� forward� was� provided� by� the� in� vivo� measurements� of� �NO� dynamics,� during�4�

activation�of� rat� somatosensory�cortex�and� the�observation� that�a� transient� increase� in� �NO�preceded�5�

CBF�change[22],�however�without�clarifying�the�source�of��NO�and�the�interdependency�of�both�events.�6�

Thus,� in� spite� of� the� intensive� research� on� �NO� over� the� last� decades,� its� role� in� the� neurovascular�7�

coupling�still�remains�elusive.�8�

In�this�work�we�aimed�to�study�the�neuronal�derived��NO�dynamics�in�connection�with�CBF�changes�in�9�

hippocampus,�identifying�the�pathway�for��NO�production�and�uncovering�the�underlying�mechanism�for�10�

vasodilation.� We� used� an� array� incorporating� a� �NO�selective� microelectrode[23],� a� microinjection�11�

pipette� and� a� laser� Doppler� probe� assembled� in� a� pre�defined� geometry,� that� allowed� to� monitor�12�

neurovascular� coupling� upon� local� glutamate� stimulation� within� �NO� diffusional� spread.� On� basis� of� a�13�

real�time� and� in� vivo� sequence� comprising� glutamate� stimulation,� �NO� production� and� CBF� (and� O2)�14�

increase,�we�demonstrate� that,� in�hippocampus,�neurovascular�coupling� is�mediated�by�nNOS�derived�15�

�NO�via�a�diffusional�connection.�16�

�17�

Materials�and�Methods�18�

�19�

Array� for� �NO�and�CBF�measurements.�The� �NO� sensors� were� fabricated� as� previously� described[24].�20�

Briefly,�a�single�carbon�fiber�(30�μm�Ø�Textron�Lowell,�MA)�was�encased�in�a�glass�capillary�and�pulled�in�21�

a�vertical�puller.�The�protruding�carbon�fiber�was�cut�to�a�tip�length�of�200±50�μm.�The�electrical�contact�22�

between� the� carbon� fiber� and� a� copper� wire� was� achieved� by� using� conductive� silver� paint� (RS,�23�

4�

Northants,� U.K.).� To� improve� their� analytical� properties� for� in� vivo� measurements� of� �NO,� the� sensors�1�

were� coated� with� Nafion®� (5%� solution,� 2� coatings� with� 4� minutes� drying� at� 170°C)� and� with� o�2�

phenylenediamine� (o�PD� 5� mM� solution� was� electropolymerized� at� a� constant� potential� of� +0.7� V� vs�3�

Ag/AgCl�during�30�minutes).�Each�sensor�was�evaluated�for��NO�sensitivity�and�selectivity�against�major�4�

interferents�(ascorbate,�nitrite,�noradrenaline�and�dopamine)�by�constant�voltage�amperometry�at�+0.9�5�

V�vs�Ag/AgCl�using�a�FAST�16�high�speed�electrochemical�system�(Quanteon,�LLC,�Nicholasville,�KY)�in�a�6�

two�electrode� configuration.� The� sensors� used� in� this� study� presented� an� average� sensitivity� of�7�

258±92pA/μM� and� selectivity� ratios� of� 28673:1,� 5732:1,� 239:1� and� 213:1� against� ascorbate,� nitrite,�8�

noradrenaline� and� dopamine,� respectively.� Cerebral� blood� flow� was� measured� using� a� laser� Doppler�9�

flowmeter� device� (Periflux� system� 5000,� Perimed,� Sweden)� to� which� a� needle� probe� (PF411;� outer�10�

diameter,� 450� �m;� fiber� separation,� 150� �m;� wavelength,� 780� nm)� was� attached.� Calibration� of� the�11�

probe�was�done�routinely�using�PF1001�motility�standard�(Perimed,�Sweden)�to�equalize�the�perfusion�12�

values�among� the�different� recordings.�The� time� constant�was� set� to�0.03� s�and� the� signal�processing�13�

unit�used�a�bandwidth�of�32�Hz.�Laser�Doppler�flowmetry�measures�CBF� in�arbitrary�units� (a.u.)�and� is�14�

therefore�used�for�measuring�relative�changes�in�CBF.�The��NO�sensor�and�the�Laser�Doppler�probe�were�15�

assembled�to�an�ejection�micropipette�using�sticky�wax�in�the�configuration�schematized�in�Figure�1.�The�16�

micropipette� was� filled� with� solutions� using� a� syringe� fitted� with� a� flexible� microfilament� (MicroFil,�17�

World�Precision�Instruments,�UK)�previously�to�brain�insertion.��18�

�19�

Array� for� O2� and� CBF� measurements.� To� record� O2� fluctuations� the� array� configuration� included� a�20�

micropipette�and�a�carbon�fiber�microelectrode�similar�to�that�used�for� �NO�recording.�Such�electrode�21�

was�evaluated�for�O2�sensitivity�by�constant�voltage�amperometry�at��0.8�V�vs�Ag/AgCl�using�a�FAST�16�22�

5�

high�speed�electrochemical� system� (Quanteon,� LLC,�Nicholasville,�KY)� in�a� two�electrode�configuration�1�

essentially�as�previously�described.�2�

�3�

In�vivo�experimental�setup.��All�the�procedures�used�in�this�study�were�performed�in�accordance�with�4�

the� European� Union� Council� Directive� for� the� Care� and� Use� of� Laboratory� animals,� 2010/63/EU,�5�

implemented� under� the� supervision� and� approval� of� the� local� Institutional� Animal� Care� and� Use�6�

Committee� of� the� animal� facility� of� Center� for� Neurosciences� and� Cell� Biology� University� of� Coimbra�7�

(ORBEA)�and� licensed�by�the�national�regulatory�office�(Direcção�Geral�de�Alimentação�e�Veterinária—8�

DGAV).� The� animals� were� submitted� to� surgery� under� anesthesia� and� body� temperature� and� blood�9�

pressure�was�controlled�during�the�experiments�as�detailed�below.�At�the�end�of� the�experiments�the�10�

animals�were�sacrificed�by�cervical�displacement�while�still�under�anesthesia.�Forty�five�male�Wistar�rats�11�

(8�10�weeks;�weight�294�±�31�g)�were�anaesthetized�by�an� intraperitoneal� injection�of�urethane� (1.25�12�

g/Kg)�and�placed� in�a�stereotaxic�apparatus�(Stoelting�Co.,�USA).�Body�temperature�was�maintained�at�13�

37ºC�using�a�deltaphase�isothermal�pad�(BrainTree�Scientific,�MA,�USA)�and�controlled�through�a�rectal�14�

thermometer.�An�incision�was�made�with�the�scalp�and�the�skin�was�reflected�to�expose�the�surface�of�15�

the�skull,�allowing�for�drilling�a�hole�(3x4�mm)�in�the�surface�overlying�the�hippocampus.�Another�hole�16�

(~1� mm� diameter)� was� drilled� in� a� site� remote� from� the� recording� area� for� insertion� of� an� Ag/AgCl�17�

reference�electrode�(200�μm�diameter).�After�removing�the�dura�matter,�the�array�was�inserted�into�the�18�

rat�hippocampus�according�to�coordinates�calculated�based�on�the�rat�brain�atlas�of�Paxinos�and�Watson�19�

(2007)� using� the� tip� of� the� microelectrode� as� reference� in� the� bregma� (anteroposterior� �4.1� � mm;�20�

mediolateral��2.8�mm�and�dorsoventral��3.7�mm).�After�the�insertion�of�the�array�into�the�hippocampus�21�

it�was�allowed�to�stabilize�for�30�minutes�before�the�beginning�of�the�experiment.�22�

�23�

6�

Experimental�Design/�Drug�application.�Solutions,�L�glutamate�20�mM,�NMDA�(N�methyl�D�aspartate)�1�

0.1� mM,� �NO� solution� 0.1� mM� or� tACPD� (trans�1�amino�cyclopentane�1,3�dicarboxylic� acid)� 0.1�1� mM�2�

(25�nL)�in�saline�buffer�(NaCl�0.9%,�pH�7.4),�were�locally�applied�from�the�tip�of�the�micropipette�using�a�3�

Picospritzer� III� (Parker� Hannifin� Corp.,� General� Valve� Operation,� USA).� A� �NO� solution� 0.1� mM� was�4�

prepared�by�diluting�a�saturated��NO�solution,�prepared�as�previously�described�[25],� in�deoxygenated�5�

saline�buffer.�Stimulations�were�performed�by�pressure�pulses�of�1s�at�7�15�psi�with�a�minimum�interval�6�

of� 15� minutes� of� recover.� Typically� three� initial� responses� were� obtained� before� pharmacology�7�

modulation� to� achieve� a� steady� state� level.� MK�801� (1� mg/Kg� in� saline),� 7�Nitroindazole� (50� mg/Kg� in�8�

DMSO),�L�NIO�(40�mg/Kg�in�DMSO),�acetylsalicylic�acid�(300�mg/Kg�in�saline)�and�caffeine�(50�mg/kg�in�9�

saline)�were�injected�intraperitoneally.�A�cocktail�of�MPEP�and�LY367385�(100�nmol�each)�was�injected�in�10�

the�lateral�ventricle�using�a�micropipette.�ODQ�(25�pmol�in�DMSO�0.5%)�and�sodium�fluoroacetate�(20�11�

μmol)�were� locally�applied�by�an�extra�micropipette�attached� to� the�previous�described�array� in�close�12�

proximity� to� the� laser� Doppler� probe.� The� effects� were� evaluated� after� 10� to� 30� min� for� ODQ� and�13�

MPEP/LY36785� and� sodium� fluoroacetate,� and� after� 20� to� 40� minutes� for� the� remaining� and� the�14�

responses�compared�with� the�corresponding�control�experiments� (with�vehicles).� �Blood�pressure�was�15�

non�invasively�measured�in�the�tail�using�a�LE5001�system�(Letica,�Scientific�Instruments).�Arterial�blood�16�

gases�and�pH�were�evaluated�in�a�Rapidlab®�1260�Blood�Gas�Analyzer�(Siemens�Healthcare�Diagnostics)�17�

from�blood�samples�collected�from�the�femoral�artery�4�to�7�h�after�anesthetic�injection.��18�

�19�

Data�Analysis�and�Statistics.�The��NO�and�CBF�recordings�were�synchronized�using�OriginPro�7.5�based�20�

on� markers� extracted� from� the� respective� software.� The� remaining� analyses� were� performed� with�21�

GraphPad�Prism�5.�Data�are�presented�as�mean�±�SEM.�Statistical�analysis�of� the�data�was�performed�22�

using�Student’s�t�test.�The� �NO�and�CBF�signals�were�characterized�in�terms�of�1)��NO�peak�flux�of�the�23�

7�

signal,�based�on�the�conversion�of�the�amperometric�currents�to��NO�fluxes�according�to�Faraday’s�law�1�

(I=n.F.�,� in� which� I� corresponds� to� the� amperometric� current,� n� corresponds� to� the� one� electron� per�2�

molecule�exchanged�for�the�oxidation�of��NO,�F�corresponds�to�the�Faraday�constant�and���is�the�flux)�3�

or�amplitude�change�(the�increase�of�CBF�beyond�the�CBF�basal�levels,�considered�100%�in�the�absence�4�

of�stimuli),� respectively;�2)�Trise,� the�time� in�seconds�necessary�to�reach�the�maximum�amplitude�after�5�

stimulation,�3)�Ttotal,�the�time�in�seconds�from�the�stimulation�point�to�return�to�basal�levels.��6�

�7�

�8�

Results�9�

�10�

Nitric�oxide�and�cerebral�blood�flow�changes:�coupling�in�space,�time,�and�amplitude�11�

We�have�previously�reported�that�a�controlled�and�localized�glutamate�stimulus�in�the�rat�hippocampus�12�

promotes�an�instantaneous�and�transient�elevation�of��NO�concentration�levels�through�the�activation�of�13�

ionotropic�glutamate�receptors[23].�By�upgrading�such�experimental�strategy,�simultaneously�measuring�14�

local�CBF,�we�observed�that�a�transient��NO�increase�induced�by�glutamate�ejection�(0.5�nmol,�25�nL,�1s),�15�

was�followed,�seconds�later,�by�a�transient�change�in�CBF�(Figure�1).��NO�production�was�characterized�16�

by�a�flux�of�3.5±1.8�fmol.s�1�with�a�time�rise�of�22±3�s�and�a�total�duration�of�64±4�s�(54�peaks�analyzed�17�

from�15� individual�experiments).�The�CBF�started�to� increase�7±2�s�after�stimulation�reaching�122±5%�18�

beyond�the�basal�level�after�62±3�s�and�returning�to�basal�levels�after�216±15�s.�19�

Likewise,� the�specific�activation�of�NMDA�glutamate�receptor,�using�the�synthetic�agonist�N�methyl�D�20�

aspartate�(NMDA),�resulted�in��NO�and�CBF�dynamics�similar�to�those�obtained�with�glutamate�and�with�21�

the�same�temporal�correlation,�thus�imparting�specificity�to�the�pathway�leading�to��NO�production.�The�22�

ejection�of�NMDA�(2.5�pmol)� resulted� in�a�transient� �NO�production�characterized�by�a� flux�of�3.5±0.5�23�

8�

fmol.s�1�with�a�time�rise�of�19±4�s�and�a�total�duration�of�78±5�s,�that�was�followed�by�an�increase�of�CBF�1�

that� lasted� 296±46s� and� reached� the� maximum� of� 104±10%� beyond� the� basal� level� after� 73±9� s� (12�2�

peaks�analyzed�from�5�individual�experiments).�Local�pressure�ejection�of�the�vehicle�(NaCl�0.9%)�caused�3�

no�change�in�either�the�baseline�oxidation�current�or�the�CBF�levels�(Supplementary�Figure�2).�4�

The� CBF� changes� induced� by� glutamatergic� activation� in� hippocampus� were� independent� of� arterial�5�

blood�pressure,�blood�gases�or�pH,�which�remained�stable�and�within�the�physiological�range�during�the�6�

time�window�in�which�experiments�were�performed,�as�randomly�assessed.�On�average,�the�values�for�7�

pH,�paCO2�and�paO2�were�7.39±0.07;�41±7�mmHg�and�108±31�mmHg,�respectively.��8�

�9�

Exogenous�nitric�oxide�mimics�glutamate�induced�CBF�changes�10�

The�temporal�coupling�between�endogenous� �NO�signal�and�the� increase� in�CBF�was�mimicked�by� the�11�

local� ejection� of� a� �NO� solution.� Figure� 2� shows� that� when� �NO� is� exogenously� applied,� a� temporal�12�

correlation�is�observed�between�the��NO�signal�and�CBF�change�that�is�similar�to�that�observed�for�the�13�

endogenous�glutamate�dependent�production�of��NO�(CBF�started�to�increase�8±2�s�after��NO�ejection,�14�

lasting�for�282±60�s�whereas��NO�temporary� increase�was�65�±�12�s,�n=3).�Nevertheless,�and�although�15�

the� �NO� flux� achieved� is� higher� when� exogenously� added� (11.3±2.7� fmol.s�1� versus� 3.8±1.6� fmol.s�1�16�

achieved�via�glutamate�stimulus),�the�CBF�change�from�the�baseline�is�weaker�than�that�observed�upon�17�

endogenous��NO�production�(55±11%�versus�122±5%�beyond�the�basal�level).�In�this�regard,�it�has�to�be�18�

considered�the�peculiar�nature�of��NO�volume�signaling�in�the�brain[26]:�following�activation�of�a�volume�19�

of� tissue,� comprising� numerous� synaptic� �NO� sources,� small� dispersed� �NO� sources� of� low� individual�20�

efficacy�can�cooperate�to�originate�an�extensive�and�strong�volume�signal,�inducing�an�increase�in�CBF.�21�

We� have� recently� shown� that� the� decay� of� �NO� when� produced� endogenously� in� hippocampus� via�22�

activation� of� NMDA� glutamate� receptors� is� much� slower� than� when� ejected� exogenously[23,� 27].�23�

9�

Therefore,�one�could�expect�a� lower�diffusional�spread�due�the� fast�decay�achieved�upon� �NO�release�1�

from�a�single�point�in�the�tip�of�the�pipette,�as�compared�with�glutamate�stimulus�and,�consequently,�a�2�

lower�effect�on�the�regional�CBF.�3�

�4�

Glutamate�induced�CBF�changes�correlate�with�oxygen�tension�changes�5�

The� local� transient� change� in� CBF� elicited� by� neuronal��NO� is� expected� to� translate� into� correlated�6�

changes�in�local�O2�tension.�By�measuring�O2�tensions,�we�observed�that�the�local�transient�changes�in�7�

CBF�elicited�by�glutamate�stimulation�are�followed,�seconds�later,�by�a�correlated�transient�elevation�in�8�

O2�tension�(Figure�3,�R2=0.69,�p=0.0016,�n=12�from�3�individual�experiments).�This�observation�supports�9�

the�prediction�that�the��NO�signal�is�sequentially�followed�by�CBF�increase�and�by�a�transitory�elevation�10�

in� O2� tension.� It� should� be� remarked� that� the� geometrical� configuration� of� the� array� and� the� physical�11�

properties�of�carbon�fiber�microelectrodes�and�LDF�probe�impose�that�the�measurement�of��NO/O2�and�12�

CBF�are�preformed�within�a�volume�of�hippocampal�tissue�likely�comprising�a�few�hundreds�of�microns�13�

of� diameter.� Within� this� volume� of� tissue,� the� fine� correlation� observed� between� O2� tension� and� CBF�14�

events,� measured� at� separate� locations� within� �NO� diffusion� field,� further� supports� the� validity� of� the�15�

approach�used�to�study�the�relationship�between��NO�and�CBF.�16�

�17�

Neuronal�derived��NO�is�the�mediator�of�neurovascular�coupling�in�hippocampus�18�

The�neurovascular�coupling�process�is�expected�to�be�prone�to�modulation�by�functional� interferences�19�

along� the� pathway� of� nNOS�dependent� �NO� production,� in� particular� at� the� level� of� NMDA� glutamate�20�

receptor� and� nNOS.� Among� several� potential� pharmacological� tools,� we� have� selected� a� glutamate�21�

NMDA� receptor� blocker� (MK�801),� a� selective� nNOS� inhibitor� (7�NI)� and� a� selective� eNOS� inhibitor� (L�22�

NIO).� The� results� obtained� are� summarized� in� Figure� 4A.� The� responses� obtained� following� drugs�23�

10�

injection� were� compared� with� the� corresponding� control� experiments� (with� vehicles).� None� of� the�1�

vehicles�used�promote�any�significant�effect�in��NO�or�CBF�responses�(Supplementary�Figure�2).�Blocking�2�

of� the� NMDA� receptor� elicited� a� significant� inhibition� of� �NO� production� (73±8%,� p=0.0017,� n=3)� that�3�

was�accompanied�by�a�dramatic�decrease�of�glutamate�induced�CBF�(74±3%,�p=0.0008,�n=3).�Likewise,�4�

the� inhibition�of�the�neuronal� isoform�of�NOS�by�7�NI� induced�a�significant� inhibition�of�both� �NO�and�5�

CBF�responses� to�glutamate� (62±10�and�83±6%,�p=0.0002�and�p=0.0025,� respectively,�n=4),�as�well�as�6�

the� decrease� in� CBF� basal� levels� (27±7%).� Conversely,� no� significant� effects� were� found� on� either�7�

induced�� or� basal� levels� upon� inhibition� of� the� endothelial� NOS� isoform� with� L�NIO� (Figure� 4C).�8�

Nevertheless,�it�was�observed�an�increase�in�mean�arterial�blood�pressure�of�the�animals�after�the�L�NIO�9�

injection,�supporting�its�efficacy�in�terms�of�eNOS�inhibition�(n=4,�MABP�90.3±1.1�to�114.9±2.5�mmHg).��10�

The�modulation�of�the�NMDA�receptor:nNOS�pathway�showed�that,�in�hippocampus,�the�increase�in�CBF�11�

was�dependent�on��NO�signal�amplitude�(Figure�4B).�Within�the�range�tested,�the�relationship�between�12�

�NO�and�CBF�showed�to�be�linear�above�a�threshold�of�0.43�fmol.s�1,�which�is�coherent�with�the�concept�13�

of� a� linear� relationship� between� neuronal� activity� and� vascular� responses� provided� by� neuroimaging�14�

studies[28].� However,� it� should� be� pointed� out� that� below� the� threshold� our� data� does� not� identify�15�

whether�the�relationship�is�linear�or�a�gating�process�is�operative.�16�

�17�

The��NO�mediated�neurovascular�coupling�encompasses�the�activation�of�soluble�guanylate�cyclase�18�

The� classical� pathway� for� vasodilation� mediated� by� �NO� involves� the� activation� of� soluble� guanylate�19�

cyclase� (sGC)� in� smooth� muscle� cells,� which� via� cGMP�dependent� protein� kinases,� promotes� the�20�

dephosphorylation�of�myosin�light�chains�and�thus�vasodilation[15].�In�order�to�unravel�the�mechanism�21�

by�which�neuronal�derived��NO�promoted�the�increase�in�CBF,�the�effect�of�ODQ,�a�selective�heme�site�22�

inhibitor� of� soluble� guanylate� cyclase,� was� evaluated.� Following� the� local� application� of� ODQ� in�23�

11�

hippocampus,� glutamate� induced� a� typical� �NO� concentration� dynamics� but� the� CBF� changes� were�1�

significantly� reduced� (67±7%,� p=0.002,� n=4)� (Figure� 5).� The� slight� inhibition� of� �NO� signal� observed� is�2�

likely� related� to� the� unspecific� inhibition� of� nNOS� by� ODQ[29]� (nNOS� being� an� hemeprotein� might� be�3�

affected� by� ODQ),� and� not� a� consequence� of� cGMP� decrease� in� neurons,� as� �NO� concentration� was�4�

resumed� in� the� following� stimulations� while� CBF� remained� inhibited,� progressively� returning� to� the�5�

control�values�obtained� in� the�absence�of�ODQ.�The�uncoupling�observed�between� �NO�concentration�6�

dynamics�and�CBF�changes�in�the�presence�of�ODQ�strongly�supports�that��NO�produced,�and�diffusing�7�

from�neurons,�reaches�vascular�smooth�muscle�cells�and�triggers�vasodilation�via�sGC�activation.�8�

�9�

The�astrocytic�pathway�is�not�explicitly�involved�in�the��NO�mediated�neurovascular�coupling��10�

Given�the�commonly�accepted�paradigm�of�astrocytes�bridging�neurons�and�vascular�cells�encompassing�11�

metabotropic� glutamate� receptors� activation� and� subsequent� production� of� arachidonic� acid�12�

metabolites[2,� 6],� we� addressed� the� potential� contribution� of� the� astrocytic� bridge� to� neurovascular�13�

coupling�in�hippocampus�under�our�experimental�conditions.�For�that�purpose,�a�cocktail�of�antagonists�14�

of�metabotropic�glutamate�receptors�(LY367385�and�MPEP)�was�injected�intracerobroventricularly�in�the�15�

rat� brain� to� inhibit� mGluR1� and� mGluR5,� respectively.� If� glutamate�triggered� [Ca2+]i� elevations� in�16�

astrocytes�are�crucial�for�neurovascular�coupling,�their�specific�inhibition�should�hamper�the�increase�of�17�

CBF�induced�by�glutamate�via�the�astrocytes.�However,�the�inhibition�of�astrocyte�mGluR�receptors�did�18�

not� elicit� any� alteration� in� the� recorded� �NO� and� CBF� signals� (Figure� 6A).� Both,� inhibition� of�19�

cyclooxygenase� activity� by� acetylsalicylic� acid� –downstream� in� the� astrocytic� pathway–� and� the�20�

impairment�of�astrocytic�metabolism�linked�to�the�inhibition�of�mitochondrial�aconitase�via�metabolism�21�

of�sodium�fluoroacetate��which�is�preferentially�uptaked�by�glial�cells[30]��had�no�effect�either.�Also,�no�22�

contribution� could� be� assigned� to� adenosine� receptors,� argued� to� intermediate� a� complex� signaling�23�

12�

process� of� neuronal� activity�induced� increase� in� cerebral� vasodilation� [31,� 32],� as� caffeine,� a�1�

nonselective� antagonist� of� adenosine� receptors[33]� also� failed� to� modulate� both� �NO� and� CBF� (Figure�2�

6A).�As�an�additional�control,�the�specific�activation�of�mGLuR�receptors�by�the�mGluR�agonist�tACPD�–3�

known�to�promote�Ca2+�elevations�in�astrocyte�somata�and�related�pathways[5]–�did�not�elicit�changes�4�

in�either��NO�production�or�CBF�(Figure�6B).�5�

�6�

�NO�dependent�CBF�increase�is�region�specific�7�

The� regional� occurrence� of� the� diffusive� connection� established� by� �NO� between� neurons� and� vessels�8�

without� the� intermediacy� of� cellular� systems� between� glutamatergic� synapses� and� vessels� in� brain�9�

regions�other�than�hippocampus�was�assessed,�for�it�has�been�previously�reported�that�the�mechanisms�10�

underlying�neurovascular�coupling�may�be�region�specific.�With�that�intent�the�experimental�approach�11�

used� in�hippocampus�was�applied� to�probe�cerebral�cortex�with� the�tip�of� the�array�positioned� in� the�12�

deeper� layers,�where�nNOS�immunoreactivity� is�predominant[34].�Whereas�data�support�the�coupling,�13�

the�profiles�of��NO�and�CBF�change�do�not�reproduce�the�phenomenon�observed�in�hippocampus�in�all�14�

its�dimensions�(Figure�7).�In�fact,�in�cerebral�cortex,�as�compared�with�hippocampus,�glutamate�induced�15�

a�weaker�production�of��NO�(0.46±0.14�fmol.s�1)�followed�by�a�less�intense�increase�in�CBF�(46±6%)�(12�16�

peaks� analyzed� from� 4� individual� experiments)� but,� quantitatively,� the� response� of� �NO� and� CBF� to�17�

glutamate�in�cerebral�cortex�was�not�proportionally�attenuated;�while��NO�production�in�cerebral�cortex�18�

was� 87�4%� lower� than� in� hippocampus� and� the� reduction� of� CBF� change� was� only� 62�5%� lower.� We�19�

would� predict� that� distinct� neuroanatomic� regions� (encompassing� variations� in� nNOS� expression,�20�

vascularization�density,�mean�distance�between�glutamatergic�neurons�and�arterioles,��NO�inactivation�21�

mechanisms)� exhibited� different� quantitative� relationships� between� �NO� and� CBF� signals� but�22�

maintaining� a� temporal� and� local� correlation,� as� observed.� Therefore,� the� kinetics� of� neuronal��NO�23�

13�

dependent� CBF� changes� may� be� intrinsically� distinct� in� hippocampus� as� compared� with� other� brain�1�

regions.�2�

Discussion�3�

The�mechanisms�that�regulate�the�synergy�between�cerebral�microcirculation�and�local�neuronal�activity�4�

have�been�debated�over�a�century�without�clear�conclusions,�in�part�because�of�the�severe�experimental�5�

limitations�to�measure�the�process�in�real�time�in�the�natural�working�environment.�The�identification�of�6�

�NO� as� a� diffusible� vasodilator� produced� at� active� neurons,� via� ionotropic� glutamate� receptors�7�

dependent�pathways,�led�to�the�hypothesis�that�it�could�be�the�mediator�coupling�the�brain�activity�to�8�

CBF[9]�but,�paradoxically,� it� introduced�a� further�difficulty.� In� fact,�because� �NO� is�small,�hydrophobic,�9�

and�overcomes�storage�and�specific�interaction�with�receptors,�it�conveys�information�associated�to�its�10�

concentration�dynamics,�a�parameter�difficult�to�measure�in�vivo.�11�

By�simultaneously�and�direct�measuring��NO�and�CBF�dynamics�in�the�hippocampus,�this�work�supports�12�

that� �NO,� generated� by� nNOS� during� glutamatergic� activation,� induces� a� coupled� increase� in� the� local�13�

CBF�(Figure�8).�Coherently,��NO�and�CBF�events�precede�an�increase�of�O2�tension�from�a�background,�a�14�

critical�prediction�of� the� neurovascular� coupling�concept.�The�observation� that�both� MK�801�and�7�NI�15�

individually� elicited� a� significant� inhibition� of� �NO� production,� and� that� this� effect� was� translated� into�16�

CBF� changes,� clearly� identifies� the� pathway� that� paves� the� neurovascular� coupling� process� in� the�17�

hippocampus�and�recognizes�nNOS�derived��NO�as�the�coupler�molecule.�Data�showing�the�abolishment�18�

of� CBF� changes� in� spite� of� the� strong� �NO� signal� under� conditions� of� sGC� inhibition� with� ODQ,�19�

mechanistically�established�the�pathway�for��NO�dependent�CBF�changes�in�the�rat�hippocampus�by�the�20�

canonic�cGMP�dependent�vasodilation�pathway�and��NO�as�a�critical�messenger.�Furthermore,�data�also�21�

supports�that�nNOS�derived��NO�participates�in�the�maintenance�of�resting�CBF,�as�7�NI�administration�22�

also� promoted� a� decrease� in� hippocampal� CBF� basal� levels.� It� should� be� noted� that� glutamate�23�

14�

microinjections� (within� the� range� used)� and� electric� stimulation� induce� equivalent� cerebrovascular�1�

effects,�as�shown�previously�in�cerebellar�cortex�upon�electric�stimulation�of�parallel�fibers[35].�2�

The�pattern�of��NO�production�and�CBF�changes�measured�accomplishes�the�fundamental�criteria�of�the�3�

neurovascular� coupling� concept:� a� temporal,� amplitude� and� spatial� relationship[36].� Besides� the�4�

temporal� and� amplitude� correlation� evident� in� the� occurrence� of� both� dynamics,� data� show� that� the�5�

increase� in� CBF� is� limited� in� space,� affecting� just� the� area� of� increased� neuronal� activity,� the�6�

hippocampus,� for� if� the� laser�Doppler�probe� is�placed� in� the�overlay�cerebral� cortex�no�CBF�change� is�7�

observed�in�response�to�glutamate.��8�

The�strategy�established�in�this�work�also�complies�with�the�criteria�for��NO�signaling�in�the�framework�9�

of� the� coupling� inasmuch� as� stimulation� of� dispersed� glutamatergic� synapses� via� nNOS� generates� a�10�

volume�signaling�that�encompasses�the�diffusion�of��NO�from�neurons�to�nearby�arterioles�to�induce�an�11�

increase� in� CBF,� thus� establishing� that,� upon� eliciting� neuronal� activation,� �NO� from� nNOS� launches� a�12�

diffusible� connection� between� neurons� and� the� vasculature.� The� concept� of� �NO�mediated� diffusible�13�

connection�is�supported�by�two�observations:�first,�in�hippocampal�slices,�in�the�absence�of�a�functional�14�

vascular�system�to�remove��NO,�the�diffusion�field�of��NO�was�measured�as�being�close�to�400�μm[37];�15�

second,�the�mean�distance�between�arterioles�and�nerve�fibers�along�the�longitudinal�axis�of�pyramidal�16�

layer� neurons� in� the� CA1� region� of� hippocampus� is� circa� 150� μm[38],� i.e.,� within� the� range� of� �NO�17�

diffusional� spread.� Furthermore,� mathematical� modeling� of� the� physicochemical� process� of� �NO�18�

production� from� an� active� neuron� in� cerebellar� slices� estimated� that� the� range� of� �NO� concentration�19�

reaching� the� nearby� blood� vessels� rise� drastically,� exceeding� c.a.� 100� fold� its� basal� levels� during�20�

stimulated�neuronal�activity[39].��21�

Among�the�wide�collection�of�proposed�mechanisms,�a�generally�accepted�paradigm�for�neurovascular�22�

coupling�supports�the�view�of�astrocytes�carrying�the�neuronal�signal�by�bridging�neurons�and�vascular�23�

15�

cells[6].�Astrocytes�are�known�to�respond�to�glutamate�via�metabotropic�glutamate�receptors�(mGluR)�1�

activation,�involving�a�transient�increase�in�[Ca2+]i�that�ends�up�in�the�synthesis�of�vasoactive�substances�2�

upon� activation� of� phospholipase� A2� and� arachidonic� acid� production.� The� vasoactive� compounds�3�

include�prostaglandin�E2� (via�cycloxygenase)[5]�and/or�epoxyeicosatrienoic�acids� (via�cytochrome�P450�4�

epoxygenase)[7,� 40].� Accordingly,� it� has� been� speculated� that� �NO� levels� may� modulate� the�5�

cerebrovascular� response� to� mGluR� activation� by� regulating� the� enzymatic� conversion� of� arachidonic�6�

acid�via�cytochrome�P450�to�either�vasodilators�or�vasoconstrictors[41].�However,�our�results�show�that,�7�

in�hippocampus,�this�pathway�has�minor�if�any�contribution�in�the�neurovascular�coupling�promoted�by�8�

glutamatergic�activation,�supporting�that�this�process�is�developed�by�different�mechanisms�depending�9�

on� the� brain� region.� Also,� it� should� also� be� considered� that� glutamate�dependent� astrocyte� signaling�10�

may�change� during�development.�Recently,� it�was� demonstrated� that�astrocytic� mGluR5� expression� is�11�

developmentally�regulated,�being�undetectable� in�adulthood,�and�that,� in�adult�mice,�astrocytes�failed�12�

to� respond� to� the� mGluR5� agonist� tACPD� with� significant� Ca2+� increase[3].� Also,� it� is� of� note� that�13�

functional� NMDA� receptors[42]� and� constitutive� NOS� expression[43]have� been� found� in� cultured�14�

astrocytes.�Therefore,�and�although�the�abolishment�of�astrocytic�metabolism�by�sodium�fluoroacetate�15�

did�not�affect�the�recorded�dynamics,�we�cannot�fully�exclude�the�potential�involvement�of�astrocytes�in�16�

the�NMDA�nNOS�pathway,�leading�to�CBF�changes.�Further�supporting�the�notion�of�region�specificity�of�17�

neurovascular� coupling� process[44],� we� observed� that� glutamatergic� activation� in� cerebral� cortex,� as�18�

compared� with� hippocampus,� resulted� in� a� significant� lower� �NO� production� of� translated� into� less�19�

dramatic�CBF�changes.�The� �NO/CBF�ratio�was,�however,� lower� in�the�cortex,� in�agreement�to�the� less�20�

outstanding�role�attributed�to�the��NO�in�this�brain�region[18�20].��Also,�the�latency�of�CBF�response�is�21�

slower� in� the� cerebral� cortex� than� in� hippocampus,� which,� in� turn,� is� longer� than� that� commonly�22�

reported� by� studies� using� electric� stimulation� [22,� 45,� 46].� Apart� the� stimulation� paradigm,� these�23�

16�

variations�can�be�related�with�differences� in�the�neurovascular�coupling�process�and�vascular�network�1�

among� regions.� In� fact,� Lauritzen� group� has� observed� slower� CBF� responses� in� cerebellum� [47,� 48]� as�2�

compared� with� the� somatosensory� system� [46].� These� observations� reinforce� the� notion� that� the�3�

kinetics� of� neuronal��NO� dependent� CBF� changes� may� be� intrinsically� distinct� in� hippocampus,� as�4�

compared�with�other�brain�regions.�5�

Following�the�formulation�of�the�hypothesis�of��NO�as�a�candidate�to�mediate�neurovascular�coupling[9]�6�

intensive� research� has� attempted� to� elucidate� its� role� in� the� process.� All� the� studies,� however,� were�7�

largely�based� in� the�pharmacological�modulation�of�NOS�activity�and,�expectedly,�controversial� results�8�

were�collected.�Assuming�as�plausible�that�different�pathways�and�molecules�might�be� involved� in�the�9�

neurovascular� coupling� phenomenon,� and� that� the� relative� contribution� of� these� pathways� can� vary�10�

among� regions,� we� should� however� be� aware� that� the� use� of� such� pharmacological� tools� without�11�

additional�verification�of�their�efficacy�can�give�rise�to�misleading�results.�For�instance,�it�was�shown�that�12�

7�NI,�a�widely�used�selective�nNOS�inhibitor,�did�not�maximally�inhibit�nNOS�activity�and�did�not�affect�13�

all�brain� regions�at� the�same�extent[49].�Additionally,�methodological�variables�such�as� the�anesthetic�14�

used� seem� to� contribute� to� the� discrepancy� of� the� results,� because� their� possible� effects� on�15�

neurotransmission� and� hemodynamics[50].� Therefore,� for� a� messenger� that� conveys� information�16�

associated�with�its�concentration�dynamics,�its�direct,�localized�and�quantitative�measurement�in�vivo�is�17�

mandatory�to�progress�in�the�understanding�of�its�role�in�the�mechanisms�of�neurovascular�coupling,�a�18�

critical� pathway� for� proper� brain� function.� Indeed,� a� study� in� cerebellar� slices,� by� simultaneously�19�

measuring��NO�dynamics�and�capillaries�local�enlargements�associated�to�treatment�with�NOS�inhibitor�20�

or�with�tetrodotoxin,�suggested�that��NO�from�neuronal�origin�is�responsible�for�the�vast�majority�of�the�21�

vasomotor�response�in�cerebellum[51].�22�

17�

Finally,� from� a� pure� conceptual� viewpoint,� the� mechanism� of� neuronal�derived� �NO,� mediating�1�

neurovascular� coupling�via�volume�signaling,�may� be�a�non�canonical� fashion� to�underlie�a�process�of�2�

vital�importance�for�the�brain�to�preserve�its�structural�and�functional�integrity.�However,�although�the�3�

mechanistic� link� to� match� blood� supply� with� the� metabolic� demands� imposed� by� increased� neuronal�4�

activity�is�based�in�diffusion�and�characterized�by�lack�of�specific�recognition�by�receptors,�it�still�remains�5�

a�highly�and�intrinsically�controlled�mechanism.�Because�red�blood�cells�constitutes�the�major�pathway�6�

for� �NO� inactivation� in� the� brain[27],� the� increase� in� the� cerebral� blood� flow� triggered� by� neuronal�7�

derived��NO�constitute�itself�a�mechanism�to�shape�the��NO�concentration�dynamics�and�interrupt�the�8�

signaling�pathway.��9�

�10�

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11�

�12�

Figure�Legends:�13�

�14�

Figure� 1:� Coupling� between� �NO� concentration� dynamics� and� CBF� changes� upon� glutamatergic�15�

activation�in�the�hippocampus.�A.�Representative�recording�of�the�simultaneous�measurements�of��NO�16�

concentration� dynamics� (bottom,� blue� line)� and� CBF� changes� (top,� red� line)� in� the� hippocampus� of�17�

urethane�anesthetized�rat�over�a�period�of�five�stimulations.�Glutamate�was�locally�applied�at�the�times�18�

indicated� by� the� arrows� (0.5� nmol,� 1� s).� B.� Detail� of� the� temporal� correlation� of� �NO� concentration�19�

dynamics� and� CBF� changes� evoked� by� local� glutamate� stimulation.� C.� Array� used� to� measure�20�

simultaneously� •NO� concentration� dynamics� and� CBF� changes� in� the� rat� hippocampus.� The� array�21�

comprises� a� •NO�selective� microelectrode,� a� microinjection� pipette� and� a� laser� Doppler� flow� probe�22�

assembled�in�a�pre�defined�geometry.�23�

�24�

22�

Figure�2:�Exogenous� �NO�mimics�glutamate�induced�CBF�changes.�Average�recordings�of�CBF�changes�1�

(red� line)� in� response�to�exogenous�and� localized� �NO�application� in� the�hippocampus� (blue� line).� �NO�2�

solution�(100�μM�in�saline)�was�locally�applied�by�pressure�ejection�(5�psi,�90�s,�1�μL).�3�

�4�

Figure� 3:� The� CBF� change� coupled� to� �NO� dynamics� is� followed� by� a� transient� increase� in� local� O2�5�

tension.�Average� recordings� of� simultaneous� measurements� of� O2� (green� line)� and� CBF� changes� (red�6�

line)� in� the� hippocampus� of� urethane�anesthetized� rat� and� temporal� correlation� with� �NO� dynamics�7�

(blue� line).� Insert� shows� the� correlation� between� the� amplitudes� of� O2� and� CBF� changes� (R2=0.69,�8�

p=0.0016,�n=12�from�3�individual�experiments).�9�

�10�

Figure� 4:� Glutamate�induced� �NO� production� and� CBF� changes� are� dependent� on� the� activation� of�11�

NMDA� receptor� and� nNOS.� A.� Effect� of� MK�801� (1� mg/kg,� NMDA� receptor� blocker),� 7�NI� (50� mg/Kg,�12�

selective�nNOS�inhibitor)�and�L�NIO�(40�mg/Kg,�selective�eNOS�inhibitor).�Data�represents�mean�±�SEM.�13�

Statistical�analysis�was�performed�by�Student’s�t�test�in�relation�to�control�experiments�(***p<�0.001).�B.�14�

Plot�of�the�linear�relationship�between�the�amplitude�of��NO�production�and�CBF�changes�obtained�by�15�

glutamatergic�activation.�The�linear�regression�showed�a�R2=0.98�and�a�slope�of�78.9%�CBF/fmol.s�1�NO�16�

(X� intercept=0.438).�C.�Average� recordings� of� the� 7�NI� and� L�NIO� effects� on� the� �NO� production� (blue�17�

line)�and�CBF�changes�(red�line)�elicited�by�glutamate�in�the�hippocampus.�18�

�19�

Figure� 5:� Neuronal� derived��NO�mediates� the� neurovascular� coupling� through� activation� of� soluble�20�

guanylate� cyclase.� Effect� ODQ,� a� selective� heme�site� inhibitor� of� soluble� guanylate� cyclase,� on�21�

23�

glutamate�induced��NO�signals�(blue�line)�and�CBF�changes�(red�line)�in�the�rat�hippocampus.�ODQ�was�1�

locally�applied�through�an�additional�pipette�attached�to�the�array�in�the�proximity�of�the�laser�Doppler�2�

probe�(25�pmol,�0.5�μL).�3�

�4�

Figure� 6:� The� astrocytic� pathway� is� not� explicitly� involved� in� the� �NO�mediated� neurovascular�5�

coupling.�A.�Effect�of�LY367385/MPEP�(100��M�each,�antagonists�of�metabotropic�glutamate�receptors,�6�

mGluR1�and�mGluR5,�respectively,�n=6),�acetylsalicylic�acid�(ASA,�300�mg/kg,�COX�inhibitor,�n=5),�sodium�7�

fluoroacetate� (SFA,� 20� μmol� locally,� glial� toxin,� n=5)� and� caffeine� (CAF,� 50� mg/kg,� nonselective�8�

antagonist�of�adenosine�receptors,�n=3)�on��NO�signals�and�CBF�changes.�Data�represents�mean�±�SEM.�9�

Statistical�analysis�was�performed�by�Student’s�t�test�in�relation�to�control�experiments�(performed�with�10�

vehicles).� B.� Representative� recording� of� tACPD� injection,� an� agonist� of� mGluR,� in� �NO� dynamics�11�

(bottom,� blue� line)� and� CBF� changes� (top,� red� line)� in� the� hippocampus� of� urethane�anesthetized� rat.�12�

tACPD� was� locally� pressure� ejected� at� the� time� indicated� by� the� arrows� (25�,� 50�,� and� 100� pmol,�13�

respectively).�14�

�15�

Figure� 7:� Coupling� between� �NO� concentration� dynamics� and� CBF� changes� upon� glutamatergic�16�

activation� in� the� cortex.� Average� recordings� of� the� simultaneous� measurement� of� �NO� concentration�17�

dynamics� (bottom,� blue� line)� and� CBF� changes� (top,� red� line)� in� the� cerebral� cortex� of� urethane�18�

anesthetized�rat�upon�glutamate�stimulation�(0.5�nmol,�1�s).�19�

�20�

24�

Figure� 8:�Nitric� oxide� is� a� critical� diffusible�messenger� between� active� neurons� and� the� local� blood�1�

vessels� in� hippocampus.� The� scheme� depicts� the� cellular� relationships� and� the� sequence� of� events�2�

underlying�the�neurovascular�coupling�mediated�by��NO�upon�glutamatergic�stimulation.��NO�transitorily�3�

produced� by� neurons� upon� stimulation� on� glutamate� NMDA� receptors� diffuses� towards� neighboring�4�

blood�vessels�where,�following�the�interaction�with�soluble�GC,�triggers�a�sequence�of�events�comprising�5�

vasodilation�and�a�transient�and�localized�increase�in�O2�tension.�6�

Highlights�7�

� Nitric�oxide�is�as�a�mediator�of�the�neurovascular�coupling�in�hippocampus�8�� Nitric�oxide�induces�a�time,�spatial�and�amplitude�coupled�increase�of�local�CBF�9�� Both�NO�and�CBF�dynamics�are�dependent�on�the�activation�of�NMDA�receptors�and�nNOS�10�� The�CBF�increase�mediated�by�neuronal�derived�NO�depends�on�the�activation�of�sGC��11�

�12�

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