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Original Contribution
Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide Cátia F. Lourenço a, Ricardo M. Santos a, Rui M. Barbosa a, Enrique Cadenas b, Rafael Radi c, João Laranjinha a,n a
Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, Health Sciences Campus, 3000-548 Coimbra, Portugal Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA c Department of Biochemistry and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 19 May 2014 Accepted 23 May 2014
The coupling between neuronal activity and cerebral blood flow (CBF) is essential for normal brain function. The mechanisms behind this neurovascular coupling process remain elusive, mainly because of difficulties in probing dynamically the functional and coordinated interaction between neurons and the vasculature in vivo. Direct and simultaneous measurements of nitric oxide (dNO) dynamics and CBF changes in hippocampus in vivo support the notion that during glutamatergic activation nNOS-derived d NO induces a time-, space-, and amplitude-coupled increase in the local CBF, later followed by a transient increase in local O2 tension. These events are dependent on the activation of the NMDAglutamate receptor and nNOS, without a significant contribution of endothelial-derived dNO or astrocyte–neuron signaling pathways. Upon diffusion of dNO from active neurons, the vascular response encompasses the activation of soluble guanylate cyclase. Hence, in the hippocampus, neurovascular coupling is mediated by nNOS-derived dNO via a diffusional connection between active glutamatergic neurons and blood vessels. & 2014 Elsevier Inc. All rights reserved.
Keywords: Nitric oxide Neurovascular coupling Neurons Hippocampus Brain Functional hyperemia Free radicals
Neuronal activity imposes a need for blood-flow-carried substrates for the brain to maintain its functional and structural integrity [1]. Intensive research during the past decades aimed to untangle the underlying mechanisms that couple neuronal activity to cerebral blood flow (CBF)—neurovascular coupling [2]. Strong evidence indicates that astrocytes are critical contributors to the process, bridging neurons and blood vessels, although several questions still persist [3] and disparate temporal events demand reconciliation [4]. Other, nonastrocytic, physiological mechanisms have been proposed to underlie the coupling between neuronal activity and the changes in CBF [5–7]. In this regard, the concept that dNO, produced upon neuronal activation, reaches blood vessels by diffusion from neurons, inducing vasodilation, has been a tempting suggestion [8–12]. In neurons, dNO is produced upon glutamatergic activation by the neuronal isoform of nitric oxide synthase (nNOS), an enzyme physically and functionally coupled to the NMDA glutamate receptors [13,14]. Because dNO is highly diffusible and overcomes specific ligand–receptor interactions, its biological action is critically determined by its concentration and temporal dynamics, as well as by the distribution of the potential
n
Corresponding author. E-mail address: [email protected] (J. Laranjinha).
targets; in this way, the profile of dNO change in time and space is translated into a biological action [15]. These unorthodox properties of dNO have raised difficulties in directly substantiating its role as a diffusible messenger in neurovascular coupling, as well as in other signaling pathways. Pharmacological approaches have provided insights into the involvement of dNO in neurovascular coupling [10,12,16,17], but also opposing evidence [18–20]. It has also been suggested that, although dNO is required, it does not directly mediate the neuronto-vessels signaling, at least in the somatosensory cortex [21]. A step forward was provided by the in vivo measurement of dNO dynamics during activation of rat somatosensory cortex and the observation that a transient increase in dNO preceded CBF change [22], without, however, clarifying the source of dNO and the interdependency of both events. Thus, despite the intensive research on dNO over the past decades, its role in neurovascular coupling still remains elusive. In this work we aimed to study the neuronal-derived dNO dynamics in connection with CBF changes in hippocampus, identifying the pathway for dNO production and uncovering the underlying mechanism for vasodilation. We used an array incorporating a dNO-selective microelectrode [23], a microinjection pipette, and a laser Doppler probe assembled in a predefined geometry, which allowed us to monitor neurovascular coupling
http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021 0891-5849/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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upon local glutamate stimulation within dNO diffusional spread. On the basis of a real-time and in vivo sequence comprising glutamate stimulation, dNO production, and CBF (and O2) increase, we demonstrate that, in hippocampus, neurovascular coupling is mediated by nNOS-derived dNO via a diffusional connection.
Materials and methods Array for dNO and CBF measurements The dNO sensors were fabricated as previously described [24]. Briefly, a single carbon fiber (30 mm Ø; Textron, Lowell, MA, USA) was encased in a glass capillary and pulled in a vertical puller. The protruding carbon fiber was cut to a tip length of 200 750 mm. The electrical contact between the carbon fiber and a copper wire was achieved by using conductive silver paint (RS, Northants, UK). To improve their analytical properties for in vivo measurements of d NO, the sensors were coated with Nafion (5% solution, two coatings with 4 min drying at 170 1C) and with o-phenylenediamine (5 mM solution was electropolymerized at a constant potential of þ0.7 V vs Ag/AgCl for 30 min). Each sensor was evaluated for dNO sensitivity and selectivity against major interferents (ascorbate, nitrite, noradrenaline, and dopamine) by constant voltage amperometry at þ0.9 V vs Ag/AgCl using a FAST-16 high-speed electrochemical system (Quanteon, Nicholasville, KY, USA) in a two-electrode configuration. The sensors used in this study presented an average sensitivity of 258 792 pA/mM and selectivity ratios of 28673:1, 5732:1, 239:1, and 213:1 against ascorbate, nitrite, noradrenaline, and dopamine, respectively. Cerebral blood flow was measured using a laser Doppler flowmeter device (Periflux System 5000, Perimed, Sweden) to which a needle probe (PF411; outer diameter, 450 mm; fiber separation, 150 mm; wavelength, 780 nm) was attached. Calibration of the probe was done routinely using a PF1001 motility standard (Perimed) to equalize the perfusion values among the various recordings. The time constant was set to 0.03 s and the signalprocessing unit used a bandwidth of 32 Hz. Laser Doppler flowmetry measures CBF in arbitrary units and is therefore used for measuring relative changes in CBF. The dNO sensor and the laser Doppler probe were assembled to an ejection micropipette using sticky wax in the configuration schematized in Fig. 1. The micropipette was filled with solutions using a syringe fitted with a flexible microfilament (MicroFil, World Precision Instruments, UK) prior to brain insertion. Array for O2 and CBF measurements To record O2 fluctuations the array configuration included a micropipette and a carbon fiber microelectrode similar to that used for dNO recording. Such electrode was evaluated for O2 sensitivity by constant voltage amperometry at 0.8 V vs Ag/AgCl using a FAST-16 high-speed electrochemical system (Quanteon) in a two-electrode configuration essentially as previously described. in vivo experimental setup All the procedures used in this study were performed in accordance with the European Union Council Directive for the Care and Use of Laboratory Animals, 2010/63/EU, implemented under the supervision and approval of the local institutional animal care and use committee of the animal facility of the Center for Neurosciences and Cell Biology University of Coimbra and licensed by the national regulatory office (Direcção Geral de Alimentação e Veterinária). The animals were submitted to surgery under anesthesia, and body temperature and blood pressure
67 were controlled during the experiments as detailed below. At the 68 end of the experiments the animals were sacrificed by cervical 69 displacement while still under anesthesia. Forty-five male Wistar 70 rats (8–10 weeks; weight 294 731 g) were anesthetized by an 71 intraperitoneal injection of urethane (1.25 g/kg) and placed in a 72 stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA). Body 73 temperature was maintained at 37 1C using a deltaphase isother74 mal pad (BrainTree Scientific, Braintree, MA, USA) and controlled 75 through a rectal thermometer. An incision was made in the scalp 76 and the skin was reflected to expose the surface of the skull, 77 allowing for drilling a hole (3 4 mm) in the surface overlying the 78 hippocampus. Another hole ( 1 mm diameter) was drilled in a 79 site remote from the recording area for insertion of an Ag/AgCl 80 reference electrode (200 mm diameter). After the dura mater was 81 removed, the array was inserted into the rat hippocampus accord82 ing to coordinates calculated based on the rat brain atlas of Paxinos and Watson [52] using the tip of the microelectrode as Q2 83 84 reference in the bregma (anteroposterior 4.1 mm, mediolateral 85 2.8 mm, and dorsoventral 3.7 mm). After the insertion of the 86 array into the hippocampus it was allowed to stabilize for 30 min 87 before the beginning of the experiment. 88 89 Experimental design/drug application 90 91 Solutions, L-glutamate 20 mM, NMDA (N-methyl-D-aspartate) 92 0.1 mM, dNO solution 0.1 mM, or tACPD (trans-1-aminocyclopen93 tane-1,3-dicarboxylic acid) 0.1–1 mM (25 nl) in saline buffer (NaCl 94 0.9%, pH 7.4), were locally applied from the tip of the micropipette 95 using a Picospritzer III (Parker Hannifin Corp., General Valve 96 Operation, USA). A 0.1 mM dNO solution was prepared by diluting 97 a saturated dNO solution, prepared as previously described [25], in 98 deoxygenated saline buffer. Stimulations were performed by pressure pulses of 1 s at 7–15 psi with a minimum interval of 99 15 min of recovery. Typically three initial responses were obtained 100 before pharmacology modulation to achieve a steady-state level. 101 MK-801 (1 mg/kg in saline), 7-nitroindazole (7-NI; 50 mg/kg in Q3102 103 dimethyl sulfoxide (DMSO)), L-NIO (40 mg/kg in DMSO), acetylsa104 licylic acid (300 mg/kg in saline), and caffeine (50 mg/kg in saline) 105 were injected intraperitoneally. A cocktail of MPEP and LY367385 106 (100 nmol each) was injected into the lateral ventricle using a 107 micropipette. ODQ (25 pmol in DMSO 0.5%) and sodium fluoroa108 cetate (20 mmol) were locally applied by an extra micropipette 109 attached to the previously described array in close proximity to 110 the laser Doppler probe. The effects were evaluated after 10 to 111 30 min for ODQ and MPEP/LY36785 and sodium fluoroacetate, and 112 after 20 to 40 min for the remaining, and the responses were 113 compared with the corresponding control experiments (with 114 vehicles). Blood pressure was noninvasively measured in the tail 115 using a LE5001 system (Letica Scientific Instruments). Arterial 116 blood gases and pH were evaluated in a Rapidlab 1260 blood 117 gas analyzer (Siemens Healthcare Diagnostics) from blood samples 118 collected from the femoral artery 4 to 7 h after anesthetic 119 injection. 120 121 Data analysis and statistics 122 123 The dNO and CBF recordings were synchronized using Origi124 nPro 7.5 based on markers extracted from the respective software. 125 The remaining analyses were performed with GraphPad Prism 5. 126 Data are presented as the mean 7 SEM. Statistical analysis of the 127 data was performed using Student's t test. The dNO and CBF signals 128 were characterized in terms of (1) dNO peak flux of the signal, 129 based on the conversion of the amperometric currents to dNO 130 fluxes according to Faraday's law (I ¼ nFΦ, in which I corresponds 131 to the amperometric current, n corresponds to the one electron 132 per molecule exchanged for the oxidation of dNO, F corresponds to
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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Fig. 1. Coupling between dNO concentration dynamics and CBF changes upon glutamatergic activation in the hippocampus. (A) Representative recording of the simultaneous measurements of dNO concentration dynamics (bottom, blue line) and CBF changes (top, red line) in the hippocampus of a urethane-anesthetized rat over a period of five stimulations. Glutamate was locally applied at the times indicated by the arrows (0.5 nmol, 1 s). (B) Detail of the temporal correlation of dNO concentration dynamics and CBF changes evoked by local glutamate stimulation. (C) Array used to measure simultaneously dNO concentration dynamics and CBF changes in the rat hippocampus. The array comprises a dNO-selective microelectrode, a microinjection pipette, and a laser Doppler flow probe assembled in a predefined geometry. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the Faraday constant, and Φ is the flux) or the amplitude change (the increase in CBF beyond the CBF basal levels, considered 100% in the absence of stimuli), respectively; (2) Trise, the time in seconds necessary to reach the maximum amplitude after stimulation; and (3) Ttotal, the time in seconds from the stimulation point to return to basal levels.
Results Nitric oxide and cerebral blood flow changes: coupling in space, time, and amplitude We have previously reported that a controlled and localized glutamate stimulus in the rat hippocampus promotes an instantaneous and transient elevation of dNO concentration levels through the activation of ionotropic glutamate receptors [23]. By upgrading such experimental strategy, simultaneously measuring local CBF, we observed that a transient dNO increase induced by glutamate ejection (0.5 nmol, 25 nl, 1 s), was followed, seconds later, by a transient change in CBF (Fig. 1). dNO production was characterized by a flux of 3.5 7 1.8 fmol s 1 with a time rise of 22 73 s and a
total duration of 647 4 s (54 peaks analyzed from 15 individual experiments). The CBF started to increase 7 72 s after stimulation, reaching 12275% beyond the basal level after 62 73 s and returning to basal levels after 2167 15 s. Likewise, the specific activation of the NMDA glutamate receptor, using the synthetic agonist NMDA, resulted in dNO and CBF dynamics similar to those obtained with glutamate and with the same temporal correlation, thus imparting specificity to the pathway leading to dNO production. The ejection of NMDA (2.5 pmol) resulted in a transient dNO production characterized by a flux of 3.5 70.5 fmol s 1 with a time rise of 19 74 s and a total duration of 78 75 s, which was followed by an increase in CBF that lasted 296 746 s and reached the maximum of 104 710% beyond the basal level after 73 79 s (12 peaks analyzed from five individual experiments). Local pressure ejection of the vehicle (NaCl 0.9%) caused no change in either the baseline oxidation current or the CBF levels (Supplementary Fig. 2). The CBF changes induced by glutamatergic activation in the hippocampus were independent of arterial blood pressure, blood gases, or pH, which remained stable and within the physiological range during the time window in which experiments were performed, as randomly assessed. On average, the values for pH, paCO2,
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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Fig. 2. Exogenous dNO mimics glutamate-induced CBF changes. Average recordings of CBF changes (red line) in response to exogenous and localized dNO application in the hippocampus (blue line). dNO solution (100 mM in saline) was locally applied by pressure injection (5 psi, 90 s, 1 ml). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and paO2 were 7.3970.07, 4177 mm Hg, and 108731 mm Hg, respectively. Exogenous nitric oxide mimics glutamate-induced CBF changes The temporal coupling between the endogenous dNO signal and the increase in CBF was mimicked by the local injection of a d NO solution. Fig. 2 shows that when dNO is exogenously applied, a temporal correlation is observed between the dNO signal and the CBF change that is similar to that observed for the endogenous glutamate-dependent production of dNO (CBF started to increase 8 72 s after dNO injection, lasting for 282 760 s, whereas the dNO temporary increase was 657 12 s, n ¼ 3). Nevertheless, and although the dNO flux achieved is higher when exogenously added (11.3 72.7 fmol s 1 versus 3.8 71.6 fmol s 1 achieved via glutamate stimulus), the CBF change from the baseline is weaker than that observed upon endogenous dNO production (55 711% versus 12275% beyond the basal level). In this regard, the peculiar nature of dNO volume signaling in the brain [26] has to be considered: after activation of a volume of tissue comprising numerous synaptic dNO sources, small dispersed dNO sources of low individual efficacy can cooperate to originate an extensive and strong volume signal, inducing an increase in CBF. We have recently shown that the decay of dNO when produced endogenously in the hippocampus via activation of NMDA glutamate receptors is much slower than when injected exogenously [23,27]. Therefore, one could expect a lower diffusional spread due to the fast decay achieved upon dNO release from a single point in the tip of the pipette, compared with glutamate stimulus, and, consequently, a lower effect on the regional CBF. Glutamate-induced CBF changes correlate with oxygen tension changes The local transient change in CBF elicited by neuronal dNO is expected to translate into correlated changes in local O2 tension. By measuring O2 tension, we observed that the local transient changes in CBF elicited by glutamate stimulation are followed, seconds later, by a correlated transient elevation in O2 tension (Fig. 3, R2 ¼ 0.69, p ¼ 0.0016, n ¼ 12 from three individual experiments). This observation supports the prediction that the d NO signal is sequentially followed by CBF increase and by a
Fig. 3. The CBF change coupled to dNO dynamics is followed by a transient increase in local O2 tension. Average recordings of simultaneous measurements of O2 (green line) and CBF changes (red line) in the hippocampus of a urethane-anesthetized rat and temporal correlation with dNO dynamics (blue line). Inset shows the correlation between the amplitudes of the O2 and CBF changes (R2 ¼ 0.69, p ¼ 0.0016, n ¼ 12 from three individual experiments). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
transitory elevation in O2 tension. It should be remarked that the geometrical configuration of the array and the physical properties of the carbon fiber microelectrodes and laser Doppler probe impose the constraint that the measurements of dNO/O2 and CBF are performed within a volume of hippocampal tissue comprising probably a few hundreds of microemters in diameter. Within this volume of tissue, the fine correlation observed between O2 tension and CBF events, measured at separate locations within the dNO diffusion field, further supports the validity of the approach used to study the relationship between dNO and CBF. Neuronal-derived dNO is the mediator of neurovascular coupling in hippocampus The neurovascular coupling process is expected to be prone to modulation by functional interferences along the pathway of nNOSdependent dNO production, in particular at the level of the NMDA glutamate receptor and nNOS. Among several potential pharmacological tools, we have selected a glutamate NMDA receptor blocker (MK-801), a selective nNOS inhibitor (7-NI), and a selective eNOS inhibitor (L-NIO). The results obtained are summarized in Fig. 4A. The responses obtained after drug injection were compared with the corresponding control experiments (with vehicles). None of the vehicles used showed any significant effect on dNO or CBF responses (Supplementary Fig. 2). Blocking of the NMDA receptor elicited a significant inhibition of dNO production (7378%, p ¼ 0.0017, n ¼ 3) that was accompanied by a dramatic decrease in glutamate-induced CBF (7473%, p ¼ 0.0008, n ¼ 3). Likewise, the inhibition of the neuronal isoform of NOS by 7-NI induced a significant inhibition of both dNO and CBF responses to glutamate (62710 and 8376%, p ¼ 0.0002 and p ¼ 0.0025, respectively, n ¼ 4), as well as a decrease in CBF basal levels (2777%). Conversely, no significant effects were found on either induced or basal levels upon inhibition of the endothelial NOS isoform with L-NIO (Fig. 4C). Nevertheless, an increase in mean arterial blood pressure (MABP) of the animals was observed after the L-NIO injection, supporting its efficacy in terms of eNOS inhibition (n ¼ 4, MABP 90.371.1 to 114.972.5 mm Hg). The modulation of the NMDA receptor:nNOS pathway showed that, in the hippocampus, the increase in CBF was dependent on dNO signal amplitude (Fig. 4B). Within the range tested, the relationship between dNO and CBF showed to be linear above a threshold
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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of 0.43 fmol s 1, which is coherent with the concept of a linear relationship between neuronal activity and vascular responses provided by neuroimaging studies [28]. However, it should be pointed out that below the threshold our data do not identify whether the relationship is linear or a gating process is operative.
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The dNO-mediated neurovascular coupling encompasses the activation of soluble guanylate cyclase The classical pathway for vasodilation mediated by dNO involves the activation of soluble guanylate cyclase (sGC) in smooth muscle
Fig. 4. Glutamate-induced dNO production and CBF changes are dependent on the activation of NMDA receptor and nNOS. (A) Effects of MK-801 (1 mg/kg, NMDA receptor blocker), 7-NI (50 mg/kg, selective nNOS inhibitor), and L-NIO (40 mg/kg, selective eNOS inhibitor). Data represent the mean 7 SEM. Statistical analysis was performed by Student's t test in relation to control experiments (nnnp o 0.001). (B) Plot of the linear relationship between the amplitudes of dNO production and CBF changes obtained by glutamatergic activation. The linear regression showed an R2 ¼ 0.98 and a slope of 78.9% CBF/fmol s 1 dNO (x intercept 0.438). (C) Average recordings of the 7-NI and L-NIO effects on the dNO production (blue line) and CBF changes (red line) elicited by glutamate in the hippocampus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Neuronal-derived dNO mediates the neurovascular coupling through activation of soluble guanylate cyclase. The effect of ODQ, a selective heme-site inhibitor of soluble guanylate cyclase, on glutamate-induced dNO signals (blue line) and CBF changes (red line) in the rat hippocampus. ODQ was locally applied through an additional pipette attached to the array in the proximity of the laser Doppler probe (25 pmol, 0.5 ml). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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cells, which, via cGMP-dependent protein kinases, promotes the dephosphorylation of myosin light chains and thus vasodilation [15]. To unravel the mechanism by which neuronal-derived dNO promoted the increase in CBF, the effect of ODQ, a selective heme-site inhibitor of soluble guanylate cyclase, was evaluated. After the local application of ODQ in the hippocampus, glutamate induced a typical dNO concentration dynamics but the CBF changes were significantly reduced (6777%, p ¼ 0.002, n ¼ 4; Fig. 5). The slight inhibition of dNO signal observed is probably related to the nonspecific inhibition of nNOS by ODQ [29] (nNOS, being a heme protein, might be affected by ODQ), and not a consequence of cGMP decrease in neurons, as dNO concentration was resumed in the following stimulations and CBF remained inhibited, progressively returning to the control values obtained in the absence of ODQ. The uncoupling observed between d NO concentration dynamics and CBF changes in the presence of ODQ strongly supports the idea that dNO produced, and diffusing from neurons, reaches vascular smooth muscle cells and triggers vasodilation via sGC activation. The astrocytic pathway is not explicitly involved in the dNO-mediated neurovascular coupling Given the commonly accepted paradigm of astrocytes bridging neurons and vascular cells, encompassing metabotropic glutamate receptor activation and subsequent production of arachidonic acid metabolites [2,6], we addressed the potential contribution of the astrocytic bridge to neurovascular coupling in hippocampus under our experimental conditions. For that purpose, a cocktail of antagonists of metabotropic glutamate receptors (LY367385 and MPEP) was injected intracerobroventricularly into the rat brain to inhibit metabotropic glutamate receptors mGluR1 and mGluR5, respectively. If glutamate-triggered [Ca2 þ ]i elevations in astrocytes are crucial for neurovascular coupling, their specific inhibition should hamper the increase in CBF induced by glutamate via the astrocytes. However, the inhibition of astrocyte mGluR receptors did not elicit any alteration in the recorded dNO and CBF signals (Fig. 6A). Neither inhibition of cyclooxygenase activity by acetylsalicylic acid—downstream in the astrocytic pathway—nor impairment of astrocytic metabolism linked to the inhibition of mitochondrial aconitase via metabolism of sodium fluoroacetate—which is preferentially taken up by glial cells [30]—had any effect either. Also, no contribution could be assigned to adenosine receptors, argued to mediate a complex signaling process of neuronal activity-induced increase in cerebral vasodilation [31,32], as caffeine, a nonselective antagonist of adenosine receptors [33], also failed to modulate either dNO or CBF (Fig. 6A). As an additional control, the specific activation of mGLuR receptors by the mGluR agonist tACPD—known to promote Ca2 þ elevation in astrocyte somata and related pathways [5]—did not elicit any changes in either dNO production or CBF (Fig. 6B).
Fig. 6. The astrocytic pathway is not explicitly involved in the dNO-mediated neurovascular coupling. (A) Effects of LY367385/MPEP (100 μM each, antagonists of metabotropic glutamate receptors mGluR1 and mGluR5, respectively, n ¼ 6), acetylsalicylic acid (ASA; 300 mg/kg, COX inhibitor, n ¼ 5), sodium fluoroacetate (SFA; 20 mmol locally, glial toxin, n ¼ 5), and caffeine (CAF; 50 mg/kg, nonselective antagonist of adenosine receptors, n ¼ 3) on dNO signals and CBF changes. Data represent the mean 7 SEM. Statistical analysis was performed by Student's t test in relation to control experiments (performed with vehicles). (B) Representative recording of injection of tACPD, an agonist of mGluR, in dNO dynamics (bottom, blue line) and CBF changes (top, red line) in the hippocampus of a urethaneanesthetized rat. tACPD was locally pressure injected at the time indicated by the arrows (25, 50, and 100 pmol, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
NO-dependent CBF increase is region specific
d
The regional occurrence of the diffusive connection established by NO between neurons and vessels without the intermediacy of cellular systems between glutamatergic synapses and vessels in brain regions other than hippocampus was assessed, for it has been previously reported that the mechanisms underlying neurovascular coupling may be region specific. With that intent the experimental approach used in the hippocampus was applied to probe the cerebral cortex with the tip of the array positioned in the deeper layers, where nNOS immunoreactivity is predominant [34]. Whereas data support the coupling, the profiles of dNO and CBF change do not reproduce the phenomenon observed in hippocampus in all its dimensions (Fig. 7). In fact, in cerebral cortex, compared with hippocampus, glutamate induced a weaker production of dNO (0.4670.14 fmol s 1) followed by a less intense increase in CBF (4676%) (12 peaks analyzed from four d
Fig. 7. Coupling between dNO concentration dynamics and CBF changes upon glutamatergic activation in the cortex. Average recordings of the simultaneous measurement of dNO concentration dynamics (bottom, blue line) and CBF changes (top, red line) in the cerebral cortex of urethane-anesthetized rat upon glutamate stimulation (0.5 nmol, 1 s). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
individual experiments) but, quantitatively, the response of dNO and CBF to glutamate in cerebral cortex was not proportionally attenuated; d NO production in cerebral cortex was 87 7 4% lower than in hippocampus and the reduction in CBF change was only 62 7 5% lower. We would predict that distinct neuroanatomic regions (encompassing variations in nNOS expression, vascularization density, mean
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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distance between glutamatergic neurons and arterioles, dNO inactivation mechanisms) exhibited different quantitative relationships between dNO and CBF signals but maintain a temporal and local correlation, as observed. Therefore, the kinetics of neuronal dNOdependent CBF changes may be intrinsically distinct in hippocampus compared with other brain regions.
Discussion The mechanisms that regulate the synergy between cerebral microcirculation and local neuronal activity have been debated over a century without clear conclusions, in part because of the severe experimental limitations to measure the process in real time in the natural working environment. The identification of d NO as a diffusible vasodilator produced at active neurons, via ionotropic glutamate receptor-dependent pathways, led to the hypothesis that it could be the mediator coupling the brain activity to CBF [9] but, paradoxically, it introduced a further difficulty. In fact, because dNO is small, is hydrophobic, and overcomes storage and specific interaction with receptors, it conveys information associated with its concentration dynamics, a parameter difficult to measure in vivo. By simultaneously and direct measuring dNO and CBF dynamics in the hippocampus, this work supports the notion that dNO, generated by nNOS during glutamatergic activation, induces a coupled increase in the local CBF (Fig. 8). Accordingly, dNO and CBF events precede an increase in O2 tension from background, a critical prediction of the neurovascular coupling concept. The observation that both MK-801 and 7-NI individually elicited a significant inhibition of dNO production, and that this effect was translated into CBF changes, clearly identifies the pathway that paves the neurovascular coupling process in the hippocampus and recognizes nNOS-derived dNO as the coupler molecule. Data showing the abolishment of CBF changes, despite the strong dNO signal under conditions of sGC inhibition with ODQ, mechanistically established the pathway for dNO-dependent CBF changes in the rat hippocampus by the canonic cGMP-dependent vasodilation pathway and dNO as a critical messenger. Furthermore, data also support the idea that nNOS-derived dNO participates in the maintenance of resting CBF, as 7-NI administration also promoted a decrease in hippocampal CBF basal levels. It should be noted that
Fig. 8. Nitric oxide is a critical diffusible messenger between active neurons and the local blood vessels in hippocampus. The scheme depicts the cellular relationships and the sequence of events underlying the neurovascular coupling mediated by dNO upon glutamatergic stimulation. dNO transitorily produced by neurons upon stimulation on glutamate NMDA receptors diffuses toward neighboring blood vessels where, after the interaction with soluble GC, triggers a sequence of events comprising vasodilation and a transient and localized increase in O2 tension.
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glutamate microinjections (within the range used) and electric stimulation induce equivalent cerebrovascular effects, as shown previously in cerebellar cortex upon electric stimulation of parallel fibers [35]. The pattern of dNO production and CBF changes measured accomplishes the fundamental criteria of the neurovascular coupling concept: a temporal, amplitude, and spatial relationship [36]. In addition to the temporal and amplitude correlation evident in the occurrence of both dynamics, data show that the increase in CBF is limited in space, affecting just the area of increased neuronal activity, the hippocampus, for if the laser Doppler probe is placed in the overlying cerebral cortex no CBF change is observed in response to glutamate. The strategy established in this work also complies with the criteria for dNO signaling in the framework of the coupling inasmuch as stimulation of dispersed glutamatergic synapses via nNOS generates a volume signaling that encompasses the diffusion of dNO from neurons to nearby arterioles to induce an increase in CBF, thus establishing that, upon elicitation of neuronal activation, d NO from nNOS launches a diffusible connection between neurons and the vasculature. The concept of a dNO-mediated diffusible connection is supported by two observations: first, in hippocampal slices, in the absence of a functional vascular system to remove d NO, the diffusion field of dNO was measured as being close to 400 mm [37]; second, the mean distance between arterioles and nerve fibers along the longitudinal axis of pyramidal layer neurons in the CA1 region of hippocampus is circa 150 mm [38], i.e., within the range of dNO diffusional spread. Furthermore, mathematical modeling of the physicochemical process of dNO production from an active neuron in cerebellar slices estimated that the range of d NO concentration reaching the nearby blood vessels rises drastically, exceeding ca. 100-fold its basal levels during stimulated neuronal activity [39]. Among the wide collection of proposed mechanisms, a generally accepted paradigm for neurovascular coupling supports the view of astrocytes carrying the neuronal signal by bridging neurons and vascular cells [6]. Astrocytes are known to respond to glutamate via mGluR activation, involving a transient increase in [Ca2 þ ]i that ends up in the synthesis of vasoactive substances upon activation of phospholipase A2 and arachidonic acid production. The vasoactive compounds include prostaglandin E2 (via cyclooxygenase) [5] and epoxyeicosatrienoic acids (via cytochrome P450 epoxygenase) [7,40]. Accordingly, it has been speculated that dNO levels may modulate the cerebrovascular response to mGluR activation by regulating the enzymatic conversion of arachidonic acid via cytochrome P450 to either vasodilators or vasoconstrictors [41]. However, our results show that, in hippocampus, this pathway makes a minor if any contribution to the neurovascular coupling promoted by glutamatergic activation, supporting that this process is developed by different mechanisms depending on the brain region. Also, it should be considered that glutamate-dependent astrocyte signaling may change during development. Recently, it was demonstrated that astrocytic mGluR5 expression is developmentally regulated, being undetectable in adulthood, and that, in adult mice, astrocytes failed to respond to the mGluR5 agonist tACPD with significant Ca2 þ increase [3]. Also, it is of note that functional NMDA receptors [42] and constitutive NOS expression [43] have been found in cultured astrocytes. Therefore, and although the abolishment of astrocytic metabolism by sodium fluoroacetate did not affect the recorded dynamics, we cannot fully exclude the potential involvement of astrocytes in the NMDA–nNOS pathway, leading to CBF changes. Further supporting the notion of region specificity of neurovascular coupling process [44], we observed that glutamatergic activation in cerebral cortex, compared with hippocampus, resulted in a significantly lower dNO production translated into less dramatic CBF changes. The dNO:CBF ratio was, however, lower in the cortex, in
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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agreement with the less outstanding role attributed to the dNO in this brain region [18–20]. Also, the latency of CBF response is slower in the cerebral cortex than in hippocampus, which, in turn, is longer than that commonly reported by studies using electric stimulation [22,45,46]. Apart from the stimulation paradigm, these variations can be related to differences in the neurovascular coupling process and vascular network among regions. In fact, the Lauritzen group has observed slower CBF responses in cerebellum [47,48] compared with the somatosensory system [46]. These observations reinforce the notion that the kinetics of neuronal dNO-dependent CBF changes may be intrinsically distinct in hippocampus, compared with other brain regions. Following the formulation of the hypothesis of dNO as a candidate to mediate neurovascular coupling [9] intensive research has attempted to elucidate its role in the process. All the studies, however, were largely based on the pharmacological modulation of NOS activity and, expectedly, controversial results were collected. Assuming as plausible that different pathways and molecules might be involved in the neurovascular coupling phenomenon, and that the relative contributions of these pathways can vary among regions, we should, however, be aware that the use of such pharmacological tools without additional verification of their efficacy can give rise to misleading results. For instance, it was shown that 7-NI, a widely used selective nNOS inhibitor, did not maximally inhibit nNOS activity and did not affect all brain regions to the same extent [49]. Additionally, methodological variables such as the anesthetic used seem to contribute to the discrepancy of the results, because of their possible effects on neurotransmission and hemodynamics [50]. Therefore, for a messenger that conveys information associated with its concentration dynamics, its direct, localized, and quantitative measurement in vivo is mandatory to progress in the understanding of its role in the mechanisms of neurovascular coupling, a critical pathway for proper brain function. Indeed, a study in cerebellar slices, by simultaneously measuring dNO dynamics and local capillary enlargement associated with treatment with NOS inhibitors or with tetrodotoxin, suggested that dNO of neuronal origin is responsible for the vast majority of the vasomotor response in cerebellum [51]. Finally, from a pure conceptual viewpoint, the mechanism of neuronal-derived dNO mediating neurovascular coupling via volume signaling may be a noncanonical way to underlie a process of vital importance for the brain to preserve its structural and functional integrity. However, although the mechanistic link to match blood supply with the metabolic demands imposed by increased neuronal activity is based on diffusion and characterized by lack of specific recognition by receptors, it still remains a highly and intrinsically controlled mechanism. Because red blood cells constitute the major pathway for dNO inactivation in the brain [27], the increase in the cerebral blood flow triggered by neuronalderived dNO itself constitutes a mechanism to shape the dNO concentration dynamics and interrupt the signaling pathway.
Appendix A.
Supplementary material
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Original Contribution
Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide Cátia F. Lourenço a, Ricardo M. Santos a, Rui M. Barbosa a, Enrique Cadenas b, Rafael Radi c, João Laranjinha a,n a
Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, Health Sciences Campus, 3000-548 Coimbra, Portugal Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA c Department of Biochemistry and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 19 May 2014 Accepted 23 May 2014
The coupling between neuronal activity and cerebral blood flow (CBF) is essential for normal brain function. The mechanisms behind this neurovascular coupling process remain elusive, mainly because of difficulties in probing dynamically the functional and coordinated interaction between neurons and the vasculature in vivo. Direct and simultaneous measurements of nitric oxide (dNO) dynamics and CBF changes in hippocampus in vivo support the notion that during glutamatergic activation nNOS-derived d NO induces a time-, space-, and amplitude-coupled increase in the local CBF, later followed by a transient increase in local O2 tension. These events are dependent on the activation of the NMDAglutamate receptor and nNOS, without a significant contribution of endothelial-derived dNO or astrocyte–neuron signaling pathways. Upon diffusion of dNO from active neurons, the vascular response encompasses the activation of soluble guanylate cyclase. Hence, in the hippocampus, neurovascular coupling is mediated by nNOS-derived dNO via a diffusional connection between active glutamatergic neurons and blood vessels. & 2014 Elsevier Inc. All rights reserved.
Keywords: Nitric oxide Neurovascular coupling Neurons Hippocampus Brain Functional hyperemia Free radicals
Neuronal activity imposes a need for blood-flow-carried substrates for the brain to maintain its functional and structural integrity [1]. Intensive research during the past decades aimed to untangle the underlying mechanisms that couple neuronal activity to cerebral blood flow (CBF)—neurovascular coupling [2]. Strong evidence indicates that astrocytes are critical contributors to the process, bridging neurons and blood vessels, although several questions still persist [3] and disparate temporal events demand reconciliation [4]. Other, nonastrocytic, physiological mechanisms have been proposed to underlie the coupling between neuronal activity and the changes in CBF [5–7]. In this regard, the concept that dNO, produced upon neuronal activation, reaches blood vessels by diffusion from neurons, inducing vasodilation, has been a tempting suggestion [8–12]. In neurons, dNO is produced upon glutamatergic activation by the neuronal isoform of nitric oxide synthase (nNOS), an enzyme physically and functionally coupled to the NMDA glutamate receptors [13,14]. Because dNO is highly diffusible and overcomes specific ligand–receptor interactions, its biological action is critically determined by its concentration and temporal dynamics, as well as by the distribution of the potential
n
Corresponding author. E-mail address: [email protected] (J. Laranjinha).
targets; in this way, the profile of dNO change in time and space is translated into a biological action [15]. These unorthodox properties of dNO have raised difficulties in directly substantiating its role as a diffusible messenger in neurovascular coupling, as well as in other signaling pathways. Pharmacological approaches have provided insights into the involvement of dNO in neurovascular coupling [10,12,16,17], but also opposing evidence [18–20]. It has also been suggested that, although dNO is required, it does not directly mediate the neuronto-vessels signaling, at least in the somatosensory cortex [21]. A step forward was provided by the in vivo measurement of dNO dynamics during activation of rat somatosensory cortex and the observation that a transient increase in dNO preceded CBF change [22], without, however, clarifying the source of dNO and the interdependency of both events. Thus, despite the intensive research on dNO over the past decades, its role in neurovascular coupling still remains elusive. In this work we aimed to study the neuronal-derived dNO dynamics in connection with CBF changes in hippocampus, identifying the pathway for dNO production and uncovering the underlying mechanism for vasodilation. We used an array incorporating a dNO-selective microelectrode [23], a microinjection pipette, and a laser Doppler probe assembled in a predefined geometry, which allowed us to monitor neurovascular coupling
http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021 0891-5849/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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upon local glutamate stimulation within dNO diffusional spread. On the basis of a real-time and in vivo sequence comprising glutamate stimulation, dNO production, and CBF (and O2) increase, we demonstrate that, in hippocampus, neurovascular coupling is mediated by nNOS-derived dNO via a diffusional connection.
Materials and methods Array for dNO and CBF measurements The dNO sensors were fabricated as previously described [24]. Briefly, a single carbon fiber (30 mm Ø; Textron, Lowell, MA, USA) was encased in a glass capillary and pulled in a vertical puller. The protruding carbon fiber was cut to a tip length of 200 750 mm. The electrical contact between the carbon fiber and a copper wire was achieved by using conductive silver paint (RS, Northants, UK). To improve their analytical properties for in vivo measurements of d NO, the sensors were coated with Nafion (5% solution, two coatings with 4 min drying at 170 1C) and with o-phenylenediamine (5 mM solution was electropolymerized at a constant potential of þ0.7 V vs Ag/AgCl for 30 min). Each sensor was evaluated for dNO sensitivity and selectivity against major interferents (ascorbate, nitrite, noradrenaline, and dopamine) by constant voltage amperometry at þ0.9 V vs Ag/AgCl using a FAST-16 high-speed electrochemical system (Quanteon, Nicholasville, KY, USA) in a two-electrode configuration. The sensors used in this study presented an average sensitivity of 258 792 pA/mM and selectivity ratios of 28673:1, 5732:1, 239:1, and 213:1 against ascorbate, nitrite, noradrenaline, and dopamine, respectively. Cerebral blood flow was measured using a laser Doppler flowmeter device (Periflux System 5000, Perimed, Sweden) to which a needle probe (PF411; outer diameter, 450 mm; fiber separation, 150 mm; wavelength, 780 nm) was attached. Calibration of the probe was done routinely using a PF1001 motility standard (Perimed) to equalize the perfusion values among the various recordings. The time constant was set to 0.03 s and the signalprocessing unit used a bandwidth of 32 Hz. Laser Doppler flowmetry measures CBF in arbitrary units and is therefore used for measuring relative changes in CBF. The dNO sensor and the laser Doppler probe were assembled to an ejection micropipette using sticky wax in the configuration schematized in Fig. 1. The micropipette was filled with solutions using a syringe fitted with a flexible microfilament (MicroFil, World Precision Instruments, UK) prior to brain insertion. Array for O2 and CBF measurements To record O2 fluctuations the array configuration included a micropipette and a carbon fiber microelectrode similar to that used for dNO recording. Such electrode was evaluated for O2 sensitivity by constant voltage amperometry at 0.8 V vs Ag/AgCl using a FAST-16 high-speed electrochemical system (Quanteon) in a two-electrode configuration essentially as previously described. in vivo experimental setup All the procedures used in this study were performed in accordance with the European Union Council Directive for the Care and Use of Laboratory Animals, 2010/63/EU, implemented under the supervision and approval of the local institutional animal care and use committee of the animal facility of the Center for Neurosciences and Cell Biology University of Coimbra and licensed by the national regulatory office (Direcção Geral de Alimentação e Veterinária). The animals were submitted to surgery under anesthesia, and body temperature and blood pressure
67 were controlled during the experiments as detailed below. At the 68 end of the experiments the animals were sacrificed by cervical 69 displacement while still under anesthesia. Forty-five male Wistar 70 rats (8–10 weeks; weight 294 731 g) were anesthetized by an 71 intraperitoneal injection of urethane (1.25 g/kg) and placed in a 72 stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA). Body 73 temperature was maintained at 37 1C using a deltaphase isother74 mal pad (BrainTree Scientific, Braintree, MA, USA) and controlled 75 through a rectal thermometer. An incision was made in the scalp 76 and the skin was reflected to expose the surface of the skull, 77 allowing for drilling a hole (3 4 mm) in the surface overlying the 78 hippocampus. Another hole ( 1 mm diameter) was drilled in a 79 site remote from the recording area for insertion of an Ag/AgCl 80 reference electrode (200 mm diameter). After the dura mater was 81 removed, the array was inserted into the rat hippocampus accord82 ing to coordinates calculated based on the rat brain atlas of Paxinos and Watson [52] using the tip of the microelectrode as Q2 83 84 reference in the bregma (anteroposterior 4.1 mm, mediolateral 85 2.8 mm, and dorsoventral 3.7 mm). After the insertion of the 86 array into the hippocampus it was allowed to stabilize for 30 min 87 before the beginning of the experiment. 88 89 Experimental design/drug application 90 91 Solutions, L-glutamate 20 mM, NMDA (N-methyl-D-aspartate) 92 0.1 mM, dNO solution 0.1 mM, or tACPD (trans-1-aminocyclopen93 tane-1,3-dicarboxylic acid) 0.1–1 mM (25 nl) in saline buffer (NaCl 94 0.9%, pH 7.4), were locally applied from the tip of the micropipette 95 using a Picospritzer III (Parker Hannifin Corp., General Valve 96 Operation, USA). A 0.1 mM dNO solution was prepared by diluting 97 a saturated dNO solution, prepared as previously described [25], in 98 deoxygenated saline buffer. Stimulations were performed by pressure pulses of 1 s at 7–15 psi with a minimum interval of 99 15 min of recovery. Typically three initial responses were obtained 100 before pharmacology modulation to achieve a steady-state level. 101 MK-801 (1 mg/kg in saline), 7-nitroindazole (7-NI; 50 mg/kg in Q3102 103 dimethyl sulfoxide (DMSO)), L-NIO (40 mg/kg in DMSO), acetylsa104 licylic acid (300 mg/kg in saline), and caffeine (50 mg/kg in saline) 105 were injected intraperitoneally. A cocktail of MPEP and LY367385 106 (100 nmol each) was injected into the lateral ventricle using a 107 micropipette. ODQ (25 pmol in DMSO 0.5%) and sodium fluoroa108 cetate (20 mmol) were locally applied by an extra micropipette 109 attached to the previously described array in close proximity to 110 the laser Doppler probe. The effects were evaluated after 10 to 111 30 min for ODQ and MPEP/LY36785 and sodium fluoroacetate, and 112 after 20 to 40 min for the remaining, and the responses were 113 compared with the corresponding control experiments (with 114 vehicles). Blood pressure was noninvasively measured in the tail 115 using a LE5001 system (Letica Scientific Instruments). Arterial 116 blood gases and pH were evaluated in a Rapidlab 1260 blood 117 gas analyzer (Siemens Healthcare Diagnostics) from blood samples 118 collected from the femoral artery 4 to 7 h after anesthetic 119 injection. 120 121 Data analysis and statistics 122 123 The dNO and CBF recordings were synchronized using Origi124 nPro 7.5 based on markers extracted from the respective software. 125 The remaining analyses were performed with GraphPad Prism 5. 126 Data are presented as the mean 7 SEM. Statistical analysis of the 127 data was performed using Student's t test. The dNO and CBF signals 128 were characterized in terms of (1) dNO peak flux of the signal, 129 based on the conversion of the amperometric currents to dNO 130 fluxes according to Faraday's law (I ¼ nFΦ, in which I corresponds 131 to the amperometric current, n corresponds to the one electron 132 per molecule exchanged for the oxidation of dNO, F corresponds to
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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Fig. 1. Coupling between dNO concentration dynamics and CBF changes upon glutamatergic activation in the hippocampus. (A) Representative recording of the simultaneous measurements of dNO concentration dynamics (bottom, blue line) and CBF changes (top, red line) in the hippocampus of a urethane-anesthetized rat over a period of five stimulations. Glutamate was locally applied at the times indicated by the arrows (0.5 nmol, 1 s). (B) Detail of the temporal correlation of dNO concentration dynamics and CBF changes evoked by local glutamate stimulation. (C) Array used to measure simultaneously dNO concentration dynamics and CBF changes in the rat hippocampus. The array comprises a dNO-selective microelectrode, a microinjection pipette, and a laser Doppler flow probe assembled in a predefined geometry. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the Faraday constant, and Φ is the flux) or the amplitude change (the increase in CBF beyond the CBF basal levels, considered 100% in the absence of stimuli), respectively; (2) Trise, the time in seconds necessary to reach the maximum amplitude after stimulation; and (3) Ttotal, the time in seconds from the stimulation point to return to basal levels.
Results Nitric oxide and cerebral blood flow changes: coupling in space, time, and amplitude We have previously reported that a controlled and localized glutamate stimulus in the rat hippocampus promotes an instantaneous and transient elevation of dNO concentration levels through the activation of ionotropic glutamate receptors [23]. By upgrading such experimental strategy, simultaneously measuring local CBF, we observed that a transient dNO increase induced by glutamate ejection (0.5 nmol, 25 nl, 1 s), was followed, seconds later, by a transient change in CBF (Fig. 1). dNO production was characterized by a flux of 3.5 7 1.8 fmol s 1 with a time rise of 22 73 s and a
total duration of 647 4 s (54 peaks analyzed from 15 individual experiments). The CBF started to increase 7 72 s after stimulation, reaching 12275% beyond the basal level after 62 73 s and returning to basal levels after 2167 15 s. Likewise, the specific activation of the NMDA glutamate receptor, using the synthetic agonist NMDA, resulted in dNO and CBF dynamics similar to those obtained with glutamate and with the same temporal correlation, thus imparting specificity to the pathway leading to dNO production. The ejection of NMDA (2.5 pmol) resulted in a transient dNO production characterized by a flux of 3.5 70.5 fmol s 1 with a time rise of 19 74 s and a total duration of 78 75 s, which was followed by an increase in CBF that lasted 296 746 s and reached the maximum of 104 710% beyond the basal level after 73 79 s (12 peaks analyzed from five individual experiments). Local pressure ejection of the vehicle (NaCl 0.9%) caused no change in either the baseline oxidation current or the CBF levels (Supplementary Fig. 2). The CBF changes induced by glutamatergic activation in the hippocampus were independent of arterial blood pressure, blood gases, or pH, which remained stable and within the physiological range during the time window in which experiments were performed, as randomly assessed. On average, the values for pH, paCO2,
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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Fig. 2. Exogenous dNO mimics glutamate-induced CBF changes. Average recordings of CBF changes (red line) in response to exogenous and localized dNO application in the hippocampus (blue line). dNO solution (100 mM in saline) was locally applied by pressure injection (5 psi, 90 s, 1 ml). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and paO2 were 7.3970.07, 4177 mm Hg, and 108731 mm Hg, respectively. Exogenous nitric oxide mimics glutamate-induced CBF changes The temporal coupling between the endogenous dNO signal and the increase in CBF was mimicked by the local injection of a d NO solution. Fig. 2 shows that when dNO is exogenously applied, a temporal correlation is observed between the dNO signal and the CBF change that is similar to that observed for the endogenous glutamate-dependent production of dNO (CBF started to increase 8 72 s after dNO injection, lasting for 282 760 s, whereas the dNO temporary increase was 657 12 s, n ¼ 3). Nevertheless, and although the dNO flux achieved is higher when exogenously added (11.3 72.7 fmol s 1 versus 3.8 71.6 fmol s 1 achieved via glutamate stimulus), the CBF change from the baseline is weaker than that observed upon endogenous dNO production (55 711% versus 12275% beyond the basal level). In this regard, the peculiar nature of dNO volume signaling in the brain [26] has to be considered: after activation of a volume of tissue comprising numerous synaptic dNO sources, small dispersed dNO sources of low individual efficacy can cooperate to originate an extensive and strong volume signal, inducing an increase in CBF. We have recently shown that the decay of dNO when produced endogenously in the hippocampus via activation of NMDA glutamate receptors is much slower than when injected exogenously [23,27]. Therefore, one could expect a lower diffusional spread due to the fast decay achieved upon dNO release from a single point in the tip of the pipette, compared with glutamate stimulus, and, consequently, a lower effect on the regional CBF. Glutamate-induced CBF changes correlate with oxygen tension changes The local transient change in CBF elicited by neuronal dNO is expected to translate into correlated changes in local O2 tension. By measuring O2 tension, we observed that the local transient changes in CBF elicited by glutamate stimulation are followed, seconds later, by a correlated transient elevation in O2 tension (Fig. 3, R2 ¼ 0.69, p ¼ 0.0016, n ¼ 12 from three individual experiments). This observation supports the prediction that the d NO signal is sequentially followed by CBF increase and by a
Fig. 3. The CBF change coupled to dNO dynamics is followed by a transient increase in local O2 tension. Average recordings of simultaneous measurements of O2 (green line) and CBF changes (red line) in the hippocampus of a urethane-anesthetized rat and temporal correlation with dNO dynamics (blue line). Inset shows the correlation between the amplitudes of the O2 and CBF changes (R2 ¼ 0.69, p ¼ 0.0016, n ¼ 12 from three individual experiments). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
transitory elevation in O2 tension. It should be remarked that the geometrical configuration of the array and the physical properties of the carbon fiber microelectrodes and laser Doppler probe impose the constraint that the measurements of dNO/O2 and CBF are performed within a volume of hippocampal tissue comprising probably a few hundreds of microemters in diameter. Within this volume of tissue, the fine correlation observed between O2 tension and CBF events, measured at separate locations within the dNO diffusion field, further supports the validity of the approach used to study the relationship between dNO and CBF. Neuronal-derived dNO is the mediator of neurovascular coupling in hippocampus The neurovascular coupling process is expected to be prone to modulation by functional interferences along the pathway of nNOSdependent dNO production, in particular at the level of the NMDA glutamate receptor and nNOS. Among several potential pharmacological tools, we have selected a glutamate NMDA receptor blocker (MK-801), a selective nNOS inhibitor (7-NI), and a selective eNOS inhibitor (L-NIO). The results obtained are summarized in Fig. 4A. The responses obtained after drug injection were compared with the corresponding control experiments (with vehicles). None of the vehicles used showed any significant effect on dNO or CBF responses (Supplementary Fig. 2). Blocking of the NMDA receptor elicited a significant inhibition of dNO production (7378%, p ¼ 0.0017, n ¼ 3) that was accompanied by a dramatic decrease in glutamate-induced CBF (7473%, p ¼ 0.0008, n ¼ 3). Likewise, the inhibition of the neuronal isoform of NOS by 7-NI induced a significant inhibition of both dNO and CBF responses to glutamate (62710 and 8376%, p ¼ 0.0002 and p ¼ 0.0025, respectively, n ¼ 4), as well as a decrease in CBF basal levels (2777%). Conversely, no significant effects were found on either induced or basal levels upon inhibition of the endothelial NOS isoform with L-NIO (Fig. 4C). Nevertheless, an increase in mean arterial blood pressure (MABP) of the animals was observed after the L-NIO injection, supporting its efficacy in terms of eNOS inhibition (n ¼ 4, MABP 90.371.1 to 114.972.5 mm Hg). The modulation of the NMDA receptor:nNOS pathway showed that, in the hippocampus, the increase in CBF was dependent on dNO signal amplitude (Fig. 4B). Within the range tested, the relationship between dNO and CBF showed to be linear above a threshold
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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of 0.43 fmol s 1, which is coherent with the concept of a linear relationship between neuronal activity and vascular responses provided by neuroimaging studies [28]. However, it should be pointed out that below the threshold our data do not identify whether the relationship is linear or a gating process is operative.
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The dNO-mediated neurovascular coupling encompasses the activation of soluble guanylate cyclase The classical pathway for vasodilation mediated by dNO involves the activation of soluble guanylate cyclase (sGC) in smooth muscle
Fig. 4. Glutamate-induced dNO production and CBF changes are dependent on the activation of NMDA receptor and nNOS. (A) Effects of MK-801 (1 mg/kg, NMDA receptor blocker), 7-NI (50 mg/kg, selective nNOS inhibitor), and L-NIO (40 mg/kg, selective eNOS inhibitor). Data represent the mean 7 SEM. Statistical analysis was performed by Student's t test in relation to control experiments (nnnp o 0.001). (B) Plot of the linear relationship between the amplitudes of dNO production and CBF changes obtained by glutamatergic activation. The linear regression showed an R2 ¼ 0.98 and a slope of 78.9% CBF/fmol s 1 dNO (x intercept 0.438). (C) Average recordings of the 7-NI and L-NIO effects on the dNO production (blue line) and CBF changes (red line) elicited by glutamate in the hippocampus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Neuronal-derived dNO mediates the neurovascular coupling through activation of soluble guanylate cyclase. The effect of ODQ, a selective heme-site inhibitor of soluble guanylate cyclase, on glutamate-induced dNO signals (blue line) and CBF changes (red line) in the rat hippocampus. ODQ was locally applied through an additional pipette attached to the array in the proximity of the laser Doppler probe (25 pmol, 0.5 ml). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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cells, which, via cGMP-dependent protein kinases, promotes the dephosphorylation of myosin light chains and thus vasodilation [15]. To unravel the mechanism by which neuronal-derived dNO promoted the increase in CBF, the effect of ODQ, a selective heme-site inhibitor of soluble guanylate cyclase, was evaluated. After the local application of ODQ in the hippocampus, glutamate induced a typical dNO concentration dynamics but the CBF changes were significantly reduced (6777%, p ¼ 0.002, n ¼ 4; Fig. 5). The slight inhibition of dNO signal observed is probably related to the nonspecific inhibition of nNOS by ODQ [29] (nNOS, being a heme protein, might be affected by ODQ), and not a consequence of cGMP decrease in neurons, as dNO concentration was resumed in the following stimulations and CBF remained inhibited, progressively returning to the control values obtained in the absence of ODQ. The uncoupling observed between d NO concentration dynamics and CBF changes in the presence of ODQ strongly supports the idea that dNO produced, and diffusing from neurons, reaches vascular smooth muscle cells and triggers vasodilation via sGC activation. The astrocytic pathway is not explicitly involved in the dNO-mediated neurovascular coupling Given the commonly accepted paradigm of astrocytes bridging neurons and vascular cells, encompassing metabotropic glutamate receptor activation and subsequent production of arachidonic acid metabolites [2,6], we addressed the potential contribution of the astrocytic bridge to neurovascular coupling in hippocampus under our experimental conditions. For that purpose, a cocktail of antagonists of metabotropic glutamate receptors (LY367385 and MPEP) was injected intracerobroventricularly into the rat brain to inhibit metabotropic glutamate receptors mGluR1 and mGluR5, respectively. If glutamate-triggered [Ca2 þ ]i elevations in astrocytes are crucial for neurovascular coupling, their specific inhibition should hamper the increase in CBF induced by glutamate via the astrocytes. However, the inhibition of astrocyte mGluR receptors did not elicit any alteration in the recorded dNO and CBF signals (Fig. 6A). Neither inhibition of cyclooxygenase activity by acetylsalicylic acid—downstream in the astrocytic pathway—nor impairment of astrocytic metabolism linked to the inhibition of mitochondrial aconitase via metabolism of sodium fluoroacetate—which is preferentially taken up by glial cells [30]—had any effect either. Also, no contribution could be assigned to adenosine receptors, argued to mediate a complex signaling process of neuronal activity-induced increase in cerebral vasodilation [31,32], as caffeine, a nonselective antagonist of adenosine receptors [33], also failed to modulate either dNO or CBF (Fig. 6A). As an additional control, the specific activation of mGLuR receptors by the mGluR agonist tACPD—known to promote Ca2 þ elevation in astrocyte somata and related pathways [5]—did not elicit any changes in either dNO production or CBF (Fig. 6B).
Fig. 6. The astrocytic pathway is not explicitly involved in the dNO-mediated neurovascular coupling. (A) Effects of LY367385/MPEP (100 μM each, antagonists of metabotropic glutamate receptors mGluR1 and mGluR5, respectively, n ¼ 6), acetylsalicylic acid (ASA; 300 mg/kg, COX inhibitor, n ¼ 5), sodium fluoroacetate (SFA; 20 mmol locally, glial toxin, n ¼ 5), and caffeine (CAF; 50 mg/kg, nonselective antagonist of adenosine receptors, n ¼ 3) on dNO signals and CBF changes. Data represent the mean 7 SEM. Statistical analysis was performed by Student's t test in relation to control experiments (performed with vehicles). (B) Representative recording of injection of tACPD, an agonist of mGluR, in dNO dynamics (bottom, blue line) and CBF changes (top, red line) in the hippocampus of a urethaneanesthetized rat. tACPD was locally pressure injected at the time indicated by the arrows (25, 50, and 100 pmol, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
NO-dependent CBF increase is region specific
d
The regional occurrence of the diffusive connection established by NO between neurons and vessels without the intermediacy of cellular systems between glutamatergic synapses and vessels in brain regions other than hippocampus was assessed, for it has been previously reported that the mechanisms underlying neurovascular coupling may be region specific. With that intent the experimental approach used in the hippocampus was applied to probe the cerebral cortex with the tip of the array positioned in the deeper layers, where nNOS immunoreactivity is predominant [34]. Whereas data support the coupling, the profiles of dNO and CBF change do not reproduce the phenomenon observed in hippocampus in all its dimensions (Fig. 7). In fact, in cerebral cortex, compared with hippocampus, glutamate induced a weaker production of dNO (0.4670.14 fmol s 1) followed by a less intense increase in CBF (4676%) (12 peaks analyzed from four d
Fig. 7. Coupling between dNO concentration dynamics and CBF changes upon glutamatergic activation in the cortex. Average recordings of the simultaneous measurement of dNO concentration dynamics (bottom, blue line) and CBF changes (top, red line) in the cerebral cortex of urethane-anesthetized rat upon glutamate stimulation (0.5 nmol, 1 s). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
individual experiments) but, quantitatively, the response of dNO and CBF to glutamate in cerebral cortex was not proportionally attenuated; d NO production in cerebral cortex was 87 7 4% lower than in hippocampus and the reduction in CBF change was only 62 7 5% lower. We would predict that distinct neuroanatomic regions (encompassing variations in nNOS expression, vascularization density, mean
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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distance between glutamatergic neurons and arterioles, dNO inactivation mechanisms) exhibited different quantitative relationships between dNO and CBF signals but maintain a temporal and local correlation, as observed. Therefore, the kinetics of neuronal dNOdependent CBF changes may be intrinsically distinct in hippocampus compared with other brain regions.
Discussion The mechanisms that regulate the synergy between cerebral microcirculation and local neuronal activity have been debated over a century without clear conclusions, in part because of the severe experimental limitations to measure the process in real time in the natural working environment. The identification of d NO as a diffusible vasodilator produced at active neurons, via ionotropic glutamate receptor-dependent pathways, led to the hypothesis that it could be the mediator coupling the brain activity to CBF [9] but, paradoxically, it introduced a further difficulty. In fact, because dNO is small, is hydrophobic, and overcomes storage and specific interaction with receptors, it conveys information associated with its concentration dynamics, a parameter difficult to measure in vivo. By simultaneously and direct measuring dNO and CBF dynamics in the hippocampus, this work supports the notion that dNO, generated by nNOS during glutamatergic activation, induces a coupled increase in the local CBF (Fig. 8). Accordingly, dNO and CBF events precede an increase in O2 tension from background, a critical prediction of the neurovascular coupling concept. The observation that both MK-801 and 7-NI individually elicited a significant inhibition of dNO production, and that this effect was translated into CBF changes, clearly identifies the pathway that paves the neurovascular coupling process in the hippocampus and recognizes nNOS-derived dNO as the coupler molecule. Data showing the abolishment of CBF changes, despite the strong dNO signal under conditions of sGC inhibition with ODQ, mechanistically established the pathway for dNO-dependent CBF changes in the rat hippocampus by the canonic cGMP-dependent vasodilation pathway and dNO as a critical messenger. Furthermore, data also support the idea that nNOS-derived dNO participates in the maintenance of resting CBF, as 7-NI administration also promoted a decrease in hippocampal CBF basal levels. It should be noted that
Fig. 8. Nitric oxide is a critical diffusible messenger between active neurons and the local blood vessels in hippocampus. The scheme depicts the cellular relationships and the sequence of events underlying the neurovascular coupling mediated by dNO upon glutamatergic stimulation. dNO transitorily produced by neurons upon stimulation on glutamate NMDA receptors diffuses toward neighboring blood vessels where, after the interaction with soluble GC, triggers a sequence of events comprising vasodilation and a transient and localized increase in O2 tension.
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glutamate microinjections (within the range used) and electric stimulation induce equivalent cerebrovascular effects, as shown previously in cerebellar cortex upon electric stimulation of parallel fibers [35]. The pattern of dNO production and CBF changes measured accomplishes the fundamental criteria of the neurovascular coupling concept: a temporal, amplitude, and spatial relationship [36]. In addition to the temporal and amplitude correlation evident in the occurrence of both dynamics, data show that the increase in CBF is limited in space, affecting just the area of increased neuronal activity, the hippocampus, for if the laser Doppler probe is placed in the overlying cerebral cortex no CBF change is observed in response to glutamate. The strategy established in this work also complies with the criteria for dNO signaling in the framework of the coupling inasmuch as stimulation of dispersed glutamatergic synapses via nNOS generates a volume signaling that encompasses the diffusion of dNO from neurons to nearby arterioles to induce an increase in CBF, thus establishing that, upon elicitation of neuronal activation, d NO from nNOS launches a diffusible connection between neurons and the vasculature. The concept of a dNO-mediated diffusible connection is supported by two observations: first, in hippocampal slices, in the absence of a functional vascular system to remove d NO, the diffusion field of dNO was measured as being close to 400 mm [37]; second, the mean distance between arterioles and nerve fibers along the longitudinal axis of pyramidal layer neurons in the CA1 region of hippocampus is circa 150 mm [38], i.e., within the range of dNO diffusional spread. Furthermore, mathematical modeling of the physicochemical process of dNO production from an active neuron in cerebellar slices estimated that the range of d NO concentration reaching the nearby blood vessels rises drastically, exceeding ca. 100-fold its basal levels during stimulated neuronal activity [39]. Among the wide collection of proposed mechanisms, a generally accepted paradigm for neurovascular coupling supports the view of astrocytes carrying the neuronal signal by bridging neurons and vascular cells [6]. Astrocytes are known to respond to glutamate via mGluR activation, involving a transient increase in [Ca2 þ ]i that ends up in the synthesis of vasoactive substances upon activation of phospholipase A2 and arachidonic acid production. The vasoactive compounds include prostaglandin E2 (via cyclooxygenase) [5] and epoxyeicosatrienoic acids (via cytochrome P450 epoxygenase) [7,40]. Accordingly, it has been speculated that dNO levels may modulate the cerebrovascular response to mGluR activation by regulating the enzymatic conversion of arachidonic acid via cytochrome P450 to either vasodilators or vasoconstrictors [41]. However, our results show that, in hippocampus, this pathway makes a minor if any contribution to the neurovascular coupling promoted by glutamatergic activation, supporting that this process is developed by different mechanisms depending on the brain region. Also, it should be considered that glutamate-dependent astrocyte signaling may change during development. Recently, it was demonstrated that astrocytic mGluR5 expression is developmentally regulated, being undetectable in adulthood, and that, in adult mice, astrocytes failed to respond to the mGluR5 agonist tACPD with significant Ca2 þ increase [3]. Also, it is of note that functional NMDA receptors [42] and constitutive NOS expression [43] have been found in cultured astrocytes. Therefore, and although the abolishment of astrocytic metabolism by sodium fluoroacetate did not affect the recorded dynamics, we cannot fully exclude the potential involvement of astrocytes in the NMDA–nNOS pathway, leading to CBF changes. Further supporting the notion of region specificity of neurovascular coupling process [44], we observed that glutamatergic activation in cerebral cortex, compared with hippocampus, resulted in a significantly lower dNO production translated into less dramatic CBF changes. The dNO:CBF ratio was, however, lower in the cortex, in
Please cite this article as: Lourenço, CF; et al. Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.021i
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agreement with the less outstanding role attributed to the dNO in this brain region [18–20]. Also, the latency of CBF response is slower in the cerebral cortex than in hippocampus, which, in turn, is longer than that commonly reported by studies using electric stimulation [22,45,46]. Apart from the stimulation paradigm, these variations can be related to differences in the neurovascular coupling process and vascular network among regions. In fact, the Lauritzen group has observed slower CBF responses in cerebellum [47,48] compared with the somatosensory system [46]. These observations reinforce the notion that the kinetics of neuronal dNO-dependent CBF changes may be intrinsically distinct in hippocampus, compared with other brain regions. Following the formulation of the hypothesis of dNO as a candidate to mediate neurovascular coupling [9] intensive research has attempted to elucidate its role in the process. All the studies, however, were largely based on the pharmacological modulation of NOS activity and, expectedly, controversial results were collected. Assuming as plausible that different pathways and molecules might be involved in the neurovascular coupling phenomenon, and that the relative contributions of these pathways can vary among regions, we should, however, be aware that the use of such pharmacological tools without additional verification of their efficacy can give rise to misleading results. For instance, it was shown that 7-NI, a widely used selective nNOS inhibitor, did not maximally inhibit nNOS activity and did not affect all brain regions to the same extent [49]. Additionally, methodological variables such as the anesthetic used seem to contribute to the discrepancy of the results, because of their possible effects on neurotransmission and hemodynamics [50]. Therefore, for a messenger that conveys information associated with its concentration dynamics, its direct, localized, and quantitative measurement in vivo is mandatory to progress in the understanding of its role in the mechanisms of neurovascular coupling, a critical pathway for proper brain function. Indeed, a study in cerebellar slices, by simultaneously measuring dNO dynamics and local capillary enlargement associated with treatment with NOS inhibitors or with tetrodotoxin, suggested that dNO of neuronal origin is responsible for the vast majority of the vasomotor response in cerebellum [51]. Finally, from a pure conceptual viewpoint, the mechanism of neuronal-derived dNO mediating neurovascular coupling via volume signaling may be a noncanonical way to underlie a process of vital importance for the brain to preserve its structural and functional integrity. However, although the mechanistic link to match blood supply with the metabolic demands imposed by increased neuronal activity is based on diffusion and characterized by lack of specific recognition by receptors, it still remains a highly and intrinsically controlled mechanism. Because red blood cells constitute the major pathway for dNO inactivation in the brain [27], the increase in the cerebral blood flow triggered by neuronalderived dNO itself constitutes a mechanism to shape the dNO concentration dynamics and interrupt the signaling pathway.
Appendix A.
Supplementary material
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