Spectral analysis of pial arteriolar diameter variability during cortical activation in rats suggests a link between vasomotion and neurovascular coupling

Testo completo

(1)TESI DI DOTTORATO NEUROSCIENZE DI BASE E DELLO SVILUPPO. UNIVERSITA’ DI PISA DIPARTIMENTO DI FISIOLOGIA UMANA “G. MORUZZI”. “Spectral analysis of pial arteriolar diameter variability during cortical activation in rats suggests a link between vasomotion and neurovascular coupling”. Relatore Candidato Pierlorenzo Marchiafava Francesco Vetri.

(2) PhD thesis Dr. Vetri Francesco INDEX…………………………………………………………….……………………-1ABSTRACT……………………………………………………………………………-3RIASSUNTO...………………………………………………………………………...-4INTRODUCTION .……………………………………………………………………-5Cerebral microcirculation .…………………………………………………………-5Control of cerebral circulation .……………………………………………….……-6Autoregulation ………………………………… ……………………………...……-6 Chemical regulation of cerebral blood flow…………………………………..……-7Effects of CO2 and H+ on cerebral vessels……………..…………………………..-7Effects of O2 on cerebral vessels……………..………………………………..…..-10Nervous regulation of cerebrovascular tone.…………………………………...…-11Role of intrinsic innervation..…………………………………….…………….…-12Role of intrinsic pathways…………………….…………..………………….....…-13Subcortical vasoactive pathways……………………………………………......…-13Local interneurons……………………………………………………………...…-16Neurovascular coupling…………………………………………………………..…-17Mechanisms of neurovascular coupling: searching for mediators………..…......-19Vasoactive ions……………………………………………………………….........-19Nitric oxide…………………………………………………………………………-20Vasoactive factors related to energy metabolism………………………….………-22Adenosine.…………………………………………………………….………........-23Prostaglandins..…………………………………………………………………….-24Epoxyeicosatrienoic acids…………………………………………………….........-25Interactions of mediators……………………………………………………..……-26Central pathways, interneurons and vasoactive neurotransmitters………...….…-27Other vasoactive factors released by neural activity………………………...….…-29Role of astrocytes in neurovascular coupling ……………………………...………-29Neuronal activity-dependent calcium elevations in astrocyte endfeet…...….……-30Propagating calcium wave between astrocytes may contribute to control microcirculation……………………………………….………………...…………-32Activation of astrocytes can also trigger arteriole constriction………...…….......-33Local versus remote vasodilation…………………………….………...……….......-37Mechanisms of neurovascular coupling: an integrated view…………………......-39Neurovascular coupling and pathology..………….……………………………….-39-1-.

(3) PhD thesis Dr. Vetri Francesco Hypertension…………………………………………………………………...……-40Alzheimer disease…………………………..…………………………………......…-41Ischemic stroke…………………………………...……………………………….…-42A final common pathway to cerebrovascular dysfunction: vascular oxidative stress…………………………………………………………………………………-43Future directions in the study of neurovascular coupling…………………………-44Vasomotion ………………………………………………………………………...…-45Evidence of vasomotion….………………………………………………………...…-46Factors influencing vasomotion…………………………………………………...…-48Cellular mechanisms underlying vasomotion………………………….……………-51Functional consequences of vasomotion………………….……………….……....…-58Pathophysiological role of vasomotion…………..……….…………….………....…-60Vasomotion and neurovascular coupling……..……….……………….………....…-61AIM OF THE THESIS…………………………………………………………………-63MATERIALS & METHODS…………………………………………………………..-64Animal preparation …………………………………………………………………..-64Sciatic nerve stimulation…………………………………………………….………..-65Somatosensory evoked potentials ……………………………………………….…..-66Intravital microscopy and vessel diameter measurement…………………….…....-66Experimental protocol……………………………………………….…………….....-69Parameter estimation……………………………………....……….……………......-69Statistical analysis……………………………...…………....……….………….…....-71RESULTS………………………………………...…………....……….……….……....-72Oscillatory pattern during basal conditions ……………………………………......-72Stimulus-related vascular response…….…………………………………...…….....-74DISCUSSION…….…………………………………………...………………………....-83Arteriolar oscillations in basal conditions…………………………………………...-84Arteriolar oscillations associated to neural activation……………………................86CONCLUSIONS…….………………………………………………….…………..…...-90BIBLIOGRAPHY…….…………………………………….……...................................-92-. -2-.

(4) PhD thesis Dr. Vetri Francesco ABSTRACT. The oscillatory pattern of pial arterioles, i.e. vasomotion, has been described since early 1980s, but the impact of neural activation on such oscillations has never been formally examined. Sciatic nerve stimulation, a well characterized model for studying neurovascular coupling, leads to a neural activity-related increase of pial arteriolar diameter in the contralateral hindlimb somatosensory cortex. Exploiting such an experimental model, the aim of the present study was to explore vasomotion and its changes during neurovascular coupling with a novel analytical approach. Indeed, to characterize oscillations, we evaluated the total spectral power in the range 0.02-2.00 Hz and subdivided this frequency interval into seven 50% overlapping frequency bands. Results indicated that only arterioles overlying the stimulated hindlimb cortex showed a significant increase of total power, unlike arterioles overlaying the whisker barrel cortex, used as control for the vascular response specificity. The total power increase was sustained mainly by marked increments in the low frequency range, with two peaks at 0.03 and 0.08 Hz, and by a wide increase in the high frequency range (0.60-2.00 Hz) in the averaged spectrum. These activity-related spectral changes suggest: i) that it is possible to assess the vascular responses by using total power; ii) the existence of at least three distinct mechanisms involved in the control of neurovascular coupling: two with a feedback frequency loop in the low frequency range and another one in the high range; iii) a potential involvement of vasomotion in neurovascular coupling. Moreover, these findings highlight the oscillatory nature of the mechanisms controlling neurovascular coupling.. -3-.

(5) PhD thesis Dr. Vetri Francesco RIASSUNTO. L’attività oscillatoria spontanea delle arteriole piali, detta vasomotion, è stata descritta agli inizi degli anni ’80, ma l’impatto dell’attivazione neuronale su tali oscillazioni non è stato analizzato in dettaglio. La stimolazione del nervo sciatico porta ad un incremento del diametro delle arteriole piali sulla corteccia somatosensoriale controlaterale nell’area di rappresentazione dell’arto posteriore. Sfruttando questo ben caratterizzato modello sperimentale per lo studio dell’accoppiamento neurovascolare, il presente studio ha lo scopo di esplorare la vasomotion e le sue modificazioni durante l’accoppiamento neurovascolare con un nuovo approccio di analisi. Infatti, per caratterizzare le oscillazioni vascolari, è stata calcolata la potenza totale dello spettro di frequenza nell’intervallo 0.02-2.00 Hz e si è suddiviso questo range in 7 bande con una sovrapposizione del 50%. I risultati indicano che solo le arteriole che si trovano al di sopra della corteccia stimolata mostrano un significativo aumento della potenza totale, a differenza delle arteriole al di sopra della zona di rappresentazione delle vibrisse, usata come controllo per la specificità della risposta vascolare. L’incremento della potenza totale era sostenuto principalmente dall’aumento nel range a bassa frequenza, con due picchi a 0.03 e 0.08 Hz, e da un diffuso aumento nel range frequenziale alto (0.60-2.00 Hz) dello spettro medio. Queste modificazioni dello spettro correlate all’attività suggeriscono: i) la possibilità di valutare le risposte vascolari usando la potenza totale; ii) l’esistenza di almeno tre meccanismi coinvolti nell’accoppiamento neurovascolare: due con una frequenza di feedback nel range a bassa frequenza ed un altro ad alta frequenza; iii) un potenziale coinvolgimento della vasomotion nell’accoppiamento neurovascolare. Inoltre, questi dati mettono in luce la natura oscillatoria dei meccanismi di controllo dell’accoppiamento neurovascolare.. -4-.

(6) PhD thesis Dr. Vetri Francesco INTRODUCTION. Cerebral microcirculation Large cerebral arteries arising from the circle of Willis branch out into smaller pial arteries and arterioles that travel on the surface of the brain across the subarachnoid space. The microcirculation is represented by vessels comprising arterioles with diameter up to 100 µm, capillaries and venules. Pial arteries give rise to penetrating arteries and arterioles that enter into the substance of the brain. These arteries (resistance vessels) consist of an endothelial cell layer, a smooth muscle cell layer, an outer layer, termed adventitia, containing collagen, fibroblasts, and perivascular nerves and a leptomeningeal membrane. Penetrating vessels are separated from the brain by the Virchow-Robin space, which contains cerebrospinal fluid. On the outer side of the Virchow- Robin space, astrocytes give rise to the glia limitans membrane. As the arterioles penetrate deeper into the brain, the Virchow-Robin space disappears and the vascular basement membrane enters into direct contact with the astrocytic end feet. Arterioles become progressively smaller, lose the smooth muscle cell layer, and become cerebral capillaries. Arterioles with a mean diameter of 8-10 µm are called terminal arterioles and control the blood distribution to capillaries. The density of brain capillaries within the brain is regionally heterogeneous and varies according to regional blood flow and regional metabolic demands. Capillaries (diameter 4-7 µm) consist of endothelial cells, pericytes, and the capillary basal lamina on which astrocytic feet are attached. Brain endothelial cells are unique in that they are not fenestrated and are sealed by tight junctions, features that underlie the blood-brain barrier. Endothelial cells play an important role in the regulation of vascular tone by releasing potent vasoactive factors, such as nitric oxide (NO), free radicals, prostacyclin, endothelium-derived hyperpolarizing. -5-.

(7) PhD thesis Dr. Vetri Francesco factor (EDHF) and endothelin (Faraci and Heistad, 1998). Pericytes have contractile properties and may modulate capillary diameter. Perivascular astrocytes surround most of the capillary abluminal surface with their end feet. There is close association between cerebral arteries, arterioles, and capillaries with nerves originating from central and peripheral sources (see “nervous regulation of cerebrovascular tone”). Venous circulation, comprising post-capillary venules and larger veins, drain the blood coming from pial circulation and the cortex. Moreover, there are artero-venous anastomosys, especially at precapillary level, which represent a complex system of blood flux deviation.. Control of cerebral circulation. Autoregulation. The blood pressure in the brain is normally regulated by intrinsic baroceptive circulatory reflexes which not seem to involve directly the cerebrovascular tone. The seno-carotideal reflex tend to maintain constant the arterial pressure at cranial level and, consequently, the cerebral blood. pressure gradient, without altering the cerebrovascular resistance. This. mechanism provides a relatively constant cerebral blood flow (CBF). In case of hypersensibility of these reflexes and altered blood pressure, the adaptation of cerebrovascular tone tend to contrast the effects of arterial pressure and pressure gradient variations. In healthy subjects this adaptation is sufficient to maintain the CBF constant in a wide range of arterial pressure variations (Lassen, 1964). This mechanism of blood flow preservation within the physiological range after blood pressure modifications is known as autoregulation. -6-.

(8) PhD thesis Dr. Vetri Francesco In conclusion, when mean arterial pressure (MABP) falls from normal levels (90mmHg) to critical levels, between 50-70 mmHg, the CBF fall down and signs and symptoms of cerebrovascular insufficiency start to compare (Lassen, 1964). When MABP reaches 35 mmHg, the CBF falls from normal levels of 50-60 to 30 ml/100 g min-1, which is an inadequate level for sustaining consciousness. In vascular pathologies, as malignant hypertension, the accommodation of cerebrovascular tone appears to be more limited and the symptoms of cerebrovascular insufficiency manifest following the proportionally minor reduction in blood pressure.. Chemical regulation of cerebral blood flow. It was demonstrated that the regulation of the cerebrovascular tone is strongly influenced by chemical factors like H+, CO2 e O2.. Effects of CO2 and H+ on cerebral vessels H+ e CO2 have marked relaxing effect on smooth muscle cells of cerebral vessels, therefore modifications of pH and CO2 concentration have marked influence on CBF and vascular resistance. The effects of PaCO2 change is generally used for measuring the response of cerebral circulation. Topical application of solutions with acid pH on brain surface produces dilation of pial arteries, while the application of alkaline solutions has constrictive effects. Solutions with variable PCO2 or [HCO3-] do not have effects on diameters of pial arterioles expect when pH modifies in the same direction. Therefore, the effect of CO2 is mediated by the variations of extracellular pH while molecular CO2 and HCO3- do not have intrinsic vasoactive capacity.. -7-.

(9) PhD thesis Dr. Vetri Francesco In fact, many experimental results demonstrate that H+ ions have direct relaxing effect on vessel muscle. The effect of pH variations on cerebral vessels is quantitatively similar to that on arterioles of skeletal muscle. The pial arterioles, indeed, dilate rapidly (in less than 10 sec.) as the response after microapplication of acidic liquor. Arterial hypercapnia dilates the pial arterioles and hypocapnia contracts them. The effect of hypercapnia is directly correlated with the diameter of the vessels; the augmented PCO2 produces, in percent, a more pronounced dilation on vessels with small diameter. Indeed, during a mild hypercapnia the dilatory effect of CO2 on large vessels is attenuated by vasoconstriction provoked by augmented activity of discharge in adrenergic fibres. Moreover, the dilation of large pial vessels, caused by hypercapnia, increases after the ablation of the cervical superior ganglion, arterial chemoreceptor or vagal denervation and after inhibition of α-adrenergic receptors. Conversely, the response to hypocapnia is not diameter-dependent and is not altered by the α-adrenergic block. The dilation of pial arterioles during hypercapnia evolves slowly. The beginning of the dilation, corresponding to a 10% increase of vessel diameter, occurs 1,5-2 minutes after starting to inhalation of 7-10% CO2 (this delay is due to the time requested for increasing PaCO2). The dilation of pial arterioles during hypercapnia is quantitatively similar in awake rabbit and in anesthetised cat. An important question is whether the local effects of CO2 can explain in toto diameter changes of cerebral vessels caused by PaCO2 alterations. Modifying the PaCO2 and the PCO2 of cerebrospinal fluid of an equal quantity but in the opposite directions, it was observed in cats that vasodilator effects of hypercapnia can be totally balanced by an equal decrease of liquor PCO2. These findings demonstrate that the local action of CO2 on pial arterioles is sufficient to explain the vessel changes provoked by PaCO2, without involving ulterior CO2 effects.. -8-.

(10) PhD thesis Dr. Vetri Francesco It is known that CO2 action is mediated by the direct effects of H+ on vessel muscle of cerebral arterioles. The [H+] close to vessel muscle depends on [HCO3-] and PCO2 of the extracellular liquid, which, in turn, depends on PaCO2 and liquor PCO2. The blood brain barrier is impermeable to H+ e HCO3 but is completely permeable to molecular CO2. When the PaCO2 augments, the molecular CO2 diffuses through the blood brain barrier, the local pH of the extracellular liquid reduces, producing dilation throughout direct vascular smooth muscle relaxation. When PaCO2 decreases, the opposite phenomenon occurs. Beside the effects on vessel diameter, PaCO2 modifications exert marked effects also on cerebral blood volume, flow and vessel resistance in all studied species. Hypercapnia increases blood flow and volume and decrease resistance, while hypocapnia induce opposite effects. Responses to PaCO2 modifications depends on many factors (brain area, autoregulatory responses, O2 consumption and age). Maximal increases of CBF occur at PaCO2 over 60-80 mmHg. The pial vessel-glia limitans arrangement has a great relevance to the reactivity of pial arterioles to vasodilating stimuli in vivo, particularly when the vascular responses are mediated by processes originating outside the vessel as hypercapnia. Evidence from multiple laboratories (Wang et al., 1995; Iadecola and Zhang, 1996) points to neuronal NO synthase (nNOS)-generated NO playing major role in adult rodent CO2-induced cerebral vasodilation in vivo (including pial arterioles). However, nNOS-containing neurons are sparsely distributed in the rat cerebral cortex and are virtually absent from the layer I. Moreover, it is unlikely that other neuronal factors influence the hypercapnic response of pial arterioles, because nitrergic innervation of these arterioles does not extend to vessels smaller than 100 μm in diameter. Furthermore, local blockade of neuronal transmission with TTX has not been found to alter hypercapnia-induced pial arteriolar dilation (Wang et al., 1995). Despite this, hypercapnia elicits a global dilation of pial arterioles, a response that is substantially. -9-.

(11) PhD thesis Dr. Vetri Francesco diminished by selective nNOS blockade (Santizo et al., 2001). Because of their unique association with neurons and pial vessels, astrocytes, via the glia limitans, may act to transmit and amplify signals arising in comparatively few cells to the entirety of the pial arteriolar system. A selective injury of the glia limitans alters hypercapnia-induced pial arteriolar dilation (Xu et al., 2004). Considering that the gap junctional protein connexin43 is abundantly expressed in the glia limitans, it has been shown that connexin43-dependent gap junctional communication could serve to facilitate the spread of vasodilating signals along the glia limitans.. Effects of O2 on cerebral vessels Arterial hypoxia induced by inhalation of gas containing low concentration of O2 causes dilation of pial arterioles and an increase of CBF. For low O2 tension, in the range 20-30 mmHg, vasodilation is marked and similar to that observed for severe hypercapnia. An increase of PaO2 over normal values induces slight constriction of pial arterioles. Data obtained by varying PO2 of cerebrospinal fluid close to pial vessels has shown that PO2 is able to alter smooth muscle activity with local mechanisms. Pial arteriole dilation during hypoxia could derive from direct relaxing effect of low PO2 on vascular muscle. The mechanism by which tissue hypoxia drives to dilation is not completely clear. A conceivable hypothesis is that such response is mediated by release of adenosine from brain parenchyma during arterial hypoxia. Other potential mediators could be K+, H+ and prostaglandins. Certainly, hypoxia activates anaerobic glycolysis and can cause acidosis, but vasodilation occurs in the early phase of arterial hypoxia, before than pH could decrease. During submaximal combined hypoxic and hypercapnic stimulations, dilating effects can sum. The response to hypoxia is more pronounced within grey matter and appears principally related to local factors. Little o moderate PaO2 decrease induces only slight CBF increase. - 10 -.

(12) PhD thesis Dr. Vetri Francesco During inhalation of gas mixture with low O2 content, reflex hyperventilation produces hypocapnia and the vasoconstriction related to this latter can totally counteract the vasodilating effect of hypoxia. Hyperoxia induced by inhalation of O2100% at 1 atmosphere in humans produces mild cerebral vasoconstriction, while at 3.5 atmosphere severe vasoconstriction occurs.. Nervous regulation of cerebrovascular tone. Cerebral vessels are richly innerved, but the role of neurogenic mechanisms in the regulation of cerebral blood flow (CBF) is still matter of debate. A general consensus is that the “extrinsic innervation” of extracerebral blood vessels (e.g. carotid and mean cerebral artery) finds its origin either in: •. the superior cervical ganglion: sympathetic innervation containing norepinephrine and neuropeptide Y (NPY),. •. the sphenopalatine and otic ganglia: parasympathetic nerves containing vasoactive intestinal peptide (VIP), acetylcholine (ACh), NO synthase and, in human, peptide histidine isoleucine or methionine,. •. the trigeminal ganglion: sensory nerves containing calcitonina gene-related peptide (CGRP), substance P (SP), neurokinin A, and pituitary adenylate-cyclase activating polypeptide.. However, upon their entry into the brain parenchyma, cerebral arteries loose their peripheral nerve supply and, once the Virchow-Robin space has vanished, receive neural input from neurons located within the brain itself, hence the appellation of “intrinsic innervation” of the brain microcirculation. The neural regulation of the microcirculation has been most. - 11 -.

(13) PhD thesis Dr. Vetri Francesco extensively studied in the cerebral cortex, where it receives afferents from subcortical pathways (Iadecola et al., 1997; Cauli et al., 2004) as well as from local cortical interneurons (Vaucher et al., 2000). Key features of perivascular nerves, whether associated with vessels located outside or inside the brain, are their lack of classical synaptic junctions at the site of contact with the blood vessels, general enrichment within less than 1 micrometer from the vessel wall, and ability to directly modulate the tone of the vessels upon stimulation (Akselrod et al., 1981).. Roles of the extrinsic innervation The main role of the sympathetic system, independent from its direct contractile or trophic effects on brain vessels, probably relates to its capacity to shift the upper limit of the autoregulation curve toward higher pressures, a response mediated in part by NPY and aimed at protecting the brain against blood pressure increases due to sympathetic activation (cfr. “Autoregulation”) (Goadsby and Edvinsson, 2002). In contrast, the parasympathetic system, a potent dilator of brain vessels upon stimulation, does not appear to play a significant role in either autoregulation or other physiological cerebrovascular responses, but its implication in pathological situations such as ischemia or migraine headache has been advanced (Goadsby and Edvinsson, 2002). The trigeminovascular pathway, which provides the unique sensory innervation to brain vessel, appears as a “protective” system that is able to restore vessel tone after vasocontractile stimuli, a response mediated by the potent vasodilator CGRP released from trigeminovascular nerves. Most recent research on the trigeminovascular system has focused on its role in migraine headache. Indeed, it was recently shown in human or animal models that cortical spreading depression, a wave of cortical depolarization that underlies migraine aura (Hadjikhani et al., 2001) and, possibly, also migraine without aura, activates trigeminovascular afferents and initiates a cascade of events that culminate into CGRP (SP. - 12 -.

(14) PhD thesis Dr. Vetri Francesco and neurokinin A) release, blood flow increase, and inflammation within the meningeal dura (Bolay et al., 2002).. Role of intrinsic pathways The neuromediators present in perivascular nerves around cortical microvessels, the changes induced in cortical perfusion after activation of their neurons of origin, as well as the distribution of specific populations of receptors within the different cellular compartments of the “neurovascular unit” and their ability to regulate microvascular tone have been well described (Akselrod et al., 1981; Iadecola, 2004). Only the most salient and recent findings will be highlighted here. However, before doing so, it is important to revisit the basic concept of the functional neurovascular unit. The latter is anatomically best described as a “neuronalastrocytic-vascular” tripartite unit (Akselrod et al., 1981). Indeed, perivascular neuronal varicosities, irrespective of the neuromediators they contain, abut primarily on astrocytic endfeet surrounding blood vessel walls, with a smaller proportion directly contacting the vessel. basal. lamina.. Such. an. arrangement implies. that. perivascularly released. neurotransmitters and mediators can activate receptors on both vascular and astroglial cells to alter the tone of brain microvessels.. Subcortical vasoactive pathways The best-studied intrinsic neural pathways that project to cortical microvessels are those originating in the nucleus basalis, locus coeruleus, or raphe nucleus, and respectively containing ACh, norepinephrine, or 5-HT. Upon electrical or chemical stimulation, these subcortical areas elicit increases or decreases in cortical cerebral blood flow (CBF). Anatomical, molecular, and pharmacological studies have provided unequivocal evidence that shows that:. - 13 -.

(15) PhD thesis Dr. Vetri Francesco 1) these neurons send projection fibers to cortical microvessels and surrounding astrocytes, 2) specific receptors for the vasoactive mediators they release exist on microvascular endothelial and/or smooth muscle cells that can either dilate or constrict cortical microvessels upon activation, 3) receptors are also found on astrocytes, thereby providing an additional means for modulation of microvascular tone following changes in neuronal activity. Specifically, projections from basal forebrain neurons to cortical microvessels and associated astrocytes, hereafter referred to as “perivascular” afferents, contain primarily ACh but also NOS, the synthesizing enzyme for the gaseous dilator NO. Coincidently, the increase in cortical perfusion elicited by stimulation of the basal forebrain is decreased after blockade of ACh receptors or inhibition of NOS activity. Muscarinic M5 receptors have been identified as those mediating cerebral vasodilatation (Elhusseiny and Hamel, 2000). However, multiple muscarinic ACh receptors also exist on astrocytes and, although not yet demonstrated, it cannot be excluded that these cells also contribute to the perfusion response through the release of vasoactive mediators (see below), as will be described below for the norepinephrine- and glutamate-mediated changes in cortical perfusion. A similar scenario has been reported for serotonergic afferents to cortical microvessels, which, depending on the rostrocaudal level of stimulation of their cells of origin within the brain stem raphe nucleus, increase or decrease cortical CBF (Akselrod et al., 1981). Cortical microvessels are also endowed with several 5-HT receptors. Despite the presence of several 5-HT receptors subtypes in astrocytes, a role for astrocytes in the 5-HT-mediated changes in cerebral microvascular tone and cortical perfusion has not yet been demonstrated (Akselrod et al., 1981).. - 14 -.

(16) PhD thesis Dr. Vetri Francesco In contrast, a role for astrocytes in mediating the decrease in cortical CBF observed following stimulation of the locus coeruleus has recently been highlighted. In fact, it is known that stimulation of noradrenergic neurons in the locus coeruleus leads to a reduction in cortical CBF and that perivascular noradrenergic afferents in the cerebral cortex target mainly perivascular astrocytes rather than microvessel walls (Mulligan and MacVicar, 2004). Recently, in cortical and hippocampal brain slices, it was evidenced that application of norepinephrine triggers increases in intracellular Ca++ concentrations ([Ca++]i) in astrocytes and perivascular astrocytic end-feet and that this response elicited constriction of the microarterioles on which the end-feet abutted (Mulligan and MacVicar, 2004). Furthermore, the. authors. were. able. to. show. that. the. contraction. was. mediated. by. 20-. hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450A derivative of arachidonic acid. However, other studies in cortical brain slices showed that a rise in astrocytic [Ca2+]i after increased neuronal activity by electrical stimulation or synaptically released glutamate induced dilation of cortical arterioles (Zonta et al., 2003b). In the latter study, the vasoactive signaling molecule corresponded to a cyclooxygenase product of arachidonic acid, likely PGE2, but could not be unequivocally demonstrated. Furthermore, it was suggested by Filosa and colleagues that suppression of [Ca2+]i oscillations and accompanying vasomotion in microarterioles, possibly due to smooth muscle hyperpolarization, was involved in coupling local perfusion to increased neuronal activity (Filosa et al., 2004). Despite apparent discrepancies between findings of microvascular contraction and dilation. mediated by. changes in astrocytic Ca2+, likely because of different experimental paradigms and the use or not of preconstricted vessels in the slices, these studies emphasize the importance of further assessing this newly identified intermediary role of astrocytes in transducing neuronal signals into vasomotor responses (Simard et al., 2003) and whether or not the endothelium is required for their vasomotor effects (Murphy et al., 1994). Furthermore, as can be appreciated, several. - 15 -.

(17) PhD thesis Dr. Vetri Francesco recent studies have used brain slices to investigate the role of astrocytes or neurons (see “neurovascular coupling”) in the regulation of microvascular tone. Although limited by the fact that brain slices are maintained in artificial conditions in which vessels are not pressurized and do not have intraluminal flow, it is conceivable that such preparations, in which neuronal-glial-vascular interactions are preserved and can be assessed in a controlled manner, offer an additional means to isolated microvessels and whole animal experiments for investigating the microcirculation. However, data deriving from such techniques must be interpreted with care.. Local interneurons A role for interneurons in the regulation of cerebrovascular tone has been proposed in the cerebral and cerebellar cortices, based on both anatomical and functional studies. Owing to the possibility of simultaneously visualizing neurons (or astrocytes) and microvessels in isolated brain slices, and assessing changes in vascular tone upon single-cell depolarization or activation, a resurgence of interest in the regulation of microvascular tone has occurred. In this respect, it was recently shown that the evoked firing of specific subsets of cortical GABA interneurons can induce either dilation or constriction of local microvessels, some of these being contacted by the stimulated interneurons (Cauli et al., 2004). Although the nature of “vasomotor” gabaergic interneurons could not be identified in all cases, those that colocalized VIP or NOS elicited dilation. while those colocalizing somatostatin (SOM) induced. contraction. The interneuron-driven vasomotor responses could be mimicked by bath application of the vasodilatory (VIP) or vasocontractile (SOM) agents colocalized with GABA within the identified interneurons and for which receptors, able to either dilate or constrict microvessels, are expressed by endothelial or smooth muscle cells of cortical microvessels (Cauli et al., 2004). Astrocytes also expressed different receptor subtypes for. - 16 -.

(18) PhD thesis Dr. Vetri Francesco either VIP or SOM, but their contribution, if any, in the dilation and constriction elicited by stimulation of these distinct subpopulations of GABA interneurons will require further investigation. Additionally, the subpopulations of GABA interneurons that elicited changes in local microvessel diameter represented distinct targets for subcortical basal forebrain ACh and brain stem 5-HT afferents. Although the serotonergic input to these cortical interneurons was comparable, the ACh afferents privileged the NOS/NPY and SOM cells. These data suggest that specific subsets of cortical interneurons could act as relays to adapt perfusion to local changes in activity following afferent signals from subcortical pathways, such as basal forebrain ACh and brain stem 5-HT pathways. This would be compatible with the earlier observation that the integrity of local interneurons is necessary for the cortical perfusion increase evoked by stimulation of vasoactive pathways remote from the cerebral cortex (Iadecola et al., 1997).. Neurovascular coupling. The increased energy demand of active neurons is also met by local increases in blood flow in the area of elevated neuronal activity. This phenomenon, which was first described by A. Mosso in the late 1800s (Mosso, 1880) and later confirmed by Roy and Sherrington (Roy and Sherrington, 1890), is a fundamental event in brain function. Local increases in blood flow result from the rapid dilation of arterioles and capillaries of a restricted area in response to an episode of high neuronal activity. As a consequence, blood flow increases in that region within a few seconds, thereby ensuring that most active neurons receive an adequate supply of oxygen and metabolic substrates for energy consumption. This phenomenon has been called “neurovascular coupling” or “functional hyperemia”.. - 17 -.

(19) PhD thesis Dr. Vetri Francesco Local accumulation of metabolic products has been initially proposed to directly control blood flow. Although under particular circumstances, such as brain hypoxia or ischemia, this process may indeed affect blood vessels, the time course of the neurovascular coupling argues against this hypothesis. Results obtained over the last few years provide conclusive support for the view that blood flow is directly coupled to neuronal activity rather than to local energy needs (Sandor, 1999; Attwell and Iadecola, 2002). The present knowledge on the multiple signaling pathways that during activation lead to the production of vasoactive factors suggests that the molecular mechanism at the basis of functional hyperemia is highly complex and may not necessarily be the same in all brain regions. Although various aspects remain to be elucidated, most recent studies highlight a central role of neuron-to-astrocyte signaling in the local control of microcirculation (Li et al., 2003). The regulation of CBF during brain activity involves the coordinated interaction of neurons, glia, and vascular cells. Thus, whereas neurons and glia generate the signals initiating the vasodilation, endothelial cells, pericytes, and smooth muscle cells act in concert to transduce these signals into carefully orchestrated vascular changes that lead to CBF increases focused to the activated area and temporally linked to the period of activation. Cerebral blood vessels have many unique structural and functional characteristics that differentiate them from vessels in other organs. Perhaps the most distinctive feature of cerebral blood vessels is their close interaction with neurons and glia. A growing body of evidence indicates that neurons, glia (astrocytes, microglia, oligodendrocytes), and vascular cells (endothelium, smooth muscle cells or pericytes) are closely related developmentally, structurally, and functionally. The term “neurovascular unit” was introduced to highlight the intimate functional relationships between these cells and their coordinated pattern of reaction to injury (Iadecola et al., 1997). The increase in CBF produced by brain activity, or functional hyperemia, is an example of the close interaction between neurons, glia, and vascular cells. In. - 18 -.

(20) PhD thesis Dr. Vetri Francesco the next paragraphs, the anatomical and functional bases of neurovascular coupling in the normal brain will be described.. Mechanisms of neurovascular coupling: searching for mediators The mechanisms underlying neurovascular coupling have been the subject of enquiry for more than a century (Iadecola, 2004), and numerous vasoactive factors have been implicated in neurovascular coupling. These include ions, NO, metabolic by-products, adenosine, prostaglandins, epoxyeicosatrienoic acids, vasoactive neurotransmitters and vasoactive factors released in response to neurotransmitters.. Vasoactive ions K+ e H+ are generated by the extracellular ionic currents induced by action potentials and synaptic transmission. Elevation of the concentration of external potassium ions to levels sufficient to depolarize Smooth muscle cells (>20 mM) induces vasoconstriction of cerebral arteries and arterioles (Horiuchi et al., 2002). Paradoxically, a modest elevation of K+ (<20 mM) is one of the most potent vasodilatory signals in the cerebrovasculature. Elevations in extracellular K+ up to 20 mM cause dilation of arterioles both in vitro and in vivo (Nguyen et al., 2000). This effect is mediated by the opening of K+ channels, mainly of the inward rectifier type (Kir2.1) on the membrane of arterial smooth muscle cells (Knot et al., 1996), leading to their hyperpolarization and subsequent vasodilation up to 60%. It has been recently shown on cortical slices that neuronal activation induced a rapid ( < 2 s latency) vasodilation that was greatly reduced by Kir channel blockade and completely abrogated by concurrent cyclooxygenase inhibition (Filosa et al., 2006). On the other hand, astrocytic endfeet exhibited large-conductance Ca2+-sensitive K+ channel (BK) currents that could be activated by neuronal stimulation. Blocking BK channels or ablating the gene encoding these channels. - 19 -.

(21) PhD thesis Dr. Vetri Francesco prevented neuronally induced vasodilation and suppression of arteriolar smooth muscle cell Ca2+, without affecting the astrocytic Ca2+ elevation (see below). These results support the concept of intercellular K+ channel–to–K+ channel signaling, through which neuronal activity in the form of an astrocytic Ca2+ signal is decoded by astrocytic BK channels, which locally release K+ into the perivascular space to activate smooth muscle cell Kir channels and cause vasodilation. Furthermore, KATP channels have been implicated in the mechanisms of neurovascular coupling (Nguyen et al., 2000), because they could mediate the vasodilation produced by agents that increase cAMP, such as adenosine or prostacyclin (Faraci and Sobey, 1998). The vasodilatory effect of increased concentrations of H+ is also mediated, at least in part, by the opening of K+ channels (Faraci and Sobey, 1998).. Nitric Oxide The neuronal isoform of NOS is present in a small population of interneurons, some of which are in close proximity to intraparenchymal blood vessels. Activation of NMDA receptors on these neurons leads to Ca2+ entry and stimulation of NOS anchored in the vicinity of NMDA receptors by postsynaptic density proteins (Christopherson et al., 1999). Because NO can diffuse for considerable distances across neighbouring cells, it can produce vasorelaxation of arteriolar smooth muscle. Several lines of evidence support a role for neuronally derived NO in neurovascular coupling. A transient burst of NO has been measured within 1 s of neuronal activation and preceding the increase in CBF (Buerk et al., 2003). The neuronal NOS-specific inhibitor, 7-nitroindazole (7-NI) reduces the cortical blood flow response to whisker stimulation by 50–60%, whereas the nonisoform-specific inhibitor N-nitro-L-arginine (LNNA) attenuates functional hyperemia in both wild-type and endothelial NOS null mice (Ayata et al., 1996) but has no effect in neuronal NOS null mice (Ayata et al., 1996; Ma et al.,. - 20 -.

(22) PhD thesis Dr. Vetri Francesco 1996). However, neuronally derived NO is not an essential mediator of the flow response. The attenuating effect of NOS inhibition on the cortical flow response to whisker stimulation is smaller in unanesthetized rats than it is in anesthetized animals (Gotoh et al., 2001), and administration of a NOS inhibitor to humans failed to significantly reduce the evoked CBF response in frontal cortex to a learning task in a PET study (White et al., 1999). Furthermore, neuronal NOS null mice have a normal cortical blood flow response to whisker stimulation, suggesting compensation by other mediators (Ma et al., 1996). Moreover, inhibition of NOS results in an increase in arterial pressure and a decrease in baseline CBF. When baseline CBF is restored after NOS inhibition by the use of either a NO donor to clamp the level of NO or a cGMP analog, the CBF response to whisker stimulation is restored (Lindauer et al., 1999). These results suggest that the presence of an adequate concentration of NO and cyclic GMP is required for an intact response, but that dynamic fluctuations in NO are not required for mediating the dynamic CBF response. Therefore, NO appears to play more of a role as a modulator, rather than a mediator, of the cortical flow response to activation. For instance, in brain slice preparations (Mulligan and MacVicar, 2004), NO might act to inhibit the formation of 20-HETE, a potent vasoconstrictor, in vascular smooth muscle from PLA2mobilized arachidonic acid at astrocyte end-feet and thereby permit vasodilation. Regional differences might also be important, in that NO appears to play a more prominent role in cerebellum (Iadecola et al., 1997). However, unlike the cerebral cortex, in cerebellum the attenuation of the CBF response cannot be reversed by NO donors and is observed also in nNOS null mice. These findings suggest that, in cerebellum, NO plays an obligatory role in the mechanisms of the vasodilation (Iadecola et al., 1997).. - 21 -.

(23) PhD thesis Dr. Vetri Francesco Vasoactive factors related to energy metabolism The brain has little energy reserve and requires a continuous supply of glucose and O2 through CBF. A sudden increase in the demand for energy during synaptic activity could result in a relative lack of O2 and glucose, which may be a factor in triggering the haemodynamic response (Attwell and Iadecola, 2002). However, the reduction in brain O2 concentration at the site of activation is small and transient and cannot account for sustained increases in flow (Ances, 2004). Furthermore, the CBF response to activation is not altered by hypoglycaemia or hypoxia, suggesting that lack of glucose or O2 is not the primary factor triggering vasodilation (Attwell and Iadecola, 2002). On the other hand, adenosine, a potent vasodilator produced during ATP catabolism (see below), is involved in neurovascular coupling in cerebellum (Li and Iadecola, 1994) and cerebral cortex (Ko et al., 1990). Lactate produced during brain activation could also be an important mediator of functional hyperemia by increasing H+ concentration and producing vasodilation (Attwell and Iadecola, 2002). Pellerin and colleagues hypothesized that astrocytes metabolize glucose through glycolysis, leading to lactate production (Pellerin et al., 1998). Lactate, in turn, is taken up by neurons and used as fuel for ATP synthesis. Recent data in hippocampal slices support this theory. Using twophoton confocal microscopy of NADH fluorescence, Kasischke et al. have investigated the spatial and temporal characteristics of energy metabolism in neurons and astrocytes during activation (Kasischke et al., 2004). They provided evidence of an early NADH decrease in neurons, reflecting oxidative metabolism, followed by a NADH increase in astrocytes, reflecting glycolytic metabolism. These observations are consistent with the hypothesis that neuronal oxidative metabolism precedes glial glycolysis and fit well with the reduction in tissue O2 (“initial dip”) observed at the onset of activation with optical imaging, functional MRI, and O2 electrodes (Ances, 2004). However, the data of Kasischke et al. do not shed light on the role of lactate in the vasodilation produced by neural activation. Rather, the lactate rise. - 22 -.

(24) PhD thesis Dr. Vetri Francesco is small and transient, and it cannot account in full for the increase in flow produced by neural activity (Attwell and Iadecola, 2002).. Adenosine A role for adenosine in mediating functional hyperemia is supported by evidence that the nonselective adenosine-receptor antagonist theophylline attenuates the increase in CBF during whisker stimulation (Villringer et al., 1994) and the vasodilation of extraparenchymal pial arterioles during sciatic nerve stimulation (Ko et al., 1990). In addition, adenosine deaminase attenuates the CBF response to whisker stimulation (Villringer et al., 1994). Evidence against a major role for adenosine is based on data that show a lack of effect of the non-selective adenosine antagonist caffeine on the cortical flow response to whisker stimulation in unanesthetized rats (Gotoh et al., 2001). However, relatively high concentrations of caffeine are required to inhibit the pial arteriolar dilation to adenosine or to sciatic nerve stimulation (Meno et al., 2005). High concentrations of caffeine probably exert nonspecific effects, such as those on ryanodine receptors, which could offset effects on adenosine receptors. Dilation of pial arterioles to exogenous adenosine is mediated primarily by high-affinity A2A receptors and, to a lesser extent, by low-affinity A2B receptors (Shin et al., 2000). Pial arteriolar dilation to topical glutamate and to sciatic nerve stimulation is attenuated by an A2A antagonist (Iliff et al., 2003). However, preliminary work indicates that the CBF response to whisker stimulation is attenuated by an A2B antagonist, rather than by an A2A antagonist, suggesting that the intraparenchymal vascular response is mediated by different adenosine receptors than is the extraparenchymal vascular response (Shi et al., 2004). In addition to direct actions of adenosine on smooth muscle, adenosine can act on astrocytes. Adenosine is derived from the breakdown of ATP in cells and by ecto-ATPase and ecto-5’nucleotidase (Joseph et al., 2003). ATP is released in synapses as a coneurotransmitter and from astrocytes through connexin hemichannels where ATP can act on adjacent astrocyte - 23 -.

(25) PhD thesis Dr. Vetri Francesco P2Y receptors to promote the spread of Ca2+ waves (Stout et al., 2002). In human astrocytoma cells, ecto-nucleotidase activity colocalizes with sites of ATP release (Joseph et al., 2003). Thus extracellular adenosine concentration is expected to be increased near sites of ATP release. In astrocytes, stimulation of A2B receptors increases cyclic AMP, glycogen synthesis, and intracellular Ca2+. Adenosine potentiates the increase of Ca2+ waves evoked by ATP through an action largely attributed to A2B receptors (Alloisio et al., 2004). In the retina, physiological activation with light induces an increase in Ca2+ in Muller glial cells, and the spread of Ca2+ waves is augmented by adenosine primarily through effects on A2B receptors (Newman, 2005). Therefore, A2B receptors are likely to play a role in astrocyte communication with the vasculature. Moreover, purinergic receptor expression is dense along perivascular end-feet (Simard et al., 2003). If ecto-ATPase and ecto-nucleotidase activity is also present at these sites along the end-feet, localized perivascular adenosine concentration could become elevated to concentrations sufficient to activate low-affinity A2B receptors during stimulation (Koehler et al., 2006).. Prostaglandins A role for prostaglandins in functional hyperemia is derived from data showing that the CBF response to whisker stimulation is reduced 40–50% by a cyclooxygenase-2 (COX-2) inhibitor or by COX-2 gene deletion in mice (Niwa et al., 1993). In contrast, a COX-1 inhibitor or COX-1 gene deletion, which attenuated acetylcholine vasodilation, did not impair the CBF response to whisker stimulation (Niwa et al., 1993). Although COX-2 is a highly inducible isoform of COX, it is constitutively expressed in somatosensory cortical neurons, including dendritic and terminal processes adjacent to perivascular astrocyte processes and, in some cases, NOS-positive interneurons (Wang et al., 2006). In contrast, expression of COX-2 in astrocytes is low. Thus a COX-2 metabolite, such as prostaglandin E2 (PGE2) released from. - 24 -.

(26) PhD thesis Dr. Vetri Francesco neurons, is postulated to act either directly on vascular smooth muscle, indirectly via perivascular astrocytes or by an interaction with NOS-containing neurons. Prostaglandins are also postulated to be released from astrocytes during activation and to mediate vasodilation. Acetylsalicylic acid inhibits vascular dilation during electrical activation in cortical slices (Zonta et al., 2003b). Cultured astrocytes release PGE2 when activated by a mGluR agonist (Zonta et al., 2003a). Moreover, administration of mGluR antagonists in vivo decreases the CBF response to whisker stimulation in the same manner that they act in slices to reduce astrocyte Ca2+ responses (Zonta et al., 2003b). However, the role of an astrocyte-based PGE2 mechanism in vivo is unclear because of the low expression of COX-2 in astrocytes and the lack of dependence of the blood flow response on COX-1 (Koehler et al., 2006).. Epoxyeicosatrienoic acids Cultured astrocytes from rat express cytochrome P (CYP) 2C11, which possesses epoxygenase activity (Alkayed et al., 1996). Conversion of arachidonic acid to epoxyeicosatrienoic acids (EETs) has been described in cultured astrocytes and in brain parenchyma (Amruthesh et al., 1993). Expression of CYP 2C11 in rat brain colocalizes with glial fibrillary acidic protein-positive astrocytes, including perivascular astrocytes. EETs differ from prostaglandins in that they are stored in the phospholipids membrane, including astrocyte membranes, and thus could be mobilized without de novo synthesis (Shivachar et al., 1995). Within astrocytes, release of Ca2+ from internal stores induced by thapsigargin causes arachidonic acid to be mobilized followed by formation of EETs and Ca2+ influx. Moreover, EETs derived from astrocytes may act intercellularly. Indeed, addition of glutamate to cultured astrocytes causes release of EETs into the media (Alkayed et al., 1997). The release of EETs evoked by a mGluR agonist is reduced by an inhibitor of KCa channels, suggesting a. - 25 -.

(27) PhD thesis Dr. Vetri Francesco link between hyperpolarization induced by KCa channel opening, sustained Ca2+ influx and release of EETs by astrocytes. In vascular smooth muscle, EETs cause hyperpolarization by opening of KCa channels and dilate cerebral arteries (Gebremedhin et al., 1992). Thus, it has been proposed that EETs might serve as an astrocyte-derived vasodilator (Harder et al., 1998). In support of this hypothesis, increases in CBF elicited by topical glutamate application to the brain surface or by dialysis of NMDA into striatal tissue are markedly reduced by inhibitors of epoxygenase activity (Alkayed et al., 1997). Furthermore, epoxygenase inhibitors applied to the cortical surface reduced the CBF response to whisker stimulation and to electrical stimulation of the forepaw (Peng et al., 2004). Therefore, an astrocyte-based epoxygenase pathway appears to be critical in the coupling of CBF to neuronal activation in cortex. Consistent with this hypothesis, antagonists of KCa channels also reduce the CBF response to whisker stimulation (Gerrits et al., 2002). Further work in vivo is needed using molecular approaches to specifically alter EET signaling to fully understand their role in neurovascular coupling.. Interactions of mediators The epoxygenase pathway has complex interactions with other mediators of functional hyperemia. In the presence of indomethacin, a COX antagonist, epoxygenase inhibition is still capable of reducing the CBF response to whisker stimulation, thereby indicating that the actions of the epoxygenase mechanism do not require COX activity (Peng et al., 2002). On the other hand, the NOS pathway does significantly interact with the epoxygenase pathway. The striatal blood flow response to NMDA was completely blocked by individually inhibiting either NOS or epoxygenase activity (Bhardwaj et al., 2000). Also, no additive effect of combined inhibition was observed with NOS and epoxygenase inhibitors on the cortical CBF response to electrical forelimb stimulation (Peng et al., 2004). The CBF response was. - 26 -.

(28) PhD thesis Dr. Vetri Francesco attenuated about 60% with either inhibitor alone or with combined inhibition. Likewise, an adenosine A2B antagonist combined with an epoxygenase inhibitor, an EET antagonist, or a mGluR antagonist produced no substantial additive inhibition beyond the 50% inhibition seen with each agent alone during whisker stimulation (Shi et al., 2004). This observation is consistent with an action of adenosine on astrocyte A2B receptors amplifying Ca2+ stimulation of EETs release. Moreover, NO has been reported to promote capacitative Ca2+ influx in astrocytes (Li et al., 2003). Combining a NOS inhibitor with the adenosine antagonist theophylline produced a small additive effect on the CBF response to whisker stimulation but did not eliminate the response (Villringer et al., 1994). Therefore, the NOS, adenosine, and EET pathways do not appear to operate by parallel, independent signaling mechanisms, consistent with an interplay at the astrocyte or smooth muscle level. Indeed, none of the combinations of drug inhibitors and antagonists has been able to completely eliminate the increase in CBF during activation. These findings suggest that other regulatory pathways are recruited when these feed-forward signaling pathways are interrupted. In this regard, it is important to recall that CO2 production is proportional to glucose consumption and that the percent increase in CBF matches the percent increase in glucose consumption. Ordinarily, the match between increased glycolysis and increased CBF results in no measurable change in tissue pH (Ueki et al., 1988). When the feed-forward signaling pathways are interrupted and the CBF response is attenuated, some increase in tissue PCO2 would be anticipated. A consequent decrease in extracellular pH might represent a contingency feedback mechanism for ensuring some degree of vasodilation during activation when other pathways are inoperative.. Central pathways, interneurons and vasoactive neurotransmitters. - 27 -.

(29) PhD thesis Dr. Vetri Francesco It has long been proposed that vasoactive neurotransmitters released during neural activity contribute to the vasodilation. These neurotransmitters could be released from neurovascular projections that terminate close to blood vessels originating from local interneurons or from distant nuclei and modulate CBF. However, the contribution of the vascular innervation to functional hyperemia has not been clearly established. The role of interneurons has recently been addressed by Cauli et al., who, using forebrain slices, have been able to show that activation of interneurons with neurovascular contacts can produce vasodilation or vasoconstriction (Cauli et al., 2004). A similar role for interneurons has been recently confirmed also in cerebellum, where the flow response evoked by somatosensory activation is dissociate from spiking of Purkinje cells and depends almost exclusively on the NO released from stellate interneurons in the cerebellar molecular layer (Iadecola et al., 1997). This study, performed on cerebellar slices, showed that stellate cells firing was sufficient to elicit both NO release and dilation of intraparenchymal and upstream pial arterioles. Taken together this data implicate that specific classes of interneurons, after depolarization, can modify the tone of vessels close to them or, as seen in cerebellum, of pial vessels. Because interneurons act as integrators of incoming afferent signals, these findings further suggest that they could also be involved in neurovascular coupling by precisely adapting perfusion to local changes in neuronal activity. However, they also raise an important issue that still remains to be fully understood: namely, how changes in the tone of brain microvessels that result in increase or decrease in local perfusion deep in the brain parenchyma, mediated directly by neuronal and/or indirectly by astroglial signaling molecules, are transmitted to upstream resistance vessels to maintain blood volume and intracranial pressure constant. In this respect, flowmediated and propagated dilation have been reported in brain vessels (Iadecola et al., 1997), but shear stress-induced contraction of cerebral resistance arteries and arterioles also occurs and this independent from the endothelium (Garcia-Roldan and Bevan, 1990). The exact. - 28 -.

(30) PhD thesis Dr. Vetri Francesco mechanisms are still a matter of debate, but endothelial factors, cytoskeletal matrix components, and gap junctions between vascular and/or astroglial cells appear to be involved (cfr. "cellular mechanisms underlying vasomotion”).. Other vasoactive factors released by neural activity Vasoactive factors can also be generated by the intracellular signaling induced by activation of neurotransmitter receptors. For example, activation of glutamate receptors produces vasodilation and increases blood flow. In neocortex and hippocampus, exogenous glutamate or NMDA dilates pial arterioles and/or cerebral microvessels (Faraci and Breese, 1993; Lovick et al., 1999). Functional hyperemia in cerebral and cerebellar cortex is inhibited by NMDA receptor blockers. Because glutamate is not vasoactive in isolated cerebral arteries, the vasodilation is mediated by vasoactive factors whose synthesis is triggered by the changes in intracellular Ca2+ associated with glutamate receptor activation. The increase in Ca2+ activates Ca2+-dependent enzymes, such as nNOS, that produce potent vasodilators. Thus the increase in CBF in the somatosensory cortex induced by sensory stimulation is associated with NO release and is attenuated by nNOS inhibitors (Faraci and Breese, 1993).. Role of astrocytes in neurovascular coupling. Astrocytes are strategically positioned to contribute to the CBF increase related to neural activity. They receive inputs from thousands of synapses and at the same time can make contact with the local vasculature throughout their endfeet. The ability of astrocytes to remove from the extracellular space around active synapses potassium ions increasingly concentrated there following high neuronal activity and to redistribute them, through the syncytium, to distal regions, was originally considered a plausible mechanism to couple neuronal activity. - 29 -.

(31) PhD thesis Dr. Vetri Francesco with dilation of vessels (Trachtenberg and Pollen, 1970). This hypothesis was substantiated in the retina where a high potassium conductance was found in astrocyte endfeet and Muller cell processes in contact with blood vessels (Paulson and Newman, 1987). The demonstration that astrocytes produce a plethora of vasoactive substances, such as nitric oxide (NO), cyclooxygenase and epoxygenase activity-derived products, and ATP hints at the possibility that the control of microcirculation by astrocytes could not be based simply on the “spatial buffering” of K+ hypothesis, but rather involves a more complex mechanism and a number of different molecules. For the first time Zonta et al. demonstrated, with electrical stimulation of cortical slices, increases in intracellular Ca2+ in astrocyte cell bodies and in astrocyte end-feet on blood vessels, activation of COX and an increase in diameter of small arterioles (Zonta et al., 2003b). The importance of this study resides in the demonstration that transient calcium increase in astrocytes are directly related to modification of cerebrovascular tone (see next paragraph). However, the brain slice preparation has several limitations. The blood vessels have no blood flow or associated shear stress and have no pressure-induced myogenic tone, so that vessel diameter changes, in these conditions, are hard to assess (Krizanac-Bengez et al., 2004).. Neuronal activity-dependent calcium elevations in astrocyte endfeet Significant evidence in support of a distinct role of astrocytes in neurovascular coupling was then obtained in a series of experiments performed mainly in brain slice preparations. In hippocampal and cortical slices it was first observed that glutamate released at active synapses triggered Ca2+ oscillations in astrocytes that increased in frequency according to increasing levels of neuronal activity (Pasti et al., 1997). While this observation demonstrates that astrocytes are sophisticated sensors of neuronal activity, it also represents a clue to the. - 30 -.

(32) PhD thesis Dr. Vetri Francesco possibility that astrocytes transfer to blood vessels information on the level of neuronal activity. Indeed, neuronal activity-dependent Ca2+ elevations in astrocytes were observed to propagate to perivascular endfeet (Zonta et al., 2003b). Such a signal provides a mechanistic basis for the graded response of the blood flow to different levels of neuronal activity, thereby strengthening the idea of a distinct astrocytic role in neurovascular coupling. Importantly, high-frequency stimulation of neuronal afferents was found to trigger both Ca2+ elevations in astrocyte endfeet and dilation of cerebral arterioles. Furthermore, Ca2+ elevations triggered in astrocytes by either t-ACPD, a mGluR agonist, or direct mechanical stimulation of individual astrocytes by a patch pipette, also evoked dilation of cortical arterioles, while inhibition by mGluR antagonists of Ca22+ oscillations evoked in astrocytes by synaptic glutamate, or the incubation with COX inhibitors that block prostaglandin synthesis, reduced neuronal activitydependent dilation of cerebral arterioles. Vasodilation appears to be mediated, at least in part, by prostaglandin E2, since astrocytes in culture were observed to release this powerful dilating agent in a pulsatile manner according to the pattern of t-ACPD-mediated Ca2+ oscillations (Zonta et al., 2003a). Results from in vivo experiments that used the same mGluR antagonists that in brain slices inhibited astrocyte mediated vasodilation corroborated the role of astrocytes in functional hyperemia (Zonta et al., 2003b). By measuring the blood flow in the somatosensory cortex by laser Doppler flowmetry, the hyperemic response evoked by forepaw stimulation was found to be markedly reduced after the systemic application of mGluR antagonists. The action of the mGluR antagonists was unrelated to unspecific effects on the intensity of neuronal stimulation since the evoked somatosensory potential was unchanged. This is in agreement with the unchanged amplitude of the Ca2+ increase triggered by neuronal afferent stimulation in neurons from brain slices in the presence of the mGluR antagonists. According to these results, a model is proposed in which astrocytes can encode different levels of neuronal. - 31 -.

(33) PhD thesis Dr. Vetri Francesco activity into defined Ca2+ oscillation frequencies that, at the level of perivascular endfeet, mediate the release of dilating agents, such as EETs and PGE2 as well as constrictive agents such as 20-HETE. Neuronal activity-dependent Ca2+ oscillations may ultimately represent the signaling system that allows blood flow to vary in a manner proportional to the intensity of neuronal activity.. Propagating calcium waves between astrocytes may contribute to control microcirculation During functional hyperemia, the dilation of arterioles in the area of activation will not increase blood flow in that region effectively unless upstream vessels also dilate. How vasodilator and vasoconstrictor responses are conveyed from the initial site of activation to distant locations is unclear (see “local vs. remote vasodilation”). A Ca2+ wave propagating between perivascular astrocytes may also be involved. The Ca2+ response in an astrocyte in contact with a blood vessel, initially evoked either by neuronal activity (Zonta et al., 2003b) or by direct electrical stimulation (Simard et al., 2003), has been indeed observed to spread to other perivascular astrocytes. Furthermore, connexin43 and purinergic receptors, i.e., the basic elements which mediated the propagation of the Ca2+ wave in cultured astrocytes, are highly expressed at astrocyte endfeet (Simard et al., 2003), and filling single astrocytes that are in the proximity of a blood vessel with Lucifer yellow results in the diffusion of the dye to other astrocyte endfeet. Through the release of vasoactive factors, activation of perivascular astrocytes by the Ca2+ wave may affect the tone of upstream and/or downstream blood vessels, thereby regulating the overall conductance of the vascular network in a defined region.. Activation of astrocytes can also trigger arteriole constriction. - 32 -.

(34) PhD thesis Dr. Vetri Francesco In hippocampal slices, Ca2+ elevations in astrocyte endfeet triggered by either photolysis of a Ca2+ caged compound or t-ACPD have been observed to evoke also arteriole constriction (Staub et al., 1995). Studies in cultured cells show that astrocytes can indeed produce, in addition to various dilating agents, constrictive agents such as the COX products PGF2α and thromboxane A2, endothelins, and 20-HETE. This latter compound, depolarizes smooth muscle cells by inhibiting the opening of K+ channels (Lange et al., 1997), and also enhances Ca2+ influx through voltage-dependent Ca2+ channels (Gebremedhin et al., 1992). The formation of 20-HETE from arachidonic acid in smooth muscle cells is proposed to mediate the constrictive action of astrocytes in the hippocampus (Staub et al., 1995). This action can also account for the constriction of cerebral blood vessels associated with spreading depression and ischemia (Dreier et al., 2001), since Ca2+ elevations and Ca2+ waves are known to occur in the astrocytes during these pathological brain conditions. The release of 20HETE from astrocytes may, however, have a role also under normal physiological conditions like the maintenance of myogenic tone in cerebral blood vessels (Lange et al., 1997). The constrictive action of 20-HETE may also control, together with that of dilating agents, the extent of neuronal activity-dependent increases in blood flow. The results reported by Mulligan and MacVicar’s study (Mulligan and MacVicar, 2004) are in conflict with those reported by Zonta et al. (Zonta et al., 2003b) in which Ca2+ elevations in astrocyte endfeet were observed to trigger dilation of cerebral arterioles. How can these conflicting results be reconciled in a unifying hypothesis? In the latter study, most, although not all, experiments were performed in cortical slices incubated with NG-nitro-L-arginine methyl ester (LNAME), a NO synthase inhibitor that blocks the tonic action of NO on arterioles and thus results in a long-lasting constriction of arterioles. In contrast, this procedure was not applied in most of the experiments described in Mulligan and MacVicar’s study in hippocampal. - 33 -.