Modulatory effects of noradrenergic and serotonergic signaling pathway on neurovascular coupling.

Dynamic changes in astrocyte Ca2+ are recognized as contributors to functional hyperemia, a critical response to increased neuronal activity mediated by a process known as neurovascular coupling (NVC). Although the critical role of glutamatergic signaling in this process has been extensively investigated, the impact of behavioral state, and the release of behavior-associated neurotransmitters, such as norepinephrine and serotonin, on astrocyte Ca2+ dynamics and functional hyperemia have received less attention. We used two-photon imaging of the barrel cortex in awake mice to examine the role of noradrenergic and serotonergic projections in NVC. We found that both neurotransmitters facilitated sensory stimulation-induced increases in astrocyte Ca2+. Interestingly, while ablation of serotonergic neurons reduced sensory stimulation-induced functional hyperemia, ablation of noradrenergic neurons caused both attenuation and potentiation of functional hyperemia. Our study demonstrates that norepinephrine and serotonin are involved in modulating sensory stimulation-induced astrocyte Ca2+ elevations and identifies their differential effects in regulating functional hyperemia.

Modulatory Effects of Noradrenergic and Serotonergic Signaling Pathway on Neurovascular Coupling.

Dynamic changes in astrocyte Ca2+ are recognized as contributors to functional hyperemia, a critical response to increased neuronal activity mediated by a process known as neurovascular coupling (NVC). Although the critical role of glutamatergic signaling in this process has been extensively investigated, the impact of behavioral state, and the release of behavior-associated neurotransmitters, such as norepinephrine and serotonin, on astrocyte Ca2+ dynamics and functional hyperemia have received less attention. We used two-photon imaging of the barrel cortex in awake mice to examine the role of noradrenergic and serotonergic projections in NVC. We found that both neurotransmitters facilitated sensory-induced increases in astrocyte Ca2+. Interestingly, while ablation of serotonergic neurons reduced sensory-induced functional hyperemia, ablation of noradrenergic neurons caused both attenuation and potentiation of functional hyperemia. Our study demonstrates that norepinephrine and serotonin are involved in modulating sensory-induced astrocyte Ca2+ elevations and identifies their differential effects in regulating functional hyperemia.

Astrocytes amplify neurovascular coupling to sustained activation of neocortex in awake mice.

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca2+ signaling in vivo by expressing a plasma membrane Ca2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca2+ is to amplify functional hyperemia when neuronal activation is prolonged.

Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Astrocytes regulate ultra-slow arteriole oscillations via stretch-mediated TRPV4-COX-1 feedback.

Very-low-frequency oscillations in microvascular diameter cause fluctuations in oxygen delivery that are important for fueling the brain and for functional imaging. However, little is known about how the brain regulates ongoing oscillations in cerebral blood flow. In mouse and rat cortical brain slice arterioles, we find that selectively enhancing tone is sufficient to recruit a TRPV4-mediated Ca2+ elevation in adjacent astrocyte endfeet. This endfoot Ca2+ signal triggers COX-1-mediated "feedback vasodilators" that limit the extent of evoked vasoconstriction, as well as constrain fictive vasomotion in slices. Astrocyte-Ptgs1 knockdown in vivo increases the power of arteriole oscillations across a broad range of very low frequencies (0.01-0.3 Hz), including ultra-slow vasomotion (∼0.1 Hz). Conversely, clamping astrocyte Ca2+in vivo reduces the power of vasomotion. These data demonstrate bidirectional communication between arterioles and astrocyte endfeet to regulate oscillatory microvasculature activity.

Whole brain irradiation in mice causes long-term impairment in astrocytic calcium signaling but preserves astrocyte-astrocyte coupling.

Whole brain irradiation (WBI) therapy is an important treatment for brain metastases and potential microscopic malignancies. WBI promotes progressive cognitive dysfunction in over half of surviving patients, yet, the underlying mechanisms remain obscure. Astrocytes play critical roles in the regulation of neuronal activity, brain metabolism, and cerebral blood flow, and while neurons are considered radioresistant, astrocytes are sensitive to γ-irradiation. Hallmarks of astrocyte function are the ability to generate stimulus-induced intercellular Ca2+ signals and to move metabolic substrates through the connected astrocyte network. We tested the hypothesis that WBI-induced cognitive impairment associates with persistent impairment of astrocytic Ca2+ signaling and/or gap junctional coupling. Mice were subjected to a clinically relevant protocol of fractionated WBI, and 12 to 15 months after irradiation, we confirmed persistent cognitive impairment compared to controls. To test the integrity of astrocyte-to-astrocyte gap junctional coupling postWBI, astrocytes were loaded with Alexa-488-hydrazide by patch-based dye infusion, and the increase of fluorescence signal in neighboring astrocyte cell bodies was assessed with 2-photon microscopy in acute slices of the sensory-motor cortex. We found that WBI did not affect astrocyte-to-astrocyte gap junctional coupling. Astrocytic Ca2+ responses induced by bath administration of phenylephrine (detected with Rhod-2/AM) were also unaltered by WBI. However, an electrical stimulation protocol used in long-term potentiation (theta burst), revealed attenuated astrocyte Ca2+ responses in the astrocyte arbor and soma in WBI. Our data show that WBI causes a long-lasting decrement in synaptic-evoked astrocyte Ca2+ signals 12-15 months postirradiation, which may be an important contributor to cognitive decline seen after WBI.

Obesity-induced cognitive impairment in older adults: a microvascular perspective.

Over two-thirds of individuals aged 65 and older are obese or overweight in the United States. Epidemiological data show an association between the degree of adiposity and cognitive dysfunction in the elderly. In this review, the pathophysiological roles of microvascular mechanisms, including impaired endothelial function and neurovascular coupling responses, microvascular rarefaction, and blood-brain barrier disruption in the genesis of cognitive impairment in geriatric obesity are considered. The potential contribution of adipose-derived factors and fundamental cellular and molecular mechanisms of senescence to exacerbated obesity-induced cerebromicrovascular impairment and cognitive decline in aging are discussed.

Limiting RyR2 Open Time Prevents Alzheimer's Disease-Related Neuronal Hyperactivity and Memory Loss but Not ß-Amyloid Accumulation.

Neuronal hyperactivity is an early primary dysfunction in Alzheimer's disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued ß-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-ß-amyloid-targeted anti-AD strategy.

Author Correction: Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.

Arteriole dilation to synaptic activation that is sub-threshold to astrocyte endfoot Ca2+ transients.

Ca(2+)-dependent pathways in neurons and astrocyte endfeet initiate changes in arteriole diameter to regulate local brain blood flow. Whether there exists a threshold of synaptic activity in which arteriole diameter is controlled independent of astrocyte endfeet Ca(2+) remains unclear. We used two-photon fluorescence microscopy to examine synaptically evoked synthetic or genetic Ca(2+) indicator signals around penetrating arterioles in acute slices of the rat neocortex. We discovered a threshold below which vasodilation occurred in the absence of endfeet Ca(2+) signals but with consistent neuronal Ca(2+) transients, suggesting endfoot Ca(2+) is not necessary for activity-dependent vasodilation under subtle degrees of brain activation.

Impact of aging on spreading depolarizations induced by focal brain ischemia in rats.

Spreading depolarization (SD) contributes to the ischemic damage of the penumbra. Although age is the largest predictor of stroke, no studies have examined age dependence of SD appearance. We characterized the electrophysiological and hemodynamic changes in young (6 weeks old, n = 7), middle-aged (9 months old, n = 6), and old (2 years old, n = 7) male Wistar rats during 30 minutes of middle cerebral artery occlusion (MCAO), utilizing multimodal imaging through a closed cranial window over the ischemic cortex: membrane potential changes (with a voltage-sensitive dye), cerebral blood volume (green light reflectance), and cerebral blood flow (CBF, laser-speckle imaging) were observed. The initial CBF drop was similar in all groups, with a significant further reduction during ischemia in old rats (p < 0.01). Age reduced the total number of SDs (p < 0.05) but increased the size of ischemic area displaying prolonged SD (p < 0.01). The growth of area undergoing prolonged SDs positively correlated with the growth of ischemic core area (p < 0.01) during MCAO. Prolonged SDs and associated hypoperfusion likely compromise cortical tissue exposed to even a short focal ischemia in aged rats.

Mitochondrial dynamics associated with oxygen-glucose deprivation in rat primary neuronal cultures.

Our objective was to investigate the mitochondrial dynamics following oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. We documented changes in morphology, protein expression, and DNA levels in mitochondria following OGD and examined the roles of mitochondrial fission [dynamin-related protein 1 (Drp1), fission protein-1 (Fis1)] and fusion [mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1)] proteins on mitochondrial biogenesis and morphogenesis. We tested the effects of two Drp1 blockers [15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and Mitochondrial Division Inhibitor (Mdivi-1)] on mitochondrial dynamics and cell survival. One hour of OGD had minimal effects on neuronal viability but mitochondria appeared condensed. Three hours of OGD caused a 60% decrease in neuronal viability accompanied by a transition from primarily normal/tubular and lesser number of rounded mitochondria during normoxia to either poorly labeled or small and large rounded mitochondria. The percentage of rounded mitochondria remained the same. The mitochondrial voltage-dependent anion channel, Complex V, and mitoDNA levels increased after OGD associated with a dramatic reduction in Drp1 expression, less reduction in Mfn2 expression, an increase in Mfn1 expression, with no changes in either OPA1 or Fis1. Although PGJ2 increased polymerization of Drp1, it did not reduce cell death or alter mitochondrial morphology following OGD and Mdivi-1 did not protect neurons against OGD. In summary, mitochondrial biogenesis and maintained fusion occurred in neurons along with mitochondrial fission following OGD; thus Mfn1 but not Drp1 may be a major regulator of these processes.

Depolarization of mitochondria in endothelial cells promotes cerebral artery vasodilation by activation of nitric oxide synthase.

OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Protection against recurrent stroke with resveratrol: endothelial protection.

Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier in vivo demonstrated that resveratrol treatment reduced blood-brain barrier disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro 'ischemia' resulting in improved viability against oxygen and glucose deprivation (39.6 ± 6.6% and 81.3 ± 9.5% in vehicle and resveratrol treated cells, respectively). An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.

Cerebral microcirculatory responses of insulin-resistant rats are preserved to physiological and pharmacological stimuli.

OBJECTIVE: Previously, we have shown that IR impairs the vascular reactivity of the major cerebral arteries of ZO rats prior to the occurrence of Type-II diabetes mellitus. However, the functional state of the microcirculation in the cerebral cortex is still being explored. METHODS: We tested the local CoBF responses of 11-13-week-old ZO (n = 31) and control ZL (n = 32) rats to several stimuli measured by LDF using a closed cranial window setup. RESULTS: The topical application of 1-100 µm bradykinin elicited the same degree of CoBF elevation in both ZL and ZO groups. There was no significant difference in the incidence, latency, and amplitude of the NMDA-induced CSD-related hyperemia between the ZO and ZL groups. Hypercapnic CoBF response to 5% carbon-dioxide ventilation did not significantly change in the ZO compared with the ZL. Topical bicuculline-induced cortical seizure was accompanied by the same increase of CoBF in both the ZO and ZL at all bicuculline doses. CONCLUSIONS: CoBF responses of the microcirculation are preserved in the early period of the metabolic syndrome, which creates an opportunity for intervention to prevent and restore the function of the major cerebral vascular beds.

Impaired vascular responses of insulin-resistant rats after mild subarachnoid hemorrhage.

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 µl) or saline was infused (10 µl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.

Effects of early aging and cerebral hypoperfusion on spreading depression in rats.

Cortical spreading depression (CSD) is a feature of stroke pathophysiology. As stroke incidence increases with age, we have examined the effects of early aging and chronic cerebral hypoperfusion on CSD in rats. Three groups were studied: Young, 2-month-old animals; Middle-aged-2VO, subjected to 8 months of bilateral carotid occlusion from 2-month-of-age; and Middle-aged-SHAM, sham-operated. At 2- and 10-month-of-age for the Young and Middle-aged groups, recurrent CSD were induced under halothane anesthesia, by sustained application of 1 M KCl to the cortex for 2 h. Propagating CSD (i.e., cortical EEG, direct current potential) and associated laser Doppler blood flow changes were recorded anteriorly. Susceptibility to CSD and event duration were both decreased by early aging (frequency: 21±0.5 and 6±0.5 CSD/h; duration: 139±7 and 63±8 s; in Young and Middle-aged-SHAM, respectively). There was also a tendency for CSD-associated hyperemia to be reduced in the Middle-aged-2VO group (8.9±2.1 vs. 32.8±12.6% × min in Young). These data suggest reduced sensitivity of the cortex to CSD elicitation with early aging, and a less responsive cerebrovascular system with chronic hypoperfusion.

Changes in pro-oxidant and antioxidant enzyme levels during cerebral hypoperfusion in rats.

Chronic cerebral hypoperfusion is a mild ischemic condition associated with a cognitive decline which is prevalent during senescence or Alzheimer's disease. Its experimental animal model compromises permanent occlusion of the common carotid arteries (2VO) in rats, which results in neuronal damage and microglia activation. Various mechanisms, including oxidative stress, have been proposed to be involved in this process. Accordingly, we set out to characterize the changes induced in the expressions of several pro-oxidant and antioxidant enzymes in cerebral hypoperfusion. Male Wistar rats were exposed to 2VO (n=30) or sham operation (n=33), while a third group served as absolute control (naive, n=16). Tissue samples from the hippocampus and frontal cortex were taken 1 and 3 days, 1 and 2 weeks and 3, 6 and 12 months following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS, respectively) and manganese superoxide dismutase (MnSOD). During the early phase of hypoperfusion, the COX-2 and eNOS enzyme levels increased in both the hippocampus and the frontal cortex, indicating the presence of excitotoxicity and vascular reactions caused by ischemia, while the expressions of nNOS, iNOS and MnSOD were less affected. There were significant reductions in most of the investigated enzyme levels 2 weeks and 3 months after 2VO induction, which may be a sign of neuronal loss. One year following 2VO onset, the eNOS expression was upregulated, which may strengthen the adaptation of the brain to cerebral ischemia.