Isabel N. Christie

Functional Oxygen Sensitivity of Astrocytes

In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2. Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca2+]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca2+ from the intracellular stores. Hypoxia-induced [Ca2+]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. SIGNIFICANCE STATEMENT Most, if not all, animal cells possess mechanisms that allow them to detect decreases in oxygen availability leading to slow-timescale, adaptive changes in gene expression and cell physiology. To date, only two types of mammalian cells have been demonstrated to be specialized for rapid functional oxygen sensing: glomus cells of the carotid body (peripheral respiratory chemoreceptors) that stimulate breathing when oxygenation of the arterial blood decreases; and pulmonary arterial smooth muscle cells responsible for hypoxic pulmonary vasoconstriction to limit perfusion of poorly ventilated regions of the lungs. Results of the present study suggest that there is another specialized oxygen-sensitive cell type in the body, the astrocyte, that is tuned for rapid detection of physiological changes in brain oxygenation.

fMRI response to blue light delivery in the naïve brain: Implications for combined optogenetic fMRI studies

The combination of optogenetics and functional magnetic resonance imaging (fMRI) is referred to as opto-fMRI. Optogenetics utilises genetic engineering to introduce light sensitive actuator proteins into cells. Functional MRI (fMRI) is a specialist form of magnetic resonance imaging concerned with imaging changes in blood flow and oxygenation, linked to regional variation in metabolic activity, in the brain. This study describes a methodological concern regarding the effects of light delivery into the brain for the purposes of opto-fMRI. We show that blue light delivery to the naïve rat brain causes profound fMRI responses, despite the absence of optogenetic activation. We demonstrate that these fMRI responses are dependent upon laser power and show that the laser causes significant heating. We identify how heating impacts upon the MR signal causing NMR frequency shifts, and T1 and T2* changes. This study brings attention to a possible confounder which must be taken into account when opto-fMRI experiments are designed.

A Critical Role for Purinergic Signalling in the Mechanisms Underlying Generation of BOLD fMRI Responses

The mechanisms of neurovascular coupling underlying generation of BOLD fMRI signals remain incompletely understood. It has been proposed that release of vasoactive substances by astrocytes couples neuronal activity to changes in cerebrovascular blood flow. However, the role of astrocytes in fMRI responses remains controversial. Astrocytes communicate via release of ATP, and here we tested the hypothesis that purinergic signaling plays a role in the mechanisms underlying fMRI. An established fMRI paradigm was used to trigger BOLD responses in the forepaw region of the somatosensory cortex (SSFP) of an anesthetized rat. Forepaw stimulation induced release of ATP in the SSFP region. To interfere with purinergic signaling by promoting rapid breakdown of the vesicular and/or released ATP, a lentiviral vector was used to express a potent ectonucleotidase, transmembrane prostatic acid phosphatase (TMPAP), in the SSFP region. TMPAP expression had no effect on resting cerebral blood flow, cerebrovascular reactivity, and neuronal responses to sensory stimulation. However, TMPAP catalytic activity markedly reduced the magnitude of BOLD fMRI responses triggered in the SSFP region by forepaw stimulation. Facilitated ATP breakdown could result in accumulation of adenosine. However, blockade of A1 receptors had no effect on BOLD responses and did not reverse the effect of TMPAP. These results suggest that purinergic signaling plays a significant role in generation of BOLD fMRI signals. We hypothesize that astrocytes activated during periods of enhanced neuronal activity release ATP, which propagates astrocytic activation, stimulates release of vasoactive substances and dilation of cerebral vasculature.

Brain metabolic sensing and metabolic signaling at the level of an astrocyte

Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals—hormones like ghrelin, glucagon‐like peptide‐1 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever‐changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension.

fMRI mapping of the visual system in the mouse brain with interleaved snapshot GE-EPI.

The use of functional magnetic resonance imaging (fMRI) in mice is increasingly prevalent, providing a means to non-invasively characterise functional abnormalities associated with genetic models of human diseases. The predominant stimulus used in task-based fMRI in the mouse is electrical stimulation of the paw. Task-based fMRI in mice using visual stimuli remains underexplored, despite visual stimuli being common in human fMRI studies. In this study, we map the mouse brain visual system with BOLD measurements at 9.4 T using flashing light stimuli with medetomidine anaesthesia. BOLD responses were observed in the lateral geniculate nucleus, the superior colliculus and the primary visual area of the cortex, and were modulated by the flashing frequency, diffuse vs focussed light and stimulus context. Negative BOLD responses were measured in the visual cortex at 10 Hz flashing frequency; but turned positive below 5 Hz. In addition, the use of interleaved snapshot GE-EPI improved fMRI image quality without diminishing the temporal contrast-noise-ratio. Taken together, this work demonstrates a novel methodological protocol in which the mouse brain visual system can be non-invasively investigated using BOLD fMRI.

Volumetric Spatial Correlations of Neurovascular Coupling Studied using Single Pulse Opto-fMRI

Neurovascular coupling describes the link between neuronal activity and cerebral blood flow. This relationship has been the subject of intense scrutiny, with most previous work seeking to understand temporal correlations that describe neurovascular coupling. However, to date, the study of spatial correlations has been limited to two-dimensional mapping of neuronal or vascular derived signals emanating from the brain’s surface, using optical imaging techniques. Here, we investigate spatial correlations of neurovascular coupling in three dimensions, by applying a single 10 ms pulse of light to trigger optogenetic activation of cortical neurons transduced to express channelrhodopsin2, with concurrent fMRI. We estimated the spatial extent of increased neuronal activity using a model that takes into the account the scattering and absorption of blue light in brain tissue together with the relative density of channelrhodopsin2 expression across cortical layers. This method allows precise modulation of the volume of activated tissue in the cerebral cortex with concurrent three-dimensional mapping of functional hyperemia. Single pulse opto-fMRI minimizes adaptation, avoids heating artefacts and enables confined recruitment of the neuronal activity. Using this novel method, we present evidence for direct proportionality of volumetric spatial neurovascular coupling in the cerebral cortex.

CNS distribution, signalling properties and central effects of G-protein coupled receptor 4

ABSTRACT Information on the distribution and biology of the G‐protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to Gs proteins and may mediate central respiratory sensitivity to CO2. Using a knock‐in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro‐trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52‐QQ57 effectively blocked GPR4‐mediated cAMP accumulation (IC50 26.8nM in HEK293cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4–7.0) resulted in a cAMP increase by ˜55% which was completely prevented by 1&mgr;M NE 52‐QQ57. The main extracellular organic acid, l‐lactic acid (LL; 1–10mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52‐QQ57 (20mgkg−1) reduced ventilatory response to 5 and 10% CO2. In anaesthetised rats, systemic administration of NE 52‐QQ57 (up to 20mgkg−1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52‐QQ57 (1mM) in vagotomised anaesthetised rats did not affect CO2‐induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52‐QQ57 blunts hypercapnic response to CO2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.