Mariel G. Kozberg

A Critical Role for the Vascular Endothelium in Functional Neurovascular Coupling in the Brain

Background The functional modulation of blood flow in the brain is critical for brain health and is the basis of contrast in functional magnetic resonance imaging. There is evident coupling between increases in neuronal activity and increases in local blood flow; however, many aspects of this neurovascular coupling remain unexplained by current models. Based on the rapid dilation of distant pial arteries during cortical functional hyperemia, we hypothesized that endothelial signaling may play a key role in the long‐range propagation of vasodilation during functional hyperemia in the brain. Although well characterized in the peripheral vasculature, endothelial involvement in functional neurovascular coupling has not been demonstrated. Methods and Results We combined in vivo exposed‐cortex multispectral optical intrinsic signal imaging (MS‐OISI) with a novel in vivo implementation of the light‐dye technique to record the cortical hemodynamic response to somatosensory stimulus in rats before and after spatially selective endothelial disruption. We demonstrate that discrete interruption of endothelial signaling halts propagation of stimulus‐evoked vasodilation in pial arteries, and that wide‐field endothelial disruption in pial arteries significantly attenuates the hemodynamic response to stimulus, particularly the early, rapid increase and peak in hyperemia. Conclusions Involvement of endothelial pathways in functional neurovascular coupling provides new explanations for the spatial and temporal features of the hemodynamic response to stimulus and could explain previous results that were interpreted as evidence for astrocyte‐mediated control of functional hyperemia. Our results unify many aspects of blood flow regulation in the brain and body and prompt new investigation of direct links between systemic cardiovascular disease and neural deficits.

Resolving the transition from negative to positive blood oxygen level-dependent responses in the developing brain

The adult brain exhibits a local increase in cortical blood flow in response to external stimulus. However, broadly varying hemodynamic responses in the brains of newborn and young infants have been reported. Particular controversy exists over whether the “true” neonatal response to stimulation consists of a decrease or an increase in local deoxyhemoglobin, corresponding to a positive (adult-like) or negative blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI), respectively. A major difficulty with previous studies has been the variability in human subjects and measurement paradigms. Here, we present a systematic study in neonatal rats that charts the evolution of the cortical blood flow response during postnatal development using exposed-cortex multispectral optical imaging. We demonstrate that postnatal-day-12–13 rats (equivalent to human newborns) exhibit an “inverted” hemodynamic response (increasing deoxyhemoglobin, negative BOLD) with early signs of oxygen consumption followed by delayed, active constriction of pial arteries. We observed that the hemodynamic response then matures via development of an initial hyperemic (positive BOLD) phase that eventually masks oxygen consumption and balances vasoconstriction toward adulthood. We also observed that neonatal responses are particularly susceptible to stimulus-evoked systemic blood pressure increases, leading to cortical hyperemia that resembles adult positive BOLD responses. We propose that this confound may account for much of the variability in prior studies of neonatal cortical hemodynamics. Our results suggest that functional magnetic resonance imaging studies of infant and child development may be profoundly influenced by the maturing neurovascular and autoregulatory systems of the neonatal brain.

Resting-state hemodynamics are spatiotemporally coupled to synchronized and symmetric neural activity in excitatory neurons

Significance Resting-state functional connectivity mapping exploits correlations in the functional magnetic resonance imaging (fMRI) blood oxygen level-dependent (BOLD) signal across the brain. However, results are difficult to interpret without an understanding of the neural correlates of these hemodynamic fluctuations. This work uses mice in which neural activity and brain hemodynamics can be mapped simultaneously. We show that resting-state hemodynamics can be predicted from spontaneous neural activity and correspond to a series of driven increases in local blood volume, coupled with spontaneous, bilaterally symmetric fluctuations in excitatory neural activity. This result provides reassurance that resting-state functional connectivity has neural origins. The network-like spontaneous neural activity visualized here represents an underexplored feature of neural activity in the awake brain. Brain hemodynamics serve as a proxy for neural activity in a range of noninvasive neuroimaging techniques including functional magnetic resonance imaging (fMRI). In resting-state fMRI, hemodynamic fluctuations have been found to exhibit patterns of bilateral synchrony, with correlated regions inferred to have functional connectivity. However, the relationship between resting-state hemodynamics and underlying neural activity has not been well established, making the neural underpinnings of functional connectivity networks unclear. In this study, neural activity and hemodynamics were recorded simultaneously over the bilateral cortex of awake and anesthetized Thy1-GCaMP mice using wide-field optical mapping. Neural activity was visualized via selective expression of the calcium-sensitive fluorophore GCaMP in layer 2/3 and 5 excitatory neurons. Characteristic patterns of resting-state hemodynamics were accompanied by more rapidly changing bilateral patterns of resting-state neural activity. Spatiotemporal hemodynamics could be modeled by convolving this neural activity with hemodynamic response functions derived through both deconvolution and gamma-variate fitting. Simultaneous imaging and electrophysiology confirmed that Thy1-GCaMP signals are well-predicted by multiunit activity. Neurovascular coupling between resting-state neural activity and hemodynamics was robust and fast in awake animals, whereas coupling in urethane-anesthetized animals was slower, and in some cases included lower-frequency (<0.04 Hz) hemodynamic fluctuations that were not well-predicted by local Thy1-GCaMP recordings. These results support that resting-state hemodynamics in the awake and anesthetized brain are coupled to underlying patterns of excitatory neural activity. The patterns of bilaterally-symmetric spontaneous neural activity revealed by wide-field Thy1-GCaMP imaging may depict the neural foundation of functional connectivity networks detected in resting-state fMRI.

Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches.

Although modern techniques such as two-photon microscopy can now provide cellular-level three-dimensional imaging of the intact living brain, the speed and fields of view of these techniques remain limited. Conversely, two-dimensional wide-field optical mapping (WFOM), a simpler technique that uses a camera to observe large areas of the exposed cortex under visible light, can detect changes in both neural activity and haemodynamics at very high speeds. Although WFOM may not provide single-neuron or capillary-level resolution, it is an attractive and accessible approach to imaging large areas of the brain in awake, behaving mammals at speeds fast enough to observe widespread neural firing events, as well as their dynamic coupling to haemodynamics. Although such wide-field optical imaging techniques have a long history, the advent of genetically encoded fluorophores that can report neural activity with high sensitivity, as well as modern technologies such as light emitting diodes and sensitive and high-speed digital cameras have driven renewed interest in WFOM. To facilitate the wider adoption and standardization of WFOM approaches for neuroscience and neurovascular coupling research, we provide here an overview of the basic principles of WFOM, considerations for implementation of wide-field fluorescence imaging of neural activity, spectroscopic analysis and interpretation of results. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’.

Reversible Switching of the Shear Modulus of Photoresponsive Liquid‐Crystalline Polymers

Manipulation makes light work: The morphology and rheological properties of a liquid-crystalline system can be dynamically manipulated with UV light by attaching photoresponsive liquid-crystalline moieties to a siloxane-based polymer. Stimulation with UV light induces a conformational change in the molecule, which disrupts the liquid-crystalline mesophase (see picture), and results in a dramatic change in its rheological properties.

Rapid Postnatal Expansion of Neural Networks Occurs in an Environment of Altered Neurovascular and Neurometabolic Coupling

In the adult brain, increases in neural activity lead to increases in local blood flow. However, many prior measurements of functional hemodynamics in the neonatal brain, including functional magnetic resonance imaging (fMRI) in human infants, have noted altered and even inverted hemodynamic responses to stimuli. Here, we demonstrate that localized neural activity in early postnatal mice does not evoke blood flow increases as in the adult brain, and elucidate the neural and metabolic correlates of these altered functional hemodynamics as a function of developmental age. Using wide-field GCaMP imaging, the development of neural responses to somatosensory stimulus is visualized over the entire bilaterally exposed cortex. Neural responses are observed to progress from tightly localized, unilateral maps to bilateral responses as interhemispheric connectivity becomes established. Simultaneous hemodynamic imaging confirms that spatiotemporally coupled functional hyperemia is not present during these early stages of postnatal brain development, and develops gradually as cortical connectivity is established. Exploring the consequences of this lack of functional hyperemia, measurements of oxidative metabolism via flavoprotein fluorescence suggest that neural activity depletes local oxygen to below baseline levels at early developmental stages. Analysis of hemoglobin oxygenation dynamics at the same age confirms oxygen depletion for both stimulus-evoked and resting-state neural activity. This state of unmet metabolic demand during neural network development poses new questions about the mechanisms of neurovascular development and its role in both normal and abnormal brain development. These results also provide important insights for the interpretation of fMRI studies of the developing brain. SIGNIFICANCE STATEMENT This work demonstrates that the postnatal development of neuronal connectivity is accompanied by development of the mechanisms that regulate local blood flow in response to neural activity. Novel in vivo imaging reveals that, in the developing mouse brain, strong and localized GCaMP neural responses to stimulus fail to evoke local blood flow increases, leading to a state in which oxygen levels become locally depleted. These results demonstrate that the development of cortical connectivity occurs in an environment of altered energy availability that itself may play a role in shaping normal brain development. These findings have important implications for understanding the pathophysiology of abnormal developmental trajectories, and for the interpretation of functional magnetic resonance imaging data acquired in the developing brain.

Neurovascular coupling and energy metabolism in the developing brain

In the adult brain, increases in local neural activity are almost always accompanied by increases in local blood flow. However, many functional imaging studies of the newborn and developing human brain have observed patterns of hemodynamic responses that differ from adult responses. Among the proposed mechanisms for the observed variations is that neurovascular coupling itself is still developing in the perinatal brain. Many of the components thought to be involved in actuating and propagating this hemodynamic response are known to still be developing postnatally, including perivascular cells such as astrocytes and pericytes. Both neural and vascular networks expand and are then selectively pruned over the first year of human life. Additionally, the metabolic demands of the newborn brain are still evolving. These changes are highly likely to affect early postnatal neurovascular coupling, and thus may affect functional imaging signals in this age group. This chapter will discuss the literature relating to neurovascular development. Potential effects of normal and aberrant development of neurovascular coupling on the newborn brain will also be explored, as well as ways to effectively utilize imaging techniques that rely on hemodynamic modulation such as fMRI and NIRS in younger populations.

Neurovascular coupling develops alongside neural circuits in the postnatal brain

ABSTRACT In the adult brain, increases in local neural activity are accompanied by increases in regional blood flow. This relationship between neural activity and hemodynamics is termed neurovascular coupling and provides the blood flow-dependent contrast detected in functional magnetic resonance imaging (fMRI). Neurovascular coupling is commonly assumed to be consistent and reliable from birth; however, numerous studies have demonstrated markedly different hemodynamics in the early postnatal brain. Our recent study in J. Neuroscience examined whether different hemodynamics in the immature brain are driven by differences in the underlying spatiotemporal properties of neural activity during this period of robust neural circuit expansion. Using a novel wide-field optical imaging technique to visualize both neural activity and hemodynamics in the mouse brain, we observed longer duration and increasingly complex patterns of neural responses to stimulus as cortical connectivity developed over time. However, imaging of brain blood flow, oxygenation, and metabolism in the same mice demonstrated an absence of coupled blood flow responses in the newborn brain. This lack of blood flow coupling was shown to lead to oxygen depletions following neural activations – depletions that may affect the duration of sustained neural responses and could be important to the vascular patterning of the rapidly developing brain. These results are a step toward understanding the unique neurovascular and neurometabolic environment of the newborn brain, and provide new insights for interpretation of fMRI BOLD studies of early brain development.

Correction to ‘Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches’

Phil. Trans. R. Soc. B 371 , 20150360 (2016; Published 29 August 2016) ([doi:10.1098/rstb.2015.0360][1]) After … [1]: http://dx.doi.org/10.1098/rstb.2015.0360

Optical Imaging and Microscopy of the Living Brain

Techniques for capturing functional information from the living brain including high-speed multispectral optical intrinsic signal imaging (MS-OISI) and dual-beam in-vivo two-photon microscopy, and their applications to understanding brain blood flow control will be described.