Investigating the neurovascular coupling in the cerebellar granular layer.

The neurovascular coupling (NVC) or functional hyperemia is the mechanism whereby neuronal activity controls cerebral blood flow (CBF). The tight coupling between neuronal activation and blood vessels diameter modifications ensures the proper supply of oxygen and nutrients to the central nervous system. In the brain, CBF adaptations are governed by vasoactive agents and their action on the vascular system (Iadecola, 2017). This phenomenon is also involved in the genesis of the blood-oxygen-level-dependent (BOLD) signals used by neuroimaging techniques, like functional magnetic resonance imaging (fMRI), to map changes in brain activity. Although being highly investigated, the interpretation of activity-dependent BOLD responses is widely debated (Hall et al., 2016). The complexity of investigating BOLD neurophysiological basis, i.e. NVC mechanisms, resulted in the inability to define a comprehensive theory for BOLD signals interpretation. In this work of thesis, the attention was focused on NVC mechanisms in the cerebellum. In this region, NVC has been previously investigated in the molecular layer, where interneurons activation has been found as the main player, unlike Purkinje cells spiking activity (Cauli et al., 2004; Thomsen et al., 2004). Surprisingly, there was no information about the role of the granular layer in cerebellar NVC before our investigations. In the granular layer, NVC is mediated by granule cells through an NMDA receptor/NO-dependent system acting on pericytes (Mapelli et al., 2017). The latter are contractile cells able to regulate the caliber of brain capillaries (Attwell et al., 2016),which are thought to be involved in the genesis of BOLD signals (Hall et al., 2016). Recent investigations using human fMRI demonstrated that cerebellar vermis lobule V and hemisphere lobule VI showed respectively linear and non-linear BOLD responses during the same motor task performance (Alahmadi et al., 2017). In mouse cerebellar slices, vermis lobule V and hemisphere lobule VI responded with different non-linear neurovascular events to several frequency patterns of neuronal activation, suggesting that NVC and thus BOLD signals might be region-dependent in the cerebellum (Gagliano et al., 2018 in preparation). In conclusion, granule cells, pericytes and capillaries may drive the basic neurovascular mechanisms of the cerebellum, but different cerebellar regions (vermis and hemisphere) could differently contribute to the genesis of cerebellar BOLD signals. These results might reflect different functions of these cerebellar areas following the same input

Investigating the neurovascular coupling in the cerebellar granular layer.

The neurovascular coupling (NVC) or functional hyperemia is the mechanism whereby neuronal activity controls cerebral blood flow (CBF). The tight coupling between neuronal activation and blood vessels diameter modifications ensures the proper supply of oxygen and nutrients to the central nervous system. In the brain, CBF adaptations are governed by vasoactive agents and their action on the vascular system (Iadecola, 2017). This phenomenon is also involved in the genesis of the blood-oxygen-level-dependent (BOLD) signals used by neuroimaging techniques, like functional magnetic resonance imaging (fMRI), to map changes in brain activity. Although being highly investigated, the interpretation of activity-dependent BOLD responses is widely debated (Hall et al., 2016). The complexity of investigating BOLD neurophysiological basis, i.e. NVC mechanisms, resulted in the inability to define a comprehensive theory for BOLD signals interpretation. In this work of thesis, the attention was focused on NVC mechanisms in the cerebellum. In this region, NVC has been previously investigated in the molecular layer, where interneurons activation has been found as the main player, unlike Purkinje cells spiking activity (Cauli et al., 2004; Thomsen et al., 2004). Surprisingly, there was no information about the role of the granular layer in cerebellar NVC before our investigations. In the granular layer, NVC is mediated by granule cells through an NMDA receptor/NO-dependent system acting on pericytes (Mapelli et al., 2017). The latter are contractile cells able to regulate the caliber of brain capillaries (Attwell et al., 2016),which are thought to be involved in the genesis of BOLD signals (Hall et al., 2016). Recent investigations using human fMRI demonstrated that cerebellar vermis lobule V and hemisphere lobule VI showed respectively linear and non-linear BOLD responses during the same motor task performance (Alahmadi et al., 2017). In mouse cerebellar slices, vermis lobule V and hemisphere lobule VI responded with different non-linear neurovascular events to several frequency patterns of neuronal activation, suggesting that NVC and thus BOLD signals might be region-dependent in the cerebellum (Gagliano et al., 2018 in preparation). In conclusion, granule cells, pericytes and capillaries may drive the basic neurovascular mechanisms of the cerebellum, but different cerebellar regions (vermis and hemisphere) could differently contribute to the genesis of cerebellar BOLD signals. These results might reflect different functions of these cerebellar areas following the same input

Non-Linear Frequency Dependence of Neurovascular Coupling in the Cerebellar Cortex Implies Vasodilation-Vasoconstriction Competition

Neurovascular coupling (NVC) is the process associating local cerebral blood flow (CBF) to neuronal activity (NA). Although NVC provides the basis for the blood oxygen level dependent (BOLD) effect used in functional MRI (fMRI), the relationship between NVC and NA is still unclear. Since recent studies reported cerebellar non-linearities in BOLD signals during motor tasks execution, we investigated the NVC/NA relationship using a range of input frequencies in acute mouse cerebellar slices of vermis and hemisphere. The capillary diameter increased in response to mossy fiber activation in the 6-300 Hz range, with a marked inflection around 50 Hz (vermis) and 100 Hz (hemisphere). The corresponding NA was recorded using high-density multi-electrode arrays and correlated to capillary dynamics through a computational model dissecting the main components of granular layer activity. Here, NVC is known to involve a balance between the NMDAR-NO pathway driving vasodilation and the mGluRs-20HETE pathway driving vasoconstriction. Simulations showed that the NMDAR-mediated component of NA was sufficient to explain the time course of the capillary dilation but not its non-linear frequency dependence, suggesting that the mGluRs-20HETE pathway plays a role at intermediate frequencies. These parallel control pathways imply a vasodilation-vasoconstriction competition hypothesis that could adapt local hemodynamics at the microscale bearing implications for fMRI signals interpretation

Interpreting BOLD: towards a dialogue between cognitive and cellular neuroscience

Cognitive neuroscience depends on the use of blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to probe brain function. Although commonly used as a surrogate measure of neuronal activity, BOLD signals actually reflect changes in brain blood oxygenation. Understanding the mechanisms linking neuronal activity to vascular perfusion is, therefore, critical in interpreting BOLD. Advances in cellular neuroscience demonstrating differences in this neurovascular relationship in different brain regions, conditions or pathologies are often not accounted for when interpreting BOLD. Meanwhile, within cognitive neuroscience, increasing use of high magnetic field strengths and the development of model-based tasks and analyses have broadened the capability of BOLD signals to inform us about the underlying neuronal activity, but these methods are less well understood by cellular neuroscientists. In 2016, a Royal Society Theo Murphy Meeting brought scientists from the two communities together to discuss these issues. Here we consolidate the main conclusions arising from that meeting. We discuss areas of consensus about what BOLD fMRI can tell us about underlying neuronal activity, and how advanced modelling techniques have improved our ability to use and interpret BOLD. We also highlight areas of controversy in understanding BOLD and suggest research directions required to resolve these issues

Identification and Functional Characterization of CNS Pericytes and the Role they Play in Neurovascular Coupling in Physiological and Pathological Conditions

Brain blood flow increases, evoked by neuronal activity, power neural computation and are the basis of BOLD functional imaging. However, it is controversial whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes. The experimental work within this thesis examines capillary pericytes, and the role they play in neurovascular coupling in physiological and pathological conditions. I show that pericytes can be identified using several protein markers and that, using the same technique, pericytes can be distinguished from other perivascular cell types. I demonstrate that pericytes respond to the neurotransmitters noradrenaline and glutamate. Noradrenaline depolarizes pericytes and constricts capillaries, and this constriction reflects pericyte contraction while glutamate, mimicking neuronal activity, hyperpolarizes pericytes and dilates capillaries, and this dilation reflects pericyte relaxation. Glutamate-evoked dilation is mediated by prostaglandin E₂ or a related compound acting at EP4 receptors, but requires nitric oxide release to suppress synthesis of the vasoconstrictor 20-HETE. In pathology, I show that pericytes die when exposed to ischaemia. This may lead to pericytes irreversibly constricting capillaries and to damage of the blood-brain barrier. Pericyte death increases on reperfusion after ischaemia, and is reduced by block of glutamate receptors or Ca2+ removal, but not by scavenging reactive oxygen species. These data establish pericytes as active regulators of capillary tone and thus as potential regulators of brain blood flow. My data also suggest prevention of pericyte death as a strategy to reduce the long-lasting blood flow decrease which contributes to neuronal death after stroke. This thesis also contains a discussion of how energy supply to the brain alters with age, and how this may affect the BOLD signal

Bayesian Comparison of Neurovascular Coupling Models Using EEG-fMRI

Functional magnetic resonance imaging (fMRI), with blood oxygenation level-dependent (BOLD) contrast, is a widely used technique for studying the human brain. However, it is an indirect measure of underlying neuronal activity and the processes that link this activity to BOLD signals are still a topic of much debate. In order to relate findings from fMRI research to other measures of neuronal activity it is vital to understand the underlying neurovascular coupling mechanism. Currently, there is no consensus on the relative roles of synaptic and spiking activity in the generation of the BOLD response. Here we designed a modelling framework to investigate different neurovascular coupling mechanisms. We use Electroencephalographic (EEG) and fMRI data from a visual stimulation task together with biophysically informed mathematical models describing how neuronal activity generates the BOLD signals. These models allow us to non-invasively infer the degree of local synaptic and spiking activity in the healthy human brain. In addition, we use Bayesian model comparison to decide between neurovascular coupling mechanisms. We show that the BOLD signal is dependent upon both the synaptic and spiking activity but that the relative contributions of these two inputs are dependent upon the underlying neuronal firing rate. When the underlying neuronal firing is low then the BOLD response is best explained by synaptic activity. However, when the neuronal firing rate is high then both synaptic and spiking activity are required to explain the BOLD signal

Concepts of neural nitric oxide-mediated transmission

As a chemical transmitter in the mammalian central nervous system, nitric oxide (NO) is still thought a bit of an oddity, yet this role extends back to the beginnings of the evolution of the nervous system, predating many of the more familiar neurotransmitters. During the 20 years since it became known, evidence has accumulated for NO subserving an increasing number of functions in the mammalian central nervous system, as anticipated from the wide distribution of its synthetic and signal transduction machinery within it. This review attempts to probe beneath those functions and consider the cellular and molecular mechanisms through which NO evokes short- and long-term modifications in neural performance. With any transmitter, understanding its receptors is vital for decoding the language of communication. The receptor proteins specialised to detect NO are coupled to cGMP formation and provide an astonishing degree of amplification of even brief, low amplitude NO signals. Emphasis is given to the diverse ways in which NO receptor activation initiates changes in neuronal excitability and synaptic strength by acting at pre- and/or postsynaptic locations. Signalling to non-neuronal cells and an unexpected line of communication between endothelial cells and brain cells are also covered. Viewed from a mechanistic perspective, NO conforms to many of the rules governing more conventional neurotransmission, particularly of the metabotropic type, but stands out as being more economical and versatile, attributes that presumably account for its spectacular evolutionary success

More than just summed neuronal activity: how multiple cell types shape the BOLD response

Functional neuroimaging techniques are widely applied to investigations of human cognition and disease. The most commonly used among these is blood oxygen level-dependent (BOLD) functional magnetic resonance imaging. The BOLD signal occurs because neural activity induces an increase in local blood supply to support the increased metabolism that occurs during activity. This supply usually outmatches demand, resulting in an increase in oxygenated blood in an active brain region, and a corresponding decrease in deoxygenated blood, which generates the BOLD signal. Hence, the BOLD response is shaped by an integration of local oxygen use, through metabolism, and supply, in the blood. To understand what information is carried in BOLD signals, we must understand how several cell types in the brain—local excitatory neurons, inhibitory neurons, astrocytes and vascular cells (pericytes, vascular smooth muscle and endothelial cells), and their modulation by ascending projection neurons—contribute to both metabolism and haemodynamic changes. Here, we review the contributions of each cell type to the regulation of cerebral blood flow and metabolism, and discuss situations where a simplified interpretation of the BOLD response as reporting local excitatory activity may misrepresent important biological phenomena, for example with regards to arousal states, ageing and neurological disease

Integration of EEG-FMRI in an Auditory Oddball Paradigm Using Joint Independent Component Analysis

The integration of event-related potential (ERP) and functional magnetic resonance imaging (fMRI) can contribute to characterizing neural networks with high temporal and spatial resolution. The overall objective of this dissertation is to determine the sensitivity and limitations of joint independent component analysis (jICA) within-subject for integration of ERP and fMRI data collected simultaneously in a parametric auditory oddball paradigm. The main experimental finding in this work is that jICA revealed significantly stronger and more extensive activity in brain regions associated with the auditory P300 ERP than a P300 linear regression analysis, both at the group level and within-subject. The results suggest that, with the incorporation of spatial and temporal information from both imaging modalities, jICA is more sensitive to neural sources commonly observed with ERP and fMRI compared to a linear regression analysis. Furthermore, computational simulations suggest that jICA can extract linear and nonlinear relationships between ERP and fMRI signals, as well as uncoupled sources (i.e., sources with a signal in only one imaging modality). These features of jICA can be important for assessing disease states in which the relationship between the ERP and fMRI signals is unknown, as well as pathological conditions causing neurovascular uncoupling, such as stroke

Development of zebrafish and computational models of neurovascular coupling in health and disease

In this thesis, I have developed a novel zebrafish model of neurovascular coupling. Combining lightsheet imaging, compound transgenic zebrafish models and custom MATLAB based analysis pipelines, I characterised the neurovascular responses (neuronal calcium increases and change in red blood cell speed) in the optic tectum in response to visual stimulation. I determined the development stage at which neurovascular coupling in zebrafish larvae develops, followed by testing the requirement for nitric oxide or astrocyte cyclo-oxygenase in my model. I then used this model to investigate factors influencing neurovascular function. I first characterized the effect of glucose exposure and the role of nitric oxide in modulating neurovascular coupling. I then examined the effect of genetic mutation of Guanosine Triphosphate cyclohydrolase (an enzyme involved in nitric oxide and dopamine production in the brain) on neurovascular coupling. Finally, I have developed a minimal mathematical model of the neurovascular unit. To demonstrate the potential of this model I have simulated the effect of high blood glucose and low nitric oxide on neurovascular coupling and show this conforms with experimental data obtained in zebrafish