This is a repository copy of More than just summed neuronal activity : how multiple cell types shape the BOLD response. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/166097/ Version: Accepted Version Article: Howarth, C., Mishra, A. and Hall, C. (2020) More than just summed neuronal activity : how multiple cell types shape the BOLD response. Philosophical Transactions of the Royal Society B: Biological Sciences, 376 (1815). ISSN 0962-8436 https://doi.org/10.1098/rstb.2019.0630 © 2020 The Authors. This is an author-produced version of a paper accepted for publication in Phil. Trans. B: Biological Sciences. Uploaded in accordance with the publisher's self-archiving policy. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. 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[email protected] https://eprints.whiterose.ac.uk/ More than just summed neuronal activity: how multiple cell types shape the BOLD response Clare Howarth1*^, Anusha Mishra2*^, Catherine Hall3*^ 1Department of Psychology, University of Sheffield, Sheffield, S1 2LT, UK 2Department of Neurology, Jungers Center for Neurosciences Research and Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR 97239, USA 3School of Psychology, University of Sussex, Brighton, UK ^ These authors contributed equally to this work *Address correspondence to Dr Clare Howarth, Department of Psychology, University of Sheffield, Cathedral Court, 1 Vicar Lane, Sheffield, S1 2LT, UK. Email: [email protected]; Dr Anusha Mishra, Department of Neurology Jungers Center for Neurosciences Research and Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR 97239, USA. Email: [email protected] or Dr Catherine Hall, School of Psychology, University of Sussex, Brighton, UK. Email: [email protected] Running title: Cellular mechanisms underlying BOLD 1 Abstract 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 (fMRI). 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, 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. Keywords BOLD fMRI, neurovascular coupling, neurometabolic coupling, astrocyte, interneuron, endothelial propagation 2 The blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI) is used as a surrogate measure of neuronal activity. However, because it is not caused directly by neuronal activity but by the disruption of the magnetic field by deoxyhaemoglobin in the blood, the BOLD signal is influenced by several factors beyond neuronal activity. These factors include the geometry of the vascular bed with respect to the magnetic field [1], the concentration of haemoglobin in the blood, blood volume, and the oxygenation state of the blood. While the oxygenation state of the blood can be altered by systemic factors such as cardiac rhythm and breathing [Das et al., this issue], oxygenation state within the brain is set by the balance between extraction of oxygen from the blood to fuel increased metabolism (neurometabolic coupling), and the supply of freshly oxygenated blood to an active brain region due to the dilation of local blood vessels (neurovascular coupling, producing functional hyperaemia). In this review, we examine the contribution of different cell types to these two processes and, therefore, to the BOLD signal to better understand what a regional change in BOLD reveals about underlying neuronal activity. Part 1: Neurovascular coupling Why does neurovascular coupling exist? The brain is energetically expensive, accounting for 20% of the body’s resting energy consumption [2]. In the cerebral cortex, the largest component of this energy is used to fuel the sodium-potassium ATPase, which reverses passive ion fluxes during action and synaptic potentials to maintain ionic electrochemical gradients [3,4]. Despite this high demand, the brain stores very low levels of energy substrates, largely in the form of glycogen, required for ATP production. Compared to other organs, the brain’s glycogen storage capacity is approximately 1/10th of that of skeletal muscle and 1/30th that of liver (from values reported in [5–9]). Therefore, the brain requires a constant supply of oxygen and glucose to drive ATP production, mostly from oxidative phosphorylation [10]. Neurovascular coupling is assumed to be necessary to increase the supply of energy 3 substrates (oxygen and glucose) in the blood, when neurons are active. In fact, the supply of oxygen during neurovascular coupling is substantially greater than that consumed by active brain regions (e.g., [11–14]), at least in neocortex, resulting in the decrease in deoxygenated haemoglobin that produces the positive BOLD signal commonly measured in fMRI studies [15]. The reason for this oversupply of oxygen remains unclear, but may involve a requirement for a large concentration gradient between the vessel and the tissue for adequate oxygen delivery [16], and the spread of hyperaemia (increased blood supply) to vessels in regions that are not themselves active but that surround and are upstream of active brain regions (see below). Alternatively, the main purpose of neurovascular coupling may not be to increase oxygen supply [17,18] but something else, such as the maintenance of stable tissue glucose concentrations to support aerobic glycolysis [19] (but also see [20], [21]), washout of waste products such as CO2 (but see [22]) and lactate (discussed in [23]), maintenance of appropriate tissue [O2]/[CO2] ratio [Buxton, this issue], or temperature regulation [24]. Whatever its purpose, the regional increase in oxygenated blood generated by neurovascular coupling is reliable enough, in healthy physiology, to generally allow an inference of increased neuronal activity from BOLD fMRI signals. However, an understanding of which cells drive the increase in cerebral blood flow (CBF, Figure 1) and which cells consume oxygen is required to fully and accurately interpret BOLD signals and to understand the limits of their utility. Neuronal subtypes. Neuronal activity is the initiator of the BOLD signal, which is often assumed to represent the aggregate activity of excitatory neurons in a brain region. Indeed, task-associated BOLD signals increase in areas of the brain where excitatory activity is expected to be increasing [25–28]. Furthermore, studies combining electrophysiological recordings or specific inhibitors of neural activity with BOLD signals [28] and haemodynamic increases [29,30] have directly demonstrated that these measures reflect an underlying increase in net neural activity. Conversely, negative BOLD responses in human subjects were observed in regions exhibiting increased GABAergic tone [31], and thus where neuronal activity may have decreased below baseline levels. The idea that increased inhibition, and 4 thus lower net neural activity, underlies negative BOLD responses is further supported by experiments in primates [32] and rodents [33–36], which show that negative BOLD and haemodynamic signals occur in areas with decreased excitatory activity [37]. While this simple interpretation, that positive and negative BOLD signals reflect increases and decreases in net activity, lends itself easily to investigations of cognitive function in humans, it may not always hold true. Pharmacological studies blocking both glutamate and -aminobutyric acid (GABA) receptors have shown that both neurotransmitters are likely involved in neurovascular coupling [30,38,39]), suggesting that haemodynamic responses (and, therefore, the BOLD signal) are elicited by a combination of signals from excitatory and inhibitory neurons. Indeed, inhibitory interneurons may play a more important role in the production of BOLD signals than was previously appreciated.