RESUMO
An ongoing controversy in brain metabolism is whether increases in neural activity cause a local and rapid decrease in oxygen concentration (i.e., the "initial dip") preceding functional hyperemia. This initial dip has been suggested to cause a transient increase in vascular deoxyhemoglobin with several imaging techniques and stimulation paradigms, but not consistently. Here, we investigate contributors to this initial dip in a distinct neuronal network, an olfactory bulb (OB) glomerulus most sensitive to a specific odorant (ethyl tiglate [ET]) and a site of strong activation and energy consumption upon ET stimulation. Combining two-photon fluorescence and phosphorescence lifetime microscopy, and calcium, blood flow, and pO2 measurements, we characterized this initial dip in pO2 in mice chronically implanted with a glass cranial window, during both awake and anesthetized conditions. In anesthetized mice, a transient dip in vascular pO2 was detected in this glomerulus when functional hyperemia was slightly delayed, but its amplitude was minute (0.3 SD of resting baseline). This vascular pO2 dip was not observed in other glomeruli responding nonspecifically to ET, and it was poorly influenced by resting pO2. In awake mice, the dip in pO2 was absent in capillaries as well as, surprisingly, in the neuropil. These high-resolution pO2 measurements demonstrate that in awake mice recovered from brain surgery, neurovascular coupling was too fast and efficient to reveal an initial dip in pO2.
Assuntos
Encéfalo , Acoplamento Neurovascular , Oxigênio , Vigília , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Camundongos , Rede Nervosa , Neurônios/fisiologia , Bulbo Olfatório/metabolismo , Oxigênio/metabolismoRESUMO
Oxygen is critical for neural metabolism, but under most physiological conditions, oxygen levels in the brain are far more than are required. Oxygen levels can be dynamically increased by increases in respiration rate that are tied to the arousal state of the brain and cognition, and not necessarily linked to exertion by the body. Why these changes in respiration occur when oxygen is already adequate has been a long-standing puzzle. In humans, performance on cognitive tasks can be affected by very high or very low oxygen levels, but whether the physiological changes in blood oxygenation produced by respiration have an appreciable effect is an open question. Oxygen has direct effects on potassium channels, increases the degradation rate of nitric oxide, and is rate limiting for the synthesis of some neuromodulators. We discuss whether oxygenation changes due to respiration contribute to neural dynamics associated with attention and arousal.
Assuntos
Oxigênio , Respiração , Humanos , Oxigênio/metabolismo , Consumo de Oxigênio/fisiologia , Encéfalo/metabolismo , Óxido Nítrico/metabolismoRESUMO
Understanding the relationships between biological processes is paramount to unravel pathophysiological mechanisms. These relationships can be modeled with Transfer Functions (TFs), with no need of a priori hypotheses as to the shape of the transfer function. Here we present Iliski, a software dedicated to TFs computation between two signals. It includes different pre-treatment routines and TF computation processes: deconvolution, deterministic and non-deterministic optimization algorithms that are adapted to disparate datasets. We apply Iliski to data on neurovascular coupling, an ensemble of cellular mechanisms that link neuronal activity to local changes of blood flow, highlighting the software benefits and caveats in the computation and evaluation of TFs. We also propose a workflow that will help users to choose the best computation according to the dataset. Iliski is available under the open-source license CC BY 4.0 on GitHub (https://github.com/alike-aydin/Iliski) and can be used on the most common operating systems, either within the MATLAB environment, or as a standalone application.
Assuntos
Software , Algoritmos , Biologia Computacional/métodos , Fluxo de TrabalhoRESUMO
The activity of mitral and tufted cells, the principal neurons of the olfactory bulb, is modulated by several classes of interneurons. Among them, diverse periglomerular (PG) cell types interact with the apical dendrites of mitral and tufted cells inside glomeruli at the first stage of olfactory processing. We used paired recording in olfactory bulb slices and two-photon targeted patch-clamp recording in vivo to characterize the properties and connections of a genetically identified population of PG cells expressing enhanced yellow fluorescent protein (EYFP) under the control of the Kv3.1 potassium channel promoter. Kv3.1-EYFP(+) PG cells are axonless and monoglomerular neurons that constitute â¼30% of all PG cells and include calbindin-expressing neurons. They respond to an olfactory nerve stimulation with a short barrage of excitatory inputs mediated by mitral, tufted, and external tufted cells, and, in turn, they indiscriminately release GABA onto principal neurons. They are activated by even the weakest olfactory nerve input or by the discharge of a single principal neuron in slices and at each respiration cycle in anesthetized mice. They participate in a fast-onset intraglomerular lateral inhibition between principal neurons from the same glomerulus, a circuit that reduces the firing rate and promotes spike timing variability in mitral cells. Recordings in other PG cell subtypes suggest that this pathway predominates in generating glomerular inhibition. Intraglomerular lateral inhibition may play a key role in olfactory processing by reducing the similarity of principal cells discharge in response to the same incoming input.
Assuntos
Potenciais de Ação/fisiologia , Rede Nervosa/fisiologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Bulbo Olfatório/citologia , Animais , Calbindina 1/metabolismo , Creatina/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Blood flow in the brain is regulated by neurons and astrocytes. Knowledge of how these cells control blood flow is crucial for understanding how neural computation is powered, for interpreting functional imaging scans of brains, and for developing treatments for neurological disorders. It is now recognized that neurotransmitter-mediated signalling has a key role in regulating cerebral blood flow, that much of this control is mediated by astrocytes, that oxygen modulates blood flow regulation, and that blood flow may be controlled by capillaries as well as by arterioles. These conceptual shifts in our understanding of cerebral blood flow control have important implications for the development of new therapeutic approaches.
Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Humanos , Neurotransmissores/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Transdução de SinaisRESUMO
The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.
Assuntos
Encéfalo/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/metabolismo , Algoritmos , Animais , Velocidade do Fluxo Sanguíneo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Cristais Líquidos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Modelos Estatísticos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Neurovascular coupling (NVC), which mediates rapid increases in cerebral blood flow in response to neuronal activation, is commonly used to map brain activation or dysfunction. Here we tested the reemerging hypothesis that CO2 generated by neuronal metabolism contributes to NVC. We combined functional ultrasound and two-photon imaging in the mouse barrel cortex to specifically examine the onsets of local changes in vessel diameter, blood flow dynamics, vascular/perivascular/intracellular pH, and intracellular calcium signals along the vascular arbor in response to a short and strong CO2 challenge (10 s, 20%) and whisker stimulation. We report that the brief hypercapnia reversibly acidifies all cells of the arteriole wall and the periarteriolar space 3-4 s prior to the arteriole dilation. During this prolonged lag period, NVC triggered by whisker stimulation is not affected by the acidification of the entire neurovascular unit. As it also persists under condition of continuous inflow of CO2, we conclude that CO2 is not involved in NVC.
Assuntos
Dióxido de Carbono , Circulação Cerebrovascular , Hipercapnia , Acoplamento Neurovascular , Vibrissas , Animais , Dióxido de Carbono/metabolismo , Acoplamento Neurovascular/fisiologia , Camundongos , Circulação Cerebrovascular/fisiologia , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Vibrissas/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Concentração de Íons de Hidrogênio , Neurônios/metabolismo , Neurônios/fisiologia , Córtex Somatossensorial/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/metabolismo , Arteríolas/fisiologia , Arteríolas/metabolismoRESUMO
Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.
Assuntos
Hormônio do Crescimento/metabolismo , Microcirculação , Hipófise/irrigação sanguínea , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipófise/citologia , Hipófise/metabolismoRESUMO
In the brain, neuronal activation triggers a local increase in cerebral blood flow, a response named functional hyperemia. The extent to which functional hyperemia faithfully reports brain activation, spatially or temporally, remains a matter of debate. Here, we used the olfactory bulb glomerulus as a neurovascular model and two-photon microscopy imaging to investigate the correlation between calcium signals in glutamatergic terminals of olfactory sensory neurons and local vascular responses. We find that, depending on odor stimulation intensity, vascular responses are differently coupled to calcium signals. Upon moderate odor stimulation, glomerular vascular responses increase accordingly with calcium signals. In contrast, in silent glomeruli neighboring strongly activated ones and in glomeruli adapting upon high odor stimulation, vascular responses are independent of or negatively coupled to presynaptic calcium signals, respectively. Hence, functional hyperemia, a key signal used in functional imaging, can be, at times, an unreliable marker of local brain activation.
Assuntos
Hiperemia , Odorantes , Bulbo Olfatório/irrigação sanguínea , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Transdução de Sinais , Animais , Sinalização do Cálcio/fisiologia , Imageamento Tridimensional , Microscopia , Bulbo Olfatório/fisiologia , Ratos , Ratos Wistar , Olfato/fisiologiaRESUMO
Olfactory sensory neurons (OSNs) expressing the same odorant receptor converge in specific glomeruli where they transmit olfactory information to mitral cells. Surprisingly, synaptic mechanisms underlying mitral cell activation are still controversial. Using patch-clamp recordings in mouse olfactory bulb slices, we demonstrate that stimulation of OSNs produces a biphasic postsynaptic excitatory response in mitral cells. The response was initiated by a fast and graded monosynaptic input from OSNs and followed by a slower component of feedforward excitation, involving dendro-dendritic interactions between external tufted, tufted and other mitral cells. The mitral cell response occasionally lacked the fast OSN input when few afferent fibers were stimulated. We also show that OSN stimulation triggers a strong and slow feedforward inhibition that shapes the feedforward excitation but leaves unaffected the monosynaptic component. These results confirm the existence of direct OSN to mitral cells synapses but also emphasize the prominence of intraglomerular feedforward pathways in the mitral cell response.
Assuntos
Rede Nervosa/fisiologia , Neurônios/classificação , Neurônios/fisiologia , Bulbo Olfatório/citologia , Sinapses/classificação , Sinapses/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Biofísica , Cromonas/farmacologia , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Rede Nervosa/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Piridazinas/farmacologia , Sinapses/efeitos dos fármacosRESUMO
Functional ultrasound (fUS) is an emerging technique that measures blood flow to report brain activity. In this issue of Neuron, Nunez-Elizalde et al. (2022) use simultaneous electrophysiological and fUS measurements to quantify the relationship between firing and fUS signals in awake mice.
Assuntos
Neurônios , Som , Animais , Fenômenos Eletrofisiológicos , Camundongos , VigíliaRESUMO
Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans in physiological and pathological vessels. With scans of broken lines, i.e., lines made of several segments with different orientations, it also allows simultaneous monitoring of vessel diameter dynamics or the activity of specific cells. Analysis of red blood cell (RBC) velocity from line-scans requires specific image-processing algorithms, as angle measurements, Line-Scanning Particle Image Velocimetry (LSPIV) or Fourier transformation of line-scan images. The conditions under which these image-processing algorithms give accurate measurements have not been fully characterized although the accuracy of measurements vary according to specific experimental parameters: the vessel type, the RBC velocity, the scanning parameters, and the image signal to noise ratio. Here, we developed mathematical models for the three previously mentioned line-scan image-processing algorithms. Our models predict the experimental conditions in which RBC velocity measurements are accurate. We illustrate the case of different vessel types and give the parameter space available for each of them. Last, we developed a software generating artificial line-scan images and used it to validate our models.
RESUMO
Significance: All functional brain imaging methods have technical drawbacks and specific spatial and temporal resolution limitations. Unraveling brain function requires bridging the data acquired with cellular and mesoscopic functional imaging. This imposes the access to animal preparations, allowing longitudinal and multiscale investigations of brain function in anesthetized and awake animals. Such preparations are optimal to study normal and pathological brain functions while reducing the number of animals used. Aim: To fulfill these needs, we developed a chronic and stable preparation for a broad set of imaging modalities and experimental design. Approach: We describe the detailed protocol for a chronic cranial window, transparent to light and ultrasound, devoid of BOLD functional magnetic resonance imaging (fMRI) artifact and allowing stable and longitudinal multimodal imaging of the entire mouse cortex. Results: The inexpensive, transparent, and curved polymethylpentene cranial window preparation gives access to the entire mouse cortex. It is compatible with standard microscopic and mesoscopic neuroimaging methods. We present examples of data on the neurovascular unit and its activation using two-photon, functional ultrasound imaging, and BOLD fMRI. Conclusion: This preparation is ideal for multimodal imaging in the same animal.
RESUMO
The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial. Here we utilized a single whisker stimulation model to map out functional hyperemia along vascular arbours from layer II/III to the surface of primary somatosensory cortex, in anesthetized and awake Thy1-GCaMP6 mice. We demonstrate that sensory stimulation triggers an increase in blood velocity within the mid-capillary bed and a dilation of upstream large capillaries, and the penetrating and pial arterioles. We report that under physiological stimulation, response onset times are highly variable across compartments of different vascular arbours. Furthermore, generating transfer functions (TFs) between neuronal Ca2+ and vascular dynamics across different brain states demonstrates that anesthesia decelerates neurovascular coupling (NVC). This spatial-temporal pattern of vascular events demonstrates functional diversity not only between different brain regions but also at the level of different vascular arbours within supragranular layers of the cerebral cortex.
Assuntos
Encéfalo/fisiologia , Córtex Cerebral/fisiologia , Circulação Cerebrovascular/fisiologia , Acoplamento Neurovascular/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Encéfalo/irrigação sanguínea , Mapeamento Encefálico/métodos , Capilares/fisiologia , Córtex Cerebral/irrigação sanguínea , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neuroimagem/métodos , Neurônios/fisiologia , Bulbo Olfatório/irrigação sanguínea , Bulbo Olfatório/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Vibrissas/fisiologia , Vigília/fisiologiaRESUMO
Adaptation is a general property of sensory receptor neurons and has been extensively studied in isolated cell preparation of olfactory receptor neurons. In contrast, little is known about the conditions under which peripheral adaptation occurs in the CNS during odorant stimulation. Here, we used two-photon laser-scanning microscopy and targeted extracellular recording in freely breathing anesthetized rats to investigate the correlate of peripheral adaptation at the first synapse of the olfactory pathway in olfactory bulb glomeruli. We find that during sustained stimulation at high concentration, odorants can evoke local field potential (LFP) postsynaptic responses that rapidly adapt with time, some within two inhalations. Simultaneous measurements of LFP and calcium influx at olfactory receptor neuron terminals reveal that postsynaptic adaptation is associated with a decrease in odorant-evoked calcium response, suggesting that it results from a decrease in glutamate release. This glomerular adaptation was concentration-dependent and did not change the glomerular input-output curve. In addition, in situ application of antagonists of either ionotropic glutamate receptors or metabotropic GABA(B) receptors did not affect this adaptation, thus discarding the involvement of local presynaptic inhibition. Glomerular adaptation, therefore, reflects the response decline of olfactory receptor neurons to sustained odorant. We postulate that peripheral fast adaptation is a means by which glomerular output codes for high concentration of odor.
Assuntos
Adaptação Fisiológica/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Nervos Periféricos/fisiologia , Olfato/fisiologia , Animais , Bulbo Olfatório/citologia , Condutos Olfatórios/fisiologia , Ratos , Ratos WistarRESUMO
Odors synchronize the activity of olfactory bulb mitral cells that project to the same glomerulus. In vitro, a slow rhythmic excitation intrinsic to the glomerular network persists, even in the absence of afferent input. We show here that a subpopulation of juxtaglomerular cells, external tufted (ET) cells, may trigger this rhythmic activity. We used paired whole-cell recording and Ca(2+) imaging in bulb slices from wild-type and transgenic mice expressing the fluorescent Ca(2+) indicator protein GCaMP-2. Slow, periodic population bursts in mitral cells were synchronized with spontaneous discharges in ET cells. Moreover, activation of a single ET cell was sufficient to evoke population bursts in mitral cells within the same glomerulus. Stimulation of the olfactory nerve induced similar population bursts and activated ET cells at a lower threshold than mitral cells, suggesting that ET cells mediate feedforward excitation of mitral cells. We propose that ET cells act as essential drivers of glomerular output to the olfactory cortex.
Assuntos
Potenciais de Ação/fisiologia , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Olfato/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Relógios Biológicos/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Forma Celular/fisiologia , Sincronização Cortical , Estimulação Elétrica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/ultraestrutura , Bulbo Olfatório/ultraestrutura , Nervo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Periodicidade , Sinapses/ultraestruturaRESUMO
The relationship between metabolism of neuronal activity, microvascular organization, and blood flow dynamics is critical for interpreting functional brain imaging. Here we used the rat dorsal olfactory bulb as a model to determine in vivo the correlation between action potential propagation, synaptic transmission, oxygen consumption, and capillary density during odor stimulation. We find that capillary lumen occupies approximately 3% of the glomerular volume, where synaptic transmission occurs, and only 0.1% of the overlying nerve layer. In glomeruli, odor triggers a local early decrease in tissue oxygen partial pressure that results principally from dendritic activation rather than from firing of axon terminals, transmitter release or astrocyte activation. In the nerve layer, action potential propagation does not generate local changes in tissue oxygen partial pressure. We conclude that capillary density is tightly correlated with the oxidative metabolism of synaptic transmission, and suggest that action potential propagation operates mainly anaerobically.
Assuntos
Potenciais de Ação/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Consumo de Oxigênio/fisiologia , Transmissão Sináptica/fisiologia , Animais , Bulbo Olfatório/irrigação sanguínea , Bulbo Olfatório/ultraestrutura , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Olfato/fisiologiaRESUMO
The amino acid gamma-aminobutiric acid (GABA) is a major inhibitory transmitter in the vertebrate central nervous system (CNS) where it can be released by neurons and by glial cells. Neuronal GABAergic signaling is well characterized: the mechanisms of GABA release, the receptors it targets and the functional consequences of their activation have been extensively studied. In contrast, the corresponding features of glial GABAergic signaling have attracted less attention. In this review, we first discuss evidence from the literature for GABA accumulation, production and release by glial cells. We then review the results of recent experiments that point toward functional roles of GABA as a "gliotransmitter".
Assuntos
Neuroglia/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.