The Correlation between Astrocytic Calcium and fMRI Signals is Related to the Thalamic Regulation of Cortical States

BOLD fMRI has been wildly used for mapping brain activity, but the cellular contribution of BOLD signals is still controversial. In this study, we investigated the correlation between neuronal/astrocytic calcium and the BOLD signal using simultaneous GCaMP-mediated calcium and BOLD signal recording, in the event-related state and in resting state, in anesthetized and in free-moving rats. To our knowledge, the results provide the first demonstration that evoked and intrinsic astrocytic calcium signals could occur concurrently accompanied by opposite BOLD signals which are associated with vasodilation and vasoconstriction. We show that the intrinsic astrocytic calcium is involved in brain state changes and is related to the activation of central thalamus. First, by simultaneous LFP and fiber optic calcium recording, the results show that the coupling between LFP and calcium indicates that neuronal activity is the basis of the calcium signal in both neurons and astrocytes. Second, we found that evoked neuronal and astrocytic calcium signals are always positively correlated with BOLD responses. However, intrinsic astrocytic calcium signals are accompanied by the activation of the central thalamus followed by a striking negative BOLD signal in cortex, which suggests that central thalamus may be involved in the initiation of the intrinsic astrocytic calcium signal. Third, we confirmed that the intrinsic astrocytic calcium signal is preserved in free moving rats. Moreover, the occurrences of intrinsic astrocytic calcium spikes are coincident with the transition between different sleep stages, which suggests intrinsic astrocytic calcium spikes reflect brain state transitions. These results demonstrate that the correlation between astrocytic calcium and fMRI signals is related to the thalamic regulation of cortical states. On the other hand, by studying the relationship between vessel–specific BOLD signals and spontaneous calcium activity from adjacent neurons, we show that low frequency spontaneous neuronal activity is the cellular mechanism of the BOLD signal during resting state

High-Field Functional MRI from the Perspective of Single Vessels in Rats and Humans

Functional MRI (fMRI) has been employed to map brain activity and connectivity based on the neurovascular coupled hemodynamic signal. However, in most cases of fMRI studies, the cerebral vascular hemodynamic signal has been imaged in a spatially smoothed manner due to the limit of spatial resolution. There is a need to improve the spatiotemporal resolution of fMRI to map dynamic signal from individual venule or individual arteriole directly. Here, the thesis aims to provide a vascular-specific view of hemodynamic response during active state or resting state. To better characterize the temporal features of task-related fMRI signal from different vascular compartments, we implemented a line-scanning method to acquire vessel-specific blood-oxygen-level-dependent (BOLD) / cerebral-blood-volume (CBV) fMRI signal at 100-ms temporal resolution with sensory or optogenetic stimulation. Furthermore, we extended the line-scanning method with multi-echo scheme to provide vessel-specific fMRI with the higher contrast-to-noise ratio (CNR), which allowed us to directly map the distinct evoked hemodynamic signal from arterioles and venules at different echo time (TE) from 3 ms to 30 ms. The line-scanning fMRI methods acquire single k-space line per TR under a reshuffled k space acquisition scheme which has the limitation of sampling the fMRI signal in real-time for resting-state fMRI studies. To overcome this, we implemented a balanced Steady-state free precession (SSFP) to map task-related and resting-state fMRI (rsfMRI) with high spatial resolution in anesthetized rats. We reveal venule-dominated functional connectivity for BOLD fMRI and arteriole-dominated functional connectivity for CBV fMRI. The BOLD signal from individual venules and CBV signal from individual arterioles show correlations at an ultra-slow frequency (< 0.1 Hz), which are correlated with the intracellular calcium signal measured in neighboring neurons. In complementary data from awake human subjects, the BOLD signal is spatially correlated among sulcus veins and specified intracortical veins of the visual cortex at similar ultra-slow rhythms. This work provides a high-resolution fMRI approach to resolve brain activation and functional connectivity at the level of single vessels, which opened a new avenue to investigate brian functional connectivity at the scale of vessels

Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI

BOLD fMRI has become a prevalent method to study cerebral sensory processing in rodent disease models, including pain and mechanical hypersensitivity. fMRI data analysis is frequently combined with a general-linear-model (GLM) -based analysis, which uses the convolution of a hemodynamic response function (HRF) with the stimulus paradigm. However, several studies indicated that the HRF differs across species, sexes, brain structures, and experimental factors, including stimulation modalities or anesthesia, and hence might strongly affect the outcome of BOLD analyzes. While considerable work has been done in humans and rats to understand the HRF, much less is known in mice. As a prerequisite to investigate mechano-sensory processing and BOLD fMRI data in male and female mice, we (1) designed a rotating stimulator that allows application of two different mechanical modalities, including innocuous von Frey and noxious pinprick stimuli and (2) determined and statistically compared HRFs across 30 brain structures and experimental conditions, including sex and, stimulus modalities. We found that mechanical stimulation lead to brain-wide BOLD signal changes thereby allowing extraction of HRFs from multiple brain structures. However, we did not find differences in HRFs across all brain structures and experimental conditions. Hence, we computed a whole-brain mouse HRF, which is based on 88 functional scans from 30 mice. A comparison of this mouse-specific HRF with our previously reported rat-derived HRF showed significantly slower kinetics in mice. Finally, we detected pronounced differences in cerebral BOLD activation between male and female mice with mechanical stimulation, thereby exposing divergent processing of noxious and innocuous stimuli in both sexes

Functional MRI Readouts From BOLD and Diffusion Measurements Differentially Respond to Optogenetic Activation and Tissue Heating

Functional blood-oxygenation-level-dependent (BOLD) MRI provides a brain-wide readout that depends on the hemodynamic response to neuronal activity. Diffusion fMRI has been proposed as an alternative to BOLD fMRI and has been postulated to directly rely on neuronal activity. These complementary functional readouts are versatile tools to be combined with optogenetic stimulation to investigate networks of the brain. The cell-specificity and temporal precision of optogenetic manipulations promise to enable further investigation of the origin of fMRI signals. The signal characteristics of the diffusion fMRI readout vice versa may better resolve network effects of optogenetic stimulation. However, the light application needed for optogenetic stimulation is accompanied by heat deposition within the tissue. As both diffusion and BOLD are sensitive to temperature changes, light application can lead to apparent activations confounding the interpretation of fMRI data. The degree of tissue heating, the appearance of apparent activation in different fMRI sequences and the origin of these phenomena are not well understood. Here, we disentangled apparent activations in BOLD and diffusion measurements in rats from physiological activation upon sensory or optogenetic stimulation. Both, BOLD and diffusion fMRI revealed similar signal shapes upon sensory stimulation that differed clearly from those upon heating. Apparent activations induced by high-intensity light application were dominated by T2∗-effects and resulted in mainly negative signal changes. We estimated that even low-intensity light application used for optogenetic stimulation reduces the BOLD response close to the fiber by up to 0.4%. The diffusion fMRI signal contained T2, T2∗ and diffusion components. The apparent diffusion coefficient, which reflects the isolated diffusion component, showed negative changes upon both optogenetic and electric forepaw stimulation. In contrast, positive changes were detected upon high-intensity light application and thus ruled out heating as a major contributor to the diffusion fMRI signal

Analysis of resting-state neurovascular coupling and locomotion-associated neural dynamics using wide-field optical mapping

Understanding the relationship between neural activity and cortical hemodynamics, or neurovascular coupling is the foundation to interpret neuroimaging signals such as functional magnetic resonance imaging (fMRI) which measure local changes in hemodynamics as a proxy for underlying neural activity. Even though the stereotypical stimulus-evoked hemodynamic response pattern with increased concentration of oxy- and total-hemoglobin and decrease in concentration of deoxy-hemoglobin has been well-recognized, the linearity of neurovascular coupling and its variances depending on brain state and tasks haven’t been thoroughly evaluated. To directly assess the cortical neurovascular coupling, simultaneous recordings of neural and hemodynamic activity were imaged by wide-field optical mapping (WFOM) over the bilateral dorsal surface of the mouse brain through a bilateral thinned-skull cranial window. Neural imaging is achieved through wide-field fluorescence imaging in animals expressing genetically encoded calcium sensor (Thy1-GCaMP). Hemodynamics are recorded via simultaneous imaging of multi-spectral reflectance. Significant hemodynamic crosstalk was found in the detected fluorescence signal and the physical model of the contamination, methods of correction as well as electrophysiological verification are presented. A linear model between neural and hemodynamic signals was used to fit spatiotemporal hemodynamics can be predicted by convolving local fluorescence changes with hemodynamic response functions derived through both deconvolution and gamma-variate fitting. Beyond confirming that the resting-state hemodynamics in the awake and anesthetized brain are coupled to underlying neural activity, the patterns of bilaterally symmetric spontaneous neural activity observed by WFOM emulate the functionally connected networks detected by fMRI. This result provides reassurance that resting-state functional connectivity has neural origins. With the access to cortical neural activity at mesoscopic level, we further explore the cortical neural representations preceding and during spontaneous locomotion

Combining Optogenetics and fMRI to Study Cerebral Networks in Animal Models

Functional magnetic resonance imaging is a well-established technique to examine brain activity and networks in animal models. In contrast to electrophysiology, optogenetics enables the control of specific cell types. A second advantage is that optogenetic stimulation can trigger excitatory as well as inhibitory effects. This study investigated optogenetic manipulation of neuronal activity and connectivity changes in mice and rats, using fMRI as an analytical method. The work addresses (1) glutamate release in mice and its use as a neurotransmitter and (2) oxytocin release in rats and its impact on neuronal networks. In fMRI, changes of metabolism and blood flow are described by the hemodynamic response function. The influence of the optogenetic stimulation on the hemodynamic response function was examined and characterized for both species. In the first part, glutamatergic neurons in the left hippocampus of mice were optogenetically stimulated via channelrhodopsin-2. Sham animals, without virus injection, were used as a control group to study unspecific effects induced by the laser stimulation. In transgenic (α-CamKII-Cre) mice, the direct hippocampal activation was investigated as well as its projections to other regions. Additionally, the impact of the dorsal-ventral position of the optical fiber on the hippocampal-prefrontal projections was investigated. We could demonstrate that the hemodynamic response measured in the hippocampus of mice reached its maximum earlier compared to the hemodynamic response function in humans. An explanation for this observation may be the smaller body size and the faster metabolism of mice. A highly significant increase of the BOLD signal was found for the optogenetic stimulation of glutamatergic neurons in the left hippocampus and its projecting areas, like the contralateral hippocampus and prefrontal regions. Furthermore, a negative correlation of prefrontal activation and the fiber depth was measured, which may be explained by the larger amount of stimulated neurons. In the second part, we optogenetically stimulated oxytocin-releasing neurons either in the amygdala or in the paraventricular nucleus of rats. We hypothesize that changes in the oxytocin associated networks of the basal ganglia and the olfactory system should result from the optogenetic stimulation. Long-term changes in connectivity were investigated. Therefore, changes in correlations between different brain regions were calculated using the resting-state measurements before and after the optogenetic stimulation. Moreover, seed regions of defined functional networks were determined and voxel-based changes in resting-state correlation to the seed region were investigated. Additionally, short-term connectivity changes were examined in a psychophysiological interaction analysis as well as the direct activation induced by the laser stimulation. We found that both channelrhodopsin-2 groups showed increased connectivity in the olfactory and basal ganglia networks compared to the control group. However, no short-term network changes were observed comparing the laser on and laser off condition. This might be explained by the hormonal characteristics of oxytocin, leading to a more global and prolonged response. To summarize, this thesis demonstrates the successful combination of optogenetics and fMRI as a tool for basic neuropsychiatric research. It proves the successful manipulation, not only of single neurons, but also of neuronal networks in vivo by optogenetic stimulation

Two-photon all-optical interrogation of mouse barrel cortex during sensory discrimination

The neocortex supports a rich repertoire of cognitive and behavioural functions, yet the rules, or neural ‘codes’, that determine how patterns of cortical activity drive perceptual processes remain enigmatic. Experimental neuroscientists study these codes through measuring and manipulating neuronal activity in awake behaving subjects, which allows links to be identified between patterns of neural activity and ongoing behaviour functions. In this thesis, I detail the application of novel optical techniques for simultaneously recording and manipulating neurons with cellular resolution to examine how tactile signals are processed in sparse neuronal ensembles in mouse somatosensory ‘barrel’ cortex. To do this, I designed a whisker-based perceptual decision-making task for head-fixed mice, that allows precise control over sensory input and interpretable readout of perceptual choice. Through several complementary experimental approaches, I show that task performance is exquisitely coupled to barrel cortical activity. Using two- photon calcium imaging to simultaneously record from populations of barrel cortex neurons, I demonstrate that different subpopulations of neurons in layer 2/3 (L2/3) show selectivity for contralateral and ipsilateral whisker input during behaviour. To directly test whether these stimulus-tuned groups of neurons differentially impact perceptual decision-making I performed patterned photostimulation experiments to selectively activate these functionally defined sets of neurons and assessed the resulting impact on behaviour and the local cortical network in layer 2/3. In contrast with the expected results, stimulation of sensory-coding neurons appeared to have little perceptual impact on task performance. However, activation of non- stimulus coding neurons did drive decision biases. These results challenge the conventional view that strongly sensory responsive neurons carry more perceptual weight than non-responsive sensory neurons during perceptual decision-making. Furthermore, patterned photostimulation revealed and imposed potent surround suppression in L2/3, which points to strong lateral inhibition playing a dominant role in shaping spatiotemporally sparse activity patterns. These results showcase the utility of combined patterned photostimulation methods and population calcium imaging for revealing and testing neural circuit function during sensorimotor behaviour and provide new perspectives on sensory coding in barrel cortex

Functional Connectivity of the Rodent Striatum

The striatum serves as the major input nucleus of the basal ganglia circuitry, important for its varied roles in cognition, motivation, and sensorimotor function. Despite decades of study, fundamental features of the striatum’s functional organization and broader role(s) within the basal ganglia circuitry remain contentious and/or poorly defined. Given the diverse and critical roles of striatal activity in normal brain function and a multitude of disease states (including neurodegenerative and psychiatric disorders), a better understanding of this nucleus’ functional organization is imperative. The use of electrophysiological tools, which predominate the field, allow for in-depth characterizations of discrete, pre-selected brain regions, but are not appropriate for delineating functional neural circuit interactions on large spatial scales in an unbiased manner. A complementary approach to these studies is the use of functional magnetic resonance imaging (fMRI), which provides global, unbiased measures of functional neural circuit and network connectivity. In the first two studies described herein (Chapters 2 and 3), we used fMRI to map the functional response patterns to electrical DBS of the rat nucleus accumbens (NAc; ventral striatum), as well as the dual striatal outputs: external globus pallidus (GPe), and substantia nigra pars reticulata. Notable findings included the presence of negative fMRI signals in striatum during stimulation of each nuclei, robust prefrontal cortical modulation by NAc- and GPe-DBS, and marked functional connectivity changes by high frequency DBS. We next used optogenetic tools to more selectively map the brain-wide responses to stimulation of GPe neurons in healthy and Parkinson’s disease model rats (Chapter 4), as well as dorsal striatal neurons and their motor cortical inputs (Chapter 5). Optogenetic stimulation of each nuclei elicited an intriguing dorsal striatal negative fMRI signal, observed during direct striatal stimulation as well as putative recruitment of both excitatory both inhibitory striatal inputs, and thus suggestive of neurovascular uncoupling. Additionally, results from our GPe experiments revealed that this signal may be compromised in certain neurological disease states (e.g., Parkinson’s disease). Collectively, the studies described in this dissertation have exploited fMRI tools to reveal novel features of striatal connectivity, which may shed light on striatal function in health and disease.Doctor of Philosoph

Use of functional neuroimaging and optogenetics to explore deep brain stimulation targets for the treatment of Parkinson's disease and epilepsy

Deep brain stimulation (DBS) is a neurosurgical therapy for Parkinson’s disease and epilepsy. In DBS, an electrode is stereotactically implanted in a specific region of the brain and electrical pulses are delivered using a subcutaneous pacemaker-like stimulator. DBS-therapy has proven to effectively suppress tremor or seizures in pharmaco-resistant Parkinson’s disease and epilepsy patients respectively. It is most commonly applied in the subthalamic nucleus for Parkinson’s disease, or in the anterior thalamic nucleus for epilepsy. Despite the rapidly growing use of DBS at these classic brain structures, there are still non-responders to the treatment. This creates a need to explore other brain structures as potential DBS-targets. However, research in patients is restricted mainly because of ethical reasons. Therefore, in order to search for potential new DBS targets, animal research is indispensable. Previous animal studies of DBS-relevant circuitry largely relied on electrophysiological recordings at predefined brain areas with assumed relevance to DBS therapy. Due to their inherent regional biases, such experimental techniques prevent the identification of less recognized brain structures that might be suitable DBS targets. Therefore, functional neuroimaging techniques, such as functional Magnetic Resonance Imaging and Positron Emission Tomography, were used in this thesis because they allow to visualize and to analyze the whole brain during DBS. Additionally, optogenetics, a new technique that uses light instead of electricity, was employed to manipulate brain cells with unprecedented selectivity