In vivo identification of brain structures functionally involved in spatial learning and strategy switch

Spatial learning is a complex behavior which includes, among others, encoding of space, sensory and motivational processes, arousal and locomotor performance. Today, our view on spatial navigation is largely hippocampus-centrist. Less is known about the involvement of brain structures up- and downstream, or out of this circuit. Here, I provide the first in vivo assessment of the neural matrix underlying spatial learning, using functional manganese-enhanced MRI (MEMRI) and voxel-wise whole brain analysis. Mice underwent place-learning (PL) vs. response-learning (RL) in the water cross maze (WCM) and its readout was correlated to the Mn2+ contrasts. Thus, I identified structures involved in spatial learning largely overlooked in the past, due to methods focused on region of interest (ROI) analyses. These structures include several sensory-related structures and differ between place-learners and response-learners, with the former (PL) comprising mostly structures involved in different properties of visual processing, such as horizontal gaze (e.g. nucleus prepositus) and saccade (e.g. fastigial nucleus), or provide vision-input and eye movement information from parahippocampal (e.g. presubiculum, perirhinal, postrhinal and ectorhinal areas) and other regions (e.g. orbital area, superior colliculus and vestibular ocular-reflex from the vestibular nucleus) likely to head-direction, grid- and place-cells; and the latter (RL) presenting structures related to more basic rodent sensory computations, like odor (e.g main and accessory olfactory bulb, cortical amygdala, piriform, endopiriform and postpiriform areas) and acoustic stimuli representation (e.g. auditory area, nucleus of the lateral lemniscus and superior olivary complex), or sensory-motor properties, such as body representation (e.g. somatosensory area – upper limbs) and head-direction signal. Add-on experiments pointed to preferential Mn2+ accumulation towards projection terminals, suggesting that our mapping was mostly formed by projection sites of the originally activated structures. This is corroborated by in-depth analysis of MEMRI data after WCM learning showing mostly downstream targets of the hippocampus. These differ between fornical afferences from vCA1 and direct innervation from dCA1/iCA1 (for PL), and structures along the longitudinal association bundle originating in vCA1 (for RL). To elucidate the pattern of Mn2+ accumulation seen on the scans, I performed c-fos expression analyses following learning in the WCM. This helped me identify the structures initially activated during spatial learning and its underlying connectivity to establish the matrix. Finally, to test the causal involvement of selected structures from our previous findings I inhibited them (through DREADDs) while mice performed the WCM task. I also focused on the causal involvement of the vHPC-mPFC circuit on strategy switch during WCM learning. I believe that this study might shed light into new brain structures involved in spatial learning and strategy switch and complement the current knowledge on these circuits’ connectivity. Moreover, I elucidated some functional mechanisms of MEMRI, clarifying the interpretation of data obtained with this method and its possible future applications

In vivo identification of brain structures functionally involved in spatial learning and strategy switch

Spatial learning is a complex behavior which includes, among others, encoding of space, sensory and motivational processes, arousal and locomotor performance. Today, our view on spatial navigation is largely hippocampus-centrist. Less is known about the involvement of brain structures up- and downstream, or out of this circuit. Here, I provide the first in vivo assessment of the neural matrix underlying spatial learning, using functional manganese-enhanced MRI (MEMRI) and voxel-wise whole brain analysis. Mice underwent place-learning (PL) vs. response-learning (RL) in the water cross maze (WCM) and its readout was correlated to the Mn2+ contrasts. Thus, I identified structures involved in spatial learning largely overlooked in the past, due to methods focused on region of interest (ROI) analyses. These structures include several sensory-related structures and differ between place-learners and response-learners, with the former (PL) comprising mostly structures involved in different properties of visual processing, such as horizontal gaze (e.g. nucleus prepositus) and saccade (e.g. fastigial nucleus), or provide vision-input and eye movement information from parahippocampal (e.g. presubiculum, perirhinal, postrhinal and ectorhinal areas) and other regions (e.g. orbital area, superior colliculus and vestibular ocular-reflex from the vestibular nucleus) likely to head-direction, grid- and place-cells; and the latter (RL) presenting structures related to more basic rodent sensory computations, like odor (e.g main and accessory olfactory bulb, cortical amygdala, piriform, endopiriform and postpiriform areas) and acoustic stimuli representation (e.g. auditory area, nucleus of the lateral lemniscus and superior olivary complex), or sensory-motor properties, such as body representation (e.g. somatosensory area ? upper limbs) and head-direction signal. Add-on experiments pointed to preferential Mn2+ accumulation towards projection terminals, suggesting that our mapping was mostly formed by projection sites of the originally activated structures. This is corroborated by in-depth analysis of MEMRI data after WCM learning showing mostly downstream targets of the hippocampus. These differ between fornical afferences from vCA1 and direct innervation from dCA1/iCA1 (for PL), and structures along the longitudinal association bundle originating in vCA1 (for RL). To elucidate the pattern of Mn2+ accumulation seen on the scans, I performed c-fos expression analyses following learning in the WCM. This helped me identify the structures initially activated during spatial learning and its underlying connectivity to establish the matrix. Finally, to test the causal involvement of selected structures from our previous findings I inhibited them (through DREADDs) while mice performed the WCM task. I also focused on the causal involvement of the vHPC-mPFC circuit on strategy switch during WCM learning. I believe that this study might shed light into new brain structures involved in spatial learning and strategy switch and complement the current knowledge on these circuits? connectivity. Moreover, I elucidated some functional mechanisms of MEMRI, clarifying the interpretation of data obtained with this method and its possible future applications

Hyperpolarized ¹³C Magnetic Resonance Spectroscopy Reveals the Rate-Limiting Role of the Blood-Brain Barrier in the Cerebral Uptake and Metabolism of l-Lactate in Vivo.

The dynamics of l-lactate transport across the blood-brain barrier (BBB) and its cerebral metabolism are still subject to debate. We studied lactate uptake and intracellular metabolism in the mouse brain using hyperpolarized <sup>13</sup> C magnetic resonance spectroscopy (MRS). Following the intravenous injection of hyperpolarized [1- <sup>13</sup> C]lactate, we observed that the distribution of the <sup>13</sup> C label between lactate and pyruvate, which has been shown to be representative of their pool size ratio, is different in NMRI and C57BL/6 mice, the latter exhibiting a higher level of cerebral lactate dehydrogenase A ( Ldha) expression. On the basis of this observation, and an additional set of experiments showing that the cerebral conversion of [1- <sup>13</sup> C]lactate to [1- <sup>13</sup> C]pyruvate increases after exposing the brain to ultrasound irradiation that reversibly opens the BBB, we concluded that lactate transport is rate-limited by the BBB, with a 30% increase in lactate uptake after its disruption. It was also deduced from these results that hyperpolarized <sup>13</sup> C MRS can be used to detect a variation in cerebral lactate uptake of <40 nmol in a healthy brain during an in vivo experiment lasting only 75 s, opening new opportunities to study the role of lactate in brain metabolism

Manganese Enhanced MRI for Use in Studying Neurodegenerative Diseases

MRI has been extensively used in neurodegenerative disorders, such as Alzheimer’s disease (AD), frontal-temporal dementia (FTD), mild cognitive impairment (MCI), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). MRI is important for monitoring the neurodegenerative components in other diseases such as epilepsy, stroke and multiple sclerosis (MS). Manganese enhanced MRI (MEMRI) has been used in many preclinical studies to image anatomy and cytoarchitecture, to obtain functional information in areas of the brain and to study neuronal connections. This is due to Mn2+ ability to enter excitable cells through voltage gated calcium channels and be actively transported in an anterograde manner along axons and across synapses. The broad range of information obtained from MEMRI has led to the use of Mn2+ in many animal models of neurodegeneration which has supplied important insight into brain degeneration in preclinical studies. Here we provide a brief review of MEMRI use in neurodegenerative diseases and in diseases with neurodegenerative components in animal studies and discuss the potential translation of MEMRI to clinical use in the future

The Physical Mechanism of Blood-Brain Barrier Opening Using Focused Ultrasound and Microbubbles

The key to effective treatment of neurological diseases resides in the safe opening of the blood-brain barrier (BBB), a specialized structure that impedes the delivery of therapeutic agents to the parenchyma. Despite the fact that several approaches have been successful in overcoming the BBB impermeability, none of them can induce localized BBB opening noninvasively except for focused ultrasound (FUS) in conjunction with microbubbles. The physical mechanism behind the opening, however, has not been identified. Insight into the mechanism can be critical for delineating the safety profile for in both small and large animals alike. Therefore the purpose of this dissertation is to first determine the physical mechanism of FUS-induced BBB opening in mice and then translate this approach to non-human primates. To accomplish this goal, an in vivo transcranial cavitation detection system was developed and tested, built in phantoms and in vivo, to monitor the behavior of the microbubbles in the FUS bean, and to determine the type of cavitation, i.e., the activation of bubbles in an acoustic field, during BBB opening. We showed that the inertial cavitation (IC), a collapse of a bubble, which can vary from a fragmentation of the bubble to shock wave and liquid jets depending on the pressure, thereby damaging the endothelial cells of the brain capillaries, was not required to induce BBB opening in mice. With this system, the role of microbubble properties, including the diameter and shell components, in the BBB opening were determined. When the BBB opens with stable cavitation (SC), i.e., relatively moderate amplitude changes in the bubble size, the bubble diameter is similar to the capillary diameter (i.e., at 4-5, 6-8 µm) while with inertial cavitation it is not (i.e., at 1-2 µm). The bubble may thus have to be in closer proximity to the capillary wall to induce BBB opening without IC. The BBB opening properties, such as volume and permeability, however, were not affected by the shell component of the microbubbles in mice. The connection between the physical and physiological mechanism was then investigated to identify the lowest peak rarefactional pressure BBB opening threshold at 1.5 MHz (0.18 MPa). A sufficiently long pulse (pulse length = 0.5 ms) was required for the SC to induce BBB opening at the lowest pressure. However, the tight junctions, the main formation of the BBB, were found not to be disrupted after sonication at both low (0.18 MPa) and high (0.45 MPa) pressures. Therefore, the transcellular pathway may be the main route of the FUS-induced BBB opening. Finally, the cavitation-guided BBB opening system was used to induce reversible BBB opening in non-human primates. This is a major step towards clinical feasibility. In conclusion, a transcranial cavitation detection system was developed, in order to characterize the physical mechanism, the role of the microbubbles, and the corresponding physiological response of the FUS-induced BBB opening

Physical principles for scalable neural recoding

Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new approaches may be required, motivating an analysis of the fundamental physical constraints on the problem. We outline the physical principles governing brain activity mapping using optical, electrical, magnetic resonance, and molecular modalities of neural recording. Focusing on the mouse brain, we analyze the scalability of each method, concentrating on the limitations imposed by spatiotemporal resolution, energy dissipation, and volume displacement. Based on this analysis, all existing approaches require orders of magnitude improvement in key parameters. Electrical recording is limited by the low multiplexing capacity of electrodes and their lack of intrinsic spatial resolution, optical methods are constrained by the scattering of visible light in brain tissue, magnetic resonance is hindered by the diffusion and relaxation timescales of water protons, and the implementation of molecular recording is complicated by the stochastic kinetics of enzymes. Understanding the physical limits of brain activity mapping may provide insight into opportunities for novel solutions. For example, unconventional methods for delivering electrodes may enable unprecedented numbers of recording sites, embedded optical devices could allow optical detectors to be placed within a few scattering lengths of the measured neurons, and new classes of molecularly engineered sensors might obviate cumbersome hardware architectures. We also study the physics of powering and communicating with microscale devices embedded in brain tissue and find that, while radio-frequency electromagnetic data transmission suffers from a severe power–bandwidth tradeoff, communication via infrared light or ultrasound may allow high data rates due to the possibility of spatial multiplexing. The use of embedded local recording and wireless data transmission would only be viable, however, given major improvements to the power efficiency of microelectronic devices

Functional Connectivity of the Rodent Brain Using Optical Imaging

RÉSUMÉ L'objectif de cette thèse de doctorat est d’appliquer la connectivité fonctionnelle dans une variété de modèles animaux, à l’aide de plusieurs techniques d’imagerie optique. Le cerveau, même au repos, montre une activité métabolique élevée : la corrélation des fluctuations spontanées lentes permet d’identifier des régions cérébrales distantes mais connectées; d’où le terme connectivité fonctionnelle. Les changements dans l’activité spontanée peuvent donner un aperçu des processus neuronaux qui comprennent la majorité de l’activité métabolique du cerveau, et constituent en conséquent une vaste source de changements reliés aux maladies. L’hémodynamique du cerveau peut être modifiée grâce à des affections neurovasculaires et avoir un effet sur l’activité au repos. Cette thèse vise la compréhension des changements de connectivité fonctionnelle induits par des maladies, à l’aide de l’imagerie optique fonctionnelle. Les techniques d’imagerie explorées dans les deux premières contributions de cette thèse sont l’Imagerie Optique Intrinsèque et l’Imagerie par Granularité Laser. Ensemble, elles peuvent estimer les changements de consommation d'oxygène, étroitement liés à l’activité neuronale. Ces techniques possèdent des résolutions temporelles et spatiales adéquates et bien adaptées pour imager la convexité du cortex cérébral. Dans le dernier article, une modalité d’imagerie en profondeur, la Tomographie Photoacoustique a été utilisée chez le rat nouveau-né. La Tomographie par Cohérence Optique et la Tomographie Laminaire Optique font également partie de la gamme des techniques d’imagerie développées et appliquées dans d’autres collaborations. La première partie des résultats mesure les changements de connectivité fonctionnelle dans un modèle d’activité épileptiforme aiguë chez le rongeur. Il y a des augmentations ainsi que des diminutions entre les corrélations homologues, avec une faible dépendance aux crises épileptiques. Ces changements suggèrent un découplage potentiel entre les paramètres hémodynamiques dans les réseaux au repos, en soulignant l’importance d’investiguer les réseaux épileptiques à l’aide de plusieurs mesures hémodynamiques indépendantes. La deuxième partie des travaux étudie un nouveau modèle de rigidité artérielle chez la souris : la calcification unilatérale de la carotide droite. L’analyse de connectivité basé sur les régions d’intérêt montre une tendance décroissante de corrélation homologue dans les cortex moteur et cingulum. L’analyse de graphes montre une randomisation des réseaux corticaux, ce qui suggère une perte de connectivité; plus spécifiquement, dans le cortex moteur ipsilateral à la carotide----------ABSTRACT The aim of this thesis is to apply functional connectivity in a variety of animal models, using several optical imaging modalities. Even at rest, the brain shows high metabolic activity: the correlation in slow spontaneous fluctuations identifies remotely connected areas of the brain; hence the term “functional connectivity”. Ongoing changes in spontaneous activity may provide insight into the neural processing that takes most of the brain metabolic activity, and so may provide a vast source of disease related changes. Brain hemodynamics may be modified during disease and affect resting-state activity. The thesis aims to better understand these changes in functional connectivity due to disease, using functional optical imaging. The optical imaging techniques explored in the first two contributions of this thesis are Optical Imaging of Intrinsic Signals and Laser Speckle Contrast Imaging, together they can estimate the metabolic rate of oxygen consumption, that closely parallels neural activity. They both have adequate spatial and temporal resolution and are well adapted to image the convexity of the mouse cortex. In the last article, a depth-sensitive modality called photoacoustic tomography was used in the newborn rat. Optical coherence tomography and laminar optical tomography were also part of the array of imaging techniques developed and applied in other collaborations. The first article of this work shows the changes in functional connectivity in an acute murine model of epileptiform activity. Homologous correlations are both increased and decreased with a small dependence on seizure duration. These changes suggest a potential decoupling between the hemodynamic parameters in resting-state networks, underlining the importance to investigate epileptic networks with several independent hemodynamic measures. The second study examines a novel murine model of arterial stiffness: the unilateral calcification of the right carotid. Seed-based connectivity analysis showed a decreasing trend of homologous correlation in the motor and cingulate cortices. Graph analyses showed a randomization of the cortex functional networks, suggesting a loss of connectivity, more specifically in the motor cortex ipsilateral to the treated carotid; however these changes are not reflected in differentiated metabolic estimates. Confounds remain due to the fact that carotid rigidification gives rise to neural decline in the hippocampus as well as unilateral alteration of vascular pulsatility; howeve

Designing Scalable Biological Interfaces

This thesis presents the analysis and design of biological interfacing technologies in light of a need for radical improvements in scalability. It focuses primarily on structural and functional neural data acquisition, but also extends to other problems including genomic editing and nanoscale spatial control. Its main contributions include analysis of the physical limits of large-scale neural recording, experimental development of a screening platform for ion-dependent molecular recording devices, characterization of the design space for molecularly-annotated neural connectomics, and new designs for high-speed genome engineering and bio-nano-fabrication. Articulating governing principles and roadmaps for these domains has contributed to the initiation of multi-institutional projects that are strategically targeted towards scalability