This thesis describes work from both the University of Cambridge in the lab of Berthold Hedwig and from the HHMI Janelia Research Campus in the lab of Michael Reiser. At the University of Cambridge, my work involved the development and demonstration of a method for electrophoretically delivering dyes and tracers for anatomical and functional imaging into animals that are not amenable to genetic labelling techniques. Using this method in locusts and crickets - model systems of particular interest for their acoustic communication - I successfully delivered polar fluorescent dyes and tracers through the sheath covering the auditory nerve, simultaneously staining both the peripheral sensory structures and the central axonal projections without destroying the nerve's function. I could label neurons which extend far from the tracer delivery site on the nerve as well as local neuron populations through the brain's surface. I used the same method to deliver calcium indicators into central neuropils for in vivo optical imaging of sound-evoked activity, as well as calling song-evoked activity in the brain. The work completed at the Janelia Research Campus began with the development of a modern version of a modular LED display and virtual reality control system to enable research on the visual control of complex behaviors in head-fixed animals. The primary advantages of our newly developed LED-based display over other display technologies are its high-speed operation, brightness uniformity and control, precise synchronization with analog inputs and outputs, and its ability to be configured into a variety of display geometries. Utilizing the system's fast display refresh rates, I conducted the first accurate characterization of the upper limits of the speed sensitivity of Drosophila for apparent motion during flight. I also developed a flexible approach to presenting optic flow scenes for functional imaging of motion-sensitive neurons. Finally, through the on-line analysis of behavioral measures, image rendering, and display streaming with low latency to multi-color (UV/Green) LED panels, I demonstrated the ability to create more naturalistic stimuli and interactive virtual visual landscapes. Lastly, I used this new visual display system to explore a newly discovered cell-type that had been implicated in higher-order motion processing from a large genetic screen of visually-guided behavior deficits. Using genetic silencing and activation methods, and by designing stimuli that modeled the optic flow encountered during different types of self-motion, colleagues in the Reiser lab and I showed that this cell-type - named Lobula Plate Columnar 1 (LPC1) - is required for the stopping behavior of walking flies caused by back-to-front translation motion but is not involved in the rotational optomotor response. Using calcium imaging, I found that LPC1 was selectively excited by back-to-front motion on the eye ipsilateral to the neuron population and inhibited by front-to-back motion on the contralateral eye, demonstrating a simple mechanism for its selectivity to translation over rotation. I also examined an anatomically similar cell type - named Lobula-Lobula Plate Columnar type 1 (LLPC1) - and found that its selectivity results from a similar but opposite calculation for the detection of front-to-back translational motion. The detection of back-to-front motion had previously been hypothesized to be useful for collision avoidance, and this work provides a neural mechanism for how this detection could be accomplished, as well as providing a platform from which to explore the larger network for translation optic flow.

Les ganglions de la base (GB) forment une boucle complexe avec le cortex et le thalamus qui est impliquée dans la sélection de l’action et le contrôle du mouvement. Les activités oscillatoires synchronisées dans le réseau des GB ont été proposées comme pouvant jouer un rôle essentiel dans la coordination du flux de l’information au sein de ces circuits neuronaux. Ainsi, leur dérégulation dans le temps et l’espace pourrait devenir pathologique. Dans la maladie de Parkinson (MP), l’expression anormalement élevée d’oscillations neuronales comprises dans les gammes de fréquences beta (β, 10-30 Hz) serait la cause des déficits moteurs (akinétique et bradykinétique) de cette maladie. Cependant, les réseaux neuronaux à l’origine des oscillations β et l’implication physiopathologique de celles-ci restent encore inconnus. Le noyau sous-thalamique (NST) est un carrefour anatomique des GB situé au centre de réseaux potentiellement impliqués dans l’émergence de ces états hyper-synchronisés. L’objectif de cette thèse était de déterminer le rôle causal des principales entrées du NST (i.e. le cortex moteur, le globus pallidus, et le noyau parafasciculaire du thalamus) dans le maintien et la propagation des oscillations β. Pour cela, nous avons développé des approches de manipulation optogénétique combinées à des enregistrements électrophysiologiques in vivo dans un modèle rongeur de la MP. L’ensemble de nos travaux démontre la contribution respective des différents circuits neuronaux interrogés et souligne l’importance du globus pallidus dans le contrôle de la propagation et du maintien des oscillations β dans l’ensemble de la boucle des GB. / The basal-ganglia (BG) form a complex loop with the cortex and the thalamus that is involved in action selection and movement control. Synchronized oscillatory activities in basal-ganglia neuronal circuits have been proposed to play a key role in coordinating information flow within this neuronal network. If synchronized oscillatory activities are important for normal motor function, their dysregulation in space and time could be pathological. Indeed, in Parkinson’s disease (PD), many studies have reported an abnormal increase in the expression level of neuronal oscillations contain in the beta (β) frequency range (15-30 Hz). These abnormal β oscillations have been correlated with two mains symptoms of PD: akinesia/bradykinesia. However, which BG neuronal circuits generate those abnormal β oscillations, and whether they play a causal role in PD motor dysfunction is not known. The subthalamic nucleus (STN) is a key nucleus in BG that receives converging inputs from the motor cortex, the parafascicular thalamic nucleus and the globus pallidus. Here, we used a rat model of PD combined with in vivo electrophysiological recordings and optogenetic silencing to investigate how selective manipulation of STN inputs causally influence BG network dynamic. Our data highlight the causal role of the globus pallidus in the generation and propagation mechanisms of abnormal β-oscillations.

La stimulation cérébrale profonde (SCP) est actuellement un traitement efficace pour la maladie de parkinson. Cette approche est maintenant fortement envisagée pour le traitement des addictions. Elle consiste à délivrer des impulsions électriques au sein d’une structure cérébrale : le noyau subthalamique. Nous avons montré dans le noyau subthalamique l’existence de signatures associée à la transition vers l’addiction et la prise compulsive de drogue, ainsi que le potentiel thérapeutique de la SCP pour réduire la consommation pathologique et compulsive de cocaïne chez des rats. Nous avons également montré le contrôle spécifique du noyau subthalamique sur la motivation pour la nourriture sucrée et les drogues d’abus. Dans l’ensemble, cette thèse devrait permettre une meilleure compréhension des mécanismes de la SCP, de son potentiel thérapeutique pour les addictions et de ses éventuels effets secondaires. / Deep brain stimulation (DBS) is currently one form of effective treatment for Parkinson’s disease. This approach is currently considered for the treatment of addiction. It consists in the delivery of small electric impulses inside a brain structure: the subthalamic nucleus. We have shown in the subthalamic nucleus the existence of signature associated with the transition to addiction and compulsive drug abuse, as well as the therapeutic potential of DBS to reduce pathological intake and compulsive cocaine abuse in rats. We also established the specific control exerted by the subthalamic nucleus on the motivation for sweet food and drug of abuse. Overall this thesis could allow a better understanding of the mechanisms of DBS, its therapeutic potential in addiction and possible side effects.

A major challenge in the field of neuroscience is to link behavior with specific neuronal circuitries and cellular events. One way of facing this challenge is to identify unique cellular markers and thus have the ability to, through various mouse genetics tools, mimic, manipulate and control various aspects of neuronal activity to decipher their correlation to behavior. The Vesicular Glutamate Transporter 2 (VGLUT2) packages glutamate into presynaptic vesicles for axonal terminal release. In this thesis, VGLUT2 was used to specifically target cell populations within the basal ganglia of mice with the purpose of investigating its connectivity, function and involvement in behavior. The motor and limbic loops of the basal ganglia are important for processing of voluntary movement and emotions. During such physiological events, dopamine plays a central role in modulating the activity of these systems. The brain reward system is mainly formed by dopamine projections from the ventral tegmental area (VTA) to the ventral striatum. Certain dopamine neurons within the VTA exhibit the ability to co-release dopamine and glutamate. In paper I, glutamate and dopamine co-release was targeted and our results demonstrate that the absence of VGLUT2 in dopamine neurons leads to perturbations of reward consumption and reward-associated memory, probably due to reduced DA release observed in the striatum as detected by in vivo chronoamperometry. In papers II and IV, VGLUT2 in a specific subpopulation within the subthalamic nucleus (STN) was identified and targeted. Based on the described role of the STN in movement control, we hypothesized that the mice would be hyperlocomotive. As shown in paper II, this was indeed the case. In paper IV, a putative reward-related phenotype was approached and we could show reduced operant-self administration of sugar and altered dopamine release levels suggesting a role for the STN in reward processes. In paper III, we investigated and identified age- and sex-dimorphisms in dopamine kinetics in the dorsal striatum of one of the most commonly used mouse lines worldwide, the C57/Bl6J. Our results point to the importance of taking these dimorphisms into account when utilizing the C57/Bl6J strain as model for neurological and neuropsychiatric disorders.

Optogenetics is a set of technologies that enable optically triggered gain or loss of function in genetically specified populations of cells. Optogenetic methods have revolutionized experimental neuroscience by allowing precise excitation or inhibition of firing in specified neuronal populations embedded within complex, heterogeneous tissue. Although optogenetic tools have greatly improved our ability manipulate neural activity, they do not offer control of neural firing in the face of ongoing changes in network activity, plasticity, or sensory input. In this thesis, I develop a feedback control technology that automatically adjusts optical stimulation in real-time to precisely control network activity levels. I describe hardware and software tools, modes of optogenetic stimulation, and control algorithms required to achieve robust neural control over timescales ranging from seconds to days. I then demonstrate the scientific utility of these technologies in several experimental contexts. First, I investigate the role of connectivity in shaping the network encoding process using continuously-varying optical stimulation. I show that synaptic connectivity linearizes the neuronal response, verifying previous theoretical predictions. Next, I use long-term optogenetic feedback control to show that reductions in excitatory neurotransmission directly trigger homeostatic increases in synaptic strength. This result opposes a large body of literature on the subject and has significant implications for memory formation and maintenance. The technology presented in this thesis greatly enhances the precision with which optical stimulation can control neural activity, and allows causally related variables within neural circuits to be studied independently.

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

<div>The visual system is responsible for processing visual input, inferring its environmental causes, and assessing its behavioral significance that eventually relates to visual perception and guides animal behavior. There is emerging evidence that visual perception does not simply mirror the outside world but is heavily influenced by contextual information. Specifically, context might refer to the sensory, cognitive, and/or behavioral cues that help to assess the behavioral relevance of image features. One of the most famous examples of such behavior is visual or optical illusions. These illusions contain sensory cues that induce a subjective percept that is not aligned with the physical nature of the stimulation, which, in turn, suggests that a visual system is not a passive filter of the outside world but rather an active inference machine.</div><div>Such robust behavior of the visual system is achieved through intricate neural computations spanning several brain regions that allow dynamic visual processing. Despite the numerous attempts to gain insight into those computations, it has been challenging to decipher the circuit-level implementation of contextual processing due to technological limitations. These questions are of great importance not only for basic research purposes but also for gaining deeper insight into neurodevelopmental disorders that are characterized by altered sensory experiences. Recent advances in genetic engineering and neurotechnology made the mouse an attractive model to study the visual system and enabled other researchers and us to gain unprecedented cellular and circuit-level insights into neural mechanisms underlying contextual processing.</div><div>We first investigated how familiarity modifies the neural representation of stimuli in the mouse primary visual cortex (V1). Using silicon probe recordings and pupillometry, we probed neural activity in naive mice and after animals were exposed to the same stimulus over the course of several days. We have discovered that familiar stimuli evoke low-frequency oscillations in V1. Importantly, those oscillations were specific to the spatial frequency content of the familiar stimulus. To further validate our findings, we investigated how this novel form of visual learning is represented in serotonin-transporter (SERT) deficient mice. These transgenic animals have been previously found to have various neurophysiological alterations. We found that SERT-deficient animals showed longer oscillatory spiking activity and impaired cortical tuning after visual learning. Taken together, we discovered a novel phenomenon of familiarity-evoked oscillations in V1 and utilized it to reveal altered perceptual learning in SERT-deficient mice.</div><div>16</div><div>Next, we investigated how spatial context influences sensory processing. Visual illusions provide a great opportunity to investigate spatial contextual modulation in early visual areas. Leveraging behavioral training, high-density silicon probe recordings, and optogenetics, we provided evidence for an interplay of feedforward and feedback pathways during illusory processing in V1. We first designed an operant behavioral task to investigate illusory perception in mice. Kanizsa illusory contours paradigm was then adapted from primate studies to mouse V1 to elucidate neural correlates of illusory responses in V1. These experiments provided behavioral and neurophysiological evidence for illusory perception in mice. Using optogenetics, we then showed that suppression of the lateromedial area inhibits illusory responses in mouse V1. Taken together, we demonstrated illusory responses in mice and their dependence on the top-down feedback from higher-order visual areas.</div><div>Finally, we investigated how temporal context modulates neural responses by combining silicon probe recordings and a novel visual oddball paradigm that utilizes spatial frequency filtered stimuli. Our work extended prior oddball studies by investigating how adaptation and novelty processing depends on the tuning properties of neurons and their laminar position. Furthermore, given that reduced adaptation and sensory hypersensitivity are one of the hallmarks of altered sensory experiences in autism, we investigated the effects of temporal context on visual processing in V1 of a mouse model of fragile X syndrome (FX), a leading monogenetic cause of autism. We first showed that adaptation was modulated by tuning properties of neurons in both genotypes, however, it was more confined to neurons preferring the adapted feature in FX mice. Oddball responses, on the other hand, were modulated by the laminar position of the neurons in WT with the strongest novelty responses in superficial layers, however, they were uniformly distributed across the cortical column in FX animals. Lastly, we observed differential processing of omission responses in FX vs. WT mice. Overall, our findings suggest that reduced adaptation and increased oddball processing might contribute to altered perceptual experiences in FX and autism.</div>

Following a stroke, normal usage of the impaired limb guides spontaneous recovery across many months or even years; however, recovery is rarely complete. Pre-clinical tools are needed to investigate stroke-induced cortical reorganization over long periods. This thesis aims to characterize stroke impairment and spontaneous recovery in parallel with a battery of behaviour tasks in a mouse model of focal stroke. Young adult Thy1-ChR2 mice were implanted with a transcranial window over the intact skull permitting cortex visualization and enabling longitudinal assessments with light-based motor mapping and intrinsic signal optical imaging. Furthermore, mice were tested on sensorimotor behavioural tasks in parallel to the mapping experiments. These experiments allowed for the quantification of impairments in the sensorimotor cortex and forelimb function while identifying regions within the sensorimotor cortex that show re-mapping associated with behavioural recovery. Following primary motor cortex-stroke induction, both sensory and motor map impairments occurred. Sensory map transient impairments recovered within the same atlas-defined regions two weeks after a primary motor cortex stroke as identified by intrinsic signal optical imaging. In contrast, motor forelimb recovery was observed four weeks after the stroke in the peri-infarct region, the supplemental motor cortex, and the contralesional motor cortex. This recovery was identified through a combination of analyses, including changes in the mapped area and the amplitude of evoked forelimb movements using light-based motor mapping. Behavioural recovery occurred four to six weeks post-stroke, depending on the sensitivity of the task in forelimb impairment. Additionally, the contralesional hemisphere and forelimb did not show impairment acutely but evoked forelimb amplitude was significantly increased by post-stroke week four for both forelimbs. As the first study to conduct within-animal longitudinal spontaneous recovery sensory and motor map experiments using bilateral forelimb and hemispheric representations, we show that 1) photothrombotic stroke impacts both forelimb representations pertained within the ipsilesional hemisphere in LBMM experiments, 2) recovery of the impaired forelimb occurs ipsilesionally and contralesionally and, 3) impairments from stroke observed through motor mapping are functionally relevant and precede behavioural recovery ranging from zero to two or more weeks depending on the motor cortex's involvement in the behavioural task.

Current T-cell tracking techniques in vivo are limited. The ability to successfully target a gene in vivo in T-cells and track movement throughout its life cycle provides an exciting opportunity to elucidate the functions of genes. The aim of this study was to test an optogentically inducible Cre recombinase as well as a self-cleaving gRNA which can find and associate with Cas9 in vivo. Mouse T-cells which consecutively produce Cas9 (Cas 9, Jackson laboratory) were transduced and transplanted in immunodeficient mice (TCRb-/-, Jackson laboratory). The optogenetic component of the system is activated upon blue light stimulation and is introduced to the T-cell through a mouse stem cell virus (MSCV). The TCRb-/- mice underwent surgery which exposed their lymph nodes to blue light pulses from a fibre optic wire, this process is referred to as blue light surgery. BLU-VIPR T-cells which express self-cleaving gRNAs reduced the relative abundance of the target protein (Thy1.2), after blue light surgery in vivo. Furthermore, the optogenetic system showed minimal leakiness when used for gene targeting using gRNAs. This suggests that the gRNAs had associated with Cas9 and were able to successfully target the Thy1.2 gene. Results from the optogentically induced Cre recombinase showed that Cre was expressed in significant amounts without blue light stimulation, suggesting some background leakiness in the BLU-VIPR system.

Synaptic plasticity describes efficacy changes in synaptic transmission and ranges in duration from tens to hundreds of milliseconds (short-term), to hours and days (long-term). Short-term plasticity plays crucial roles in synaptic computation, information processing, learning, working and short-term memory as well as its dysfunction in psychiatric and neurodegenerative diseases. The main aim of my PhD thesis was to determine the molecular mechanisms of different forms of presynaptic plasticity. Short-term facilitation increases neurotransmitter release in response to a high-frequency pair (paired-pulse facilitation; PPF) or train (train facilitation; TF) of presynaptic stimuli. Synaptotagmin 7 (Syt7) has been shown to act as residual calcium (Ca$_{res}$) sensor for PPF and TF at various synapses. Syt7 also seems to be involved in recovery from depression, whereas its role in neurotransmission remains controversial. My aim was to express Syt7 in a synapse where it is not normally found and determine how it affects short-term synaptic plasticity. Immunohistochemistry indicated that Syt7 is not localized to cerebellar climbing fibers (CFs). Wild-type (WT) and Syt7 knockout (KO) recordings at CF to Purkinje cell (CF-PC) synapses established that at near-physiological external calcium (Ca$_{ext}$) levels both genotypes displayed similar recovery from paired-pulse depression. In low Ca$_{ext}$,WT CF-PC synapses showed robust PPF, which turned out to be independent of Syt7. All my experiments strongly suggested that WT CFs do not express native Syt7, but display low Ca$_{ext}$ CF-PC PPF and TF. Thus, channelrhodopsin-2 and Syt7 were bicistronically expressed via AAV9 virus in CFs. This ectopic Syt7 expression in CFs led to big increases in low-Ca$_{ext}$ CF-PC facilitation, more than doubling PPF and more than tripling TF. While overexpression of Syt7 might turn out to have an effect on the initial release probability (pr), the observed CF-PC facilitation increase still critically depended on presynaptic Syt7 expression. And when comparing only cells in a defined EPSC1 amplitude range, the Syt7-induced increase in low-Ca$_{ext}$ PPF could not be accounted for by changes in initial pr, suggesting a general role for Syt7 as calcium sensor for facilitation. Another form of short-term plasticity, post-tetanic potentiation (PTP), is believed to be mediated presynaptically by calcium-dependent protein kinase C (PKC) isoforms that phosphorylate Munc18-1 proteins. It is unknown how generally applicable this mechanism is throughout the brain and if other proteins might be able to modulate PTP. Combining genetic (PKCαβy triple knockout [TKO] and Munc18-1SA knock-in [Munc18 KI] mice, in which Munc18- 1 cannot get phosphorylated) with pharmacological tools (PKC inhibitor GF109203), helped us show that PTP at the cerebellar parallel fiber to Purkinje cell (PF-PC) synapse seems to depend on PKCs but seems mostly independent of Munc18-1 phosphorylation. In addition, compared to WT animals, genetic elimination of presynaptic active zone protein Liprin-α3 led to similar PF-PC PTP and paired-pulse ratios (PPRs). At the hippocampal CA3-CA1 synapse previous pharmacological studies suggested that PKC mediates PTP. A genetic approach helped to show that calcium dependent PKCs do not seem to be required for CA3-CA1 PTP. Pharmacologically inhibiting protein kinase A as well as genetically eliminating Syt7 also had no effect on CA3-CA1 PTP. In addition, Ca IM-AA mutant mice, in which Ca$_{v}$2.1 channels have a mutated IQ-like motif (IM) so that it cannot get bound by calcium sensor proteins any more, not only displayed regular PTP, but also normal PPF and TF at CA3-CA1 synapses. In conclusion, my PhD thesis helped further characterize different forms of presynaptic plasticity, underlined that short-term synaptic plasticity can be achieved through diverse mechanisms across the Mammalian brain and supported a potentially general role for synaptotagmin 7 acting as residual calcium sensor for facilitation.