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Development of optogenetically controllable human neural network models can provide an investigative system that is relevant to the human brain. Conventional cultures of neural networks in two-dimensions (2D) have major limitations of scale. For instance, the soma of neurons in 2D is unrealistically flattened and both axon and dendrite outgrowth is restricted. Using a combination of tissue engineering techniques and the inclusion of optogenetically modified human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), the development of a three-dimensional (3D) human neural culture model within a defined 3D microenvironment is investigated in this study. Light-sensitive neurons were successfully generated by transducing Channelrhodopsin-2 (ChR2) into human iPSC-derived NPCs and neuroblastoma cells (SH-SY5Y) using lentiviral transduction. The use of neuron specific promoters for synapsin-1 (SYN1) and calcium-calmodulin kinase II (CaMKII) in driving the expression of ChR2-Yellow Florescent Protein (YFP) within the mixed neuronal populations from hiPSC-derived neurons (Axol cells) were compared. Viability of the cells at 7 day-post-infection was 80% - 97% in all conditions tested. In line with published literature, transduction efficiency of neurons at day 14 was found to be 3% - 7% for plasmids containing the SYN1 promoter and 2% - 5% for plasmids containing the CaMKII promoter. An increase in promoter driven ChR2-YFP expression was evaluated over 28 days as the neural subpopulations matured. Stably ChR2 expression continued through-out higher passages (≥ P₁₀) and possibly for periods up to several months. Both SYN1 and CaMKII promoters were found to drive the expression of ChR2 in Axol cells targeting inhibitory and excitatory neurons, respectively. 3D culture systems to support cell growth and optogenetic application were developed and characterised. Alginate hydrogel functionalised with short peptide sequence arginine-glycine-aspartate (RGD), and small molecules such as Rho Kinase inhibitor (ROCKi) and ZVAD were incorporated to increase the viability of human pluripotent stem cells (hPSCs). Investigation of cell response reveals that a flow rate of 3 ml/min and an alginate concentration of 1.8% (w/v) are optimal and that stem cell survival is significantly improved through incorporation of RGD and ROCKi. Interestingly, ChR2-YFP expression of Axol and SY5Y cells was detectable when transferred to the 3D culture system. The optogenetically modified neurons were found responsive to light stimulation, showing firing patterns and calcium events typical of early developing neurons (e.g. mixed and burst waves; single and multipeak spikes). Neuronal activities were assessed using calcium imaging. Higher numbers of calcium events were associated with CaMKII driven ChR2-YFP expression than with SYN1 in Axol cells. However, calcium activity in SH-SY5Y cells was most noticeable in neurons expressing ChR2-YFP driven by the SYN1 promoter. In primary rodent neuronal cultures, synchronous calcium firing with repetitive action potentials (APs) resulted from ChR2-YFP expression was driven by both SYN1 and CaMKII promoter upon light stimulation. By combining multi-approaches, we report for the first time on the generation of an in vitro hiPSC-derived neural network model in 3D using functionalised alginate hydrogel and involving optogenetic targeting. Expression of ChR2-YFP was found driven by both SYN1 and CaMKII promoter in the RGD-alginate bead system that cultured with Axol cells.

The understanding and treatment of brain disorders as well as the development of intelligent machines is hampered by the lack of knowledge of how the brain fundamentally functions. Over the past century, we have learned much about how individual neurons and neural networks behave, however new tools are critically needed to interrogate how neural networks give rise to complex brain processes and disease conditions. Recent innovations in molecular techniques, such as optogenetics, have enabled neuroscientists unprecedented precision to excite, inhibit and record defined neurons. The impressive sensitivity of currently available optogenetic sensors and actuators has now enabled the possibility of analyzing a large number of individual neurons in the brains of behaving animals. To promote the use of these optogenetic tools, this thesis integrates cutting edge optogenetic molecular sensors which is ultrasensitive for imaging neuronal activity with custom wide field optical microscope to analyze a large number of individual neurons in living brains. Wide-field microscopy provides a large field of view and better spatial resolution approaching the Abbe diffraction limit of fluorescent microscope. To demonstrate the advantages of this optical platform, we imaged a deep brain structure, the Hippocampus, and tracked hundreds of neurons over time while mouse was performing a memory task to investigate how those individual neurons related to behavior. In addition, we tested our optical platform in investigating transient neural network changes upon mechanical perturbation related to blast injuries. In this experiment, all blasted mice show a consistent change in neural network. A small portion of neurons showed a sustained calcium increase for an extended period of time, whereas the majority lost their activities. Finally, using optogenetic silencer to control selective motor cortex neurons, we examined their contributions to the network pathology of basal ganglia related to Parkinson’s disease. We found that inhibition of motor cortex does not alter exaggerated beta oscillations in the striatum that are associated with parkinsonianism. Together, these results demonstrate the potential of developing integrated optogenetic system to advance our understanding of the principles underlying neural network computation, which would have broad applications from advancing artificial intelligence to disease diagnosis and treatment.

L’orientation spatiale et la fonction de navigation sont des processus contrôlés par des circuits et éléments neuronaux bien précis. Le présubiculum, aire cortical de transition de la région parahippocampique, est situé entre l’hippocampe et le cortex entorhinal. Le présubiculum est impliqué dans la navigation spatiale à la fois chez l’animal et l’Homme. Plus de la moitié des neurones du présubiculum sont des cellules de direction de la tête qui déchargent en fonction de la direction prise par la tête de l’animal. Le présubiculum est un carrefour majeur pour le transfert d’information de direction de la tête et de l’information visuelle aux régions de la formation hippocampique et parahippocampique et sous-corticale. Malgré son importance fonctionnelle, le traitement de l’information au sein du circuit présubiculaire à 6 couches reste encore peu connu. Au cours de ma thèse, j’ai étudié les éléments inhibiteurs qui composent le microcircuit présubiculaire à partir de tranches aigües de cerveau de souris en utilisant la technique du patch-clamp. J’ai caractérisé les propriétés anatomique et électriques des interneurones ainsi que leur connectivité locale et à distances avec d’autres régions corticales.Dans un premier temps, j’ai étudié la diversité des interneurones exprimant la parvalbumine et la somatostatine à partir de lignées de souris transgéniques exprimant une protéine fluorescente dans les interneurones. J’ai montré l’existence des cellules en panier à décharge rapide exprimant la parvalbumine et des cellules de Martinotti à bas seuil d’activation exprimant la somatostatine. J’ai également décrit un troisième groupe atypique avec des propriétés électriques intermédiaires et des morphologies hétérogènes. L’existence de ce groupe transitionnel pourrait s’expliquer par la présence d’interneurones exprimant à la fois la parvalbumine et la somatostatine. Ainsi, le microcircuit inhibiteur du présubiculum semble partager toute la complexité des autres aires corticales. Dans un second temps, je me suis intéressée à l’intégration des entrées thalamiques par les neurones excitateurs et inhibiteurs dans les couches superficielles du présubiculum à l’aide de la technique du double patch-clamp. J’ai montré que les axones thalamiques innervent sélectivement les couches superficielles et plus particulièrement, contactent directement les cellules de projection vers le cortex entorhinal ainsi que les interneurons exprimant la parvalbumine dans la couche 3 du présubiculum. En revanche, les interneurons exprimant la somatostatine sont indirectement recrutés par les cellules pyramidales du microcircuit. Ces interneurones joueraient un double rôle à la fois dans l’inhibition latérale et le maintien d’une décharge soutenue des cellules principales. Du fait de la forte probabilité de connexion entre les cellules principales et les interneurones exprimant la parvalbumine, ces derniers seraient impliqués dans l’inhibition de type feed-forward. Mon travail de thèse a permis d’apporter des connaissances fondamentales concernant l’inhibition au sein du présubiculum. Il a permis de dévoiler une diversité d’interneurones GABAergiques et de montrer l’existence de circuits neuronaux canoniques de type « feedforward » et « feedback » qui seraient recrutés à différents moments de la signalisation de la direction de la tête. / Spatial orientation and navigation are controlled by specific neuronal circuits and elements. The presubiculum, a transitional cortical area of the parahippocampal formation, is located between the hippocampus and the entorhinal cortex, and it participates in spatial navigation in animals and humans. More than half of presubicular neurons are head direction cells that fire as a function of the directional heading. The presubiculum is thought to be a crucial node for transferring directional heading information to the entorhinal-hippocampal network, and feeding back visual landmark information to upstream regions of the head directional circuit. Despite its functional importance, information processing within the 6-layered presubicular microcircuit remains not completely understood. During my PhD, I studied inhibitory neurons of the presubicular microcircuit in the slice preparation using patch-clamp recordings. I characterized their anatomo-physiological properties as well as their functional connectivity with local principal neurons. In the first part, I examined the diversity of two major populations of GABAergic neurons, the parvalbumin (PV) and somatostatin (SOM) expressing interneurons in mouse presubiculum. Using transgenic mouse strains Pvalb-Cre, Sst-Cre and X98, where interneurons were fluorescently labeled, I showed the existence of typical PV fast-spiking basket-like interneurons mainly in the Pvalb-Cre line and SOM low-threshold spiking Martinotti cell-like interneurons in the X98 and Sst-Cre line. Unsupervised cluster analysis based on electrophysiological parameters further revealed a transitional group containing interneurons from either Pvalb-Cre or Sst-cre lines with quasi-fast-spiking properties and heterogeneous morphologies. A small subpopulation of ~6% of interneurons co-expressed PV and SOM in mouse presubiculum. The presubiculum appears to share the whole complexity of other cortical areas in term of inhibition. In the second part, I investigated the integration of thalamic inputs by principal neurons as well as PV and SST interneurons in the presubiculum using double patch-clamp recordings. I found that thalamic axons selectively innervated superficial layers and made direct synaptic contacts with pyramidal neurons that project to medial entorhinal cortex and also with PV interneurons in superficial layer 3. In contrast, SST interneurons were indirectly recruited by presubicular pyramidal cells in a facilitating and frequency dependent manner. They may mediate lateral inhibition onto nearby principal cells, and at the same time, preserve sustained firing of principal neurons. In paired recording experiments, I found that PV cells inhibit neighboring pyramidal neurons with a high connection probability. PV interneurons are rapidly recruited by thalamic excitation and mediate feed-forward inhibition in presubicular pyramidal neurons. My PhD work brought fundamental knowledge about the presubicular inhibitory microcircuit. It has unraveled different populations of GABAergic interneurons and revealed canonical feedforward and feedback inhibitory motifs that are likely to be recruited at different times during head direction signaling.

Comment les odeurs contrôlent-elles le comportement animal ? Dans ma thèse, j'ai utilisé des manipulations optogénétiques et chimiogénétiques in vivo de l'activité neurale combinées à des analyses comportementales pour explorer l'organisation de circuits cérébraux impliqués dans des comportements olfactifs chez la souris. J'ai mis au point un test de conditionnement aversif olfactif indépendant de l'intensité des odeurs. J'ai démontré que les souris pouvaient généraliser une réponse aversive en présentant différentes concentrations d'odeurs. J’ai ensuite testé si les souris pouvaient apprendre cette tâche en inactivant les interneurones exprimant la parvalbumine dans le cortex olfactif (piriforme). J'ai trouvé que l’inactivation des cellules PV, n'était pas suffisante pour abolir l'aversion aux odeurs acquise, ce qui suggère que des composants de circuits neuronaux supplémentaires contribuent à la perception de l'odeur indépendamment de sa concentration. Ensuite, j'ai tenté de comprendre la constitution relative des différentes voies neurales du piriforme dans ce comportement d’aversion apprise. À l'aide d'outils génétiques et viraux, j'ai ciblé des sous-populations distinctes de neurones piriformes, et j'ai constaté que l'activité neurale induite par la lumière dans les cellules du piriforme projetant vers le bulbe olfactif et vers le cortex préfrontal, mais pas dans les cellules du piriforme projetant vers l’amygdale corticale et vers le cortex entorhinal latéral était suffisante pour supporter le conditionnement aversif. Ces résultats contribuent à mieux comprendre les propriétés fonctionnelles des circuits neuronaux corticaux pour l'olfaction. / How do odors control animal behavior ? In my thesis, I have used in vivo optogenetic and chemogenetic manipulations of neural activity combined with behavioral analyses to explore the organization of brain circuits involved in olfactory behaviors in mice. In the first part of the thesis, I established an odor intensity-independent olfactory conditioning task. I demonstrated that mice were able to generalize a learned escape behavior across a range of different odor concentrations. I then tested if by silencing Parvalbumin-expressing interneurons in the olfactory (piriform) cortex, a candidate cell population for mediating odor concentration invariance, mice would fail to learn the task. I found that silencing PV cells was not sufficient to abolish learned aversion, suggesting that additional neural circuit components contribute to concentration-invariant odor perception. Next, I asked whether different piriform neural output pathways differed in their ability to support learned aversion. Using viral-genetic tools, I targeted distinct subpopulations of piriform neurons and I found that light-induced neural activity in only piriform principle cells could drive a behavioral response. Furthermore, I tested the sufficiency of subpopulations of piriform projection neurons to drive learned aversion. I found that photostimulation of olfactory bulb- and prefrontal cortex-projecting piriform neurons was sufficient to support aversive conditioning, but not the photostimulation of cortical amygdala- and lateral entorhinal cortex-projecting piriform neurons. Together, these results provide new insights into the functional properties of cortical neural circuits for olfaction.

There is growing knowledge that neuronal circuitry undergoes alteration throughout development. Experience plays a key role in the reorganization of neuronal circuitry through the various mechanisms of learning. For example, when an animal is deprived of sensory input such as light in one or both sides of the eye, it can result in blindness on that side. In a study of rats placed in either isolated or enriched environments, those placed in enriched environments performed better on learning tests (maze test) than those placed in isolated environment. There was increased neurogenesis, synaptogenesis, myelination and angiogenesis in rats placed in enriched environments. These were all as a result of learning, which induces neuroplasticity in the nervous system. The goals of this study were to determine how evoked movement is altered by changes in key parameters of light stimulation: intensity and period and to determine if one hour of light (optogenetic) stimulation could give rise to plastic changes in the nervous system as indicated by alterations in spontaneous motility. To ascertain how evoked motor activity influences neuronal activity through learning and experience, optogenetics was employed to evoke movement in an embryonic chick at embryonic day nine (E9) after electroporation of a channelrhodopsin variant, ChIEF, into the neural tube. I first attempted to determine the optimal intensity needed to cause neuroplasticity in an embryonic chick by varying current to a LED light to produce three different light intensities. A protocol of 5 pulses of light with a period of 2 seconds was used to illuminate the right leg of 5 embryonic chicks with each intensity. To determine the optimal period of stimulation, I varied the period to 3 s and 4 s with one animal. Stimulation for an hour with a training protocol of 1800 pulses/hour (with a period of 2 s) of blue light (470 nm) was then used to illuminate the right thigh of the embryonic chick. There were varied responses to light of all intensities used for stimulation, but high light intensity (maximum - 100%) seemed to have produced the best responses in terms of producing the largest joint angle changes and shortest latencies of movement in all joints of the leg of embryonic chick. Movements of the hip and ankle joints were the most robust. This was closely followed by those of the mid (83.33%) intensity. Therefore, it can be inferred that the greater the intensity of light, the better the response. The training protocol did not produce significant changes in embryonic activity. There were some decreases in joint angles and variable spontaneous movement duration in all animals used but there could be some changes going on at the neuronal or muscular level which were beyond the scope of this study to investigate. It is my hope that this study will provide some knowledge pertinent to the treatment or management of neurodevelopmental disorders that may result in paraplegia or Erb's palsy.

abstract: Optical Fibers coupled to laser light sources, and Light Emitting Diodes are the two classes of technologies used for optogenetic experiments. Arizona State University's Flexible Display Center fabricates novel flexible Organic Light Emitting Diodes(OLEDs). These OLEDs have the capability of being monolithically fabricated over flexible, transparent plastic substrates and having power efficient ways of addressing high density arrays of LEDs. This thesis critically evaluates the technology by identifying the key advantages, current limitations and experimentally assessing the technology in in-vivo and in-vitro animal models. For in-vivo testing, the emitted light from a flat OLED panel was directly used to stimulate the neo-cortex in the M1 region of transgenic mice expressing ChR2 (B6.Cg-Tg (Thy1-ChR2/EYFP) 9Gfng/J). An alternative stimulation paradigm using a collimating optical system coupled with an optical fiber was used for stimulating neurons in layer 5 of the motor cortex in the same transgenic mice. EMG activity was recorded from the contralateral vastus lateralis muscles. In vitro testing of the OLEDs was done in primary cortical neurons in culture transfected with blue light sensitive ChR2. The neurons were cultured on a microelectrode array for taking neuronal recordings. / Dissertation/Thesis / ICMS response in front and hind limb / Optogenetic response using iLEDs and OLEDs / iLED vs iLED coupled to optical fiber response / Masters Thesis Bioengineering 2015

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-26T20:08:35Z No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-29T20:35:41Z (GMT) No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) / Made available in DSpace on 2016-04-29T20:35:41Z (GMT). No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) Previous issue date: 2015-06-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Tinnitus is the perception of a sound in the absence of a corresponding physical stimulus. It is not clear yet what mechanisms are involved in tinnitus and how it starts and/or becomes chronic. Due to the relationship between tinnitus and somatosensory trauma/stimuli, the dorsal cochlear nucleus (DCN), a region known to integrate somatosensory and auditory pathways, has been identified as a potential key structure in the generation of phantom sound perception. Here, we target specific neuronal populations in the DCN to allow further investigation on how this region may contribute to the generation of tinnitus signals that spread to other auditory areas. We examined the expression of optogenetic proteins (Channelrhodopsin 2 - ChR2; and enhanced Archaerhodopsin 3.0 - eArch3.0), targeting neurons expressing Calmoduline Kinase II alpha (CaMKIIa) promoter in wild-type C57/Bl6 mice and neurons expressing nicotinic acetylcholine receptor subunit alpha-2 promoter (ChRNA2) in ChRNA2- Cre transgenic C57/Bl6 mice, using local virus injection, verified by fluorescence microscopy. Unit responses were differentiated based on their electrophysiological response to sound. We then investigated if firing of neurons expressing optogenetic tools can be controlled in vivo and if the same neurons also fire action potentials in response to precisely timed sound stimulation. Both in vivo and preliminary in vitro data shows that neurons expressing ChR2 do respond to sound, and that they furthermore also can respond to light stimulation with a stable and similar waveform. Moreover, in vivo data shows that neurons expressing eArch3.0, responding to sound, will decrease their firing rate when exposed to green light. Thereby showing that optogenetic tools can be used functionally in the DCN, it is possible to further test tinnitus theories by, for example, producing an increased firing rate in the DCN, trying to mimic tinnitus; or inhibiting increased spontaneous firing rate in the DCN of animals with noise-induced or salycilate-induced tinnitus.

Various small molecules act as neurotransmitters and orchestrate neural communication. Growing evidence suggests that not only classical neurotransmitters but also several small molecules, including amino acid derivatives, modulate synaptic transmission. As conditions of acute and chronic pain alter neuronal excitability in the nucleus accumbens, we hypothesized that small molecules released in the nucleus accumbens might play important roles in modulating the pain sensation. However, it is not easy to identify possible pain modulators owing to the absence of a method for comprehensively measuring extracellular small molecules in the brain. In this study, through the use of an emerging metabolomics technique, namely ion chromatography coupled with high-resolution mass spectrometry, we simultaneously analyzed the dynamics of more than 60 small molecules in brain fluids collected by microdialysis, under both the application of pain stimuli and the administration of analgesics. We identified N-acetylaspartylglutamate as a potential pain modulator that is endogenously released in the nucleus accumbens. Infusion of N-acetylaspartylglutamate into the nucleus accumbens significantly attenuated the pain induced by the activation of sensory nerves through optical stimulation. These findings suggest that N-acetylaspartylglutamate released in the nucleus accumbens could modulate pain sensation.