Drosophila larval crawling is an attractive system to study patterned motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors including head casts, turning, and feeding. It is likely that some neurons are used in all these behaviors (e.g. motor neurons), but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We defined 10 phenotypic categories that could uniquely be evoked upon stimulation, and provide further in depth analysis of two of these categories to understand the origins of the evoked behaviors. We first identified the evolutionarily conserved Even-skipped+ interneuron phenotype (Eve/Evx). Activation or ablation of Eve+ interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting left-right synchronous timing. TEM reconstruction places the Eve+ interneurons at the heart of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction We then went on to identify a unique pair of descending neurons dubbed the ‘Mooncrawler’ descending neurons (McDNs) to be sufficient to generate reverse locomotion. We show that the McDNs are present at larval hatching, function during larval life, and are remodeled during metamorphosis while maintaining basic morphological features and neural functions necessary to generate backwards locomotion. Finally, using serial section Transmission Electron Microscopy (ssTEM) to map neural connections to upstream and downstream elements provides a mechanistic view of how sensory information is received by the McDNs and transmitted to the VNC motor system to perform backwards locomotion. Finally, we show that these McDNs are the same as those identified in recent work in Drosophila adults (Bidaye et al. 2014) to be sufficient to generate reverse locomotion. This dissertation includes previously published, co-authored material.

Neural circuitry, or how neurons connect across brain regions to form functional units, is the fundamental basis of all brain processing and behavior. There are several neural circuit analysis tools available across different model organisms, but currently the field lacks a comprehensive method that can 1) target post-synaptic neurons using a pre-synaptic driver line, 2) assess post-synaptic neuron morphology, and 3) test behavioral response of the post-synaptic neurons in an isolated manner. This work will present FLIPSOT, or Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango, which is a method developed to fulfill all three of these conditions. FLIPSOT uses a pre-synaptic driver line to drive trans-Tango, triggering heat-shock-dependent expression of post-synaptic optogenetic receptors. When heat shocked for a suitable duration of time, optogenetic activation or inhibition is made possible in a randomized selection of post-synaptic cells, allowing testing and comparison of function. Finally, imaging of each brain confirms which neurons were targeted per animal, and analysis across trials can reveal which post-synaptic neurons are necessary and/or sufficient for the relevant behavior. FLIPSOT is then tested within Drosophila melanogaster to evaluate the necessity and sufficiency of post-synaptic neurons in the Drosophila Heating Cell circuit, which is a circuit that functions to drive warmth avoidance behavior. FLIPSOT presents a new combinatory tool for evaluation of behavioral necessity and sufficiency of post-synaptic cells. The tool can easily be utilized to test many different behaviors and circuits through modification of the pre-synaptic driver line. Lastly, the success of this tool within flies paves the way for possible future adaptation in other model organisms, including mammals. / Doctor of Philosophy / The human brain is made up of billions of neurons, each of which are interconnected in various ways to allow communication. When a group of connected neurons work together to carry out a specific function, that group is known as a neural circuit. Neural circuits are the physical basis of brain activity, and different circuits are necessary for all bodily functions, including breathing, movement, regulation of sleep, memory, and all senses. Disruptions in neural circuits can be found in many brain-related diseases and disorders such as depression, anxiety, and Alzheimer's disease. One example of a neural circuit is that of temperature sensation. When someone holds a cube of ice, temperature-sensing neurons in the hand pass signals along neurons in the spine until they reach the brain. There, the signals are carried to various brain regions to be processed and recognized as cold, and eventually, pain. When the sensory signals of cold and pain grow too prominent to ignore, the person may move to avoid the feeling. In this case, the brain will send signals back down to neurons responsible for movement in the arm, allowing the person to drop the ice cube. Avoidance of temperatures that are too warm or cold is an evolutionary trait that is important in preventing the body from harm. Even in a relatively simple system like temperature sensation, neural circuits can be complex and difficult to study, especially in higher order organisms such as mammals. For this reason, it can be beneficial to use simpler animals such as Drosophila melanogaster, or the common fruit fly. Flies have far fewer neurons than humans, meaning their neuronal connections are also significantly less complicated, and there are many genetic tools available in flies that aren't available in mammalian models such as mice. Additionally, flies are inexpensive, easy to raise, and grow quickly, making them ideal for troubleshooting new tools and replicating experiments. Though somewhat different in anatomy, fly brain function is similar enough to humans and other mammals that findings can often be applied across species. Studies in flies can also be applied in other insects, such as mosquitoes, which are notorious for carrying deadly diseases. Though there are several available tools in flies to study neural circuits, many tools are better for usage in sensory neurons themselves than in the neurons that carry signals in the brain afterward. This work presents a new tool, abbreviated as FLIPSOT, that modifies and combines several existing genetic methods in order to help examine those higher order neurons. FLIPSOT allows users to determine which higher order neurons are important in leading to behavioral responses, as opposed to carrying the signal to other brain regions, such as those associated with memory. Then, FLIPSOT is implemented in a warmth-sensing neural circuit known as the Heating Cell (HC) circuit and used to identify the higher order neurons needed for fly warmth avoidance. Development of tools such as FLIPSOT helps to expand our knowledge in the fields of neural circuits and behavior. Genetic tools can also be more easily tested in flies prior to attempting to implement them in other organisms, such as mice. Finally, studying temperature in flies can help create a deeper understanding of how temperature sensation works in all animals, including humans.

No description available.

Highly organised and differentiated neural circuits form and unite to link the cortex with the basal ganglia and thalamus to mediate movement, cognition and behaviour. Previous assertions that the basal ganglia primarily acted to filter cortical information to facilitate motor outputs only have since given way to an understanding of the basal ganglia as a relay and gating structure with functionally and structurally segregated inputs, functions and outputs. Thus, cortical – basal ganglia circuits can be segregated into three broadly separable functional domains mediating motor (primary and supplementary motor cortex (SMA) and putamen), cognitive (dorsolateral prefrontal cortex (dlPFC) and caudate), and limbic (ventromedial prefrontal cortex and ventral striatum (VS)) processes. In addition, cognitive and behavioural programs that pass through the cortical – basal ganglia circuitry can be subject to filtering by the subthalamic nucleus (STN), which receives direct projections from the cortex. This work first demonstrated the functional organisation of segregated intrinsic cortical – basal ganglia circuits in humans, alongside a detailed map of functional subzones within STN, a small and technically inaccessible midbrain structure. The behavioural relevance of the defined cortical – basal ganglia circuits was investigated by examining the cognitive constructs of impulsivity and compulsivity. Waiting impulsivity, a tendency towards rapid premature responses that has been associated with compulsive drug use, was associated with connectivity between limbic regions including subgenual anterior cingulate cortex, VS and STN. However, motor impulsivity, in the form of stopping ability, was associated with motoric regions including pre-SMA and STN. Compulsivity was captured as deficits in: reversal learning, implicating lateral orbitofrontal cortex; attentional shifting, implicating dlPFC; and habit learning, implicating SMA. Neural circuit changes were also examined in individuals with alcohol dependence and binge drinkers. Waiting impulsivity was elevated in both groups and the functional connectivity, microstructural integrity and anatomical connectivity of the neural circuit underlying waiting impulsivity were associated with problematic drinking behaviours in both groups. Together, this work establishes that discrete functional subzones of small subcortical regions can be differentiated in humans and that their behavioural correlates can be similarly mapped. The definition of intrinsic network architecture underlying a particular behaviour and the demonstration its disturbance in psychiatric groups will crucially inform the development of future diagnostic and therapeutic models.

How behaviors are generated by neural circuits is one of the central questions in neurobiology. Under standard culture conditions, Caenorhabditis elegans travel by propagating sinusoidal waves, moving primarily forward, punctuated by brief runs of backing. How these behaviors are generated and altered is not well understood. Using a combination of behavioral analyses and neuronal imaging, I reveal that an activity imbalance between cholinergic A- and B-motoneurons is the key determinant of directional locomotion. Furthermore, heterotypic gap junctions that couple command interneurons and motoneurons of the backward motor circuit, mediated by innexins UNC-7 in AVA and UNC-9 in A-motoneurons, respectively, establish the B>A activity pattern required for forward movement. Loss of this coupling results in both the hyperactivation of AVA backward interneurons revealing the unregulated, endogenous activity of A-motoneurons. With equal A-motoneuron activity levels as B-motoneurons, innexin mutant animals exhibit irregular body bending (kinking) instead of executing forward motion, as well as increased backing. Through a genetic screen, I identified two stomatin-like proteins as regulators of innexin UNC-9 activity that affect C. elegans’ directional movement. The loss of function of stomatin-like unc-1 leads to the same kinker phenotype as unc-7 or unc-9 mutants. Like UNC-9, UNC-1 functions primarily in the A-motoneurons to allow forward motion, suggesting that UNC-1 is required for effective UNC-7-UNC-9 coupling between AVA and A-motoneurons. Dominant mutations in UNC-1, and another stomatin-like protein STO-6, exhibit genetic interactions with these innexin mutants. These mutations partially restore the forward movement of unc-7 mutants, in an UNC-9-dependent manner, indicating that they regulate UNC-9 channel activity in motoneurons to re-establish the B>A-motoneuron activity pattern in the absence of heterotypic gap junctions between interneurons and motoneurons. These studies describe a role of gap junctions as regulators of circuit dynamics by establishing an imbalanced motoneuron activity pattern that favors forward motion, which can be modulated by upper layer inputs. This study also identifies stomatin-like regulators of innexin hemichannel and gap junction function. Future work will focus on understanding mechanisms through which these stomatins regulate the activity of specific innexin channels in C. elegans motoneurons, as well as their contribution to the dynamic output of the C. elegans motor circuit.

How behaviors are generated by neural circuits is one of the central questions in neurobiology. Under standard culture conditions, Caenorhabditis elegans travel by propagating sinusoidal waves, moving primarily forward, punctuated by brief runs of backing. How these behaviors are generated and altered is not well understood. Using a combination of behavioral analyses and neuronal imaging, I reveal that an activity imbalance between cholinergic A- and B-motoneurons is the key determinant of directional locomotion. Furthermore, heterotypic gap junctions that couple command interneurons and motoneurons of the backward motor circuit, mediated by innexins UNC-7 in AVA and UNC-9 in A-motoneurons, respectively, establish the B>A activity pattern required for forward movement. Loss of this coupling results in both the hyperactivation of AVA backward interneurons revealing the unregulated, endogenous activity of A-motoneurons. With equal A-motoneuron activity levels as B-motoneurons, innexin mutant animals exhibit irregular body bending (kinking) instead of executing forward motion, as well as increased backing. Through a genetic screen, I identified two stomatin-like proteins as regulators of innexin UNC-9 activity that affect C. elegans’ directional movement. The loss of function of stomatin-like unc-1 leads to the same kinker phenotype as unc-7 or unc-9 mutants. Like UNC-9, UNC-1 functions primarily in the A-motoneurons to allow forward motion, suggesting that UNC-1 is required for effective UNC-7-UNC-9 coupling between AVA and A-motoneurons. Dominant mutations in UNC-1, and another stomatin-like protein STO-6, exhibit genetic interactions with these innexin mutants. These mutations partially restore the forward movement of unc-7 mutants, in an UNC-9-dependent manner, indicating that they regulate UNC-9 channel activity in motoneurons to re-establish the B>A-motoneuron activity pattern in the absence of heterotypic gap junctions between interneurons and motoneurons. These studies describe a role of gap junctions as regulators of circuit dynamics by establishing an imbalanced motoneuron activity pattern that favors forward motion, which can be modulated by upper layer inputs. This study also identifies stomatin-like regulators of innexin hemichannel and gap junction function. Future work will focus on understanding mechanisms through which these stomatins regulate the activity of specific innexin channels in C. elegans motoneurons, as well as their contribution to the dynamic output of the C. elegans motor circuit.

High precision is critical for normal neural circuit function, but that precision is not innate. The location, strength, and number of inputs in a neural circuit are modified in early postnatal development in a process called refinement. The refinement of long-range excitatory projections is well-known, but less is known about the refinement of long-range inhibitory projections. What we do know about inhibitory projection refinement comes from the glycinergic medial nucleus to the trapezoid body to lateral superior olive (MNTB-LSO) projection of the auditory brainstem. During early postnatal life, the MNTB-LSO projection undergoes morphological and physiological refinement. Notably, the MNTB-LSO projection transiently expresses vesicular glutamate transporter 3 (VGLUT3) and synaptotagmin 1 (Syt1), transiently releases glutamate, and undergoes glutamate-dependent refinement. However, it remains uncertain whether glutamate release is specific to the auditory brainstem or could be a more general phenomenon of inhibitory projections. To shed light on this question, I investigated another inhibitory projection of the hindbrain, the GABAergic Purkinje projection of the cerebellum. The Purkinje projection shares key characteristics with the MNTB-LSO projection, including its inhibitory nature, location in the hindbrain, obvious topographic organization, heterogeneity of the target cells, and expression of VGLUT3 transcript and protein. In this thesis, I sought to determine: 1) whether the expression profile of VGLUT3 and Syt1 in the Purkinje projection matches that of the MNTB-LSO projection, and whether the Purkinje projection also releases glutamate, 2) whether the expression profile of synaptic vesicle protein 2 (SV2) isoforms, SV2B and SV2C, matches the expression profile of other synaptic vesicle proteins in the Purkinje and MNTB-LSO projection, and 3) whether the Purkinje projection undergoes postnatal morphological refinement like the MNTB-LSO projection. I found that like the MNTB-LSO projection, the Purkinje projection transiently expresses VGLUT3 and Syt1, releases glutamate in early postnatal life, and may undergo morphological refinement. / Dissertation / Doctor of Philosophy (PhD) / Everything you do, whether it be playing your favorite sport or begrudgingly reading this thesis, requires neural circuits, which are the basic functional unit of the nervous system. How neurons are wired together is crucial for their role in executing a task. But how these neurons fine-tune their connections – in a process called refinement, by getting the right connections to the right location, of the right strength, and of the right number – is an open-ended question in neuroscience. Refinement is more well-studied in excitatory projection neurons, but we know very little about how refinement occurs in inhibitory projection neurons. I compare some of the unusual characteristics of what we do know about inhibitory refinement in the auditory brainstem to another famous projection of the hindbrain, the Purkinje projection. Understanding more about the refinement of inhibitory projections gives key insights into how neural circuits function and how they facilitate complex behaviours.

<p>This Thesis details the design, fabrication and characterization of organic semiconductor field effect transistors with silicon oxide-nitride-oxide-semiconductor (SONOS) gates for use in spiking analog neural circuits. The results are divided into two main sections. First, the SONOS structures, parallel plate capacitors and field effect transistors, were designed, fabricated and characterized. Second, these results are used to model spiking analog neural circuits. The modeling is achieved using PSPICE based software.</p> <p>The initial design work begins with an analysis of the basic SONOS structure. The existence of the ultrathin layers of the SONOS structure is confirmed with the use of Transmission Electron Microscopy (TEM) and Energy Dispersive Spectroscopy (EDS) scans of device stacks. Parallel plate capacitors were fabricated prior to complete transistors due to the significantly less processing required. The structure and behaviour of these capacitors is similar to that of the transistor gates which allows for the optimization of the structures prior to the fabrication of the transistors. These capacitors were fabricated using the semiconductor materials of; crystalline silicon, amorphous silicon, Zinc Oxide, copper phthalocyanine (CuPc) and tris 8-hydroxyquinolinato aluminium (AlQ3). These devices are then subjected to standard capacitance voltage (C-V) analysis. The results of this analysis demonstrate that the inclusion of SONOS structures in the capacitors (and transistors) result in a hysteresis which is the result of charge accumulation in the nitride layer of the SONOS structure. This effect can be utilized as an imbedded memory. Standard control devices were fabricated and analysed and no significant hysteresis effect was observed. The hysteresis effect is only observed after the SONOS devices are subject to high voltages (approximately 14 volts) which allows tunneling through a thin oxide layer into traps in the silicon nitride layer. This analysis was conducted to confirm that the SONOS structure causes the memory effect, not the existence of interface states that can be charged and discharged.</p> <p>The next step was to design and fabricate amorphous semiconductor field effect transistors with and without the SONOS structure. First FETs without the SONOS gates were fabricated using amorphous semiconductor materials; Zinc Oxide, CuPc and AlQ3 and then the devices were characterized. This initial step confirmed the functionality of these basic devices and the ability to fabricate working control samples. Next, SONOS gate TFTs were fabricated using CuPc as the semiconductor material. The characterization of these devices confirmed the ability to shift the transfer characteristics of the devices through a read and write mechanism similar to that used to shift the C-V characteristics of the parallel plate capacitors. Split gate FETs were also produced to examine the feasibility of individual transistors with multiple gates.</p> <p>The results of these characterizations were used to model spiking analog neural circuits. This modeling was carried out in four parts. First, representative transfer and output characteristics were used to replicate analog spiking neural circuits. This was carried out using standard PSPICE software with the modification of the discrete TFT device characteristics to represent the amorphous CuPc organic transistors. The results were found to be comparable to circuits using crystalline silicon transistors. Second, the SONOS structures were modeled closely matching the characterized results for charge and voltage shift. Third, a simple Hebbian learning circuit was designed and modeled, demonstrating the potential for imbedded memories. Lastly, split gate devices were modeled using the device characterizations.</p> / Doctor of Philosophy (PhD)

The Neuregulin 1 (NRG1)-ErbB4 signalling pathway is implicated in critical processes for the development and function of neuronal circuits. Post mortem studies have reported that elevated expression of NRG1 type 1 isoform is associated with schizophrenia. Importantly previous behavioural studies in mice that overexpress the NRG1 type 1 isoform (NRG1^tg-type-I) have suggested a schizophrenia endophenotype including impairment in the hippocampus-dependent spatial working memory, prepulse inhibition (PPI) of the startle reflex and alterations in the gamma band rhythmogenesis This study aims to reveal the cellular targets of the NRG1-ErbB4 signalling pathway and putative alterations in the function of the hippocampal network in NRG1^tg-type-I mice. Immunocytochemical analysis showed that the NRG1 receptor ErbB4 is predominantly localized in interneurons comprising parvalbumin positive (PV) and cholecystokinin (CCK) expressing cells. Comparison of the density of ErbB4-positive cells between the hippocampus of wild type (WT) and NRG1^tg-type-I mice suggested that NRG1 over-expression resulted in decreased number of ErbB4 immunopositive hippocampal interneurons. This is consistent with the proposed role of the NRG1-ErbB4 signalling in the migration of GABAergic cells during neurodevelopment and with the NRG1-mediated internalisation of the ErbB4 receptors. CCK- positive cells are a major target of NRG1-ErbB4 signalling, and therefore the NMDA receptor and AMPA receptor components of glutamatergic transmission were analysed in this population of cells by performing whole cell recordings of evoked and miniature excitatory post synaptic currents. Glutamatergic neurotransmission in CCK-positive cells was found to be compromised in the hippocampus of NRG1^tg-type-I mice. This change was attributed to hypofunction of NMDA receptors but not AMPA receptors post-synaptically. Next, the inhibitory output of CCK-positive cells to pyramidal cells was examined. Analysis of the optogenetically elicited inhibitory post synaptic currents (IPSCs) did not reveal any changes in the properties of the GABAergic synapse formed by these cells due to NRG1 over-expression Finally, the effects of this NMDA receptor hypofunction in the recurrent inhibition were analysed by performing whole cell recordings during the gamma relevant optogenetic entrainment of the hippocampal network. It was found that the disynaptic inhibition, a key synaptic interaction for the generation of gamma oscillations, depends on the NMDA receptors and was altered in the hippocampus of NRG1^tg-type-I mice. Together these data point out a key modulatory role of the NRG1-ErbB4 signalling in the neurodevelopment of cortical microcircuits and a link between ErbB4 and NMDA receptor function with a possible association to schizophrenia pathogenesis.

Network dynamics are thought to be the substrate of brain information processing and of mental representations. Moreover, network-wide dysfunctions are recognized to be at the core of several psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. The development of monosynaptically-restricted Rabies virus, G-deleted Rabies virus (ΔG-Rabies), has greatly facilitated the anatomical investigation of neural circuits, revealing the network synaptic structure upstream of defined neuronal populations. However, the inherent cytotoxicity of the Rabies virus largely restrains its use to the mere structural characterisation of neural networks. To overcome this limitation, I generated novel tools that allow the manipulation of neural networks for the entire life of the animal, without affecting neuronal and circuit properties. I first developed a viral system obtained by engineering the Rabies virus genome to eliminate its cytotoxicity. This led to the generation of a Self-inactivating Rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. I showed that SiR provides a virtually unlimited temporal window for the functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology. To further expand our ways of intervening on neural networks function I then developed a completely virus-free system, named Genetically-Encoded TransSynaptic Shuttle (GETSS), which is the only specific genetically-encoded transsynaptic tracer to date. In this thesis, I established novel approaches that provide, for the first time, the functional and genetic access to traced network elements in vivo for the lifetime of the animal, with no cytotoxic effects, no changes in the electrophysiological properties of the traced neurons and no adverse effects on network function. This opens new horizons in the functional investigation of neural circuits and potentially represent the first approaches to experimentally monitor neural circuit remodelling in vivo.