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.

Antibiotics and their corresponding resistance genes act as a tool to control bacterial survival. Antibiotic resistance is used to select for desired engineered cells, and study how pathogens acquire resistance to continue infection. Here, we develop tools to control the expression of antibiotic resistance genes using light. To accomplish this, we use optogenetics, the regulation of cellular behavior using light as a direct and programmable input for gene expression. We develop an optogenetic recombinase in Escherichia coli through split-protein engineering techniques, and characterize the behavior of our best candidate in detail: a split Cre recombinase that responds to blue light. We apply this optogenetic system to control the expression of resistance genes for four antibiotics: ampicillin/carbenicillin, kanamycin, chloramphenicol, and tetracycline. By varying the expression levels of these genes, we tune the concentrations at which bacteria can survive before and after light exposure. We then apply this system to improve production of fatty acids. Finally, we make progress toward characterizing the impact of resistance activation timing on bacterial survival. This work creates tools that are broadly useful for spatiotemporal control of bacterial survival, and enables precise studies on how bacterial resistance spreads at the single-cell level. / 2024-08-29T00:00:00Z

The autonomic nervous system (ANS) controls most involuntary functions of the body. Dysfunctions of the ANS can be life-threatening. However, several critical questions related to cardiovascular and breathing regulations remain unclear. One of the open questions is how the system lose control of the vascular tones under certain circumstances. Using the septic shock model induced by lipopolysaccharide (LPS) in isolated and perfused mesenteric arterial rings, we found the vascular hyporeactivity is attributed to the decreased vasoconstriction to α-adrenoceptor agonists. The endotoxin-induced vasodilation can be intervened with endothelin-1 (ET-1), serotonin (5-HT) or vasopressin, which have never been used in clinical treatment. It is unclear how the excitability of endothelium affects vascular tones. Using optogenetics and transgenic mice with channelrhodopsin expression in endothelial cells (ECs), we found selective activation of the ECs induces a fast, robust, reproducible and long-lasting vasoconstriction in isolated and perfused hearts and kidneys. Breathing control by the ANS within the brain becomes abnormal in certain genetic diseases, such as Rett syndrome with defected norepinephrine (NE) system in locus coeruleus (LC). The LC neurons are hyperexcitable while NE release is deficient. Using optogenetics and double transgenic mice with Mecp2 null and channelrhodopsin expression in LC neurons, we found the NE-ergic modulation of hypoglossal neurons was impaired in transgenic mice, which cannot be improved with optostimulation, suggesting that LC neuronal hyperexcitability may not benefit the NE modulation in Rett syndrome. Collectively, our results provide insight into the autonomic dysfunctions using experimental interventions that have barely been used before.

The circuit of recurring inhibition between motor neurons and Renshaw cells in the spinal cord has been known for around 70 years, though no determined function has been outlined as of yet. Renshaw cells are thought to be part of the central pattern generator in the spinal cord establishing them as an important part of the animal’s locomotive properties. In this study we aimed to investigate the role of Renshaw cells in locomotion with the help of optogenetics and electrophysiology. Halorhodopsin was inserted into the genome of mice and driven to expression with Cre recombinase in Renshaw cells. The spinal cord of P3 mice was extracted and by inducing fictive locomotion with appropriate neurotransmitters we could inhibit the Renshaw cells in action with a green laser, opening the halorhodopsin channels for Cl- ions. In previous experiments where the ability of Renshaw cells to release inhibitory neurotransmitters was inactivated, no effect was observed in either behavioral experiments or electrophysiological experiments. In a system where the effect of Renshaw cells was knocked out acutely with optogenetics there was no discernible change in fictive locomotion cycle length, frequency or amplitude. Nor was there an effect on alternation. The access of light to the Renshaw cells area might have been limited during the experiment considering the angle of light delivery and strength of the laser. Furthermore, the maturity of Renshaw cells at P3, the exclusive ability of the marker used to target Renshaw cells and the observed nature of neonatal inhibitory neurons acting as excitatory neurons could all be called into question about whether they contributed to these results or not.

Astrocytes are the most abundant non-neuronal cells in vertebrate brains. Although Drosophila melanogaster has fewer astrocytic cells relative to neuronal and other glial cell populations, they, like vertebrate astrocytes, are located in synaptic regions, organized into exclusive, minimally-overlapping domains, and play developmental roles in synaptogenesis. But, do Drosophila astrocytes have parallel roles in the regulation of synaptic signaling? Preliminary electron microscopic (EM) data indicates that astrocytic processes are located at a greater distance, on average, from Drosophila synapses than they are from vertebrate synapses, thus raising questions about their capacity to alter synaptic signals. Do astrocytic cells and processes occupy stereotyped synaptic regions across repeating segmental structures and across individuals? In the studies presented here, we have addressed these questions directly in the ventral nerve cord (VNC) of the third-instar larva. We collected the first whole-cell patch-clamp recordings from Drosophila astrocytes. These indicate that intrinsic membrane properties, such as low membrane resistance, high capacitance, a hyperpolarized resting potential relative to neurons, a passive current-voltage relationship, coupling to other astrocytic cells, and an absence of voltage-gated currents, are shared between astrocytes of highly divergent species. Next, we optogenetically activated of a group of glutamatergic pre-motor neurons and showed that astrocytes respond with a glutamate transporter current that is mediated by Eaat1, and that acute, pharmacological and chronic, genetic blockades of this transporter have subsequent effects on the decay of post-synaptic motor neuron currents. Then, we used three-dimensional EM to locate the pre-motor glutamatergic neurons that were activated in the physiological study and measured the distance from each presynaptic site to the nearest astrocytic process. We found that these distances vary 100-fold even along a single neurite and that these structures are rarely in direct contact, but that no synapse is positioned greater than one micron from an astrocytic process. Thus, it is in this anatomical configuration that the regulation of post-synaptic currents by Eaat1 occurs. Finally, we generated a library of single, fluorescently-labeled astrocytes that were co-labeled with fiduciary landmarks, and used this library to compare the placement of astrocyte cell bodies and arbors across VNC segments and individuals. We found substantial variation in the gross shape, size, and territory covered by astrocytes, and conclude that their neuropil domains are not reliably stereotyped. Given the consistent placement of neuronal connectome elements, this indicates that signals of a specific synapse are not regulated by a designated astrocyte. Together, these findings reveal new functional parallels between Drosophila and vertebrate astrocytes. These findings argue for the relevance and applicability of mechanistic discovery in Drosophila astrocytes, and set the stage for further inquiry into the genetic determinants of astrocyte morphology and physiology.

A large percentage of individuals with intractable epilepsies have an accompanying cortical malformation, the underlying cellular mechanisms of which are poorly understood. It is known however that in an animal model for one such malformation, polymicrogyria, epileptogenesis occurs most easily from an adjacent area termed the paramicrogyral region (PMR). Previous studies implicate SS interneurons as a potential contributor to this pathology, which lead to our hypothesis: in PMR, SS interneurons exert a higher modulatory influence on excitatory pyramidal cells, as compared to the same by SS interneurons within homologous control cortex. Using a freeze-lesion model for polymicrogyria in transgenic mice that selectively express either Channelrhodopsin or Archaerhodopsin optogenetic channels in these cells, we assessed the contribution of SS interneurons as it potentially differs between layer V of PMR and control cortex. These studies provided the following biological examples in support of previous extrapolations that indicate SS over-activation within PMR: (1) SS interneuron mediated evocation of inhibitory events in layer V excitatory neurons is more robust in PMR than in control. Similarly, electrically-evoked inhibitory events in these excitatory neurons trend towards being larger, signifying a larger contribution by interneurons. (2) SS interneuron mediated suppression of electrically-evoked responses trends towards being stronger in PMR; and (3) the selective silencing of SS interneurons might not impart an effect on spontaneous inhibitory postsynaptic events.

Epilepsy has been previously attributed to either increased excitation or decreased inhibition. With this closed frame of mind, modern medicine has been unable to develop a permanent treatment against the mechanisms of epilepsy. In order to treat patients with intractable seizures, especially those caused by developmental malformations, it is essential to understand the entirety of mechanisms that could possibly play a role in the abnormal cortical function. One such developmental malformation is known as polymicrogyria. Epileptogenesis occurs in an area laterally adjacent to this malformation known as the paramicrogyral region (PMR). Past studies have narrowed down the potential cause of this increased network excitation to a certain type of inhibitory interneuron, the somatostatin (SS) interneuron. Additionally, previous studies have shown an increase in the mGlu5 receptor on this interneurons in the PMR region only and not in control tissue, meaning that targeting these receptors as treatment will not affect normal functioning tissue. These results lead to our hypothesis: blockade of the mGluRs will decrease the 2 activity of SS interneurons and thereby prevent the generation of epileptiform activity and increased SS output in malformed cortex. Utilizing the freeze-lesion model for microgyria in transgenic mice expressing Channelrhodopsin optogenetic channels in SS interneurons, we assessed the contribution of these SS interneurons in four different animal groups: control or PMR treated with either Gabapentin, a current AED (antiepileptic drug), or MTEP, an mGlu5 receptor antagonist. We tested the effects of these two drugs on SS interneuron output to determine whether they decrease the over activation in the PMR that has been previously studied. The following study revealed no correlation between Gabapentin-treated animals and a decrease in epileptiform activity. Additionally, no significant difference was seen between the MTEP-treated groups in the protocols that were measured.

Active sensing, which incorporates closed-loop behavioral selection of information during sensory acquisition, is an important feature of many sensory modalities. We used the rodent whisker tactile system as a platform for studying the role cortical sensory areas play in coordinating active sensing motions. We examined head and whisker motions of freely moving mice performing a tactile search for a randomly located reward, and found that mice select from a diverse range of available active sensing strategies. In particular, mice selectively employed a strategy we term contact maintenance, where whisking is modulated to counteract head motion and sustain repeated contacts, but only when doing so is likely to be useful for obtaining reward. The context dependent selection of sensing strategies, along with the observation of whisker repositioning prior to head motion, suggests the possibility of higher level control, beyond simple reflexive mechanisms. In order to further investigate a possible role for primary somatosensory cortex (SI) in coordinating whisk-by-whisk motion, we delivered closed-loop optogenetic feedback to SI, time locked to whisker motions estimated through facial electromyography. We found that stimulation regularized whisking (increasing overall periodicity), and shifted whisking frequency, changes that emulate behaviors of rodents actively contacting objects. Importantly, we observed changes to whisk timing only for stimulation locked to whisker protractions, possibly encoding that natural contacts are more likely during forward motion of the whiskers. Simultaneous neural recordings from SI show cyclic changes in excitability, specifically that responses to excitatory stimulation locked to whisker retractions appeared suppressed in contrast to stimulation during protractions that resulted in changes to whisk timing. Both effects are evident within single whisks. These findings support a role for sensory cortex in guiding whisk-by-whisk motor outputs, but suggest a coupling that depends on behavioral context, occurring on multiple timescales. Elucidating a role for sensory cortex in motor outputs is important to understanding active sensing, and may further provide novel insights to guide the design of sensory neuroprostheses that exploit active sensing context.

One central question of neuroscience asks how a neural system can generate the diversity of complex behaviors needed to meet the range of possible demands placed on an organism by an ever changing environment. In many cases, it appears that animals assemble complex behaviors by recombining sets of simpler behaviors known as behavioral primitives. The crawling behavior of the nematode worm Caenorhabditis elegans represents a classic example of such an approach since worms use the simple behaviors of forward and reverse locomotion to assemble more complex behaviors such as search and escape. The relative simplicity and well-described anatomy of the worm nervous system combined with a high degree of genetic tractability make C. elegans an attractive organism with which to study the neural circuits responsible for assembling behavioral primitives into complex behaviors. Unfortunately, difficulty probing the physiological properties of central synapses in C. elegans has left this opportunity largely unfulfilled. In this dissertation we address this challenge by developing techniques that combine whole-cell patch clamp recordings with optical stimulation of neurons. We do this using transgenic worms that express the light-sensitive ion channel Channelrhodopsin-2 (ChR2) in putative pre-synaptic neurons and fluorescent protein reporters in the post-synaptic neurons to be targeted by electrodes. We first apply this new approach to probe C. elegans circuitry in chapter II where we test for connectivity between nociceptive neurons known as ASH required for sensing aversive stimuli, and premotor neurons required for generating backward locomotion, known as AVA. In chapter III we extend our analysis of the C. elegans locomotory circuit to the premotor neurons required for generating forward locomotion, known as AVB. We identify inhibitory synaptic connectivity between ASH and AVB and between the two types of premotor neurons, AVA and AVB. Finally, we use our observations to develop a biophysical model of the locomotory circuit in which switching emerges from the attractor dynamics of the network. Primitive selection in C. elegans may thus represent an accessible system to test kinetic theories of decision making. This dissertation includes previously published co-authored material.

The ability to take in one’s environment, integrate relevant information, and then act appropriately is an incredibly complex feat that organisms do continuously. Disruption in the ability to think and act clearly, or cognitive dysfunction, is a debilitating aspect of neuropsychiatric diseases like schizophrenia. The prefrontal cortex and the striatum are key brain regions for functional and dysfunctional cognition, but the way that they interact to allow for cognitive processing is poorly understood. To get at these questions, I manipulated and recorded from medial frontal and striatal neurons—frontostriatal ensembles—while rats engaged in interval timing, an elementary cognitive function that engages both areas. I report four main results. First, ramping activity—a gradual, consistent change in neuronal firing rate across time—is observed throughout frontostriatal ensembles. Secondly, medial frontal areas dynamically reflect changing temporal conditions during learning and precede these same changes in striatal areas. Thirdly, interval timing and striatal ramping activity are disrupted when the medial frontal cortex is inactivated. Finally, this behavioral impairment can be reduced by optogenetic stimulation of frontostriatal terminals. My results support the view that striatal neurons integrate medial frontal activity and suggest a possible mechanism—ramping activity—through which neurons might represent the passage of time. These observations elucidate temporal processing in frontostriatal circuits and provide insight into how the medial frontal cortex exerts top-down control of cognitive processing in the striatum. My hope is that these findings will contribute to a clearer understanding of basic cognitive processing and might inform future approaches to treatments that address cognitive dysfunction.