abstract: Estrogen-containing hormone therapy (HT) is approved for treatment of symptoms associated with menopause by the Food and Drug Administration. A common estrogen used in HT is 17β-estradiol (E2). Rodent models of menopause, and some clinical work as well, suggest a cognitively-beneficial role of E2. However, as of the 2017 statement released by the North American Menopause Society, HT is not currently advised for use as cognitive therapy in healthy, menopausal women, given that the data so far from existing clinical studies are not yet definitive. Indeed, the delivery of E2 treatment can be optimized to yield more consistent results on cognitive function, particularly considering that exogenously administered E2 gets rapidly metabolized and cleared from the body. Further, E2-containing HT must include a progestogen if prescribed to women with a uterus to oppose its undesired uterine stimulating effects, such as increased endometrial hyperplasia and cancer risks. Studies have shown that the addition of a progestogen to E2 treatment can attenuate the effects of E2 on cognition and brain variables associated with cognitive function. Thus, a brain-specific delivery platform of E2 treatment that would minimize the hormone’s effects in the periphery while maintaining the beneficial cognitive effects is desirable. To achieve this goal, my dissertation work bridged two distinct scientific fields – behavioral neuroendocrinology and polymeric drug delivery – with the overarching aim of targeting the delivery of E2 to the brain to achieve maximal cognitively-beneficial effects with minimal undesired uterine stimulation. This aim was addressed via three distinct delivery strategies: 1) combining E2 with a cognitively-beneficial progestogen, 2) encapsulating E2 in polymeric nanoparticles, and 3) solubilizing E2 using cyclodextrins for intranasal administration. Findings revealed that although all E2-containing treatments increased uterine horn weights, a marker of uterine stimulation, in middle-aged ovariectomized rats, some E2 treatment formulations yielded memory improvements, others were neutral in their effects on memory, and some impaired memory. Together, data from this dissertation set the stage for targeted E2 delivery research to optimize the cognitive therapeutic effects of E2 in the context of menopause while minimizing peripheral burden, leading to translationally relevant clinical implications for women’s health. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2018

<p> Initiation of alcohol use typically begins during adolescence in humans, with males reporting higher binge-drinking levels. In rodent models, the long-term developmental consequences of adolescent alcohol (ethanol) exposure include retention of some adolescent-like phenotypes such as anxiety-related responding into adulthood. Recently, it has been shown that neuromodulation via the oxytocin system selectively alleviates social anxiety in adult males following adolescent intermittent ethanol (AIE) exposure. In the current study, we investigated whether an oxytocin agonist would also result in anxiolytic effects in a nonsocial anxiety-related task, novelty induced hypophagia. Both adult males and females exhibited enhanced anxiogenic-like responding during task training following AIE exposure. The nonpeptide oxytocin agonist WAY-267464 increased anxiolytic responding in males, but increased anxiogenic responding in females, independent of adolescent exposure. We further assessed glutamate receptor expression to provide further insight into oxytocin&rsquo;s mode of action in males. Multiple effects were noted for NMDA and AMPA receptor plasticity within the lateral septum and amygdala, but not the ventral hippocampus. Overall, these findings further validate oxytocin&rsquo;s anxiolytic mechanism of action in males and further suggest responses may involve regulation of glutamate receptor subtypes.</p><p>

Animal research involving the effects of anxiolytics on theta oscillations has focused on changes in theta frequency in the hippocampus, rather than effects in medial entorhinal cortex (MEC), which provides the cortical input to the hippocampus and is the source of Type I (movement-related) theta rhythm. Neurons coding spatial location, including “grid cells,” are found in the MEC and aspects of their spatial modulation have been linked to theta rhythm in different ways. Theta frequency recorded in the local field potential (LFP) is also strongly correlated with running speed and is used in specific computational models of grid cell firing. Manipulating theta frequency through administration of anxiolytics offers a unique method of examining regulation of the LFP frequency in the MEC, along with the effects of changes in theta frequency on grid cells and other cell types in the region. In addition, the role of the medial septum (MS), which is necessary for theta rhythm in both the MEC and hippocampus, can be investigated by infusing anxiolytics directly into the MS. In this thesis, two separate anxiolytic drugs were tested: a serotonin 1A receptor agonist, 8-OH-DPAT, and a classic benzodiazepine, diazepam, the results of which are described in chapters 2 and 3, respectively. Systemic injections of either drug caused a reduction in theta frequency across all running speeds, resulting in a decrease in the y-intercept of the linear fit to the plot of theta frequency over different running speeds. However, only MS infusion of 8-OH-DPAT, not diazepam, significantly decreased the y-intercept in the MEC. Together, these results expand detection of anxiolytic drug action on theta frequency to a new structure, the MEC, when drugs are given systemically, but demonstrate a dissociation between drug types in their ability to produce effects when infused into the MS. Grid cell firing patterns were unaffected and very few effects were found in single unit firing across different cell types. Overall, these results support predictions made by specific computational models and highlight the involvement of the MEC in the anxiolytic-induced decrease in theta frequency phenomenon uniquely tied to the action of these drugs.

The brain is a complex organ that not only gathers copious amounts of information, but also interprets and reacts to information gathered. In the present study we sought to understand how relevant information for a complex experience (cocaine) was transmitted from regions throughout the brain to cocaine-activated striatal cells producing an overall phenotype expected from mice under a strong cocaine experience. To accomplish this, Arc/Ai14 mice were first exposed to repeated prior intra-peritoneal cocaine injections, after which striatal injections of AAV-DIO-TVA adeno-associated virus (AAV) were performed. Post AAV injection, cocaine-activated cells were TRAPed, and finally (EnvA)SAD-ΔG-GFP rabies virus (RV) was injected into the striatum allowing for brain wide monosynaptic retrograde tracing of inputs (cocaine-activated inputs indicated by yellow fluorescence) onto cocaine-activated striatal MSNs. While data is still being tabulated, preliminary data suggests an increase in co-connectivity between cocaine-activated orbitofrontal and medial cortical neurons (retrosplenial/cingulate) and cocaine-activated striatal cells. Thus, preliminary data suggest that chronic cocaine pre-exposure lead to corticostriatal circuit refinement.

While we can learn to produce stereotyped movements and maintain this ability for years, it is unclear how populations of individual neurons change their firing properties to coordinate these skills. This has been difficult to address because there is a lack of tools that can monitor populations of single neurons in freely behaving animals for the durations required to remark on their tuning. This thesis is divided into two main directions- device engineering and systems neuroscience. The first section describes the development of an electrode array comprised of tiny self-splaying carbon fibers that are small and flexible enough to avoid the immune response that typically limits electrophysiological recordings. I also describe the refinement of a head-mounted miniature microscope system, optimized for multi-month monitoring of cells expressing genetically encoded calcium indicators in freely behaving animals. In the second section, these tools are used to answer basic systems neuroscience questions in an animal with one of the most stable, complex learned behaviors in the animal kingdom: songbirds. This section explores the functional organization and long-term network stability of HVC, the songbird premotor cortical microcircuit that controls song. Our results reveal that neural activity in HVC is correlated with a length scale of 100um. At this mesocopic scale, basal-ganglia projecting excitatory neurons, on average, fire at a specific phase of a local 30Hz network rhythm. These results show that premotor cortical activity is inhomogeneous in time and space, and that a mesoscopic dynamical pattern underlies the generation of the neural sequences controlling song. At this mesoscopic level, neural coding is stable for weeks and months. These ensemble patterns persist after peripheral nerve damage, revealing that sensory-motor correspondence is not required to maintain the stability of the underlying neural ensemble. However, closer examination of individual excitatory neurons reveals that the participation of cells can change over the timescale of days- with particularly large shifts occurring over instances of sleep. Our findings suggest that fine-scale drift of projection neurons, stabilized by mesoscopic level dynamics dominated by inhibition, forms the mechanistic basis of memory maintenance and and motor stability.

No description available.

A key feature of many neurodegenerative diseases is the pathological loss of neurons that participate in generating behavior. to mimic the neuronal degeneration procedure of a functioning neural circuit, we designed a computer-automated system that algorithmically detects and sequentially laser-ablates constituent neurons from a neural network with single-cell precision while monitoring the progressive change of the network function in real time. We applied this cell-specific cumulative lesion technique to an advantageous experimental model, the preBotzinger Complex (preBotC), the mammalian respiratory central pattern generator (CPG) that can be retained in thin slice preparations and spontaneously generates breathing-related motor activity in vitro . as a consequence, we sought to investigate the issue: how many neurons are necessary for generating respiratory behavior in vitro ? This question pertains to whether and how progressive cell destruction will impair, and possibly preclude, behaviorally relevant network function. Our ablation system identifies rhythm-generating interneurons in the preBotC based on genetically encoded fluorescent protein markers or imaged Ca 2+ activity patterns, stores their physical locations in memory, and then randomly laser-ablates the neuron targets one at a time in sequence, while continuously measuring changes to respiratory motor output via hypoglossal (XII) nerve electrophysiologicallyin vitro. A critical feature of the system is custom software package dubbed Ablator (in Python code) that detects cell targets, controls stage translation, focuses the laser, and implements the spot-lesion protocol automatically. Experiments are typically carried out in three steps: 1) define the domain of lesion and initialize the system, 2) perform image acquisition and target detection algorithms and maps populations of respiratory neurons in the bilateral volumes of the slice, 3) determine the order of lesions and then spot-lesion target neurons sequentially until all the targets are exhausted. Here we show that selectively and cumulatively deleting rhythmically active inspiratory neurons that are detected via Ca 2+ imaging in the preBotC, progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120+/-45 neurons stopped spontaneous respiratory rhythm, and our data suggest ∼82% of the rhythm generating neurons remain un-lesioned. Similarly, destruction of 85+/-45 homeodomain transcription factor Dbx1-derived (Dbx1+) neurons, which were hypothesized to comprise the rhythmogenic core of the respiratory CPG in the preBotC, precludes the respiratory motor behavior in vitro as well. The fact that these two estimates of the size of the critical rhythmogenic core in the preBotC are different can be reconciled considering that the Ca2+ imaging method identifies ∼50% inhibitory neurons, which are found in the preBotC but are not rhythmogenic. Dbx1+, on the other hand, identifies only excitatory rhythmogenic neurons. Serial ablations in other medullary respiratory regions did not affect frequency, but diminished the amplitude of motor output to a lesser degree. These data support the hypothesis that cumulative single-cell ablations caused a critical threshold to be crossed during lesioning, after which rhythm generation in the respiratory network was unsustainable. Furthermore, this study provides a novel measurement that can help quantify network properties of the preBotC and gauge its susceptibility to failure. Our results in turn may help explain respiratory failure in patients with neurodegenerative diseases that cause progressive cell death in the brainstem respiratory networks.

abstract: Vagal Nerve Stimulation (VNS) has been shown to be a promising therapeutic technique in treating many neurological diseases, including epilepsy, stroke, traumatic brain injury, and migraine headache. The mechanisms by which VNS acts, however, are not fully understood but may involve changes in cerebral blood flow. The vagus nerve plays a significant role in the regulation of heart rate and cerebral blood flow that are altered during VNS. Here, the effects of acute vagal nerve stimulation using varying stimulation parameters on both heart rate and cerebral blood flow were examined. Laser Speckle Contrast Analysis (LASCA) was used to analyze the cerebral blood flow of male Long–Evans rats. In the first experiment, results showed two distinct patterns of responses to 0.8mA of stimulation whereby animals either experienced a mild or severe decrease in heart rate. Further, animals that displayed mild heart rate decreases showed an increase in cerebral blood flow that persisted beyond VNS. Animals that displayed severe decreases showed a transient decrease in cerebral blood flow followed by an increase that was greater than that observed in mild animals but progressively decreased after VNS. The results suggest two distinct patterns of changes in both heart rate and blood flow that may be related to the intensity of VNS. To investigate the effects of lower levels of stimulation, an additional group of animals were stimulated at 0.4mA. The results showed moderate changes in heart rate but no significant changes in cerebral blood flow in these animals. The results demonstrate that VNS alters both heart rate and cerebral blood flow and that these effects are dependent on current intensity. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

BACKGROUND: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss. Efficiently targeting mechanisms of axonal degeneration in MS has the potential to reduce disability but remains an unmet need. Prior research has identified the protein sterile alpha and TIR motif containing 1 (SARM1) as a critical factor that promotes axonal destruction in the program of axonal degeneration known as Wallerian degeneration. SARM1 inactivation reduces axonal degeneration in a variety of contexts including traumatic and toxic injury, but it remains unknown to what extent SARM1 is involved in axonal degeneration triggered by CNS inflammation. METHODS: To test the hypothesis that SARM1 inactivation will reduce the burden of axonal degeneration associated with CNS inflammatory disorders, we first induced mice to have EAE and compared inflammation (CD3) and axonal damage (SMI-31/32, Beta APP) as compared to healthy control mice. We then studied experimental allergic encephalomyelitis (EAE) in Sarm1 knockout (KO) and wild type (WT) mice. We used mice hemizygous for the Thy1-YFP transgene to study axonal damage. Degenerating axons were identified by focal swelling or fragmentation. Beta-APP was also used as a marker of axonal injury. RESULTS: EAE mice had greater inflammation and axonal injury as compared to healthy mice. Sarm1 KO mice are susceptible to developing EAE, with incidence comparable to WT littermates. Analysis of YFP+ axons and Beta-APP showed that Sarm1 KO mice had axonal damage reduced compared to WT littermates. CONCLUSION: Sarm1 is highly expressed in the brain. Preliminary data suggest that SARM1 inactivation may minimize axonal degeneration in CNS inflammatory disorders such as EAE. Further studies are needed to confirm the long-term benefit.

No description available.