A central theme of this dissertation is nervous system plasticity. Activity-dependent plasticity and dopaminergic modulation are two processes by which neural circuits adapt their function to developmental and environmental changes. These processes are involved in basic cognitive functions and can contribute to neurological disorder. An important goal in modern neurobiology is understanding how genotypic variation influences plasticity, and leveraging the quantitative genetics resources in model organisms is a valuable component of this endeavor. To this end I investigated activity-dependent plasticity and dopaminergic modulation in Drosophila melanogaster larvae using neurobiological and genetic approaches. Larval mechanosensory behavior is described in Chapter 2. The behavioral experiments in that chapter provide a system to study mechanisms of plasticity and decision-making, while the electrophysiological characterization shows that sensory-motor output depends on neural activity levels of the circuit. This system is used to investigate activity-dependent plasticity in Chapter 3, i.e., habituation to repetitive tactile stimuli. In Chapter 4, those assays are combined with pharmacological manipulations, genetic manipulations, and other experimental paradigms to investigate dopaminergic modulation. Bioinformatics analyses were used in Chapter 5 to characterize natural genetic variation and the influence of single nucleotide polymorphisms on dopamine-related gene expression. The impact and suggested future directions based on this work are discussed in Chapter 6. Dopamine also modulates cardiomyocytes. Chapter 7 describes biochemical pathways that mediate dopaminergic modulation of heart rate. The final two chapters describe neurobiology research endeavors that are separate from my work on dopamine. Experiments that have helped characterize a role for Serf, a gene that codes for a small protein with previously unknown function, are described in Chapter 8. In the final chapter I describe optogenetic behavioral and electrophysiology preparations that are being integrated into high school classrooms and undergraduate physiology laboratories. Assessment of student motivation and learning outcomes in response to those experiments is also discussed.

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD.

Autism spectrum disorder (ASD) is associated with functional abnormalities of the anterior cingulate cortex (ACC), a brain area that mediates social behavior. Given evidence of a role of inflammation in ASD, markers of pro-inflammatory and anti-inflammatory microglia were studied using postmortem ACC tissues from ASD and age-matched typically developed control donors. Gene expression levels of pro-inflammatory (CD68, HLA-DRA, IL1B, NOS2, PTGS2) and anti-inflammatory (ARG1, IGF1, MRC1, PPARG) microglial genes were measured using quantitative real-time PCR. Additionally, brain sections were immunohistochemically stained for a microglial marker. Expression levels of IGF1 were modestly higher, while the expression of MRC1 was modestly lower in ASD donors when compared to control donors. No other differences in gene expression levels between the two groups of donors were observed. Statistical significance for changes in expression levels IGF1 and MRC1 did not survive correction for multiple comparisons. Further research on anti-inflammatory microglial involvement in ASD is warranted.

The olfactory system is of great interest in research due to the olfactory epithelium’s regenerative capability and as a potential as a source of neural stem cells. The olfactory sensory neurons are constantly being replaced by the stem cells that lie at the base of the olfactory epithelium. These stem cells also remain intact after an injury to the epithelium and lead to the regeneration of the olfactory epithelium. We have developed a fate mapping technique to trace axonal regrowth from newly born olfactory sensory neurons using an inducible Cre-ERT2 model after chemical ablation by the drug methimazole. Our data shows that newly generated olfactory sensory neurons labeled 1 day after chemical ablation by injection of 4-HO-tamoxifen extend an axon that reaches the olfactory bulb and extend to the glomeruli in a timeline that is consistent with control mice that received 4-HO-tamoxifen but were injected with saline 1 day prior. In addition, we assessed the functional recovery of the olfactory epithelium by testing the ability of mice to find a hidden cookie after methimazole injection. Mice were tested at 3 and 14 days post methimazole. There was a severe impairment in the ability to find a hidden cookie at 3 days post methimazole. The mice tested at 14 days post methimazole showed an improvement in the ability to find the cookie but the latency to find the cookie was still significantly higher than controls. In conclusion, while we demonstrate that axons extend to the olfactory bulb and the glomeruli earlier than 14 days, our behavioral data suggest that there must be a critical number of axons that must reach each specific glomerulus to regain function of the olfactory system.

Maternal behavior is the complex array of caregiving behaviors females display towards offspring. In rats, the transition to motherhood depends on the action of various hormones, especially estradiol near parturition, which primes the maternal circuitry to respond to pups upon first encounter at parturition with appropriate maternal behavior. Although virgin rats avoid pups, new mothers are highly motivated to interact with pups, and their maternal behavior depends on the functional interaction between the medial preoptic area (mPOA) and the ventral tegmental area (VTA). However, a precise mapping of the VTA-projecting mPOA neurons remains to be elucidated. To determine whether pup-responsive neurons in the mPOA project to the VTA, we injected the retrograde tracer Fluorogold (FG) into the VTA of new mother and virgin female rats. Six days later, females were exposed to 3 pups for 5 minutes, and their brains processed to visualize FG and c-Fos immunostaining. In addition, we further characterized the molecular phenotype of these neurons by performing immunohistochemistry against estrogen receptor alpha (Esr1). As expected, the behavior of postpartum and virgin females toward pups was different. Mothers readily approached pups and displayed maternal behavior, whereas virgins avoided interaction with pups. Despite these disparate responses to pups, no differences were found in the number and distribution of mPOAc-Fos→VTA neurons. In addition, in both postpartum and virgin females, a significant proportion of these pup-responsive mPOA→VTA projecting neurons also express Esr1. Further functional interrogation of these c-Fos+/Esr1+ mPOA→VTA neurons in virgins and mothers might elucidate distinct circuit dynamics potentially underlying their behavioral differences towards pups.

In the amphibian auditory system, a subset of hair cells is known to be frequency tuned via electrical resonance. This tuning is thought to contribute to frequency selectivity of the information leaving the auditory periphery via the auditory afferent fibers. At the same time, while most, if not all, afferent fibers are shown to be frequency tuned, electrical resonance has only been experimentally demonstrated in a subset of amphibian auditory hair cells. In this thesis, we validate and use a novel Zap current method to probe the electrical resonance of the bullfrog amphibian papilla hair cells. We uncover the existence of two previously unknown types of electrically resonant auditory hair cells. We then show the existence of resonant hair cells across the length of amphibian papilla, with the range of frequency tuning that is nearly indistinguishable from that previously reported in the of auditory fibers. Therefore, this work further validates amphibian hair cell frequency resonance as the possible mechanism underlying frequency selectivity of the subsequent stages in auditory signal transduction.

The medial amygdala (MeA) is a central node in the interwoven circuits that regulate social behavior based on pheromones. Aromatase-expressing (arom+) neurons in the MeA are key for the establishment and maintenance of sex differences. Here, we characterized the intrinsic electrophysiological properties of arom+ neurons and non-aromatase (arom-) neurons in the MeA of male and female mice. Most electrophysiological properties were similar for arom+ neurons in the MeA between sexes, but the relative refractory period was twice as large in female mice. We also show that the firing pattern and firing frequency is markedly different between arom+ and arom- neurons. The activity of MeA neurons could be modulated by estradiol, which reduced activity in arom+ neurons in males. The differences between arom+ and arom- neurons were observed in both sexes suggesting that aromatase expression delineates a neural population in the MeA with similar and unique electrophysiological properties.

Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by motor neuron loss. Although pathological mutations exist in >15 genes, the mechanism(s) underlying ALS are unknown. FUS is one such gene and encodes the nuclear RNA-binding protein (RBP), fused in sarcoma (FUS), which actively shuttles between the nucleus and cytoplasm. Intriguingly, nearly half of the ALS mutations identified in FUS cause this protein to mislocalize, suggesting that FUS localization is relevant to disease. Here, we found that excitotoxicity, a neuronal stress caused by aberrant glutamate signaling, induces the rapid redistribution of FUS and additional disease-linked RBPs from the nucleus to the cytoplasm. As excitotoxicity is pathologically associated with ALS, it was notable that the nuclear egress of FUS was particularly robust. Further, ALS-FUS variants that predominantly localize to the nucleus also undergo redistribution. Thus, we sought to understand the purpose underlying FUS translocation and the potential relevance of this response to disease. As calcium dysregulation is strongly associated with neurodegenerative disorders, we examined the contribution of calcium to FUS egress. In addition to global changes to nucleocytoplasmic transport following excitotoxic insult, we observed that FUS translocation caused by excitotoxicity is calcium mediated. Moreover, we found that dendritic expression of Gria2, a transcript encoding an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit responsible for regulating calcium permeability, is FUS-dependent under conditions of stress. Together, these observations support the premise that FUS has a normal function during excitotoxic stress and that glutamatergic signaling may be dysregulated in FUS-mediated ALS.

Axons are injured after stroke, spinal cord injury, or neurodegenerative disease such as ALS. Most axons do not regenerate. A recent report suggests that not all neurons are poor regenerators, but rather a small subset can regenerate robustly. What intrinsic property of these regenerating neurons allows them to regenerate, but not their neighbors, remains a mystery. This subtype-specific regeneration has also been observed in Drosophila larvae sensory neurons. We exploited this powerful genetic system to unravel the intrinsic mechanism of subtype-specific neuron regeneration. We found that neuron bursting activity after axotomy correlates with regeneration ability. Furthermore, neuron bursting activity is necessary for regeneration of a regenerative neuron subtype, and sufficient for regeneration of a non-regenerative neuron subtype. This optogenetically-induced regeneration is dependent on a bursting pattern, not simply overall activity increase. We conclude that neuron bursting activity is an intrinsic mechanism of subtype-specific regeneration. We then discovered through a reverse genetic screen that an L-type voltage gated calcium channel (VGCC) promotes neuron bursting and subsequent regeneration. This VGCC has high expression in the regenerative neuron and weak expression in the non-regenerative neuron. This suggests that VGCC expression level is the molecular mechanism of subtype-specific neuron regeneration. Together, our findings identify a cellular and molecular intrinsic mechanism of subtype-specific regeneration, which is why some neurons are able to regenerate while the majority of neurons do not. Perhaps VGCC activation or neuron activity pattern modulation could be used therapeutically for patients with nerve injury.

Olfactory sensory neurons (OSNs) are contained within the olfactory epithelium (OE) and are responsible for detecting odorant molecules in the air. The exposure of OSNs to the external environment is necessary for their function, but it also leaves them exposed to potentially harmful elements and thus results in a high turnover rate. Despite the high turnover, the olfactory sense is maintained throughout life through the division of a population of stem cells that produce new OSNs both during normal turnover and after an injury occurs in the OE. When new OSNs are born, they must extend axons from the OE to the olfactory bulb (OB) where they make specific synaptic contacts. To determine the timeline of axon extension in normal turnover and after a methimazole-induced injury, we used fate-tracing utilizing an inducible Cre-LoxP model in which a fluorescent reporter was expressed by neuronal precursors and subsequently used to track axonal growth as the OSNs matured. Our results show that axon extension in both conditions follow the same timeline. However, markers of synaptic connectivity in the OB were delayed after injury. The delay in synaptic connectivity was also corroborated with delays in olfactory behavior after injury, which took 40 days to recover to control levels. Additionally, we investigated the process of removal of axonal debris created after an injury. Immunohistochemical analysis after injury indicated upregulation of IBA1+ cells within the 3 olfactory nerve layer of the OB, suggesting a role of microglia in this process. These microglia also showed an activated morphology and some had very large cell bodies with multiple nuclei. Furthermore, qPCR analysis of post-injury OB tissue shows upregulation of the CD11b receptor that is expressed on microglia. Our results have also shown upregulation of components of the complement pathway after injury, which is suggestive of a mechanism that underlies axonal debris removal after injury in the OB. Taken together, these results shed light on the process by which the olfactory system is able to recover after injury and could lead to discovery of mechanisms that could translate to treatments for injuries in other areas of the nervous system.