Spinocerebellar ataxia type 7 (SCA7) is a dominantly inherited, progressive neurodegenerative disease, which falls in a family of disorders caused by an expanded CAG repeat within the gene’s protein-coding region. This type of mutation produces an abnormal poly-glutamine tract within the diseased protein, leading to errors in folding and the formation of characteristic intracellular aggregates. SCA7 results in neurodegeneration within the hindbrain as well as photoreceptor degeneration in the retina, resulting in a loss of motor coordination and vision. To further characterize SCA7 pathogenesis, this study investigated the possibility of disease reversibility using a SCA7 murine model. At 24 weeks, cre-mediated recombination was induced to excise the mutant gene, effectively ceasing mutant gene expression in animals. Prior to and following gene excision, animals were observed for phenotypic changes in gait, spine curvature, hind limb clasping, ability to walk on a cage ledge, and performance on a Rotarod test. All animals were followed through 44 weeks of age, or the average lifespan of SCA7 mice. Following gene excision, SCA7 animals improved in all measured parameters of motor coordination, showing some reversal of disease pathology. Utilizing immunohistochemistry techniques, cerebellar sections from mice (aged 43-44 weeks) were analyzed for the pathology of Purkinje cell dendrites and Bergmann glia processes, two cerebellar cell types previously found to be significantly affected in ataxic animals. When compared with untreated SCA7 mice, there was a decrease in the degree of degeneration in both cell types of SCA7 animals that underwent gene excision treatment. These results suggest that continued expression of the mutant gene is necessary for SCA7 pathology to progress and that treatment, even after the onset of disease symptoms, may have the potential to ameliorate the disease phenotype.

The long-term aim of the project is to study molecular substrates underlying deafferentation-induced cell death in the avian cochlear nuclei. Unilateral cochlea removal (CR) in chicks deprives excitatory input into the nucleus magnocellularis (NM), which induces the death of approximately 30% of NM neurons in two days. Previous studies have demonstrated a uniform decrease in protein synthesis, RNA synthesis, ribosome integrity, and ribosomal RNA (rRNA) in NM neurons 3 hours following the surgery. After 6-12 hours, a subpopulation of NM neurons existed which lacked these biochemical processes altogether. This subpopulation underwent cell death by 2 days after CR, while the remaining neurons atrophied, but their synthetic activity recovered to near normal levels. Intracellular events that determine the fate of NM neurons in early time points remain unknown. A candidate for investigation is elongation factor 2 (EF2), which is required for the translation elongation step of protein synthesis. Phosphorylation inactivates EF2, thus slowing or stopping protein synthesis. Since phosphorylation can occur very rapidly, this mechanism may be responsible for the rapid changes in protein synthesis observed following cochlea removal. Our preliminary data have shown differential changes in the amount of total and phosphorylated EF2 (p-EF2) protein in deafferented NM neurons 1 hour after CR. Quantification analysis of the time course of EF2 and p-EF2 will be conducted at a series of early (0.5-6 h) and later (1-2 days) time points. In addition, the relationship of changes in EF2 and p-EF2 to protein synthesis and ribosome integrity will be examined using double labeling of p-EF2 with a ribosome maker. Data derived from this study may shed light on a better understanding of cellular mechanisms underlying neuronal cell death and lead to possible pharmacological targets for the prevention and treatment of hearing loss.

Ether-a-go-go (Eag) channels are a family of voltage-gated K+ channels that are expressed throughout the nervous system and have a well-documented role in disease. Eag-1, the founding member of the Eag channel family, is expressed in neural tissue, is important in cellular excitably, and may have a role in behavior. Eag-1 is also expressed in most cancerous tumors, and is clinically used as a tumor marker. Very little is known about the physiology and significance of Eag-1, including its specific role in brain tissue and its regulation pathways. Eag family channels are comprised of four subunits, each having six transmembrane domains (S1-S6). Much like Shaker family K+ channels, the S4 domain is the voltage sensor and S5-S6 form the pore. Unlike Shaker channels, however, Eag family channels have large intracellular domains that include a PAS domain, a calmodulin binding site, and a site analogous to a cyclic nucleotide-binding domain (CNBD). These intracellular domains constitute about two thirds of the channel; however, the role these domains may play in channel gating is unknown. To study Eag-1 channel structure and function, the mouse form of this channel was expressed in Xenopus oocytes, and their currents were recorded using inside-out patch clamp. Wild-type mEag-1 exhibits activation and deactivation, but no apparent inactivation. However, deletion of the intracellular N-terminus uncovers an inactivated channel state at depolarizing potentials. This new inactivation state was characterized using electrophysiology. Application of the membrane impermeable cysteine-modifying reagent MTSES to the intracellular side of the N-terminal mutant abolishes inactivation. The kinetics of the N-terminal deleted channel has been modeled and combined with information from other Eag family channels to frame understanding of the channel. Together, these results suggest that the N-terminus may act as a ligand that binds to the CNBD to prevent inactivation of the wild-type channel.

In many regions of the developing nervous system, spontaneous synchronous activity (SSA) plays a role in synaptogenesis, cell positioning, ion channel development, and neuronal migration. The hindbrain is the posterior aspect of the three primary divisions of the developing vertebrate brain. It develops into the cerebellum, pons, and medulla thereby coordinating complex muscular movements and autonomic functions. In previous studies, our lab has identified a single pacemaker region which drives SSA in mouse hindbrain. This pacemaker region is a unique cluster of serotonergic (5-HT) neurons located in the dorsal raphe of the developing hindbrain. Using intracellular calcium imaging to visualize global electrical activity, we have observed SSA, primarily propagating rostro-caudally along the midline, in hindbrains at embryonic day 11.5 (E11.5). Furthermore, we have shown that the frequency, amplitude, and distribution of SSA in this region, as well as the number of serotonergic neurons, can be modulated in culture during the period of serotonergic neuron specification. We are currently examining the effects of maternal and acute exposure to Prozac, a serotonin selective reuptake inhibitor (SSRI), on the development of SSA in this region. Preliminary studies indicate that maternal and acute exposures to Prozac increase the number of pacemaker regions and the frequency of SSA. Our results also suggest that maternal exposure to Prozac during the 24-hour critical period preceding E11.5 may increase the number of serotonergic neurons located within the pacemaker region and change the spatial distribution and length of axonal projections from these neurons. Together, this data indicates that maternal exposure to Prozac modulates the morphology of the pacemaker region and the SSA it regulates.

Animals control their movement by integrating sensory information from a range of modalities, including visual and mechanical information. In flies, modified hindwings, called halteres, that flap out of phase with the forewings provide gyroscopic sensory information that complements visual information. When in flight, Coriolis forces act on the halteres to produce deflections. Sensory organs at the base of the halteres interpret the deflections and rapidly integrate information from the Coriolis forces into the flight pattern. Halteres respond to stimuli much faster than the visual system, allowing flies to alter their wingstrokes within milliseconds. While recent research establishes a neural basis for the encoding of Coriolis forces in halteres, the biomechanical stimuli that occur naturally on halteres remain unknown. We seek to quantify the strain patterns on halteres and ultimately explore the link between biomechanical inputs and the neural circuits. To do so, we calculate the flexural stiffness of collected halteres by applying point forces to the halteres and measuring their resulting deflections using digital photography. The flexural stiffness is critical to determining the strain that is placed on the sense organs at the base of the halteres, which stimulate neurons. We digitize high-speed videos of oscillating halteres to determine their resonant frequencies. Data from these experiments, as well as high-speed video of halteres in free-flight, is used in a three-dimensional finite element model. The finite element model enables us to find the strain patterns on the halteres, as these patterns cannot be determined experimentally. Through construction of this model, we expect to quantify the direct stimulus to the nervous system. When coupled to known neural circuits of flies, these data will indicate what stimuli are required for specific neural outputs.

Control of movement in animals requires the coordination of input from multiple sensory systems with output of multiple actuators. This coordination is particularly crucial in the control of flight in insects. While wing motions are thought to dominate flight forces, relatively little attention has been given to the role of other control surfaces, including abdominal motions in flight control– either as an aerodynamic rudder or as a way of shifting the center of mass relative to the center of lift. To understand the potential role of abdominal flexion in flight control, prior studies have focused on either tethered preparations or on observations of natural abdominal motions during flight maneuvers. However, recent advances in radio technology, along with the development of neural and muscle stimulation methods provides new methods for understanding movement control. We use radio telemetric methods to deliver stimuli to the ventral nerve cord in the hawkmoth Manduca sexta. Stimulation of the ventral nerve cord elicits motor neuron activation of muscles controlling the position of the abdomen. This remote control of abdominal motions provides direct tests of the potential role of the abdomen in determining flight trajectories. We used a PIC combined with an ultra-miniature radio receiver to deliver pulse-width modulated, bipolar stimulation (100 Hz, duty cycle 50%, ±2.5 – 3V) to freely flying moths. We stimulated the moth by placing tungsten electrodes and Flexible Multisite Electrodes (FMEs) in contact with the ventral nerve cord. Animals showed a change in pitch (nose up or nose down) or yaw (lateral roations) in response to that stimulation and resulting abdominal flexion. These results show that we can elicit abdominal movement remotely in freely-flying animals (both open air and wind tunnel), and further indicate that such abdominal flexion is indeed involved in the control of flight paths.

The timing and strength of muscle activation underlies the control of animal movement. This is particularly important in maneuverability of animals in complex environments. Insect flight is an ideal model for examining motor control because we have considerable insight into the mechanical basis for flight and muscle activity is easily recorded and analyzed. Wing motions are produced primarily by two pairs of power muscles driven at a constant phase and modulated by a suite of highly variable steering muscles. However, phase of activity in power muscles could have important control consequences, since the timing of muscle activity relative to wing strokes is an essential component of muscle function and can significantly alter work output. We hypothesized that the control of turning maneuvers is a product of steering muscles, while power muscles express their presumed constant timing of activation. We examined left-right timing differences of bilateral power muscles, specifically the dorso-ventral muscles (DVMs) and dorso-longitudinal muscles (DLMs), in induced turns to determine if visual induced neural feedback could significantly alter power muscle function. We visually stimulated tethered hawkmoths with a projected vertical-banding pattern and recorded action-potentials from DVMs and DLMs using implanted tungsten electrodes. We found that both pairs of power muscles varied significantly in their phase of activation during visually induced maneuvers (p< 0.001). The time separation of the muscles was significantly correlated with the visual stimulus (p< 0.001) with a delay (~100ms) consistent with visual processing. These results reject the hypothesis that power muscles maintain an invariant power-generating function. We found that Manduca alter the timing of power muscles to significantly modulate muscle work output. By exploring the means of controlling maneuverability, we begin to unravel the complexities of locomotor control for bio-inspired applications.