Neural networks in the hindbrain and spinal cord require a proper balance between excitation and inhibition. Identification of zebrafish mutants that have defects in motor output mediated by these networks can allow entrée into underlying network mechanisms. techno trouser ( tnt) mutants demonstrate abnormal motor behavior. Two days after fertilization, wild-type larvae perform an escape response consisting of a large-amplitude body bend away from touch stimuli followed by smaller amplitude body bends to swim away. tnt mutants perform an initial large amplitude body bend away from touch stimuli, but the following smaller amplitude body bends are interrupted by several, abnormal, large amplitude body bends. Four days after fertilization, wild-type larvae exhibit faster escape behavior, whereas tnt mutants are nearly paralyzed and shorter along the rostral-caudal axis. We used meiotic mapping and candidate gene analysis to reveal that the tnt mutation disrupts slc1a2b , which encodes EAAT2, a glutamate transporter expressed in glial cells. Lesion analysis, in situ hybridization, and in vivo electrophysiological recordings all support a model in which reduced slc1a2b function results in exuberant excitation of neurons, initially in the hindbrain and later in development in the spinal cord, to produce the large-amplitude body bends and subsequent paralysis of tnt mutants. Since disruption of human EAAT2 is thought to promote several different neurological diseases, including epilepsy and amyotrophic lateral sclerosis, tnt mutants provide a new tool to understand these disorders.

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)

Parkinson’s disease (PD) is a movement disorder characterized by a massive loss of dopaminergic neurons of nigrostriatal origins. Several genes associated with familial cases of PD encode proteins that are direct components of the 26S Ubiquitin Proteasome System (UPS) or interact with enzymes involved in the UPS. Of these genes, parkin, and its product Parkin, an ubiquitin E3 ligase, is the most well characterized. Loss-of-function mutations in parkin result in the “early onset” PD known as Autosomal Recessive Juvenile Parkinsonism (AR-JP). Most research has focused on studying in what ways do nigrostriatal dopaminergic neurons differ from other neurons in the brain and how and why do these cells die in PD. In the following report I describe studies addressing the equally important alternative question: How do other neurons of the brain differ from nigrostriatal dopaminergic neurons that allow them to survive in AR-JP? I hypothesize that another E3 ligase provides redundant functions to Parkin in surviving neurons but that this redundant UPS enzyme is absent from dopaminergic neurons of the SNC. One protein that could possibly provide such a redundant function is the Parkin-like E3 ligase, Ariadne-1. Ariadne-1 and Parkin share significant sequence identity and similarity; they share the RING-IBR-RING signature domain; they share some UPS E2 enzymes; and they bind some of the substrates. In this dissertation I show Ariadne-1 to be a component of LB in post-mortem human tissue of various neurodegenerative disease. Then, in rats, I determine that Ariadne-1 is present as both mRNA and protein in cells of the SNC. Furthermore, Ariadne-1 is globally expressed throughout the mammalian brain and this expression is restricted to neurons and absent from glial cells and white matter tracts. I also find that only a subset of nigrostriatal dopaminergic neurons express Ariadne-1. Then, using the PD model of unilateral striatal lesioning of mice, I determine that Ariadne-1 expression actually correlates more closely with an increased susceptibility to oxidative stress-induced cell death. Lastly, using two different parkin-/- mice, I determine that, in the absence of Parkin, Ariadne-1 expression correlates with a measurable advantage to dopaminergic neurons of the SNC.

When fractionated using an HPLC ion exchange column, three distinct peaks (peak I, II, and III) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla cells, suggesting the presence of at least three different phosphatases. These phosphatases showed different activities toward phosphocasein in the presence of Mg$\sp{2+}$ and Mn$\sp{2+}$. Peak III, which represents about 50% of the active enzyme activity when phosphocasein is used as substrate, showed a molecular weight of 140 KDa as determined by HPLC gel filtration and has been identified as a type 2A protein phosphatase. Okadaic acid, a phosphatase inhibitor (specific for type 2A and type 1) and tumor promoter, was employed to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. After 3 days cultured in the presence of 50 ng/ml NGF, 20-25% of the PC12 cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC$\sb{50}$ of about 7 nM. This inhibition was rapidly reversed after wash-out of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells indicating that continual phosphatase activity is required to maintain neurites. A 27-mer oligonucleotide was synthesized as a hybridization probe to isolate clones encoding the sequence of protein phosphatases from a bovine adrenal medulla cDNA library. A cDNA clone encoding the full length of the catalytic subunit of protein phosphatase type 2A has been isolated. The deduced protein sequence (309 residues, 35.63 KDa) is 99.7% identical to that of phosphatase 2A$\alpha$ form from rabbit skeletal muscle, human liver and porcine kidney and differs by only one amino acid (Arg-55 vs. Cys-55). At the nucleotide level, the clone showed 97% identity with that of the catalytic subunit of protein phosphatase type 2A$\alpha$ from human liver. Sequence comparison of bovine adrenal medulla clone with phosphatase type 1 from rabbit skeletal muscle and type 2B from mouse brain identifies six highly conserved domains in the three enzymes that are expected to be crucial for the catalytic activity of protein phosphatase.

Eph receptors and their ephrin ligands play important roles in guiding mouse and Xenopus cranial neural crest (CNC) cells to their destinations. My objective was to determine if Ephs and ephrins also regulate avian CNC pathfinding. By double labeling for Eph or ephrin RNA and a neural crest marker protein, I was able to clearly distinguish neural crest from ectoderm and head mesenchyme and show that avian CNC cells express EphA3, 4, and 7 and EphB 1 and 3 and migrate along pathways bordered by non-neural crest cells expressing ephrin-B 1. Surprisingly, avian CNC cells also express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Consistent with these findings, explanted avian CNC cells are labeled by both ephrin-B I and EphB2 Fc fusion proteins. Given the choice between growing out onto substrate-bound fibronectin (FN) or FN plus clustered Fc protein in the stripe assay, these cells show no preference for either condition. Conversely, given the choice between FN or FN plus clustered ephrin-B1 or EphB2 Fc fusion protein, the cells strongly localize to stripes containing only FN. This response is mitigated in the presence of soluble ephrin-B1/Fc or EphB2/Fc, but not in the presence of soluble Fc alone. These findings show that avian CNC cells have a mutually exclusive distribution with non-neural crest cells expressing ephrin-B 1 and EphB2 RNA in situ and are repelled from ephrin-B1 and EphB2 protein in vitro, suggesting that their migration is guided by both forward signaling through a variety of Eph receptors as stimulated by ephrin-B1 and reverse signaling through ephrin-B2 as stimulated by EphB2. I further explore the phylogeny of Ephs and ephrins and show that these genes diversified at different times in evolutionary history, such that the ancestral chordate likely had a single receptor for two different ligands.

Eph receptors and their ephrin ligands play important roles in guiding mouse and Xenopus cranial neural crest (CNC) cells to their destinations. My objective was to determine if Ephs and ephrins also regulate avian CNC pathfinding. By double labeling for Eph or ephrin RNA and a neural crest marker protein, I was able to clearly distinguish neural crest from ectoderm and head mesenchyme and show that avian CNC cells express EphA3, 4, and 7 and EphB 1 and 3 and migrate along pathways bordered by non-neural crest cells expressing ephrin-B 1. Surprisingly, avian CNC cells also express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Consistent with these findings, explanted avian CNC cells are labeled by both ephrin-B I and EphB2 Fc fusion proteins. Given the choice between growing out onto substrate-bound fibronectin (FN) or FN plus clustered Fc protein in the stripe assay, these cells show no preference for either condition. Conversely, given the choice between FN or FN plus clustered ephrin-B1 or EphB2 Fc fusion protein, the cells strongly localize to stripes containing only FN. This response is mitigated in the presence of soluble ephrin-B1/Fc or EphB2/Fc, but not in the presence of soluble Fc alone. These findings show that avian CNC cells have a mutually exclusive distribution with non-neural crest cells expressing ephrin-B 1 and EphB2 RNA in situ and are repelled from ephrin-B1 and EphB2 protein in vitro, suggesting that their migration is guided by both forward signaling through a variety of Eph receptors as stimulated by ephrin-B1 and reverse signaling through ephrin-B2 as stimulated by EphB2. I further explore the phylogeny of Ephs and ephrins and show that these genes diversified at different times in evolutionary history, such that the ancestral chordate likely had a single receptor for two different ligands.

Plasticity in spinal networks has been proposed as a means to permit motor skill learning and recovery after central nervous system disorders. This plasticity is significantly driven by input from the periphery (Wolpaw & Carp, 2006). For instance, attenuation of soleus Hoffmann (H) reflex can last beyond the period of different types of conditioning via putative presynaptic inhibition (Brooke et al., 1997). Interestingly, rhythmic arm cycling can also attenuate soleus H-reflex via interlimb connections and presynaptic pathways (Frigon, Collins, & Zehr, 2004). However, it remains to be studied if this attenuation is maintained beyond the period of arm cycling. In this study, we hypothesized that excitability of H-reflex pathway would remain suppressed after cessation of arm cycling. Subjects were seated with their trunk and feet fixed at a neutral position. Using an arm ergometer, they cycled at 1Hz for 30min. H-reflexes were evoked via stimulation of the tibial nerve in the popliteal fossa at 5 minute intervals. These intervals began prior to the cycling and continued during cycling and up to 30 minutes iv after termination of cycling (n=12). Besides soleus muscle, electromyography was recorded from tibialis anterior, vastus lateralis and biceps femoris. Stimulation was set to evoke an M-wave which evoked an H-reflex on the ascending limb of the recruitment curve (size was 75% Hmax) obtained prior to cycling. The M-wave amplitude was maintained throughout all trials by monitoring and adjusting the level of stimulation intensity. All H-reflex and M-wave data were normalized to the averaged Mmax to reduce inter –subject variability. The main result was that the suppression of H-reflex amplitude persisted beyond the period of arm cycling. H-reflex amplitudes were significantly (p<0.05) smaller up to 20 min after arm cycling had stopped. This suggests that arm cycling can induce plastic adaptation in the soleus H-reflex pathway that persists well beyond the period of conditioning. Also, in an additional experiment (n=8), the prolonged effect of arm cycling combined with superficial radial (SR) nerve stimulation was investigated. Interestingly, this cutaneous nerve stimulation cancelled out the prolonged suppression of H-reflex amplitude induced by arm cycling. Since SR nerve stimulation facilitates soleus H-reflex via reductions in the level of Ia presynaptic inhibition (Zehr, Hoogenboom, Frigon, & Collins, 2004), persistence in presynaptic inhibitory pathways is suggested as an underlying neural mechanism. These results have relevance for optimizing rehabilitation techniques in the treatment of spasticity which is known to be related to the H-reflex size (Levin & Hui-Chan, 1993).

Evidence suggests that the basic neural elements controlling and coupling the arms and the legs in humans during coordinated rhythmic movements are similar to that observed in quadrupedal animals. Further, it is possible that these interlimb connections may be exploited to assist in locomotor rehabilitation after neurotrauma. Specifically, the effect of arm activity on leg neural circuitry has great implications for walking retraining. However, our understanding of the neuromechanics of rhythmic arm movement as well as the neuronal connections between arms and legs active during rhythmic movement is lacking. Greater knowledge on details of interlimb coupling and combined neural and mechanical measurement of rhythmic arm movement are necessary to optimize parameters of interlimb coupling for use in walking rehabilitation. The primary goals of this thesis were to further our understanding of neural interlimb connections during combined arm and leg rhythmic movement and conduct neuromechanical investigations of rhythmic arm movement. First, this thesis developed a method for multiple parameter analysis of the Hoffmanreflex recruitment curve. A sigmoid function was found to be a reliable analysis technique that mimics the physiologically based prediction of the input/output relation of the ascending limb of the recruitment curve. This technique provided a baseline for evaluation of neural interactions between the arms and the legs during rhythmic movement and was utilized during following experiments. Second, the effect of rhythmic leg cycling on reflexes within, and corticospinal projections to, stationary arm muscles was examined. Rhythmic leg cycling significantly suppressed H-reflexes in forearm muscles. Additionally, sub-threshold transcranial magnetic stimulation (TMS) facilitation of H-reflexes was similar during leg cycling as during static contraction suggesting a considerable sub-cortical component. These results supports the hypothesis of a loose, but significant, neural coupling between the arms and the legs during rhythmic movement. Third, we used a reduced walking model of combined arm and leg cycling to examine the separate and combined effects of rhythmic arm and leg movement on the modulation of lower limb H-reflexes with and without stimulating a nerve innervating the hand. The suppressive effect of arm movement was less than that for leg movement and combined arm and leg rhythmic movement, which were generally equivalent. For H-reflexes conditioned by cutaneous input to the hand, amplitudes during combined arm and leg movement instead were in between those for modulation produced by arm movement and leg movement alone. Further a significant contribution for arm movement was revealed only in trials when hand stimulation was used to condition H-reflex amplitudes. Therefore a measurable interaction between neural activity regulating arm and leg movement during locomotion is specifically enhanced when cutaneous input from the hand is present. Fourth, we explored interlimb interactions during a locomotor-like, 3 limb stepping paradigm involving movement of both arms and one leg while eliciting an H-reflex in the stationary test limb. The conditioning effect of contralateral leg movement, bilateral arm movement, and combined bilateral arm and contralateral leg movement on H-reflex amplitude was evaluated at different phases across all tasks. Significant interactions between arm and leg activity could be revealed using the 3-limb paradigm. Further, across phases we observed differential suppressive effects of separate and combined arm and leg movement suggesting phase dependent contributions of arm and leg activity to overall 3-limb suppression. These results support the role of the arms in modulating activity in the legs during human locomotor tasks. Fifth, the mechanical effects of stimulating a cutaneous nerve innervating the dorsum of the hand during arm cycling were quantified. The results show that mechanical responses to cutaneous stimulation of the hand during arm cycling are related to the task and phase and consistent with the anatomical location of the stimulus (local sign). Therefore, these responses are comparable to functionally relevant responses in the legs during lower limb rhythmic movement. However, unlike the responses in the lower limbs, the mechanical responses cannot be easily described in the neuromechanical context of arm cycling. Therefore we suggest that the superimposed task constraints and control variable of arm cycling limit the kinematic reflex expression and make it difficult to decipher the true functional role of the reflexes. Overall, these results provide evidence for mechanical correlates to neural responses during arm cycling and further support parallels between the neural regulation of arm and leg rhythmic movement. Sixth, a combined neural and mechanical measurement approach was used to compare three rhythmic arm movement tasks: arm cycling; arm swing while standing; and arm swing while treadmill walking. The results highlight important neural and mechanical features that distinguish differences between tasks. Overall, differences in neural control between tasks (i.e., pattern of muscle activity) reflected changes in the mechanical constraints unique to each task while the results are consistent with conserved common central motor control mechanisms operational for arm cycling, arm swing while walking, and arm swing alone yet appropriately sculpted to demands unique to each task. Taken together the data in this thesis suggest that, in addition to understanding details of neural interlimb coupling, mechanical considerations may play an important role in the coordination of locomotor movements. Additionally, the use of rhythmic arm movement as a locomotor adjunct in rehabilitation is revealed through combined neural and mechanical measurement.