We are studying the pharmacology of the human delta-opioid receptor stably expressed in Chinese hamster ovary cells (hDOR/CHO). The delta-opioid receptor mediates analgesia, without the negative side effects noted by mu- and kappa-opioid agonists. However, tolerance to delta-opioid agonists still occurs. One mechanism of drug tolerance in CHO cells involves a compensatory response by adenylyl cyclase VI. This response, termed adenylyl cyclase (AC) superactivation, arises from the observation that acute hDOR activation leads to inhibition of AC, whereas chronic activation of the receptor (greater than 4 hours) leads to a compensatory increase in AC activity, effectively negating the acute inhibition normally seen in the presence of the delta-opioid agonist. The increased AC activity also causes an overshoot of cAMP formation upon the removal of the agonistic inhibitory influence. The loss of receptor-mediated AC inhibition after chronic agonist treatment is thought to contribute to in vivo drug tolerance, and the resulting cAMP overshoot may contribute to opioid withdrawal. In studying this phenomenon, we have demonstrated a requirement for G-protein betagamma subunits (Gbetagamma) by expressing scavengers of Gbetagamma, such as alpha-transducin and phosducin. Additionally, we have shown AC VI phosphorylation by chronic agonist treatment, which may cause AC superactivation, and that this phosphorylation is sensitive to calmodulin antagonists and inhibitors of the atypical protein kinase C enzymes. We have also recently postulated the involvement of Raf-1 kinase. Inhibitors of Raf-1, as well as pathways that lead to activation of this enzyme, significantly attenuate the cAMP overshoot, suggesting that hDOR-activated Raf-1 can phosphorylate and superactivate AC VI in CHO cells.

Space is a fundamental property of nature, so it is not surprising that the processing of spatial information involves many brain structures, including the posterior parietal cortex (PPC) and hippocampus. The PPC represents body position in multiple frames of reference, aligning the reference frames of different parts of the body to cooperate in the performance of a task. Hippocampal "place cells" increase their firing rates in specific locations, and place cell responses can be based on information encoded in multiple reference frames, including external, sensory-based reference frames or internal, memory-based frames. The computation of whole-body trajectories is common to all navigation tasks, but little is known about how this computation is performed. Rats were trained to run to distant targets that were presented either randomly or as segments of sequences. When rats were trained to make direct trajectories to distant targets that were presented in random order, no evidence was found that hippocampal place cells encoded distant goals. When rats learned a sequence of goals that contained a repeated segment, hippocampal place cell activity along the repeated segment remained the same. This showed that differential hippocampal codes were not required for rats to differentiate overlapping sequential contexts. Differential hippocampal codes could be formed, however, if rats learned sequences of goals under specific conditions, reflecting differential activity from neural structures outside the hippocampus. At the initiation of trajectories, the phase of hippocampal theta was reset at the peak acceleration of the rat. The activity of some parietal neurons was modulated by hippocampal theta, though the magnitude of modulation was not as great as that for hippocampal units, and the preferred firing phase of parietal units differed from that of theta-modulated place cells. Two classes of units in PPC responded differentially during the early and late stages of trajectories: one class with an increased firing rate during the early stages of trajectories, and the other with an increased firing rate during the late stages. These results provide a foundation for future studies of parieto-hippocampal interactions during trajectory planning.

Intercellular signaling is vital for the coordinated development of multicellular organisms. This is true for all tissue types including those that constitute the nervous system. The research described here explores the role of intercellular signaling in two vital steps of nervous system development, axon pathfinding and synapse maturation/modulation. The first chapter of this dissertation explores the role of the Transforming Growth Factor-Beta signaling pathway in the modulation of synaptic function and morphology at the Drosophila melanogaster neuromuscular junction. The second chapter explores the role of the glypican Dally-like in the process of axon outgrowth and axon guidance in the same organism. Glypicans are members of the heparan sulfate proteoglycan family of molecules and are required for the proper signal transduction of various intercellular pathways including TGF-beta. Therefore both of the projects that compose this research explore the important role of intercellular signaling in development of the nervous system.

The functional reconstruction of interrupted CNS pathways in mature mammals was studied in adult hamsters in which retinal ganglion cells (RGCs) regenerated axons through a peripheral nerve graft directed from the stump of the cut optic nerve to the superior colliculus (SC). Visually elicited unitary responses of both pre- and postsynaptic origin were recorded in the reinnervated SC and were discriminated by selectively abolishing postsynaptic activity using GABA microiontophoresis. Activity from individual regenerated axonal arbors and from the neurons within their terminal field of innervation was confined to the superficial retinorecipient SC layers and could be recorded over a range of up to 200 $ mu$m in all planes. Analysis of such electrophysiological recordings suggests that the terminal arborizations of single regenerated RGC axons can establish functional synapses with several SC neurons and that the convergence of inputs from regenerated RGC axons is not required for the activation of SC neurons in response to light. Functional synapses established between regenerated RGC axons and SC neurons persisted for up to 60 weeks after grafting. Visually elicited responses were recorded in the contralateral SC from the regenerated projections of as many as 63 different RGCs. The extent of functional innervation by regenerated retinal axons accessing the SC at a single entry point covered up to 0.8 mm and 1.2 mm in the caudo-rostral and latero-medial planes, respectively. Despite large scale targeting errors in the functional reinnervation of the SC, the re-established retinal projections in the SC were not randomly deployed along the caudo-rostral axis. There was an overall tendency for the relative naso-temporal position of any two RGCs to be appropriately represented by their respective regenerated inputs in the SC along its caudo-rostral axis. This suggests that regenerating RGC axons may recognize caudo-rostral positional markers as they reinnervate the SC.

The prion protein (PrP) is known for its implication in a number of neurological disorders. Interestingly, overexpression of Bcl-2 in these PrP-/- cells protects against serum deprivation, suggesting that PrP may function like Bcl-2 to prevent cells from undergoing apoptosis. / Based on this idea, this thesis demonstrates that PrP is a potent protective agent against Bax-mediated death of cultured human primary neurons. This neuroprotective function appears to require the presence of the octapeptide repeat region of PrP. Surprisingly, while the majority of PrP resides on the cell surface as a glycophosphatidyl inositol (GPI)-anchored protein, the GPI anchor is not required for the neuroprotective function of PrP. In contrast, PrP needs trafficking past the cis-Golgi to be effective against Bax since its neuroprotective function is sensitive to brefeldin A and monensin treatments. / On the other hand, we have established a yeast-based assay to analyse the function of PrP against Bax. (Abstract shortened by UMI.)

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 (T634) and/or threonine 641 increases during long-term potentiation or associative learning in rodents. We have raised an antibody to a peptide from PKC Apl I phosphorylated at T613 and T620 (sites homologous to T634 and T641). This antibody recognizes PKC Apl I only when it is phosphorylated at T613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at T613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at T613 in neurons is resistant to both membrane translocation and downregulation. Replacement of T613 with alanine increases the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.

This study seeks to understand the development of neurons constituting the vertebrate spinal locomotory network, which is responsible for the intrinsic generation of rhythmic motor activities. Interneurons (INs) of the V2 class of ventral spinal INs in chick and mouse embryos are sub-divided into V2a and V2b. The V2 sub-classes are of opposite neurotransmitter phenotypes, with the V2a identified as excitatory glutamatergic INs and the V2b being inhibitory GABAergic INs. Their specification during development has been attributed to expression of the transcription factor Foxn4. However, recent over-expression experiments in the chick gave contradicting results as to whether Foxn4 is sufficient to induce V2b INs and if it is also able to suppress the V2a fate. I utilized the much simpler model of embryonic zebrafish with fewer neurons to validate the role of foxn4. I first characterized the expression of different foxn4 transcripts at different developmental stages in the zebrafish and confirmed the expression of an embryonic specific transcript and found that all transcripts are down-regulated in the adult fish. I also determined the cellular identity of V2b INs in the zebrafish, as double in situ hybridization and immunohistochemistry confirmed these to be GABAergic ventral INs. In an attempt to alter the V2a and V2b populations, I either over-expressed foxn4 mRNA or knocked-down its translation using a translation blocking antisense morpholino oligonucleotide. My results indicated no change in V2a or V2b IN numbers when foxn4 was knocked-down, but there was a switch from V2a to V2b when foxn4 was over-expressed. Future experiments could hopefully address if altered / Cette étude a pour but d'étudier le développement neuronal du réseau locomoteur spinal chez les vertébrés responsable de la génération intrinsèque des activités motrices rythmiques. Les interneurones (INs) de la classe V2 de la moelle épinière ventrale des embryons de poussins et de souris sont divisés en groupes V2a et V2b. Les sous-classes V2 ont des phénotypes de neurotransmetteurs opposés, avec les V2a étant identifiés comme étant glutamatergiques et excitateurs tandis que les V2b sont GABAergiques et inhibiteurs. Leur spécification durant le développement a été attribué à l'expression du facteur de transcription Foxn4. Par contre des expériences récentes de sur-expression chez le poussin ont données des résultats contradictoires à savoir si le Foxn4 est suffisant pour induire les INs V2b et aussi pour supprimer la destinée V2a. Je me suis servie du modèle beaucoup plus simple de l'embryon du poisson zébré avec moins de neurones afin de valider le rôle de la foxn4. Dans un premier temps j'ai caractérisé l'expression de différents transcrits à des stages différents du développement chez le poisson zébré. J'ai ainsi confirmé l'expression d'un transcrit embryonnaire spécifique et j'ai trouvé que tous les transcrits sont supprimés chez le poisson adulte. J'ai aussi déterminé l'identité cellulaire des INs V2b chez le poisson zébré par double marquage par hybridisation in situ et par immunocytochimie, ce qui a confirmé que ces INs ventraux sont GABAergiques. Afin d'altérer les populations V2a et V2b j'ai soit surexprimé l'ARNm du foxn4 ou réduit son expression par injection d'un oligonucléotide morpholino antisens a

Feed-forward (anticipatory) postural adjustments are known to initiate voluntary movements and displace the CoM within the base of support (BoS). These changes in muscular activity of the supporting limbs precede the onset of voluntary movements. In contrast, postural responses to unexpected surface perturbations occur at short latency after standing balance is disturbed. These compensatory (feedback-based) postural responses are characterized by directionally tuned muscle activity, and directionally limited ground forces. When these feedback responses were faced with a change in initial stance width, the directional tuning remained consistent and EMG magnitudes decreased as the BoS widened. In addition, with a wide stance, the force constraint exhibited a more dispersed pattern. This study sought to better understand the interaction between varying size of base of support and feed-forward postural control mechanisms. Postural musculature, ground forces under both limbs and body kinematics were measured as subjects were asked to stand in a narrow, natural and wide stance and reach towards 13 distinct targets. Results showed spatially tuned muscle activity as well as similar EMG magnitudes between stances in almost all muscles, however, lateral force magnitude and maximum lateral center of mass displacement increased as stance width widened. Force vectors in each limb were limited to 2 directions, displaying a similar force constraint strategy to postural feedback responses. These results suggest that both feedback and feed-forward modes of postural control may share similar underlying organizational strategies in terms of the neural control. / Les ajustements posturaux anticipateurs sont connus pour initier les mouvements volontaires et pour déplacer le centre de masse (CM) à l'intérieur de la base de support (BdS). Ces changements dans l'activité musculaire des membres porteurs precèdent le déclenchement des mouvements volontaires. Par contre, les réponses posturales à des perturbations inattendues de la surface se produisent dans un court laps de temps après que l'équilibre ait été perturbé. Ces ajustements posturaux compensatoires (à base de 'feed-back' ou rétroaction) sont caractérisés par des muscles actifs directionnels, et des forces dont les directions sont limitées. Il a été montré que quand ces ajustements ont à répondre à une changement d'écart de jambes initial, l'organisation directionnelle spécifique des muscles reste inchangée et la magnitude des électromyographes (EMG) diminue alors que la base de support s'agrandit. Par ailleurs, plus l'écart de jambes est significatif, plus les forces exercées au sol se dispersaient. Cette étude cherchait à mieux comprendre l'intéraction entre les bases de support variées et le mécanisme proactif des contrôles posturaux. Les musculatures posturales, les forces de réactions au sol sous les 2 membres et les cinématiques du corps ont été mesurées alors qu'on demandait aux sujets de se mettre debout avec des écartements étroits, naturels et écartés des jambes puis ensuite de pointer vers 13 cibles distinctes. Les résultats ont montré une organisation directionnelle spécifique des muscles, aussi bien que des magnitudes d'EMG similaires entre les écartements de jambes pour tous les muscles. Pourtant, la magnitude d

The pathophysiology of Alzheimer's disease (AD) implicates several pathogenic processes, but their relative importance remains a highly debated issue. The purposes of the present studies were (1) to evaluate the implication of the over expression, in transgenic mice, of a mutated form of the amyloid precursor protein (V717FhAPP), or of the cytokine Transforming Growth factor-beta1 (TGF-beta1), on cerebral and hippocampal innervation, and (2) to determine if TGF-beta1 can affect vasomotor responses of cerebral blood vessels. While the mice over-expressing TGF-beta1 did not show cholinergic denervation, the mice over-expressing V717FhAPP showed selective cholinergic deficits in the CA3 region of the hippocampus. On the other hand, acutely increased levels of TGF-beta1 differentially affected the efficacy and/or potency of the vessel responses to serotonin (5-HT), endothelin (ET) and acetylcholine (ACh). These results suggest that the overproduction of V717FhAPP is a sufficient factor for cholinergic denervation and that TGF-beta1 can alter some aspects of cerebrovascular function.

Stress-activated protein kinases (SAPKs) were previously implicated in the phosphorylation of the neurofilament heavy subunit (NFH). This study presents direct evidence that stress-induced phosphorylation of NFH in both differentiated PC12 cells and cultured sensory neurons is inhibited by CEP-1347 (KT7515), a specific inhibitor of SAPK activation. In addition, long-term treatment of unstressed sensory neurons with CEP-1347 decreased the phosphorylation state of NFH in neurites. CEP-1347 differentially inhibited the activation of various SAPK isoforms in neuronal cell body and neurite fractions. Specifically, activation of a 55 kDa SAPK isoform in the neurite fraction was highly sensitive to CEP-1347 inhibition, with an IC50 of about 0.02 muM, while IC50 values for other SAPK species were at least 10-fold higher. The data indicate that SAPKs are involved in both constitutive phosphorylation of axonal NFH and stress-induced phosphorylation of perikaryal NFH in cultured sensory neurons and that SAPKs in the two neuronal compartments are activated by different signaling pathways.