• A study on the interaction of Alzheimer's disease beta amyloid protein with cultured mouse neuroblastoma cell line NB41A3

      Zhao, Xiaohong (1994)
      The $\beta$ amyloid protein is the primary constituent of amyloid plaques in the brains of Alzheimer's disease patients. The generation of $\beta$ amyloid protein from $\beta$ amyloid precursor protein and its interaction with neuronal cells were studied in the mouse neuroblastoma cell line NB41A3. Immunoreactivity to the carboxyl terminal of the precursor protein was detected among the membrane proteins of these cells, indicating that $\beta$ amyloid precursor protein is produced by NB41A3 cells. Also amyloid precursor protein carboxyl terminal immunoreactivity was observed in the conditioned medium of the cells, demonstrating various cytosolic peptide fragments are secreted during the cellular processing of the $\beta$ amyloid precursor protein. Synthetic $\beta$ amyloid peptide was shown to negatively affect NB41A3 neuroblastoma cells as judged by decreasing cell numbers, decreasing amount of cell protein, and release of the cytosolic enzyme, lactic dehydrogenase, into the medium. At the ultrastructural level, internal damage to the nucleus could be observed. Synthetic $\beta$ peptide showed specific binding with neuroblastoma cells. The internalization of the $\beta$ peptide into the cells suggest a direct mechanism for $\beta$ amyloid protein toxicity in vivo. This research contributes to the knowledge of the processing of Alzheimer's disease $\beta$ amyloid precursor protein in NB41A3 cells and demonstrates that NB41A3 cell provides a practical in vitro model for studying the mechanism of Alzheimer's disease and amyloid toxicity.
    • Effect of high latitude on the variability of human event-related brain potentials

      Bush, Anita Marie; Geist, Charles R.; Barnes, Brian M.; Connor, William; Ebbesson, Sven O.; Emery, Scott; Murphy, Edward C. (1993)
      European researchers (Anderson, Chambers, Myhre, Nicholson, & Stone, 1984) at 69 degrees north latitude, have reported seasonal changes in the human electroencephalogram, EEG. Other researchers from the North American mid-latitudes (Deldin, Duncan, & Miller, 1989a, 1989b), using event-related brain potentials, ERPs, have reported changes associated with ambient light. Similar changes in the event-related brain potentials of humans have not been reported from the high latitudes nor by longitudinal methods. This dissertation longitudinally investigated ERP variability in normal humans living at high latitude. One hundred and twelve residents of Fairbanks, Alaska participated in single-trial ERP testing, both auditory and visual, to evaluate criteria of validity and reliability. Subsequently, eight normal humans participated in auditory and visual ERP testing every month for twelve consecutive months. The longitudinally-studied subjects were residents of the Alaskan subarctic, where testing occurred, and each experienced naturally-occuring photoperiod variability of 3.31 hours in December to 21.98 hours in June. Dependent variables included P3 amplitude and latency in both sensory modalities. Independent variables included: age, estimated cranial volume, length of last sleep epoch, subjective wakefulness, ambient photoperiod, and ambient geomagnetic field. Distribution of the longitudinal ERP data satisfied Shapiro-Wilke criteria for normality. The GLM-ANOVA for ordered repeated measures was used and a month effect was observed in P3 amplitude and latency, as well as a month-by-sensory mode interaction. The principal components method of factor analysis evaluated the factor weights of the independent variables. Variables unique to each subject (e.g., age, cranial volume, length of last sleep) were more heavily weighted in the first factor. The environmental variables became heavily weighted in the second factor, and descriptors of the earth's natural geomagnetic activity were weighted more heavily than ambient photoperiod. Following PCA analysis, the environmental variables of geomagnetic field and photoperiod were separately investigated. No seasonal variability in the ERP characteristics was detected when using a photoperiod-based definition of season. However, variability in auditory P3 amplitude was observed when groupings were defined based on geomagnetic criteria.
    • Primary afferent projections in a diver, the muskrat

      Delisa, Susan Manette; Ebbesson, S. O. E. (1989)
      In a preliminary search for primary afferent connections involved in the diving response, cutaneous afferents from the nose were traced in muskrats and compared with those in rats, and with projections from the soft palate, posterior pharynx and larynx. Horseradish peroxidase (HRP) was injected into the skin or mucosa, under anesthesia. After 48 h survival, the deeply anesthetized animal was transcardially perfused and the brain was frozen and sectioned transversely in a cryostat. The sections were reacted for HRP according to standard techniques, using tetramethylbenzidine; alternate sections were Nissl stained. HRP-labeled structures were mapped using darkfield photomicrographs and camera lucida drawings. Cutaneous afferents from the nose in the muskrat project densely to layers I-II of the ventral and dorsolateral parts of the caudal subnucleus of the spinal trigeminal nucleus (Sp5C) and sparsely to layers V-VI of Sp5C, sparsely to the ventromedial part of the interpolar subnucleus of the spinal trigeminal nucleus (Sp5I), moderately to the oral subnucleus of the spinal trigeminal nucleus (Sp5O)--particularly the dorsomedial part, possibly overlapping with the nucleus of the solitary tract, and with processes of labeled cells of other lateral facial nucleus extending into ventromedial Sp5O,--moderately to the principal trigeminal nucleus (Pr5); and to the paratrigeminal nucleus (Pa5). Projections in the rat were the same, except that little or no labeling of layers V-VI of Sp5C, dorsomedial Sp5O, or Pa5 was present. Projections from the soft palate to layers I-II of rostral Sp5C, Sp5O, Sp5I, and Pr5 were similar to those from the nose in the muskrat. Heavy projections from the soft palate, and less dense projections from the posterior pharynx and larynx, to Pa5 also were found. Those regions receiving dense projections from the nose, overlapping projections from the various sites, and more highly developed projections from the nose in the muskrat than in the rat, are of particular interest for further investigation of the neural substrate underlying the diving response. The projections traced from the nose correspond particularly with nociceptive and thermoreceptive projections, which suggests that thermoafferent function may be involved in the elicitation of the diving response.
    • Reconstruction of the damaged central nervous system and spine

      Helm, Gregory Anthony (1996)
      The field of neuronal transplantation has received a great deal of interest since the 1970's and is currently considered a possible treatment option for both neurodegenerative diseases and spinal cord injury. In this dissertation, fetal neostriatal transplants grafted into the lesioned striatum were studied in both the rat and rhesus monkey. Golgi-impregnation and immunohistochemical techniques were extended to the light and electron microscopic levels to determine the detailed anatomy of the developing striatal implants. Choline acetyltransferase immunoreactive and substance P-like immunoreactive neurons within the rat striatal transplants were morphologically and ultrastructurally similar to normal striatal neurons. When the striatal grafting studies were extended into the rhesus monkey, normal neuronal maturation was demonstrated three months postoperative, both at the light and electron microscopic levels, using various neuroanatomical techniques. From these studies it can be concluded that fetal striatal grafts may be a useful treatment option for Huntington's disease, although numerous difficulties including neuronal degeneration and transplant rejection need to be addressed before this approach is applied in the clinical setting. In a second group of experiments, various approaches to improve autologous bone spinal fusions were studied. Specifically, the utilization of demineralized bone matrix, Type I collagen gel, and recombinant human bone morphogenetic proteins were evaluated for their effects on autologous bone spinal fusions in canines. The study demonstrated that recombinant human bone morphogenetic protein has a strong effect on the amount of bone deposition at the fusion site and, in addition, increases the number of vertebral levels which solidly fuse. The Type I collagen gel appeared to improve the interface between the autologous bone grafts and the host bone, while the demineralized bone matrix had a strong negative effect on the autologous bone graft fusions. Spinal fusion operations in the future will be much more successful if these various methods to improve spinal arthrodesis are utilized to their full potential. It is now clear that reconstruction of the central nervous system and its bony coverings is a real possibility in the very near future, although extensive clinical studies need to be performed before they are widely used in the neurosurgery community.
    • Structure-Function Relationship Of The Developing Acetylcholine Receptor In Amphibians

      Sullivan, Michael Patrick; Duffy, Larry (2001)
      The nicotinic acetylcholine receptor is responsible for the transfer of signals from the peripheral nervous system to skeletal muscle, resulting in movement. Despite the importance of the acetylcholine receptor, many questions remain unanswered about the relationship between the structure and function of the receptor. The purpose of our research was to explain certain features of the relationship between the amino acid sequence and the function of the amphibian nicotinic acetylcholine receptor. We focused on describing the structural elements underlying the physiologic changes that occur during development and re-innervation of damaged mature muscle. We used molecular biological techniques to alter the amino acid sequence of the receptor and then studied the effects of these alterations using the electrophysiological technique of single channel recording. Our research resulted in the discovery of critical residues involved in two important characteristics of receptor function, the conductance and open time of the ion channel. These results offer new molecular insights into the classic observation that synaptic currents become briefer in duration during the course of muscle development in vertebrates.
    • Structure-Function Studies Of The Serotonin Type -3 Receptor Ligand -Binding Domain

      Joshi, Prasad Ramesh; Schulte, M. K. (2005)
      The serotonin type-3 receptor (5-HT3R) is widely distributed in peripheral and central nervous systems. This pentameric protein is a member of the Cys-loop superfamily of ligand gated ion channels and plays a role in mediating physiological processes in nervous, cardiovascular, and digestive systems. The ligand-binding domain of this receptor is extracellularly located and is composed of multiple putative loop structures. Based on structural and sequence homology with other members of the superfamily, it has been proposed that at least six such loops (loops A to F) contribute to the ligand-binding domain. Binding of agonist initiates a conformational change which is transduced to the channel, leading to channel opening (gating). The aim of this study was to elucidate the contribution of residues in loops B and E to the mechanism of channel gating in the 5-HT3R. To this end, the three critical tyrosine residues in the loop E region were characterized employing site-directed mutagenesis, electrophysiological studies as well as radio-ligand binding assays involving two structural classes of 5-HT3R agonists. In addition, structure/function analysis of the loop B region was carried out alanine-scanning mutagenesis. Experimental data were correlated with molecular modeling studies. These studies show that the hydroxytryptamine and phenylbiguanide class of compounds utilize different mechanisms of ligand binding and gating in the 5-HT3R. Studies involving the loop B reveal that this region plays a critical role in ligand binding and channel gating. Data obtained from comparison of ground state and agonist-bound models of the 5-HT 3R was correlated with biochemical data. Taken together, these data suggest that agonist interaction with loop B region probably initiates a conformational wave that results in intra- and inter- subunit hydrogen bonding interactions. Our data suggest that these interactions play a critical role in agonist-induced channel opening.