Synaptogenesis is a complex process involving several steps that ultimately results in the matching of neurotransmitters released from the presynaptic nerve terminals with the appropriate receptors expressed by the postsynaptic neuron. In this thesis, I examined one step in the process of synaptogenesis, the expression of postsynaptic receptors. Cultured neonatal rat nodose neurons are a good model for these studies; in vivo, nodose neurons do not form synaptic contacts, but interestingly, when they develop alone in dissociated tissue culture most neurons express nicotinic acetylcholine receptors (nAChRs) and form de novo cholinergic synapses. First, I investigated the factors that influence nAChR expression by nodose neurons. I determined that in vivo, only 40% of nodose neurons have functional nAChRs, however, when grown alone in culture more than 80% express nAChRs and the majority have ACh current densities comparable to those on neurons that normally received cholinergic innervation. I found that ganglionic satellite cells control nAChR expression by nodose neurons, whereas trophic factors, such as nerve growth factor (NGF) and neurotrophin-3 (NT-3), can up-regulate ACh current density in the absence of the satellite cell influence. Second, as several different genes encode nAChRs, I was interested in determining the nAChR transcripts expressed by nodose neurons; mRNA for $ alpha3, alpha5, alpha7, beta2, beta3$ and $ beta4$ were detected by RNase protection assay. In addition, these changes in differentiated properties suggested that nodose neurons were developing a phenotype resembling that of autonomic neurons. Consequently, for comparison, I examined nAChR expression by rat sympathetic neurons, both at the functional and molecular level. Superior cervical ganglion (SCG) neurons express five nAChR transcripts: $ alpha3, alpha5, alpha7, beta2$ and $ beta4.$ To address which subunits are incorporated into the functional receptor, I used both electrophysiolog

Birth in our Canadian society is a daily phenomenon that occurs approximately 1,100 times per day, but unfortunately not always within the normal time frame, after 39-40 weeks of pregnancy. The present thesis addresses the question: What factors initiate premature labour? / Although the process is incompletely understood, it is known that immune and endocrine factors as well as the fetus itself contribute to the initiatory signals that bring about parturition. Indeed, cytokines, oxytocin, and steroids have been shown to affect the tone, amplitude and frequency of myometrial contractions. Given that their effects can be abolished by the addition of cyclooxygenase (Cox) inhibitors, the changes, in vivo, of Cox-1 and Cox-2 gene expression during pregnancy and the effects of cytokines and oxytocin on prostaglandin synthesis were examined. / Analysis by Polymerase Chain Reaction and Northern blotting indicated that uterine Cox-2 transcript levels increased approaching delivery and that the Cox-1 mRNA levels remained relatively unchanged throughout pregnancy. Immunohistochemical staining for the Cox-2 and OTR proteins revealed both to be co-expressed in the myometrium and in endometrial epithelial cells. Within the stroma, no OTR staining was found but intense staining for Cox-2 was noticed during labour. / Following the in vivo work, the effects of cytokines and oxytocin on various cell lines were tested. When IL-1$ beta$ and TNF-$ alpha$ were added to CUS-V2, a stromal cell line I developed, an increase in Cox-2 mRNA levels and an approximately 2 fold increase in PgF$ sb{2 alpha}$ released per 24 hours was observed. Under the same conditions, no changes in the levels of Cox-1 or GAPDH mRNA were observed. The effect of oxytocin was more pronounced. Its addition to CHO cells expressing constitutively the rat oxytocin receptor led to a 107 fold increase in the amount of PgE$ sb2$ released in a 24 hour period and a increase in Cox-2 protein levels as determined by Western blot analysis. Therefore, since uterine oxytocin and oxytocin receptor, in the rat, are regulated by estrogen and progesterone, and these steroids are in turn regulated by placental born factors, a clearer understanding is emerging of their interaction. These three systems might synergistically or additively interact to produce an amplified signal that initiates strong synchronous contractions and a delayed, but a parallel, increase in cervical compliance by inducing prostaglandins.

Immunohistochemical studies have shown opioid peptides in sympathetic ganglia and preganglionic neurons. An inhibitory action of opioids has been demonstrated in some central and peripheral synapses. However, a physiological role of endogenous opioids in sympathetic ganglia has not been well characterized. The present study investigated endogenous opioid action in the SCG of the cat. The results obtained show that an endogenous opioid is released from preganglionic axon terminals, in a frequency range that matches the natural activity of sympathetic preganglionic neurons, and inhibits ganglionic transmission by acting on post-synaptic opiate receptors of the $ mu$ and $ delta$ subtypes coupled to PTX-sensitive G protein. The size of the store of the endogenous opioid in the preganglionic axon terminals is small and readily exhaustible. Protein synthesis and axonal transport are required for maintenance of the store. The opioid inhibition is under control of the PKC system. This study provides the first thorough characterization of the properties of an opioid-mediated inhibitory mechanism in a well-defined synapse.

Atrial natriuretic factor (ANF), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of natriuretic peptides that may be involved, among other functions, in the maintenance of proper fluid and electrolyte balance and blood pressure control, as both circulating hormones and neuropeptides. The studies presented in this thesis were undertaken primarily to provide a deeper insight into the mechanisms of action of natriuretic peptides on the central nervous system (CNS). Using autoradiographic and cross-linking techniques, the precise cellular localization, specificity, and subtypes of natriuretic peptide binding sites in selected areas of the rat CNS were established. ANF binding sites are evident primarily on the basolateral plasmalemma of the epithelial cells in the choroid plexus and on plasmalemma of axons, dendrites, and astrocytes in the areas postrema. This cellular localization is consistent with the notion that circulating natriuretic peptides may exerts their effects via a CNS locus. Furthermore, evidence is provided that ANF binding sites are not only specific to ANF, but also to BNP, whereas CNP exhibits a distinct receptor selectivity. Finally, these studies provide a regional localization of three subtypes of natriuretic peptide receptor. In contrast to binding sites with characteristics of natriuretic peptide receptors A and B, which are detected in a number of CNS areas, natriuretic peptide receptor C is detected in only a few CNS structures. These studies are expected to contribute to a better understanding of the mechanism of natriuretic peptides action on the CNS.

The hypothalamic supraoptic nucleus (SON) contains both vasopressin-secreting (AVP) and oxytocin-secreting (OT) magnocellular neurons which release their respective hormones from the posterior pituitary gland into the systemic circulation. A direct relationship has been demonstrated in earlier physiological studies between the excitability of SON neurons and hormone release from the neurohypophysis. Catecholamines have been shown to activate SON neurons, thereby increasing the action potential frequency to the neural lobe. The present study examined the ability of catecholamines to evoke release of vasopressin and/or oxytocin from the neural lobe of an intraarterially perfused preparation of rat hypothalamo-neurohypophysis. / Experiments were carried out on male Long-Evans rats. Drugs (noradrenaline, dopamine, D1 and D2 dopamine receptor agonists) were delivered both into the perfusion medium and directly over the SON. Fractions of effluent medium were collected from the area of the neurohypophysis and tested for hormone release using a selective radioimmunoassay. Noradrenaline (NA) in concentrations of 10$ sp{-5}$ to 10$ sp{-4}$ M stimulated AVP release from neural lobe. The $ alpha sb1$-adrenergic receptor antagonist, prazosin (50 nM in perfusion experiments; 1$ mu$M in local injection experiments) blocked the noradrenaline-stimulated release. Dopamine (10$ sp{-4}$ to 10$ sp{-3}$ M), D1 (SKF 38393, 0.5 mM) and D2 (QNP 0.5 mM) dopamine receptor agonists also increased the release of AVP. In the presence of prazosin, DA, QNP and SKF 38393 still evoked AVP release. Therefore, it is proposed that both noradrenaline and dopamine stimulate the release of AVP in this preparation; the responses to dopamine appear to be mediated by both D1 and D2 dopamine receptors, presumably located at the level of the cell somata/dendrites, possibly also on axon terminals in the neural lobe.

Two models of electrical conduction in the cardiac ventricles are considered. The first model considered is that of a strand of ventricular muscle which uses the one-dimensional cable equation with the Beeler-Reuter model to represent the transmembrane currents. The effect of periodic stimulation on the strand is numerically simulated, and it is found that as simulation frequency is increased, the rhythms of synchronization are successively encountered. It is shown that this sequence of rhythms can be accounted for by considering the response of the strand to premature stimulation. This involves deriving a one-dimensional finite-difference equation or "map" from the response to premature stimulation, and then iterating this map to predict the response to periodic stimulation. / The second model states that the highly ramified His-Purkinje system is reminiscent of a fractal branching structure, and that the ventricular myocardium is activated in a "fractal" (time-scale invariant) fashion, since it is activated via the His-Purkinje system. A 1/$f sp alpha$ power spectrum can sometimes be linked to fractal processes. The averaged power spectrum of single QRS complexes falls off as 1/$f sp alpha$ ($ alpha sim$ 4).

Studies were designed to investigate the biochemical mechanism and hormonal regulation of dilatation of the uterine cervix at parturition. The suitability of the guinea pig animal model was established by demonstrating collagenolysis in the uterine cervix similar to the changes reported in women by light and electron microscopy. By 50 days gestation, there was a 50% decrease in collagen content in the cervix. At parturition (68 $ pm$ 2 days) there was a 6-fold increase in procollagenase, a 26-fold increase in the tissue inhibitor of metalloproteinase (TIMP) and a 2-fold increase in net collagenase activity in cervical extracts. Cervices in organ culture obtained at birth produced 2.9 times more procollagenase, 1.6 times more TIMP and a 10-fold increase in net collagenase activity when compared to nonpregnant or 25 days pregnant animals. Estradiol stimulated the production of procollagenase, TIMP and net collagenase activity in cervical organ cultures. Using primary monolayer cervical cell cultures derived from 50 day pregnant guinea pigs, procollagenase enzyme and its mRNA were stimulated up to 2-fold by recombinant human interleukin 1$ beta$ (IL-1$ beta$), estrogens and progesterone. Procollagenase production was completely abolished by cycloheximide and by actinomycin D indicating the need for translation and transcription respectively. The mechanism of signal transduction of procollagenase was also investigated. A rabbit polyclonal antiserum (R4718) that specifically reacts with epitopes on denatured and degraded $ alpha$2 chain of guinea pig type I collagen was used to demonstrate degradation of type I collagen in the extracellular matrix of the dilated cervix at parturition. Physiological concentrations of 17$ beta$-estradiol stimulated degradation of type I collagen in the nonpregnant cervix in organ culture. This effect was completely blocked by progesterone (100 $ mu$M). These studies indicate that cervical dilatation at parturition involves estrogen-indu

No description available.

The development of the hematopoietic system occurs in two waves: a first wave of primitive erythropoiesis, which consists in the production of a single lineage, primitive erythrocytes, and a second wave of definitive hematopoiesis, which describes the generation of many specialized blood cell types from common hematopoietic stem cells. Whereas definitive hematopoiesis is fairly well understood, involves signals from the environment and the expression of lineage-specific transcription factors, the molecular mechanisms regulating primitive erythropoiesis remain to be defined. The aim of this thesis was to clarify the roles of the Stem Cell Leukemia (SCL) gene and Vascular Endothelial Growth Factor (VEGF) during primitive and definitive hematopoiesis. Although gene targeting experiments indicate essential roles for VEGF/Flk-1 signaling and SCL at the onset of hematopoiesis, their exact functions remain elusive. This work has revealed that different thresholds of VEGF are required for the migration of hematopoietic precursors from mesoderm to sites of hematopoiesis and for their subsequent expansion. Furthermore, it shows that SCL, a basic helix-loop-helix transcription factor, acts downstream of VEGF signaling to ensure the survival of primitive erythrocytes. During definitive hematopoiesis, conditional knock-out experiments establish a non-redundant role for SCL during erythroid and megakaryocytic differentiation. Yet, it remains unclear whether SCL is essential for commitment to these lineages. Results presented in this thesis suggest that SCL is not involved in commitment to these pathways, but rather acts to consolidate and expand the erythroid and megakaryocytic compartments, following lineage choice. Finally, despite the central role for SCL during hematopoietic development, the mechanisms regulating its tissue specific expression remain unknown. This work provides molecular and functional evidence that demonstrate that the homeodomain- / Taken together, this work has elucidated molecular mechanisms which underlie cell fate decisions. It describes how the activity of a master regulator of erythroid differentiation, SCL, is regulated both by signals from the environment and at the transcriptional level, through combinatorial interactions between lineage-specific transcription factors.

Photoreceptor sensitivity to the e-vector of polarized light was measured with micropipettes filled with Lucifer Yellow. The terminals of the dye injected cells were located in the two plexiform layers of the lamina ganglionaris (first optic ganglion). Receptors with greater sensitivity to horizontally polarized light projected their terminals to the proximal plexiform layer (epl2) and those with greater sensitivity to vertically polarized light projected their terminals to the distal plexiform layer (epl 1). The e-vector sensitivity of several other cell types was also measured with micropipettes filled with Lucifer Yellow. The sensitivity to rotating polarized light was also measured in several cell types in the lamina ganglionaris and the chiasm. Tangential cells Tan1 and Tan 2 were recorded from and filled with dye for morphological identification. T-neurons (T1), which are single cartridge cells, were also impaled and their response to both rotating and stationary polarized light was measured. From the results presented a new model of information channeling is proposed. The model suggests that extracolumnar units are primary channels of polarization sensitivity information to the SFs. (Abstract shortened by UMI.)