<p>Ovulation is the result of the positive feedback action of estrogen, either alone or in combination with progesterone, and the integrated sensory input from the external environment onto the neural circuits controlling gonadotrophin secretion. During the dioestrus II in the 4-day cycling rat the ovaries secrete an increasing quantity of estrogen which stimulates neurons in the preoptic area (POA) to trigger the surge of luteinizing hormone releasing hormone into the capillaries of the hypophysical portal plexus. However, the mechanisms whereby estrogen stimulates this increased activity are, as yet, unknown.</p> <p>The binding of estrogen to specific target areas within the brain may induce changes in the metabolic function of neurons or it may directly or indirectly affect the electrical properties of neuronal membranes. The latter possibility has been tested by observing the affects of an intravenous (i.v.) injection of estrogen on the spontaneous activity and responsiveness to iontonphoretically applied putative neurotransmitters of identified preoptic neurons.</p> <p>Experiments I and II were undertaken in order to establish certain parameters of the experimental design. Experiment I demonstrated that halothone anaesthesia inhibits ovulation in the cycling rat by an action on the neuroendocrine circuits which control gonadotrophin secretion. In experiment II measurements were made of the plasma concentration of estrogen following various doses of an i.v. injection of estrogen or progesterone would stimulate an increased secretion of LH in estrogen primed ovariectomized rats. Measurements of serum LH concentrations one and five hours after the injection showed there was no stimulation of LH under these conditions.</p> <p>Experiment III was undertaken to identify neurons in the POA which are thought to implicated in a neuroendocrine circuit controlling gonadotrophin secretion and which may be target neurons for the positive feedback action of estrogen. Cells in the POA, which receive an input from the stria terminals have been identified by stimulating this pathway at its point of convergence in the amygdala. Antidromic stimulation techniques were employed to identify cells in the POA which make a direct connection with the basal hypothalamus.</p> <p>Experiment IV constitutes the major objective of the research, which was to thought to be implicated in a neuroendocrine circuit controlling gonadotrophin secretion and which may be target neurons for the positive feedback action of estrogen. Cells in the POA which receive an input from the stria terminalis have been identified by stimulating this pathway at its point of convergence in the amygdala. Antidromic stimulation techniques were employed to identify cells in the POA which make a direct connection with the basal hypothalamus.</p> <p>Experiment IV constitutes the major objective of the research, which was to investigate 1) the sensitivity of antidromically identified preoptic neurons to iontophoretically applied putative neurotransmitters; the cells were inhibited by both the cataecholamines, dopamine (DA) and norepinephrine (NE) but were unresponsive to acetylcholine applied iontophoretically; b) to test the possible membrane effects of estrogen on those antidromically identified cells. Two indices of a change of the excitability of preoptic neurons were used (i) the effects of an i.v. injection of estrogen on the rate of spontaneous activity and (ii) changes in the response of a neuron to electrophoretically applied DA or NE following the injection of estrogen or progesterone in primed and unprimed animals. The results of the first series of experiments demonstrated that estrogen can decrease the rate of spontaneous activity in identified preoptic units. Some cells showed a long lasting depression in their spontaneous activity following the steriod injection while others showed a short term decrease in the rate of unit discharge five minutes after estrogen administration. In the second series of experiments no changes were observed in the dose response curves of iontophoretically applied DA or NE thirty minutes after the steroid injection in either group of animals. The negative finding could be due to the technical limitations of this type of experiment or to the fact that estrogens do not affect the response of neurons to synaptic inputs in which these amines may function as neural transmitters.</p> / Doctor of Philosophy (PhD)

<p>Early in the development of the embryo, cells lose their ability to differentiate into any adult type of cell and become determined. A cell which has achieved a certain degree of differentiation can ultimately reproduce to form only a limited number of cell types along the original line of differentiation: for example, a primitive mesenchymal cell can give rise to only connective tissues such as muscle or bone.</p> <p>Apart from the primordial germ cells (those cells which later give rise to oocytes in the female and spermatocytes in the male), all cells have begun to specialize, with a consequent narrowing of their potential, very early in gestation.</p> <p>Teratomas are neoplasms which contain many kinds of tissues which are foreign to the organ in which they arise. There is controversy over whether these tumours arise from the growth of cells which have escaped early developmental controls, or whether they arise from the proliferation of the only multipotential cell in the body, the germ cell. Rather than following their normal path of differentiation to oocytes or spermatocytes, they might fulfil their potential to form almost any kind of tissue. Thus tumours composed of skin, bone, muscle, nervous tissue, and glands are found in adult testes and ovaries.</p> <p>Since the germ cells arise in the embryonic yolk sac and travel up the umbilical cord to the developing gonads, there is a theory that these multipotential cells may occasionally become lost and much later, often in adulthood, begin their proliferation in sites along the midline: at the base of the spine, in the chest and throat.</p> <p>The experiments in this thesis explore an animal model in which teratomas can be produced by the grafting of 7½-day mouse embryos to the gonads of adult hosts. According to the literature the tumour which develops as a result of this operation is identical to those arising spontaneously in a highly inbred strain of mouse. Those experimental tumours which contain areas of undifferentiated tissues can be transplanted to other hosts; sometimes retaining their embryonic nature for many generations.</p> <p>Whole embryos develop as teratomas in up to 80% of grafts to male hosts. The embryos can be cut in such a way that one half contains extra-embryonic material (that which eventually forms the placenta) and also the yolk sac containing the germ cells, and the other half which contains only sonatic tissue.</p> <p>Grafts of the somatic parts result in teratomas in 74% of the testes, a rate similar to tumours from whole embryos, whereas grafts of the extra-embryonic parts containing germ cells resulted in only 1 small teratoma out of 35 grafts.</p> <p>In this experimental model, it has been demonstrated that the primordial germ cells of the embryo are not responsible for the development of the neoplasms, but rather it is the somatic cells which are giving rise to these tumours. One must ask whether the controls which govern normal differentiation may become faulty and allow somatic cells to retain or regain their pluripotential nature making it possible, when conditions are right, to proliferate to form the "monstrous" tumour, teratoma.</p> / Doctor of Philosophy (PhD)

<p>Repeated electrical stimulation of certain brain sites in animals leads to the progressive development of clinical seizures, a phenomenon known as kindling. The basic neuronal mechanisms underlying this effect may be similar to those involved in human epilepsy, but are not well understood. We have examined the properties of single neurones and populations of neurones in hippocampal slices derived from rats kindled in the hippocampus, fimbria/fornix, perforant path, or amygdala, and compared them with those of unstimulated animals.</p> <p>At the neuronal population level, tissue from kindled rats did not display an increased tendency to generate spontaneous epileptiform activity. This as also the finding in ionic substitution experiments, where the levels of extracellular potassium were increased as high as 9 mEq/1. Electrical stimulation of afferents to the CA1 region evoked synaptically-mediated population responses which were similar to those of controls, except for tissue derived from rats kindled in the fimbria or in the amygdala. In these experiments, there was a tendency for multiple population spikes to appear in the responses to stimulation and this may reflect an increased excitability of the neuronal population.</p> <p>Intracellular studies of hippocampal CA1 neurones from amygdala-kindled rats revealed that the basic activity and passive membrane properties did not differ from those of controls. However, the amplitude of the afterhyperpolarization following synaptic activation was increased in these neurones. In addition, the response to intracellular injection of depolarizing current was altered in some neurones, observed as an increased action potential discharge during the initial phase of depolarization.</p> <p>These findings are discussed in terms of an apparent immunity of the hippocampus towards the development of chronic epileptogenic properties, and the ionic and membrane mechanisms which may be responsible for the observed differences between kindled and unkindled tissue.</p> / Doctor of Philosophy (PhD)

<p>Sera raised in rabbits against adult rat kidney has long been known to be teratogenic when administered to pregnant rats at a time when the embryos are in the gastrulation stage. The main hypothesis postulated on the mode of action of teratogenic kidney antiserum is that the antibodies directly interfere with placental development and function; thus resulting in a nutritional deficit to the embryo. A nutritional deficit is believed to interfere with embryonic morphogenesis. To date no investigations have been performed on placental function and development.</p> <p>In the present investigation, histological and histochemical techniques were used to study the effects of kidney antiserum administered to the pregnant rat on the development and function of the fetal placental structures.</p> <p>The results of this study have demonstrated that both the visceral yolk sac and chorio-allantoic placentae were adversely affected. The effects of kidney antiserum on the visceral yolk sac is consistent with an interpretation of an inhibition of nutrient uptake. The development of the chorio-allantoic villi and the allantoic vasculature was markedly retarded. The effects of kidney antiserum on the fetal placental structures suggest that the embryo undergo a nutritional deficit.</p> <p>Glycogen and protein content of the maternal placenta was also investigated. The glycogen content was significantly lower in experimental rats as compared to controls. However, this difference was not noted until 72 hours after the administration of the kidney antiserum. This was a time when the embryos were not very susceptible to teratogenic antiserum insult.</p> <p>Localization of kidney antibodies was also performed. The antibodies were demonstrated to be present within the embryo proper, amnion, parietal yolk sac, chorion and allantois.</p> <p>Numerous Feulgen positive necrospherules were also observed within the embryo proper within 12 hours after the administration of kidney antiserum.</p> <p>The observations of the present study is thus consistent with the interpretation that the cause of kidney antiserum teratogenesis is an effect of kidney antibodies on placental function and development resulting in a nutritional deficit to the embryo and/or is a direct effect of kidney antibodies on the integrity of the cells of the embryo proper.</p> <p>Both control and experimental trophoblast shells contained islets of cells. These islets, not previously reported in the literature, contained mitotic figures. It is suggested that these islets are the proliferation sites of the trophoblast shell.</p> / Doctor of Philosophy (PhD)

<p>Infection by Pichinde virus, a member of the arenavirus group, was studied in vivo in Syrian hamsters, and in vitro in cell culture. Emphasis in the in vivo studies was placed on the mechanism of resistance to virus infection in adult hamsters. Two hamster strains were found to differ in their susceptibility to lethal Pichinde virus infection. LVG/Lak hamsters were found to be 100% susceptible to low doses of Pichinde virus during the first 6 days of life, but after 8 days of life, mortality was uncommon. Peak serum virus titers in animals infected at 3 days of life were 4 logs greater than in animals infected at 12 days. MHA/Lak hamsters, in contrast, were found to be susceptible to lethal virus infection both as newborns and as adults. Peak serum virus titers of greater than 10⁸ PFU/ml were observed 8 days after infection of adult MHA hamsters compared with less than 10³ PFU/ml in their LVG counterparts. Cultured primary kidney cells and peritoneal macrophages from either hamster strain supported Pichinde Virus replication equally well in vitro. Antibody levels measured by complement-fixation test were similar at 13, 21, and 30 days after infection. Cyclophosphamide immunosuppression, administered 3 days after infection, rendered adult LVG animals susceptible while slightly increasing the mortality among MHA hamsters. These findings suggest that immunologic factors maturing early in life in LVG hamsters and deficient in MHA hamsters limit Pichinde virus infection. The relationship of these observations with previously reported arenavirus diseases is discussed.</p> <p>The antigenic structure of Pichinde virus was examined. Lysates of BHK₂₁ cells, infected with Pichinde virus and harvested 48-96 hours after infection contained virus-specific antigens detectable by complement-fixation (CF) test. Immunodiffusion analysis of the lysates demonstrated two antigens which differed in their properties of heat and proteolytic enzyme resistance. The antigen accounting for the major proportion of the CF antigen activity was a heat stable, pronase resistent protein of 20-30,000 molecular weight estimated by gel filtration. The minor antigen was heat labile and susceptible to proteolysis. Antiserum prepared against partially purified major antigen, yielded a band of identity when tested against antiserum prepared against virus "cores" obtained by NP-40 solubilization of purified Pichinde virus. Disruption of purified virus by treatment with 1% NP-40 and 50 ug/ml RNase liberated 2 soluble antigens which identified with those demonstrated in lysates of infected cells. The liberated antigenic activity was shown to contain 3 of the 4 polypeptides found in the virion. These findings suggested that the antigens detectable by CF and immunodiffusion were components of the virion core. Major antigen derived from infected cells was purified by rate-zonal sedimentation, isoelectric focusing and gel filtration. Two low molecular weight polypeptides were demonstrable in the purified antigen.</p> <p>Since multiple segments of RNA exist in the Pichinde virion, it was of interest to determine, whether antigen synthesis and virus production could be dissociated in the infected cells. In Vero cells infected by Pichinde virus, antigen on the cell surface, and production of infectious virus shut down 48-96 hours after infection, whereas antigen detectable in the cytoplasm of infected cells appeared stable for over 5 days. In BHK₂₁ cells, actinomycin D (AD) at dosage levels of 1-4 ug/ml reduced virus yields by greater than 95% while reducing antigen detectable by CF by only 30%. The quantity of antigen produced was independent of AD dosage within the range tested. Both the major and the minor antigens were identified in lysates of AD treated cells. The observed decrease in infectious virus production could not be attributed to increased cell associated virus or to greater production of defective interferring particles. Surface antigen, demonstrable by immunofluorescence and by antibody binding assay was found to accumulate of infected cells incubated with AD. These findings suggest that Pichinde virus replication in AD treated BHK₂₁ cells is blocked at the level of virus maturation.</p> / Doctor of Philosophy (PhD)

<p>Changes in motor control processes associated with advancing age have been the focus of considerable research interest. However, the effect of age on the processes involved in motor skill acquisition have received little attention. In this thesis, two experiments are reported that examine the differences in motor learning between young and older normal adults. The strategy was to evaluate the age-related effects on learning of knowledge of results (KR) variables. The learning task in both experiments was a multisegment task with spatial/temporal requirements. The KR variables evaluated were, (1) random and blocked KR schedules, and (2) different schedules of relative frequency of KR. In general, while the findings indicated large performance differences between age groups, there was no difference between age groups in how the KR variables were used to learn the task. The results suggest that despite declines in performance that accompany age, there seems to be no decline in the capabilities of older adults to use these KR variables to acquire and retain this motor skill.</p> / Doctor of Philosophy (PhD)

<p>Partial denervation of a shared target tissue leads to 'collateral' sprouting of the remaining nerve fibres supplying the same region. The mechanisms responsible for this widespread phenomenon are unknown, but it has generally been believed that products from the degenerating fibres constitute the sprouting stimulus. Recently it was shown that a similar sprouting of intact nerves occurred in the salamander hind-limb when the axoplasmic transport in one of the neighbouring nerves was blocked by colchicine. This block did not, however, appear to interfere with the functioning of the treated nerve. On this evidence it was proposed that nerves release factors which are concerned in the regulation of their peripheral fields. The experiments of this thesis test this hypothesis further. In particular this study quantitatively examines the organization of the cutaneous mechanoreceptors of the salamander hindlimb, and the effects of various nerve treatments on this organization.</p> <p>A mechanical prodder of 10-50μ tip diameter was used to determine the sensory threshold of the skin at selected spots. The mechanoreceptors were found to be all rapidly-adapting. An analysis of the distribution of their thresholds across the skin points to the existence of a single population of mechanosensory endings of similar threshold which are fairly uniformly distributed in the plane of the skin. A simple model, based on the results, indicates that these receptors have receptive field sizes of about 50μ, and are spaced approximately 150-200μ apart. Direct inspection of data from systematically surveyed skin areas gave findings which were consistent with these estimates. These quantitative results made it possible to establish that the blocking of fast axoplasmic transport does not affect the sensitivity or distribution of the individual mechanosensory endings of the treated nerve. Nevertheless new sprouts appeared from the adjacent nerves, and it seemed that there was a tendency for these to grow preferentially to the site of the still-functioning endings of the treated nerve. Most significantly, after partial denervation of the skin the number of newly sprouted endings quantitatively matched the number of endings lost by nerve section.</p> <p>It was concluded that there is a control mechanism which continuously regulate the density of the skin innervation. This control system probably involves the mutual interaction of substances continuously secreted from the target tissue, which cause nerves to sprout, and factors released from the nerve endings which offset the effects of the growth stimulus. The nerve field density reflects an equilibrium state between the neural and tissue influences.</p> / Doctor of Philosophy (PhD)

<p>The development of murine gonads commences in both males and females as indifferent appearing blastemata around the 10th day of embryogenesis. Within the following two days, the, gonadal blastemata became heavily populated with the primordial germ cells which migrate in from extragonadal sites.</p> <p>The process of gonadal sex differentiation is first evident in males where the appearance of sex cords marks testicular histogenesis. Ovarian development is largely marked by the fact that female gonads do not resemble testes and also by the early transformation of oogonia into oocytes.</p> <p>It has been proposed by a number of investigators that the absence of the primordial germ cells in the gonadal blastemata during the indifferent stage of gonadogenesis would result in the failure of sex cord differentiation to take place within the gonadal soma. Other investigators have proposed that gonadal sex differentiation is not dependent upon the presence of germ cells within the gonadal soma.</p> <p>To resolve this controversy mice carrying the alleles Steel/Steel Dickie (S1/S1ᵈ) were selected since the Steel mutation is known to severely affect the primordial germ cell line prior to its populating the gonadal ridges before sex cord differentiation. The experiment therefore is a natural one not requiring chemical or physical ablation of the germ cell line at the time it is first histochemically identifiable in sites far removed from the gonadal anlages.</p> <p>Primordial germ cells were identified histochemically by an azo dye technique in 27 day 9, 33 day 10, 33 day 11 and 21 day 12 embryos which were obtained from matings between mature WC/ReJ-S1/+ females and C57B1/6J-S1ᵈ/+ males. One fourth of the embryo zygous S1/S1ᵈ mutation. Failure to the germ cell population to increase after 9 days gestation occurred in 26% of the embryos. These embryos were classified as the mutant S1/S1ᵈ group. The primordial germ cells of the mutants, though few in number, were found to follow a normal migratory pattern to the gonadal ridges. Gonadogenesis was studied in 227 fetuses of 12-18 days gestation and in animals from birth to maturity. The genetic sex of fetuses was determined by the presence or absence of sex chromatin in amnion cells. Prior to day 14, genotypes were established as normal or mutant according to the germ cell population present in one gonad from each fetus. After day 14 the genotypes were determined from red blood cell samples, the S1/S1ᵈ fetuses displaying the macrocytic anemia characteristic of this mutation.</p> <p>The ratio of mutant males to mutant females was the normal 1:1, and the number of mutants identified was within the expected 25% frequency. Despite a paucity of germ cells, mutant gonads differentiated according to their genetic sex. Although mutant gonads are composed almost entirely of somatic tissue they grow at the same rate as the somatic component of normal gonads up to day 16 post coitus in males, and to the day of birth in females. The overall pattern of growth was similar to that seen in normals. The Mullerian and Wolffian ducts as well as the external genitalia also developed according to gonadal sex in the mutants.</p> <p>In those mutant gonads which contained few germ cells, they grew and differentiated in the same manner as did those of the normals up to birth. By maturity, no mutant germ cells were found to have differentiated beyond prophase of meiosis I.</p> <p>It is concluded that sexual differentiation and gonadogenesis can take place in the absence or at least in the near absence of germ cells. The Steel mutation appears to act in preventing the proliferation of the primordial germ cells and their capacity to complete meiosis I of gametogenesis. The mutation, however, does not affect the evolution of the germ cells from their source nor the capacity of the germ cells to migrate to the gonadal blastemata.</p> / Doctor of Philosophy (PhD)

<p>Denervation of skeletal muscle fibres is known to lead to a marked alteration of their normal characteristics. The mechanism for this is not clear, but there is great interest in the possibility that the changes are the result of the cutting off of neurotrophic factors which normally continuously act on the muscle fibres. To study this question, colchicine, a drug known to interfere with neurorial transport, was injected through the epineurium of one sciatic nerve in rats, in an attempt to prevent the postulated neurotrophic factors from reaching the extensor digitorum longus (EDL) muscle, without interfering with impulse activity in the nerve, or the consequent activity of the muscle. Following this procedure, ipsilateral EDl muscle fibres were found to exhibit extrajunctional sensitivity to acetylcholine ( ACh ), tetrodotoxin ( TTX ) resistant action potentials, and lowered membrane potentials ( RMP ); all characteristic changes observed in denervated skeletal muscle fibres. These fibres however, were shown not to be denervated, since they displayed miniature end-plate potentials (m.e.p.p.s) and neuromuscular transmission was normal.</p> <p>Unexpectedly, the contralateral EDL muscle fibres which were examined as a routine control, also displayed the features of "denervation" following an injection of colchicine into the ipsilateral sciatic nerve, and were virtually indistinguishable from the ipsilateral EDL fibres. It thus appeared possible that nerve injections of colchicine were acting systemically, and indeed this was shown to be the case when similar denervation-like changes were caused bilaterally after intraperitoneal (I.P.) injections of the same dose of colchicine.</p> <p>Although thesis results showed that colchicine was acting systemically, it was still possible that it, was effective by blocking neuronal transport in the muscle nerves. Axoplasmic transport was therefore measured in the sciatic nerve of rats, following colchicine injections both systemically and into the contralateral nerve. At no time was there any evidence of an impairment of transport, even though both EDL muscles contained fibres which displayed extrajunctional sensitivity to ACh. lt was concluded that extrajunctional sensitivity to ACh following colchicine injections was not due to an interference with axoplasmic transport.</p> <p>In the injected sciatic nerve, an impairment of transport was consistently observed and this generally was associated with a detectable paresis and a small percentage (5-7%) of apparently denervated EDL muscle fibres. In addition, the indirectly evoked tetanic tensions produced by the ipsilateral EDL muscles decreased in comparison to the contralateral EDL muscles, commencing about 2 days following the sub-epineural injection of colchicine. From these observations, it was suggested that the demonstrable impairment of axoplasmic transport in the ipsilateral sciatic nerve caused the impairment of neuromuscular transmission on that side.</p> <p>Colchicine was also chronically applied to rat sciatic nerves by the use of drug-impregnated silicone rubber nerve-cuffs. This procedure prevented the systemic effect of colchicine, but the mechanical presence of the cuff resulted in a usually transitory and variable block of impulse conduction in the nerve. Since muscle inactivity per se also leads to changes previously believed to be due to denervation, the changes that were observed with colchicine-cuffs did not constitute unequivocal evidence supporting neurotrophic effects on the ipsilateral EDL muscle fibres.</p> <p>It was concluded from the results presented in this thesis that the results of experiments involving colchicine injections or drug-impregnated nerve-cuffs do not unequivocably support the existence of a neurotrophic control of skeletal muscle fibre properties.</p> <p>Finally, preliminary experiments in which colchicine was used in association with denervation indicated that the most likely mechanism of action of colchicine was a direct one on the muscle fibre membrane itself.</p> / Doctor of Philosophy (PhD)

<p>This research investigated absorption, distribution and utilization of Zn and Cu in developing piglets as a model for human premature infants, specifically focusing on the impact of diet (amount and ratios of minerals), drugs (dexamethasone) and stage of development on mucosal cell membrane Zn and Cu transport and body Zn and Cu storage. In 32 and 52 h old piglets, velocity of Zn transport across intestinal brush border membranes (BBM) was higher than in 10 and 20 d old piglets but occurred via a nonsaturable mechanism. Cu transport across BBM was greater in the 20 d group than in 10 d, 32 h and 52 h groups and occurred mainly via a saturable mechanism in 32 h, 10 d and 20 d groups. Plasma Zn and Cu were significantly lower but tissue Zn, Cu and metallothionein (MT) contents were higher in the younger groups than the 10 and 20 d groups. Competition for transport between elements may also be important in infants since Cu transport across BBM was suppressed by Zn but enhanced by Fe at the ratios of Zn:Cu and Fe:Cu which are similar to those in premature infant formulas. Further studies indicated that Fe mainly increases Cu binding to BBM. High dietary Zn intake did not alter Zn transport across BBM but induced Zn accumulation in the intestinal mucosa and liver in piglets. These results are valuable in determining appropriate upper limits for Zn, Cu and Fe contents in infant formula. Exogenous dexamethasone (DEX) when used therapeutically to induce lung maturation in premature infants may compromise Zn and Cu status. Zn influx across BBM was significantly greater but Zn efflux was significantly lower in DEX treated compared to control piglets. The effect of DEX on Zn influx was abolished by dietary Zn supplement. Intestinal Cu uptake was also enhanced by DEX treatment. However, DEX-induced mucosal uptake of Zn and Cu did not appear to result in increased net Zn and Cu absorption since intestinal MT was induced by DEX. This study provides evidence that DEX treatment alters Zn and Cu metabolism in early life. Zn and Cu status should be carefully monitored in premature infants receiving long-term DEX therapy.</p> / Doctor of Philosophy (PhD)