<p>One approach to the study of how nerves can establish appropriate connections, and of possible "plasticity" intrinsic to the connections themselves, is to partially denervate a target tissue and examine the reestablishment of functional connections by nerves. Cells or tissues whose normal nerve supply has been lesioned can become reinnervated by two means: the sprouting of new collaterals from nearby intact nerves, or the regeneration of the damaged fibres. Both mechanisms have been reported as involved in the recovery of function after nerve lesions in mammals.</p> <p>In the course of experiments on the reinnervation of skin in rabbits, however, evidence was obtained suggesting that intact low-threshold mechanosensory nerves in adult mammals may not sprout into denervated skin. The investigations described in this thesis were undertaken to examine this possibility further, and in particular to use the rat to study the ability of such nerves to establish functional connections in denervated skin.</p> <p>Direct electrophysiological recording from cutaneous nerve bundles was used to detect impulses generated by tactile stimulation of the skin. The area of skin supplied by a nerve (its low-threshold mechanosensory receptive field) and in particular the number of touch domes (specific sensory structures in the skin) innervated by that nerve were determined after various manipulations; both the area of the field and the population of touch domes were used as measures of the distribution in the skin of functional endings. Intact low-threshold mechnosensory nerves in the rat were found to sprout into adjacent denervated skin only during a remarkably brief "critical period" that begins at about 15 days of age and ends at about 20; such sprouting could neither be evoked nor did it continue after this age. Moreover, during the critical period, the intact low-thershold mechanosensory nerves often failed to sprout into denervated skin except that available within the same ("parent") dermatome. These intact nerves therefore are under constraints both in time and space, which operate to limit the extent of functional sprouting and also the location of such newly established endings. Regenerating nerves however were found to be subject to neither of these constraints, and even in the adult they would freely grow across dermatomal borders to establish functional endings in denervated skin. Of special significance, when the regenerating nerves arrived at skin that during the critical period had been reinnervated by sprouts of intact nerves, the regenerating fibres replaced funtionally the earlier sprouted endings; those endings within the usual territory of the intact nerve that had sprouted were not displaced however.</p> <p>It was concluded that intact and regenerating nerves are differentially regulated, intact nerves, but not regenerating ones, are subject to temporal and spatial constraints in their ability to establish functional endings in denervated skin. The endings which are established in response to denervation of skin during the critical period however incompletely suppress some quality of the target which allows regenerating nerves to recognize it, and to engage successfully in competition for its innervation.</p> / Doctor of Philosophy (PhD)

<p>Schizophrenia (SCZ) is a debilitating mental illness that affects roughly I % of the world's population. Current theories about the etiology of this disease highlight dismptions in dopamine (DA) and glutamate. However, a more recent theory, the 'synaptic hypothesis' proposes that the fundamental pathology of this illness involves disruptions in synaptic transmission. The synapsins are a family of neuron specific phosphoproteins that play an important role in neurotransmitter release, synapse formation and maintaining a reserve pool of synaptic vesicles. Previous research has suggested that synapsin II has a role in the etiology of SCZ. For example, synapsin II mRNA is significantly reduced in the medial prefrontal cortex (MPFC) of patients, and synapsin II knockout rats display a variety of behavioural abnormalities which mimic human SCZ. Considering that SCZ may result from changes at the synapse, we wanted to further elucidate the role of synapsin Il by measuring protein expression in post-mortem PFC samples. Overall, our results revealed that synapsin Ira and lIb are not significantly different betv,Ieen patients and controls, however, we hypothesize that synapsin Il expression has been nOlmalized in patients due to antipsychotic drug (APD) use. In fact, we discovered that treatment with atypical APDs significantly increases synapsin Il in the prefrontal cortex (PFC) of patients, which may underlie the beneficial effects of these dmgs. Another objective of our work was to investigate the expression of various presynaptic proteins in post-mortem samples from patients with Parkinson's disease (PD). Parkinson's disease, like SCZ, is an illness which involves dysregulated dopaminergic transmission and synaptic dysfunction. Therefore, we hypothesized that synapsin II might also be disrupted in patients with PD. Our results demonstrated that synapsin IIa and lIb are significantly reduced in the substantia nigra (SN), but not the striatum (STR) or PFC of patients, when compared to controls. Further, no changes were observed in the other synapsins (I or III), or synaptophysin, which suggests that synapsin II dysregulation may be specific to disorders which involve disruptions in dopamine (DA).</p> / Master of Science (MS)

<p>It is becoming increasingly clear from physiological and histochemical observations that many skeletal muscles are not used in an all-or-none fashion, but rather that some parts of the muscle are more active in one movement than another. Such a functional compartmentalization" could have an appropriate anatomical correlate; for example, within the pool of motoneurons supplying an individual muscle, subsets of motoneurons could be spatially grouped according to the location of their peripheral muscle fields. For a number of technical reasons, this possibility is difficult to investigate in the typical skeletal muscle. The cutaneous trunci muscle (CTM) of the rat, however, is ideally suited for such an investigation. This vast thin sheet of muscle is inserted into the deep surface of back and flank skin and is reflexly activated by nociceptive information from the overlying skin. A punctate stimulus evokes a localized contraction of the CTM in the immediate vicinity of the stimulus: the reflex activation of this muscle is therefore organized behaviorally into functional compartments. The simplest expectation would be that the peripheral nerves that drive the CTM reflex are segmentally organized. While this is true for the sensory nerves, I find that it is not the case for the motor ones. I have used electrophysiological and histochemical techniques to examine the pattern of motor innervation of the CTM and retrograde tracers to study its motoneuron pool. Interestingly, all the CTM motoneurons are located in the cervical spinal cord, several segments rostral to even the most rostral sensory input that activates the CTM. My findings indicate that there is a spatial organization within the CTM motoneuron pool; this organization corresponds to the pattern of motor nerve innervation of the muscle, and moreover, seems appropriate for the compartmentalized nature of the reflex activation of the muscle by cutaneous sensory nerves.</p> / Doctor of Philosophy (PhD)

<p>Previous studies have shown that in the rat the sprouting of low-threshold ("touch''-sensitive) nerves into adjacent denervated skin can occur only during a brief "critical period" of post-natal life that ends at about 20 d of age. Additionally, for any segmental nerve this sprouting occurred only in the region of that nerve's sensory dermatome. One objective of my thesis was to examine whether the sprouting of high-threshold ("pain"-sensitive) cutaneous nerves is also temporally and spatially constrained.</p> <p>The presence of high-threshold nerve endings in skin was detected behaviourally by pinching with toothed forceps; normally this elicits a reflex contraction of an underlying skeletal muscle, causing visible skin puckering. Denervated skin failed to respond in this way for the first 2-3 weeks after nerve section, but then a gradual recovery of the response occurred spreading from the adjacent innervated region of skin progressively further into the deprived area. That this recovery was due to sprouting was shown by histological examination of skin, and the findings of nerve fibres within the dermis in sensitive but not insensitive skin. The sprouting occurred in the adult animal, and proceeded beyond the dermatome border of a nerve, showing that intact high-threshold nerves are not under any obvious spatial and temporal constraints.</p> <p>During the examination for the presence of high-threshold nerve sprouting I noted that repeated skin pinching apparently accelerated the return of high-threshold sensitivity. The second main objective of the thesis was to examine what this phenomenon depended upon. In animals that were repeatedly stimulated physiologically every 4 d, all of the denervated skin became reinnervated by 20 d, compared to the 40 d or more needed for unstimulated animals. The accelerating affect was mimicked by direct electrical excitation of the remaining nerve. The results revealed that it was the latency for sprouting that was affected; once begun, the rate of sprouting was normal. The effects of electrical stimulation of the intact nerve were prevented if TTX was applied so as to block impulses proceeding centrally from the site of electrical stimulation, but not if they were only prevented from proceeding peripherally. A hypothetical mechanism to explain the effects of impulse activity in causing acceleration of sprouting is provided.</p> / Doctor of Philosophy (PhD)

<p>The aim of this study was to provide a better understanding of muscle function in healthy adult subjects (n=46) and in patients with myotonic muscular dystrophy (MMD; n=25) and limb-girdle muscular dystrophy (LGMD; n=20). Evoked and volitional contractions were examined in two opposing muscle groups in the leg, the ankle plantar-flexors (PF) and dorsi-flexors (DF). The following contractile properties were investigated under isometric conditions: (1) twitch torque, (2) twitch speed, (3) twitch potentiation, (4) voluntary muscle strength, (5) extent of motor unit activation during maximum voluntary effort and (6) muscle fatigue. In both control and dystrophic populations, striking contractile differences were observed between the PF and DF muscle groups. The DF muscles differed from the PF muscles in demonstrating smaller twitches, briefer contraction and half-relaxation times, marked twitch potentiation, more complete motor unit activation, and greater susceptibility to fatigue. Muscle fatigue was found to be caused by peripheral, rather than central failure; it occurred at the level of excitation-contraction coupling and/or the contractile machinery.</p> <p>Comparisons of muscle contractile properties between patients with muscular dystrophy (MMD and LGMD) and their respective matched controls have disclosed that in any patient at a given stage, the dystrophic process may sometimes spare a muscle group while destroying another, regardless of their functions and fibre-type compositions. Furthermore, both groups of dystrophic patients showed normal twitch potentiation and muscle fatigue behaviour.</p> <p>Apart from providing comprehensive information on normal and dystrophic skeletal muscles, this study constitutes a non-invasive and quantitative method for monitoring the time course of human muscular dystrophy and for assessing the benefits of any future therapy.</p> / Doctor of Philosophy (PhD)

<p>Brainstem auditory evoked potentials (BAEPs), the time locked scalp recorded electrical responses to auditory stimuli are clinically useful for diagnosing and monitoring the disorders of neurological system. The first part of the thesis presents the development and evaluation of computer based statistical classifiers to recognize normal and patients' BAEPs. The classifiers using time domain features; namely latencies of peaks V, IV and III of BAEPs, gave the optimal performance as measured by accuracy (85.3%), sensitivity (80%) among other indices. Power spectra of BAEPs in normals show three main frequency bands. In a second formulation of classifiers, three features, each feature representing the total power in each of the three frequency bands were used. The classifiers designed with these frequency domain features yielded a performance with accuracies upto 77%. Both of these classifiers may be used to assist the clinician while assessing the BAEPs.</p> <p>The second part of the thesis examines the effects of cooling on canine spinal cord blood flow (SCBF). The SCBF is an important physiological variable altered during spinal cord injury (SCI). Localized cooling of the spinal cord improves functional recovery after SCI. Hence, the effects of cooling on SCBF in normal canine cord were studied. A computerized system to measure the SCBF using hydrogen polarography at two control sites and two cooled sites was developed. In five dogs, SCBF decreased to 50% of the normothermic values during cooling of the cord to a temperature of 16 degrees Celsius. The SCBF did not change at control sites. It is postulated that following SCI decreased SCBF due to localized cooling tends to prevent the outpouring of edema fluid and other toxic factors from the injured vessels, preserving the cord function. Thus, the finding of decreased SCBF during spinal cord cooling is clinically relevant.</p> / Doctor of Philosophy (PhD)

<p>Mechanosensory transduction changes both during development and after the application of vincristine, a neurotoxic agent. Specifically, cutaneous sensitivity mediated by Type I slowly adapting cutaneous mechanoreceptors (touch domes) changes over the course of development in two ways. The first is a reduction in the density of touch domes in the skin from birth to adulthood. The second mechanism involves an increase in threshold to mechanical stimulation from birth to old age. In control adult animals, administration of the neurotoxic drug vincristine increased mechanosensory threshold within 24 hours after treatment. This raised threshold was maintained for two weeks but there was a return to control values by three weeks post-treatment. Measurements of response latency indicated that the rise in receptor threshold could occur without impulse propagation being impaired in the axon. Both the morphological and physiological state of the axon twenty-four hours after vincristine treatment indicated that vincristine modified sensory transduction at the level of the touch domes or of the fine nerve terminals abutting it. Vincristine may have produced the temporary high threshold state of the receptor by direct action on the receptor or indirectly by interfering with axoplasmic flow. If the latter is the case, this observation provides indirect evidence for the existence of a functional connection between the flow of trophic factors in the nerve fibres supplying touch domes and the mechanosensory transduction process in the receptor. Possible mechanisms for the changes observed in receptor properties during ageing and drug treatment are presented. It is proposed that during ageing a functional deafferentation occurs; the implications of this proposed process on the ageing nervous system are discussed. It is suggested that similar mechanisms may underlie threshold changes seen during ageing and after the administration of vincristine.</p> / Doctor of Philosophy (PhD)

<p>This work intended to study cell membrane properties of cells from genetically hypertensive animals in culture. The spontaneously hypertensive rat from the Wistar-Kyoto strain (SH rats) was used as a model. Genetically related Wistar-Kyoto (WKY) and unrelated Spague-Dawley (SD) rats were used as controls. Neurones from the Superior Cervical Ganglia (SCG) were cultured in vitro, with and without target cells from the vasculature (aorta, A VSMC, and mesenteric artery, MA VSMC). Passive and active membrane electrical properties from SCG neurones and A and MA VSMC were studied with microelectrodes via injection of long-duration (400 msec.) depolarizing and hyperpolarizing square pulses of current.</p> <p>Most of WKY and SD SCG neurones fired one or two action potentials upon depolarization. The majority of SH SCG neurones was found to have lost their accommodative properties and fired three or more action potentials upon depolarisation. In all strains, firing abilities were inhibited in presence of A VSMC. The firing frequencies distributions remained unchanged in SD and WKY cocultures, but was shifted to lower values in the presence of MA VSMC in SH co-cultures. Multiple firing was found to be initiated by the absence of or lack of activation of a calcium-dependent potassium channel and carried by a regenerative calcium current. This cellular dysfunction is thought to be present in all cell types in genetically hypertensive individuals, and a general framework is provided which tentatively explains the development, establishment and complex phenotype of essential hypertension.</p> / Doctor of Philosophy (PhD)

5-HT4 receptors are expressed in colonic epithelium, and activation with 5-HT4 receptor agonists causes a number of responses, including mucus secretion from goblet cells, chloride secretion from enterocytes, and 5-HT release from enterochromaffin cells. We tested whether this receptor could serve a protective role in models of colitis and under basal conditions. Male CD-1 mice (Charles River, Canada) were administered dextran sodium sulfate (DSS; 4% w/v in tap water, MW: 40,000) or trinitrobenzene sulfonic acid (TNBS; 7.5mg/mL in 50% ethanol by enema) on day 0. Treatment with the 5-HT4 receptor agonist, tegaserod (1 mg/Kg), or agonist plus the antagonist, GR113808 (1 mg/Kg), began either 24 hours after colitis induction and continued daily for 6 days (prevention paradigm), or 5 days after colitis was induced and continued for 10 days (recovery paradigm). To test for an action of 5-HT4 receptors under basal conditions, the antagonist, GR113808 was administered to normal mice by daily enema for 10 days. Colitis was evaluated using disease activity index (DAI) and histological damage scores (HDS). Possible protective mechanisms such as improved epithelial barrier function were evaluated by cell proliferation by Ki-67 immunostaining, whereas cell migration and resistance to oxidative stress were explored in CaCo-2 cells. We also tested the effects of tegaserod and/or GR113808 on colonic motility in guinea pigs, a well described model of colonic function. Treatment with tegaserod by enema in both DSS and TNBS-inflamed animals significantly attenuated the development of colitis, and accelerated recovery from established colitis, and these effects were blocked by 5-HT4 antagonist treatment. This effect was not seen when tegaserod was administered by intraperitoneal injection. TNBS-induced dysmotility in guinea pigs was significantly reversed by 5-HT4 receptor agonist treatment, but dysmotility persisted in animals treated with the agonist plus antagonist. We observed significant increases in the proportion of epithelial cells that were Ki-67 positive in DSS-inflamed mice treated with the agonist, and this effect was blocked by the antagonist. In CaCo-2 cells, 5-HT4 receptor activation accelerated cell migration into scratches on cell cultures, and increased resistance to oxidative stress-induced apoptosis, and these effects were blocked by the antagonist. Furthermore, treatment with the antagonist alone resulted in significant increases in disease activity index, histological damage scores and bacterial translocation in mice, and led to disrupted motility patterns in guinea pig distal colon. 5-HT4 receptor stimulation reduced the development of, and accelerated the recovery from, inflammation. These effects likely involved improved wound healing and resistance to oxidative stress. Interestingly, inhibition of 5-HT4 activity in normal animals resulted in inflammation, decreased epithelial proliferation and disrupted motility. Taken together, these data suggest that activation of mucosal 5-HT4 receptors has a protective effect in the normal and the inflamed colon.

Deficits within the brain microcirculation contribute to poor patient outcome following aneurysmal subarachnoid hemorrhage (SAH). However, the underlying pathophysiology is not well understood. Intra-cerebral (parenchymal) arterioles are encased by specialized glial processes, called astrocyte endfeet. Ca2+ signals in the endfeet, driven by the ongoing pattern of neuronal activity, regulate parenchymal arteriolar diameter and thereby influence local cerebral blood flow. In the healthy brain, this phenomenon, called neurovascular coupling (NVC), matches focal increases in neuronal activity with local arteriolar dilation. This ensures adequate delivery of oxygen and other nutrients to areas of the brain with increased metabolic demand. Recently, we demonstrated inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from SAH model animals. This pathological change, which would restrict blood flow to active brain regions, was accompanied by an increase in the amplitude of spontaneous Ca2+ events in astrocyte endfeet. It is possible that the emergence of higher amplitude endfoot Ca2+ events shifts the polarity of NVC after SAH by elevating levels of vasoactive agents (e.g. K+ ions) within the perivascular space. In the first aim of this dissertation we tested whether altered endfoot Ca2+ signaling underlies the inversion of NVC after SAH. Brain injury is often associated with increased levels of extracellular purine nucleotides (e.g. ATP). A recent study found that ATP levels in the cerebrospinal fluid of aneurysmal SAH patients were roughly 400-fold higher than that of non-SAH controls. Astrocytes express a variety of purinergic (P2) receptors that, when activated, could trigger a spike in intra-cellular Ca2+. It is possible that enhanced signaling via astrocyte P2 receptors underlies the change in endfoot Ca2+ signaling after SAH. In the second aim of this dissertation we determined the role of purinergic signaling in the generation of high-amplitude spontaneous endfoot Ca2+ events after SAH. Parenchymal arteriolar diameter and endfoot Ca2+ dynamics were recorded simultaneously in fluo-4-loaded rat brain slices using combined infrared-differential interference contrast and multi-photon fluorescence microscopy. We report that SAH led to a time-dependent emergence of spontaneous endfoot high-amplitude Ca2+ signals (eHACSs) that were only present in brain slices exhibiting inversion of NVC. Depletion of intracellular Ca2+ stores abolished spontaneous endfoot Ca2+ signals, including eHACSs, and restored arteriolar dilation in SAH brain slices to two downstream elements in the NVC signaling cascade, (1) increased endfoot Ca2+ and (2) elevated extracellular K+. We next tested the role of purinergic signaling in the generation of SAH-induced eHACSs by recording endfoot activity before and after treatment with the broad-spectrum purinergic receptor antagonist, suramin. Remarkably, suramin selectively abolished eHACSs and restored vasodilatory NVC in SAH brain slices. Desensitization of Ca2+-permeable ionotropic purinergic (P2X) receptors had no effect on eHACSs after SAH. However, eHACSs were selectively blocked using a cocktail of inhibitors targeting Gq-coupled purinergic (P2Y) receptors. Collectively, our results support a model in which SAH leads to an emergence of P2Y receptor-mediated eHACSs that cause inversion of NVC. Further, we identify the FDA-approved drug, suramin, as a potential therapy to be used in the treatment of aneurysmal SAH.