Spinal cord injuries (SCI) sever communication between supraspinal centers and the central pattern generator (CPG) responsible for locomotion. Because the CPG is intact and retains the ability to initiate locomotor activity, it can be accessed electrically and pharmacologically. The goal of this thesis was to identify and characterize a novel spinal cord surface site along the sacral dorsal column (sDC) for electrically evoking locomotor-like activity in the neonatal rat spinal cord. Stimulation of the sDC robustly activated rhythmic left-right alternation in flexor-related ventral roots that was dependent on the activation of high-threshold C fiber afferents. The C fibers synapsed onto spinal neurons, which project to the lumbar segments as part of a pathway dependent on purinergic, adrenergic, and cholinergic receptor activation. In ventral roots containing only somatic efferents, rhythmic activity was rarely recruited. However, in ventral roots containing both autonomic and somatic efferents, sacral dorsal column stimulation recruited autonomic efferent rhythms, which subsequently recruited somatic efferent motor rhythms. The efferent rhythms revealed a half-center organization with very low stimulation frequencies, and the evoked alternating bursts entrained to the stimuli. Similar entrainment was seen when sDC stimuli were applied during ongoing neurochemically-induced locomotor rhythms. The rhythmic patterns evoked by sDC stimulation operated over a limited frequency range, with a discrete burst structure of fast-onset, frequency-independent peaks. In comparison, neurochemically-induced locomotor bursts operated over a wide frequency range and had slower time to peaks that varied with burst frequency. The overall findings support the discovery of an autonomic efferent pattern generator that is recruited by sacral visceral C fiber afferents. It is hoped that this research will advance the understanding of afferent activation of the lumbar central pattern generator and potentially provide insight useful for future development and design of neuroprosthetic devices.

In this dissertation I introduce a simple experimental approach for studying afferent pain fibre physiology. I developed an en bloc dural-skull preparation that pairs electrophysiological stimulations, pharmacological manipulations, and the UV photolysis of caged compounds in and around selectively identified individual C-fibre nociceptors with microfluorometric imaging of Ca2+ responses. This allows the observation of physiological functioning in individual nociceptive fibre free nerve endings. I show high-resolution functional imaging of single action potential-evoked fluorescent transients, as well as sub- and supra-threshold calcium signaling events within individual nociceptive fibre terminations. Utilizing the dural-skull preparation I was able to identify a peripheral mechanism of action in the terminals of CGRP nociceptive fibres for an effective migraine therapeutic, the selective 5-HT1 receptor agonist, sumatriptan. I found sumatriptan to cause an approximately 40% reduction in the amplitude of action potential-evoked Ca2+ transients in the peripheral terminals of CGRP nociceptive fibres that was mediated selectively through the inhibition of N-type Ca2+ channels. Observations from this study support a peripheral site of action for sumatriptan in inhibiting the activity of dural pain fibres and adds to our understanding of the mechanisms that underlie the clinical effectiveness of 5-HT1 receptor agonists such as sumatriptan. While μ-opioid receptor agonists remain the most powerful analgesics for the treatment of severe pain, their mechanism of action in peripheral primary afferent pain fibres remain to be established. Further exploiting the dural-skull preparation I found activation of μ-opioid receptors in individual CGRP terminals had a dual modulatory effect; inhibition of N-type Ca2+ channel signaling and a frequency dependent, BKCa channel-mediated, suppression of action potential firing. These results establish possible anti-nociceptive mechanisms of μ-opioid receptor activation in the peripheral terminals of CGRP nociceptive fibres and identify new pathways to target for peripherally mediated analgesia. The development and subsequent testing of the dural-skull preparation in this dissertation displays its utility and opens up a new window for studying nociceptive fibre physiology and pathophysiology.

Diss. (sammanfattning) Umeå : Umeå Universitet, 2009. / Härtill 5 uppsatser.

Human motor control is a complex process involving both central and peripheral components of the nervous system. Type Ia afferent input contributes to both motor unit recruitment and firing frequency, however, whether maximal force production is dependent on this input is unclear. Therefore, chapter 2 examined maximal and explosive force production of the knee extensors following prolonged infrapatellar tendon vibration; designed to attenuate the efficacy of the homonymous Ia afferent-α-motoneuron pathway. Despite a marked decrease in H-reflex amplitude, indicating an attenuated efficacy of the Ia afferent-α-motoneuron pathway, both maximal and explosive force production were unaffected after vibration. This suggested that maximal and explosive isometric quadriceps force production was not dependent upon Ia afferent input to the homonymous motor unit pool. Voluntary movements are linked with various modulations in ongoing neural oscillations within the supraspinal sensorimotor system. Despite considerable interest in the oscillatory responses to movements per se, the influence of the motor parameters that define these movements is poorly understood. Subsequently, chapters 3 and 4 investigated how the motor parameters of voluntary contractions modulated the oscillatory amplitude. Chapter 3 recorded electroencephalography from the leg area of the primary sensorimotor cortex in order to investigate the oscillatory responses to isometric unilateral contractions of the knee-extensors at four torque levels (15, 30, 45 and 60% max.). An increase in movement-related gamma (30-50 Hz) activity was observed with increments in knee-extension torque, whereas oscillatory power within the delta (0.5-3 Hz), theta (3-7 Hz), alpha (7-13 Hz) and beta (13-30 Hz) bands were unaffected. Chapter 4 examined the link between the motor parameters of voluntary contraction and modulations in beta (15-30 Hz) oscillations; specifically, movement-related beta decrease (MRBD) and post-movement beta rebound (PMBR). Magnetoencephalography (MEG) was recorded during isometric ramp and constant-force wrist-flexor contractions at distinct rates of force development (10.4, 28.9 and 86.7% max./s) and force output (5, 15, 35 and 60%max.), respectively. MRBD was unaffected by RFD or force output, whereas systematic modulation of PMBR by both contraction force and RFD was identified for the first time. Specifically, increments in isometric contraction force increased PMBR amplitude, and increments in RFD increased PMBR amplitude but decreased PMBR duration. Physical fatigue arises not only from peripheral processes within the active skeletal muscles but also from supraspinal mechanisms within the brain. However, exactly how cortical activity is modulated during fatigue has received a paucity of attention. Chapter 5 investigated whether oscillatory activity within the primary sensorimotor cortex was modulated when contractions were performed in a state of physical fatigue. MEG was recorded during submaximal isometric contractions of the wrist-flexors performed both before and after a fatiguing series of isometric wrist-flexions or a time matched control intervention. Physical fatigue offset the attenuation in MRBD observed during the control trial, whereas PMBR was increased when submaximal contractions were performed in a fatigued state.

Véritable enjeu de santé publique, l’obésité et ses complications seraient la conséquence d’un état inflammatoire chronique de bas-grade qui pourrait résulter de la présence dans le sang de composés bactériens, les lipopolysaccharides (LPS), état appelé endotoxémie métabolique. Le premier objectif de cette thèse était de comprendre pourquoi les LPS, initialement contenus dans le microbiote, sont capables de traverser l’intestin et d’entrer dans le système sanguin. Mon second objectif était d’étudier l’impact de la composition du microbiote dans le contrôle de la satiété par le nerf vague, lien de communication entre l’intestin et le cerveau. Pour cela, un modèle de rats soumis à un régime obésogène a été utilisée.Mes travaux ont montré que la consommation d’un régime obésogène induisait une perte de la fonction de barrière intestinale au niveau de l’iléon caractérisée par une baisse des défenses mucosales et une augmentation de la perméabilité au LPS. L’obésité est également caractérisée par une altération du comportement alimentaire, avec notamment une réduction de la sensibilité aux signaux de satiété. Nous avons montré que ni l’obésité ni le pourcentage de lipides du régime n’étaient responsables de cette perte de sensibilité aux signaux de satiété mais que l’altération du microbiote en serait le contributeur principal. Ainsi, l’endotoxémie métabolique serait le résultat d’une augmentation du passage transepithelial de LPS, qui, une fois dans le sang, pourraient atteindre, entre autres, le nerf vague où ils perturberaient les signaux intestinaux de satiété. / A real public health issue, obesity and its associated metabolic and behavioral disorders are the consequences of a state of low grade chronic inflammation that might originate from the presence in host plasma of gut-derived bacteria components, lipopolysaccharides (LPS). This present state is called metabolic endotoxemia. The first aim of my thesis was to understand why, in diet-induced obesity (DIO), LPS initially contained in the gut lumen, are able to cross the intestine and enter into the circulatory system. My second aim was to investigate the effect of gut microbiota composition and LPS on the satiety regulation by the vagus nerve, the main communication pathway between the gut and the brain. To answer these questions, we have mainly used a DIO rat model.We showed that consumption of WD induced a loss of ileal barrier function characterized by a reduction in mucosal defenses associated to elevated LPS permeability. Obesity is also characterized by an alteration in feeding behavior including a decreased sensitivity to intestinal satiety signals. We showed that neither obesity nor the lipid percentage of the diet triggers loss of sensitivity to satiety signals but that gut microbiota alterations could rather be the main driver. Hence, metabolic endotoxemia could result from an increased transepithelial passage of LPS, which once spread in the blood could reach, among other things, the vagus nerve where they could disrupt intestinal signals of satiety.

While much is known about the influence of ventricular afferent neurons on cardiovascular function in the dog, identification of the neurochemicals transmitting cardiac afferent signals to central neurons is lacking. Accordingly, we identified ventricular afferent neurons in canine dorsal root ganglia (DRG) and nodose ganglia by retrograde labeling after injecting horseradish peroxidase (HRP) into the anterior right and left ventricles. Primary antibodies from three host species were used in immunohistochemical experiments to simultaneously evaluate afferent somata for the presence of HRP and markers for two neurotransmitters. Only a small percentage (2%) of afferent somata were labeled with HRP. About half of the HRP-identified ventricular afferent neurons in T3 DRG also stained for substance P (SP), calcitonin gene-related peptide (CGRP), or neuronal nitric oxide synthase (nNOS), either alone or with two markers colocalized. Ventricular afferent neurons and the general population of T3 DRG neurons showed the same labeling profiles; CGRP (alone or colocalized with SP) being the most common (30-40% of ventricular afferent somata in T3 DRG). About 30% of the ventricular afferent neurons in T2 DRG displayed CGRP immunoreactivity and binding of the putative nociceptive marker IB4. Ventricular afferent neurons of the nodose ganglia were distinct from those in the DRG by having smaller size and lacking immunoreactivity for SP, CGRP, and nNOS. These findings suggest that ventricular sensory information is transferred to the central nervous system by relatively small populations of vagal and spinal afferent neurons and that spinal afferents use a variety of neurotransmitters.

The central nervous system is largely responsible for receiving sensory information from the environment and determining motor output. Yet, centrally-derived behavior and sensation depends on the optimal maintenance of the cells, tissues, and organs that feed and support these functions. Most of visceral regulation occurs without conscious oversight, making the spinal cord a key site for integration and control. How the spinal cord modulates output to our organs, or sensory information from them, is poorly understood. The overall aim of this dissertation was to better understand spinal processing of both visceral sensory information to and sympathetic output from the spinal cord. I first established and validated a HB9-GFP transgenic mouse model that unambiguously identified sympathetic preganglionic neurons (SPNs), the spinal output neurons for the sympathetic nervous system. Using this model, I investigated the electrophysiological similarities and diversity of SPNs, and compared their active and passive membrane properties to those in other animal models. My results indicate that while many of the same characteristics are shared, SPNs are a heterogeneous group that can be differentiated based on their electrophysiological properties. Since descending monoaminergic pathways have particularly dense projections to sympathetic regions of the spinal cord, I next examined the modulatory role that the monoamines have on spinal sympathetic output. While each neuromodulator tested had a unique signature of action, serotonin and norepinephrine appeared to increase the excitability of individual SPNs, while dopamine had more mixed actions. Since many autonomic reflexes are integrated by the spinal cord, I also questioned whether these reflexes would be similarly modulated. I therefore developed a novel in vitro spinal cord and sympathetic chain preparation, which allowed for the investigation of visceral afferent-mediated reflexes and their neuromodulation by monoamines. This preparation exposed a dichotomy of action, where sympathetic and somatic motor output is generally enhanced by the monoamines, but reflexes mediated by visceral input are depressed. Utilizing the spinal cord and sympathetic chain preparation, I also investigated how the spinal cord modulates visceral sensory information. One of the most powerful means of selectively inhibiting afferent information from reaching the spinal cord is presynaptic inhibition. I hypothesized that both spinal visceral afferents and descending monoaminergic systems would depress transmission of visceral afferents to the spinal cord. My results demonstrated that activity in spinal visceral afferents can lead to spinally generated presynaptic inhibition, and that in addition to depressing synaptic transmission to the spinal cord, the monoamines also depress the intrinsic circuitry that generates this activity-dependent presynaptic inhibition. Taken together, my results indicate that descending monoaminergic pathways act to limit the amount of visceral sensory information reaching the central nervous system and increase sympathetic output, resulting in an uncoupling of output from visceral sensory input and transitioning to a feed-forward, sympathetically dominant control strategy. This combination offers complex modulatory strategies for descending systems.

Obesity is pandemic. Pharmacological treatment development depends on modeling the regulation of feeding, particularly by free fatty acids (FFA). Most models have been employed in the rat in vivo, and show FFA-stimulated intestinal satiety signals are dependent on the fat’s acyl chain-length, involve cholecystokinin (CCK) secretion, and are mediated by vagal afferents. I hypothesized that an in vitro mouse model could be employed, with sensitivity to measure afferent responses to nutrient stimuli. Male C57BL/6N mice were killed, the intestine harvested en bloc, and a jejunal section dissected with neurovascular mesenteric arcade emanating centrally. The tissue was placed in a Krebs-superfused chamber, the lumen cannulated with the outlet open to drain, and Krebs or other mediators were continuously perfused intraluminally. The dissected afferent nerve was placed in a suction electrode for extracellular recording. Afferent responses to distension and the perfusion of mediators (e.g. CCK or FFA) were tested. Preparations from normal mice (no surgery), or from mice following chronic subdiaphragmatic vagotomy or sham operation, were used to assess vagal afferent contributions. Luminally-perfused CCK (100 nM) increased afferent firing. This response was abolished with the CCK-1 receptor antagonist lorglumide (10 µM). The short-chain fatty acid (SCFA) sodium butyrate (30 mM) potentiated firing. The long-chain fatty acid (LCFA) sodium oleate (1-300 mM) activated concentration-dependent firing (EC50=25.35 mM) that was significantly greater at 30 mM than that evoked by butyrate. Lorglumide (30 µM) abolished the oleate (30 mM) response. The L-type Ca2+ channel (LTCC) inhibitor nicardipine (3 µM), intraluminally, potentiated the oleate response, while bath application abolished it. Vagotomy attenuated the oleate response. Vagotomy abolished the intraluminal CCK (100 nM) response, and attenuated the response to bath-superfused CCK. These findings support FFA chain-length-dependent mesenteric afferent activation and CCK involvement in oleate-induced firing, and suggest LTCC mediation of excitatory and inhibitory oleate response transduction pathways. The murine oleate response was shown to be mostly vagally-mediated, with some spinal contribution, and both vagal and spinal contributions to CCK responses were suggested. These data provide a basis for further investigation in vitro of cellular and molecular mechanisms of afferent satiety signals, and ultimately of obesity pathogenesis. / Thesis (Master, Physiology) -- Queen's University, 2010-06-29 15:56:08.387

Local control of regional blood flow is exerted mainly through the arterioles. An adequate minute-to-minute regulation of blood perfusion of the kidney and the pancreas is obtained by the modulation of arteriolar reactivity, which will influence the organ function. The importance of purinergic signaling in this concept has been addressed, with special emphasis on the role of the adenosine A1 receptor. The effects of adenosine on two specialized vascular beds, namely the renal glomerulus and the pancreatic islets, have been examined. Characteristic for these regional circulations is their very high basal blood flow, but with somewhat different responses to vasoconstrictor and vasodilator stimuli. By adapting a unique microperfusion technique it was possible to separately perfuse isolated single mouse arterioles with attached glomeruli or pancreatic islets ex vivo. Microvascular responses were investigated following different additions to the perfusion fluid to directly examine the degree of dilation or constriction of the arterioles. This has been performed on transgenic animals in this thesis, e.g. A1 receptor knockout mice. Also effects of P2Y receptors on islet arterioles were examined in both normoglycemic and type 2 diabetic rats. Furthermore, interference with adenosine transport in glomerular arterioles were examined.. Our studies demonstrate important, yet complex, effects of adenosine and nucleotide signaling on renal and islet microvascular function, which in turn may influence both cardiovascular and metabolic regulations. They highlight the need for further studies of other purinergic receptors in this context, studies that are at currently being investigated.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / INTRODUÃÃO: A amifostina (WR-2721) tem sido largamente estudada como agente citoprotetor em diferentes ÃrgÃos e contra os mais diversos agressores do organismo humano. Recentemente, um efeito gastroprotetor deste fÃrmaco foi observado em modelo de lesÃo gÃstrica induzida por indometacina (MOTA et al., 2007). OBJETIVOS: Este trabalho investigou o efeito da amifostina na lesÃo gÃstrica por etanol e o papel dos neurÃnios sensoriais aferentes, grupos sulfidrÃlicos nÃo-protÃicos, Ãxido nÃtrico, canais de potÃssio sensÃveis ao ATP e ciclooxigenase-2 nesse processo. MÃTODOS: Ratos Wistar foram tratados com amifostina (22,5, 45, 90 ou 180 mg/kg, v.o. ou s.c.). ApÃs 30 minutos, os animais receberam etanol absoluto (5 ml/kg v.o.). Decorridos 60 minutos da administraÃÃo de etanol, os animais foram sacrificados. Foram realizados estudos macroscÃpicos e histolÃgicos, bem como dosagem de grupos sulfidrÃlicos nÃo-protÃicos e de hemoglobina em fragmentos de estÃmago. Outros grupos foram prÃ-tratados com L-NAME (10 mg/kg i.p.), glibenclamida (10 mg/kg v.o.), celecoxibe (10 mg/kg v.o.) ou salina. ApÃs 30 minutos os ratos receberam amifostina (90 mg/kg v.o. ou s.c.) e depois de mais 30 minutos etanol absoluto (5 ml/kg), com sacrifÃcio ocorrendo 60 minutos depois. Um grupo de animais foi desensibilizado com capsaicina (125 mg/kg s.c.) entre 10 a 14 dias antes do protocolo de tratamento com amifostina. RESULTADOS: A amifostina preveniu de forma significativa o dano macroscÃpico causado por etanol no estÃmago nas doses de 45, 90 e 180 mg/kg quando administrada por via oral e 90 e 180 mg/kg quando utilizada por via subcutÃnea. Os parÃmetros histolÃgicos, edema, hemorragia e perda de cÃlulas epiteliais, tambÃm foram reduzidos (p<0,05) com o uso de amifostina. Os animais que receberam apenas etanol apresentaram nÃveis reduzidos de GSH no estÃmago. A amifostina reverteu esse efeito atravÃs de um estÃmulo à produÃÃo de novo de GSH ou pela prevenÃÃo do consumo destes grupos. A gastroproteÃÃo da amifostina na lesÃo induzida pelo etanol foi revertida pela administraÃÃo prÃvia de doses neurotÃxicas de capsaicina, mas nÃo pelo uso de L-NAME, glibenclamida ou celecoxibe. CONCLUSÃES: A amifostina protege a mucosa gÃstrica contra a injÃria induzida pelo etanol atravÃs de um aumento dos nÃveis de GSH e estimulaÃÃo de neurÃnios sensoriais aferentes no estÃmago. Esse efeito parece ser independente da ativaÃÃo de canais de potÃssio sensÃveis ao ATP e da atividade de Ãxido nÃtrico sintase e ciclooxigenase-2 / INTRODUCTION: Amifostine (WR-2721) has been widely tested as a cytoprotective agent against a number of aggressors in different organs. Recently, a gastroprotective effect was observed for this drug in a model of indomethacin-induced gastric injury (MOTA et al., 2007). OBJECTIVES: We investigated the effect of amifostine on ethanol-induced gastric injury and the role played by afferent sensory nerves, non-protein sulfhydryl groups, nitric oxide, ATP-sensitive potassium channels and cyclooxygenase-2 in the mechanism. METHODS: Wistar rats were treated with amifostine (22.5, 45, 90 or 180 mg/kg, p.o. or s.c.). Thirty minutes after amifostine administration, the animals were given 100% ethanol (5 ml/kg p.o.). Sixty minutes after ethanol administration the animals were euthanized. Macroscopic and histological studies were carried out and stomach fragments were retrieved and submitted to analysis for non-protein sulfhydryl groups and hemoglobin. Some animals were pretreated with L-NAME (10 mg/kg i.p.), glibenclamide (10 mg/kg p.o.), celecoxib (10 mg/kg p.o.) or saline solution. Thirty minutes after pretreatment the animals were given amifostine (90 mg/kg p.o. or s.c.) and, after another 30 minutes, 100% ethanol (5 ml/kg). The animals were euthanized 60 minutes later. Other rats were desensitized with capsaicin (125 mg/kg s.c.) 10-14 days before amifostine treatment. RESULTS: Amifostine treatment significantly reduced ethanol-induced macroscopic stomach injury at 45, 90 and 180 mg/kg p.o. and at 90 and 180 mg/kg s.c. The histological parameters (edema, hemorrhage and epithelial cell loss) were also reduced (p<0.05) when the animals were treated with amifostine. Animals receiving ethanol only presented reduced GSH levels in the stomach. Amifostine reverted this effect either by stimulating de novo GSH production or by preventing the consumption of GSH. Amifostine-promoted gastroprotection against ethanol-induced stomach injury was reversed by pretreatment with neurotoxic doses of capsaicin, but not by L-NAME, glibenclamide or celecoxib. CONCLUSIONS: Amifostine protects against ethanol-induced gastric injury by increasing GSH levels and stimulating the afferent sensory nerves in the stomach independently of ATP-sensitive potassium channels activation, nitric oxide synthase and cyclooxygenase-2 activity