Retrograde tract tracing combined with immunohistochemical techniques were used to identify the origin of orexin A-immunoreactive (OrA-ir) fibers in the rat medulla. One to 5 days following injection of the fluorescent dye Fluorogold into the dorsal medulla, labeled neurons were found in the lateral half of the lateral hypothalamus, paraventricular, perifornical, dorsomedial, dorsal and posterior hypothalamic nuclei. Labeling the same sections with OrA antisera revealed a concentration of OrA-ir neurons in the perifornical and dorsomedial regions of the tuberal hypothalamus. A maximum of 10% of Fluorogold-labeled hypothalamic neurons were OrA-ir and 15% of OrA-ir hypothalamic neurons contained Fluorogold. Our results demonstrate that a fraction of OrA-ir neurons in the tuberal hypothalamus project to areas of the medulla that are involved in autonomic functions.

The caudal solitary complex (cSC) is a cardiorespiratory integrative center in the dorsal medulla oblongata that plays a vital role in the central CO2-chemoreceptive network. Neurons in this area respond to hypercapnic acidosis (HA) by a depolarization of the membrane potential and increase in firing rate, however a definitive mechanism for this response remains unknown. Likewise, CO2-chemoreceptive neurons in the cSC respond to hyperoxia in a similar fashion, but via a free radical mediated mechanism. It remains unknown if the response to increased pO2 is merely an increase in redox signaling, or if it’s the result of a pathological state of redox stress. Importantly, free radical production is known to be stimulated by increasing pO2, and can be exacerbated downstream by the addition of CO2 and its subsequent acidosis. Conditions of hyperoxia in combination with HA can therefore become detrimental in several scenarios, including O2 toxicity seizures in divers and stranded submariners, as well as in cases of ischemia-reperfusion injury and sleep apneas. As such, we sought to not only determine how O2 and CO2 interact to affect cellular excitability in the cSC, but also if these cells exhibited increases in redox signaling and/or stress. We employed sharp-electrode intracellular electrophysiology to study whole-cell electrical responses to varied combinations of hyperoxia (0.4 0.95/1.95 ATA O2) and HA (0.05 0.1 ATA CO2). Additionally, we used fluorescence microscopy under similar conditions to study changes in the production rates of various free radicals, including superoxide (˙O2-), nitric oxide (˙NO), and a downstream aggregate pool of CO2/H+-dependent reactive oxygen and nitrogen species (RONS). Finally, we used several colorimetric assays to measure markers of oxidative and nitrosative stress, including malondialdehyde, 3-nitrotyrosine, and protein carbonyls. Our hypothesis for these experiments was that hyperoxia and HA alone could produce effects, but would be more pronounced when used together. As such, we saw that ~89% of cells tested that were sensitive to both hyperoxia and HA showed larger firing rate responses to HA during an increased background O2 (0.9 and/or 1.9 ATA) after showing a smaller response or no response to HA during control levels O2 (0.4 ATA). Additionally, we noted that the rate of ˙O2- fluorescence increased in response to hyperoxia, but only during pharmacological inhibition of its reactions with ˙NO and SOD. Likewise, the rate of ˙NO fluorescence increased during hyperoxia compared to control O2, but only during pharmacological scavenging of ˙O2-. Downstream, our aggregate pool of RONS showed increased rates of fluorescence during both hyperoxia alone and HA in control O2, however the most prominent increases were seen during hypercapnic hyperoxia. Finally, no significant effects were seen when probing for markers of redox stress in response to hyperoxia and hypercapnic hyperoxia. Overall, these results suggest that the increased excitability seen in cSC neurons during hypercapnic hyperoxia is the result of physiological redox signaling rather than pathological redox stress. Further research needs to be done to determine how this redox mechanism is specifically resulting in increased cellular excitability.

A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus, subcoeruleus (dorsal part), or substantia nigra (areas involved in RBD and/or PD). These results did not provide support for RBD as a prodromal phase of PD within the systemic proteasome inhibitor-based model and add to a growing body of research reporting inconsistent findings using this model. We suggest that systemic PSI exposure in rats does not produce a viable model of RBD or PD. Whether RBD is an early symptom in the progression of PD remains to be established. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-04-28 12:04:50.613