Adiponectin (ADP) is an adipokine, which acts as an insulin sensitizing hormone. Recent studies have shown that adiponectin receptors (AdipoR1, AdipoR2) are present in the CNS; however, there is some debate as to whether or not ADP crosses the blood brain barrier (BBB). Circumventricular organs (CVO) are CNS sites outside the BBB, and thus represent sites at which circulating adiponectin may act to influence the CNS without having to cross the BBB. The subfornical organ (SFO) is a CVO that is responsive to a number of different circulating satiety signals including amylin, CCK, and ghrelin. We report here that the SFO also shows a high density of mRNA for both adiponectin receptors. These observations support the concept that the SFO may be a key player in sensing circulating ADP. To test the hypothesis that ADP influences the excitability of SFO neurons, we used current-clamp electrophysiology on dissociated SFO neurons to observe changes in membrane potential. ADP (10 nM) application effected the excitability of SFO neurons, where the cells either depolarized (8.9±0.9 mV, 21 of 97 cells) or hyperpolarized (-8.0±0.5 mV, 34 of 97 cells). Using single-cell RT-PCR we found that the majority of the responsive neurons expressed AdipoR1 or R2 and the non-responsive neurons expressed neither. In view of the recognized role of ADP in the regulation of energy balance, we next examined the effects of food deprivation for 48 hours on ADP signaling in the SFO. Our previous microarray analysis of SFO showed increases in AdipoR2 mRNA, with no significant change in AdipoR1 mRNA. We have also assessed the effects of such changes in receptor expression on ADP signaling in SFO neurons using calcium imaging and patch clamp techniques. In SFO neurons obtained from control animals, ADP induced increases in intracellular Ca2+ were observed in 25% of cells, while following food deprivation 0% of cells showed this response. Furthermore, 77% of neurons from starved animals showed clear depolarization, while no hyperpolarizing responses were observed. The results presented in this study suggest that adiponectin modulates the excitability of SFO neurons and that the response to ADP changes during starvation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-17 18:07:35.099

Universiẗat, Diss., 2007--Giessen.

Zugl.: Giessen, Univ., Diss., 2007

Universiẗat, Diss.--München, 1995.

Tübingen, Univ., Diss., 2007.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 105-120).

Frankfurt (Main), Univ., Diss., 2001.

Universiẗat, Diss., 2003--Kiel.

abstract: Patients with Alzheimer's disease (AD) exhibit a significantly higher incidence of unprovoked seizures compared to age-matched non-AD controls, and animal models of AD (i.e., transgenic human amyloid precursor protein, hAPP mice) display neural hyper-excitation and epileptic seizures. Hyperexcitation is particularly important because it contributes to the high incidence of epilepsy in AD patients as well as AD-related synaptic deficits and neurodegeneration. Given that there is significant amyloid-β (Aβ) accumulation and deposition in AD brain, Aβ exposure ultimately may be responsible for neural hyper-excitation in both AD patients and animal models. Emerging evidence indicates that α7 nicotinic acetylcholine receptors (α7-nAChR) are involved in AD pathology, because synaptic impairment and learning and memory deficits in a hAPPα7-/- mouse model are decreased by nAChR α7 subunit gene deletion. Given that Aβ potently modulates α7-nAChR function, that α7-nAChR expression is significantly enhanced in both AD patients and animal models, and that α7-nAChR play an important role in regulating neuronal excitability, it is reasonable that α7-nAChRs may contribute to Aβ-induced neural hyperexcitation. We hypothesize that increased α7-nAChR expression and function as a consequence of Aβ exposure is important in Aβ-induced neural hyperexcitation. In this project, we found that exposure of Aβ aggregates at a nanomolar range induces neuronal hyperexcitation and toxicity via an upregulation of α7-nAChR in cultured hippocampus pyramidal neurons. Aβ up-regulates α7-nAChRs function and expression through a post translational mechanism. α7-nAChR up-regulation occurs prior to Aβ-induced neuronal hyperexcitation and toxicity. Moreover, inhibition of α7-nAChR or deletion of α7-nAChR prevented Aβ induced neuronal hyperexcitation and toxicity, which suggests that α7-nAChRs are required for Aβ induced neuronal hyperexcitation and toxicity. These results reveal a profound role for α7-nAChR in mediating Aβ-induced neuronal hyperexcitation and toxicity and predict that Aβ-induced up-regulation of α7-nAChR could be an early and critical event in AD etiopathogenesis. Drugs targeting α7-nAChR or seizure activity could be viable therapies for AD treatment. / Dissertation/Thesis / Ph.D. Neuroscience 2011

Introduction: Beat-to-beat variability in cycle length exists in spontaneously beating cardiac preparations of varying complexities from the level of the isolated whole heart to the single sinoatrial nodal cell (SANC). The nature of this variability is poorly characterised as are its fundamental physiological origins. Methods: Recordings of spontaneous electrical activity were made from hearts in vivo, during Langendorff-perfusion, and from single SANC. Heart rate variability (HRV) was calculated in the time- and frequency-domains at baseline and in response to pharmacological mediators that interfered with critical processes involved in automaticity (catecholamines, carbachol, ivabradine, zatebradine, ryanodine and thapsigargin). In addition, a novel 2D technique for imaging Ca2+ fluorescence in spontaneously beating, fluo4-AM loaded, patched single sinoatrial nodal cells was developed to investigate the biophysical behaviour of Ca2+ during pacemaking to see if variability in this was responsible for SANC HRV. Results: Under baseline, temperature-stable conditions, levels of HRV were greatest in vivo (human > rat). SANC exhibited slightly lower levels of HRV, whereas HRV levels expressed by Langendorff-perfused hearts were the least (rabbit > rat), although still comprised a significant proportion of the variability witnessed in vivo. Anaesthetising in vivo rabbits decreased HRV to levels similar to those seen in the Langendorff-perfused heart. HRV was decreased by catecholamines and by ryanodine/thapsigargin in the Langendorff heart. Conversely, HRV was increased by carbachol, ivabradine, zatebradine and ryanodine in SANC. Heart rate changes had a marked effect on levels of HRV. 2D Ca2+ imaging of SANC showed that diastolic local Ca2+ releases (LCRs) occurred earlier than previously thought, with early LCRs having characteristics that were distinct from later LCRs. Mean time of occurrence of all the LCRs within a given diastole closely predicted the duration of the cycle. The rate of restitution of the whole cell Ca2+ transient (used as a surrogate for the pumping function of SERCA) in turn closely predicted the mean time of occurrence of LCRs. Tight synchronisation of the electrical activity of the cell with the biophysical behaviour of Ca2+ appeared to predict shorter cycle lengths. Isoprenaline increased LCR amplitude, though did not increase LCR number, size or duration. Isoprenaline caused LCRs to occur earlier, and synchronised their occurrence and the rate of pumping of Ca2+ back into the sarcoplasmic reticulum. Finally, LCRs were found to preferentially recur in certain regions of the cell, dubbed hotspots. Isoprenaline favoured hotspot production. Conclusion: Whilst greatest in vivo, significant HRV exists in spontaneously beating cardiac preparations devoid of a functioning autonomic nervous system. Studies in SANC indicate that the origin of this is likely to be variability in release of LCRs from the SR via ryanodine receptors. This in turn is controlled by SR refilling kinetics via SR Ca2+ pumping. The coupled system of membrane- and Ca2+-pacemaker clocks are so heavily intertwined that myriad factors will come to bear on generating such variability, including the amount of Ca2+ available for pumping and the phosphorylation state of key proteins, to the extent that variability in no one process can take the credit for generating such HRV.