Glucose is the primary metabolic signal reflecting the current energy state of the body. Glucose influences the excitability of neurons in the area postrema (AP), a circumventricular organ (CVO), prompting my interest in investigating whether the subfornical organ (SFO), another sensory CVO can also detect glucose. Using patch-clamp electrophysiology, we investigated the influence of changing glucose concentrations on the excitability of SFO neurons. In dissociated SFO neurons, altering the bath concentration of glucose (1mM, 5mM, 10mM) influenced the excitability of 49% of neurons tested (n=67). Glucose-inhibited (GI, hyperpolarized by increased glucose or depolarized by decreased glucose) and glucose-excited (GE, depolarized by increased glucose or hyperpolarized by decreased glucose) neurons were observed. GI neurons (27%, n=18) depolarized in response to decreased glucose (n=10, mean 4.6 ± 1.0 mV) or hyperpolarized in response to increased glucose (n=8, mean -4.4 ± 0.8 mV). In contrast, GE neurons (22%, n=15) depolarized in response to increased glucose (n=9, mean 6.4 ± 0.4) or hyperpolarized in response to decreased glucose (n=6, mean -4.8 ± 0.6 mV). These data show that glucose acts on a subpopulation of SFO neurons to produce both excitatory and inhibitory actions. Using voltage-clamp recordings two groups of SFO neurons were identified: those producing an outward current (GI) and those producing an inward current (GE) in response to increasing concentrations of glucose from 1 to 10 mM (n=23). The mean glucose-induced inward current had a reversal potential of -24 ± 12 mV (mean input resistance 2.0 ± 0.4 GΩ, n= 5), suggesting it may be mediated by a NSCC. The mean glucose-induced outward current (mean input resistance 1.7 ± 0.3 GΩ, n=7) had a mean reversal potential of -78 mV ± 1.2 mV (n = 5), suggesting it may be mediated by an activation of either K+ or Cl-current (ECl = -67 mV, EK = -89 mV). The SFO has projections to the PVN, a regulator of energy balance. I investigated the effects of increasing concentrations of glucose (1 to 10 mM) on the membrane potential of dissociated SFO neurons projecting to the PVN. Thirty percent of SFO-PVN neurons tested (n=10) responded with membrane hyperpolarizations (mean -4.2 ± 0.8 mV, n=3) suggesting a proportion of these cells are GI neurons. These data indicate that SFO neurons are glucose-responsive, which supports a role for the SFO as a regulator of energy balance. / Thesis (Master, Physiology) -- Queen's University, 2009-09-24 20:20:33.319

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

No description available.

Leptin is an adipokine that acts centrally to regulate feeding behaviour, energy expenditure and autonomic function via activation of its receptor (ObRb) in nuclei in the central nervous system (CNS). This thesis investigates the involvement of two sensory circumventricular organs (CVOs), the subfornical organ (SFO) and area postrema (AP), in mediating the central effects of leptin using a variety of experimental approaches. We first show that acute electrical stimulation of the SFO elicits feeding in satiated rats, supporting a role for this specialized CNS structure in the control of food intake. We then demonstrate, using RT-PCR, the presence of ObRb mRNA in SFO and, using whole cell current clamp electrophysiology, reveal that leptin influences the excitability of individual SFO neurons, causing both excitatory and inhibitory responses. Furthermore, we find that leptin activates the same SFO neurons activated by amylin. Given the association between obesity and hypertension and the well-established role of the SFO in cardiovascular regulation, we show that leptin microinjection into the SFO decreases blood pressure in young rats, effects that are abolished in leptin-resistant, diet induced obese rats, suggesting that leptin-insensitivity in the SFO of obese, leptin-resistant, individuals may contribute to obesity-related hypertension. Our studies also show that the medullary AP expresses ObRb and that leptin influences the excitability of AP neurons. Furthermore, we show that leptin and amylin act on the same subpopulation of neurons in the AP. Finally, our preliminary AP/SFO lesion studies reveal that animals with these lesions exhibit a profound decrease in body weight and food intake and no longer exhibit decreases in body weight in response to peripheral leptin administration. In summary, the data presented in this thesis suggest the SFO and AP to be important in body weight homeostasis and in mediating the central effects of leptin. In addition, these areas appear to be important in the integration of multiple signals derived from peripheral sources. Furthermore, the fact that the SFO appears to be involved in leptin effects on both energy balance and cardiovascular regulation attest to the integrative nature of the SFO in the control of diverse physiological functions. / Thesis (Ph.D, Physiology) -- Queen's University, 2012-10-15 14:57:15.387

The subfornical organ (SFO) is a sensory circumventricular organ, lacking a proper blood-brain barrier. Neurons of the SFO are exposed directly to the ionic environment and circulating signaling molecules in the plasma, providing a unique window for communication of physiological status from the periphery to the central nervous system (CNS). The SFO is recognized as a key site for hydromineral balance, cardiovascular regulation and energy homeostasis. Neuropeptide Y (NPY) is a potent stimulator of food intake when released centrally, and has well-documented pressor effects when released peripherally. It has been demonstrated that the SFO expresses NPY receptors, however the effects of NPY on SFO neurons has never been investigated. The aim of this study was to determine the effects of NPY on the electrophysiological properties of SFO neurons dissociated from Sprague Dawley rats. Using whole cell patch clamp techniques in the current-clamp configuration, we report that 300 nM NPY caused 16% of SFO neurons to depolarize and 26% to hyperpolarize. The remaining neurons were insensitive to NPY. These effects were dose-dependent with a combined EC50 of 3.7 nM. Specific NPY receptor antagonists were applied, suggesting that the Y5 receptor predominately elicited a hyperpolarizing effect, while the Y1 receptor had a mixed response that was predominately hyperpolarizing, and the Y2 receptor had a mixed response that was predominately depolarizing. Using the voltage-clamp configuration, it was also observed that NPY caused an increase in the voltage-gated K+ current density as well as a shift in membrane activation of the persistent Na+ current, mediating the hyperpolarizing and depolarizing effects, respectively. These findings indicate that NPY elicits electrophysiological changes on SFO neurons, suggesting that the SFO is a key site of action for NPY in mediating energy regulation and/or cardiovascular output. / February 2017

Little is known regarding the neurophysiological mechanisms by which the neuropeptide prokineticin 2 (PK2) regulates circadian rhythms. Using whole-cell electrophysiology, we have investigated a potential role for regulation of neuronal excitability by PK2 on neurons of the area postrema (AP), a medullary structure known to influence autonomic processes in the central nervous system. In current-clamp recordings, focal application of 1µM PK2 reversibly influenced the excitability of the majority of dissociated AP cells tested, producing both depolarizations (38%) and hyperpolarizations (28%) in a concentration-dependent manner. Slow voltage ramps and ion substitution experiments revealed a PK2-induced Cl- current was responsible for membrane depolarization, while hyperpolarizations were the result of inhibition of an inwardly rectifying non-selective cation current. In contrast to these differential effects on membrane potential, nearly all neurons that displayed spontaneous activity responded to PK2 with a decrease in spike frequency. These observations are in accordance with voltage-clamp experiments showing that PK2 caused a leftward shift in Na+ channel activation and inactivation gating. Lastly, using post hoc single cell RT-PCR technology, we have shown that 7 out of 10 AP neurons depolarized by PK2 were enkephalin-expressing cells. The observed actions on enkephalin neurons indicate PK2 may have specific inhibitory actions on this population of neurons in the AP acting to reduce their sensitivity to incoming signals. These data suggest that PK2 regulates the level of AP neuronal excitability and may impart a circadian influence on AP autonomic control. / Thesis (Master, Physiology) -- Queen's University, 2009-08-18 11:18:05.977

There are two distinct communication systems in the brain, term wiring and volume transmission (Agnati et al., 2010). Volume transmission refers to a way of communication lacking any wire-like channel connecting the source of signal and its target. This way of signaling is the focus of the current thesis. Portal systems are one aspect of volume transmission in which they provide a pathway for diffusible signaling between bodily fluids (blood and cerebrospinal fluid) and the nervous system. A portal system entails two capillary beds linked by connecting veins. This connection allows signals from one capillary bed to be transported to a target in high concentrations without being diluted in the systemic circulation (Dorland, 2020). For the past decades, the only identified portal system in the brain is pituitary portal system (Popa, 1930; Popa & Fielding, 1933). Here, hypothalamic neurosecretions are released into the fenestrated capillaries of median eminence, a circumventricular organ, and transported to the capillaries of anterior pituitary via portal veins. The median eminence, due to its location on the surface of the ventricle and its contact to the cerebrospinal fluid, is categorized as a circumventricular organ. According to the classification (Oldfield & McKinley, 2015), there are three sensory circumventricular organs in the brain, all are characterized by fenestrated capillaries allowing contact between brain parenchyma and blood. For this reason, the circumventricular organs are known as “windows to the brain” (Gross et al., 1987). Whether other circumventricular organs also form portal systems is unknown. This thesis examines whether sensory circumventricular organs, specifically the organum vascular organ of the lamina terminals, the subfornical organ and the area postrema, bear portal systems. Although there have been prior studies of the vascularity of these CVOs in many species (reviewed in Duvernoy and Risold (2007)), the tissue preparation methods available limited the possibility of tracking small vessels over relatively large volumes in these structures. In the present work, to preserve the blood vessel structure, brain clearing and light sheet microscopy were combined to acquire volumetric images of the regions containing the circumventricular organs. In vivo two-photon microscopy was used to study the blood flow of the sensory circumventricular organs and the adjacent neuropil. The results indicate that organum vasculosum of the lamina terminalis is connected to the brain’s clock located in the suprachiasmatic nucleus by portal vessels. The direction of blood flow is from the suprachiasmatic nucleus to the organum vasculosum of the lamina terminalis, and speed of blood flow is faster during the night compared to the day. Volumetric imaging of the suprachiasmatic nucleus also shows portal veins emerging from the rostral shell region of this nucleus. Also, the subfornical organ connects to the septofimbrial nucleus and the triangular nucleus of the septum via portal veins. The arrangement of the vasculature of the area postrema differs from the other sensory CVOs: the AP and the nucleus of the solitary tract share a common capillary bed directly joining the vasculature of these morphologically distinct nuclei. In summary, there are multiple portal systems connecting the circumventricular organs. These newly discovered portal systems represent new pathways for diffusible signaling, bridging the systemic circulation, cerebrospinal fluid, circumventricular organs and the portal veins connected regions.

Les services de Neurochirurgie du Groupement Hospitalier Est de Lyon ont une expérience reconnue pour l’exérèse des tumeurs des régions périventriculaires du système nerveux central notamment au niveau de la région pinéale. Dans ce contexte neurochirurgical favorable et avec l’opportunité d’utiliser des techniques de biologie moléculaire, notre objectif a été l’identification de marqueurs diagnostiques pour chaque type tumoral par une étude transcriptomique en microarray, la caractérisation d’un sous-type de tumeur du parenchyme pinéal (TPP) pléïomorphe, l’évaluation de la synthèse de mélatonine par les TPP et l’étude du transcriptome de certains organes circumventriculaires (OCV) microdisséqués chez le rat. L’analyse du transcriptome des tumeurs périventriculaires a permis de regrouper les tumeurs par leur signature moléculaire et d’identifier des marqueurs diagnostiques pour chaque type tumoral. De nouveaux marqueurs pronostiques potentiels (HoxD13, Prame, CD24 et Pou4f2 et gènes de la voie Aurora kinase B) sont proposés en vue d’améliorer la classification des TPP. Pour ces dernières, une étude multicentrique a permis de caractériser un sous-type tumoral, les tumeurs pléïomorphes, souvent surgradées. L’étude des TPP ex vivo et in vivo montre leur capacité de synthèse de mélatonine. Toutefois, nous n’avons pu obtenir une lignée de cellules tumorales stable. La microdissection des OCV du rat, parfois vestigiaux chez l’homme et qui pourraient être à l’origine de tumeurs périventriculaires, a permis d’étudier leur transcriptome et de mettre en évidence des marqueurs nouveaux ou déjà associés dans la littérature à ces structures / Neurosurgery of periventricular tumors, especially of pineal region tumors, is well developed at the Neurosurgical Hospital in Lyon. Taking opportunity of this background, our objective was identification of new diagnostic markers for each of these tumors using microarray transcriptome analysis, characterisation of a pleomorphic pineal parenchyma tumor (PPT) subtype, evaluation of melatonin synthesis in PPTs and the microarray analysis of molecular signature of some of circumventricular organs (CVO) after their laser microdissection in rat. The microarray analysis of periventricular tumors allowed molecular classification of the tumours and revealed different diagnostic markers for each type of tumors. Potentially new prognostic candidate genes (HoxD13, Prame, CD24 and Pou4f2 and Aurora kinase B pathway genes) are proposed for improvement of PPT classification. A PPT multicenter study allowed the characterisation of a pleomorphic subtype frequently managed as a higher grade tumour in the literature. The study of PPT ex vivo and in vivo showed their preserved capacity for melatonin production. However a stable PPT cell line culture could not be obtained. The laser microdissection of OCV in rat, sometimes vestigial in humans and potentially at the origin of the periventricular tumors, associated with a microarray study highlighted some potentially new or already described specific markers of these structures