University of Vermont COBRE 3
"Center for Neuroscience Excellence"

Pilot Projects, Years 11-16
P30 GM 103498, 7/1/2011-6/30/2016

Pilot Project 1 - 2011 to 2012

Title: "Endothelial Ca2+ Signals and Blood Brain Barrier Permeability Following Traumatic Brain Injury"
Investigator: Kalev Freeman, M.D., Ph.D., Research Assistant Professor of Surgery


            Traumatic brain injury (TBI) affects more than 1.7 million Americans each year, and survivors may experience long-term neurocognitive problems. Disruption of the blood brain barrier (BBB) may play a role in outcomes after traumatic brain injury (TBI), due to resulting cerebral edema and vasospasm. The fundamental mechanisms that alter BBB function after TBI are unknown. The BBB consists of a matrix of tight junctions and interconnecting endothelial cells (EC) that are responsible for BBB integrity. ECs also play a key role in vasodilatory pathways, which are activated by EC Ca2+ signals to oppose vasoconstriction. Vasogenic brain edema can result from the opening of the endothelial barrier due to endothelial contractions activated by controlled cortical injury. Experimental brain injury also leads to increased production of reactive oxygen species (ROS), which can disrupt endothelial tight junctions and interfere with BBB electrical resistance and permeability. Paradoxically, the BBB may act as a target for ROS-mediated cellular damage, but it also acts to impede transport of potentially therapeutic antioxidant compounds. Reversible changes in BBB permeability have also been linked to mobilization of extracellular Ca2+ in cerebral ECs, but the role of EC Ca2+ in BBB permeability is unknown.
             In a rodent model of TBI, we found that mesenteric arteries exhibit increased myogenic tone and impaired endothelium-mediated dilations, similar to the reported effects of hypertension. Contrary to expectation, we found that Ca2+ signals were profoundly elevated in arteries from TBI animals. This suggested that Ca2+ signals are uncoupled from vasodilatory responses. Mechanisms responsible for EC Ca2+ signals include the vanilloid transient receptor potential cation-channel (TRPV4) and intracellular inositol trisphosphate receptors (IP3Rs). TRPV4 opening causes local Ca2+ entry from the extracellular environment, whereas IP3R activation causes release of intracellular Ca2+ stores as propagating waves or discrete pulsars in endothelial projections. Using high-speed confocal microscopy, Ca2+ imaging produces patterns unique to each molecular signaling pathway, which can be analyzed to determine the channel involved in creating the signal. TRPV4 signals are of particular interest, because moderate level of TRPV4 channel activation causes vasodilation and higher activity may affect barrier function in other vascular tissue beds. The applicant's K08 project seeks to understand the molecular basis and functional consequences of endothelial Ca2+ signals in mesenteric resistance vessels after TBI. We now propose a completely novel, yet complementary project that exploits the availability of cerebral vessels from the same animals to address a fundamental neuroscience question: how does acute neural injury affect the blood brain barrier?
             The goal of this project is to understand the changes in cerebrovascular endothelial Ca2+ signals, vasodilation, and BBB permeability after acute brain injury. We hypothesize that TBI causes alterations in cerebral EC Ca2+ signaling that produce functional consequences on vasodilation and BBB electrical resistance. If an acute brain injury can cause long-term changes in the endothelium of a vascular bed far removed from the brain, as demonstrated by our pilot data, it is likely that endothelial cells in the cerebral vasculature are also affected. Our rational for focusing on TRPV4 channel is that activation of this Ca2+ channel has been linked to altered barrier function in other tissues. If our hypothesis is correct, the results expected from this pilot project will support a novel mechanism that could explain the neuropathology, including edema, vasospasm, and altered neurovascular coupling, that follows acute brain injury.

Pilot Project 2 - 2011 to 2012

Title: "The Involvement of Lynx2 in Nicotinic Acetylcholine Receptor Signaling in the Prefrontal Cortex and Amygdala; Possible Relationship to Psychitatric Disorders"
Investigator: Donna Toufexis, Ph.D., Assistant Professor, Department of Psychology


            Aversive psychological stressors that trigger central fear systems also produce changes in autonomic nervous system (ANS) control cANSing sympathetic activation and parasympathetic withdrawal (i.e. reduced vagal tone). This relationship between cardiac vagal control and fear and anxiety has led to several models relating anxiety to vagal tone, and to autonomic adaptability and flexibility. Specifically, these models predict that high levels of ANS adaptive variability maintain cardiovascular (CV) health and that this is compromised by chronic or high anxiety states and therefore constitute a very real vulnerability towards CV illness in individuals who are suffering from psychopathologies like panic disorder, general anxiety disorder and post-traumatic stress disorder. Coordinated control of both the behavioral, as well as the ANS, response to threatening or aversive stimuli is mediated bilaterally within the forebrain by the amygdala. The amygdala receives significant inhibitory input from medial prefrontal cortex (mPFC) . Thus, alterations in amygdala neurotransmission would likely simultaneously affect both behavior and ANS activity.
             Nicotinic acetylcholine receptors (nAChRs) mediate a wide variety of functions in the nervous system. nAChRs are ligand-gated ion channels that mediate fast, excitatory neurotransmission in response to acetylcholine. In the peripheral nervous system cholinergic signaling drives autonomic ganglia and nAChR activation mediates vagal tone. In the adult CNS, projections from cholinergic neurons in the basal forebrain cholinergic system activate presynaptic nAChRs and influence the release of transmitters such as glutamate, GABA, serotonin, and dopamine, and thereby coordinate complex processing in the cerebral cortex governing processes including; attention, memory, cognition and emotional behavior. In fact, nAChR activation has been shown to modulate glutamate, GABA, and norepinephrine afferent to and within particular amygdalar sub-regions. Hence, the nAChR system may be a viable future candidate in which to explore the control of ANS adaptive variability related to the amygdala's co-regulation of emotional behavior.
             Nicotinic signaling can be modulated by proteins of the Ly-6/ urokinase-type plasminogen activator receptor (Ly6-UPAR) family, sometimes called Ly-6 neurotoxin-like (lynx) molecules. Murine lynx1 and lynx2 are tethered to the membrane and modulate nAChR activation and signaling. Transgenic knockout (KO) mice lacking lynx2 display greater responses to acetylcholine and display enhanced fear and anxiety in a number of behavioral tests including the amygdala-mediated Pavlovian fear-conditioning paradigm. Moreover, lynx2 mRNA is highly expressed though out the amygdala and in brain regions like the PFC. Furthermore, microarray gene analysis from mPFC punches taken after the extinction of fear-conditioning in mice showed increased lynx2 expression suggesting that reducing nAChR activation via lynx2 in projections to the amygdala are involved in fear extinction.
             The availability of transgenic lynx2 knockout mice at UVM allows a unique opportunity to further investigate this protein's role in modulating nAChRs afferents to, and nAChRs within, the amygdala and tying together the function of the nicotinic acetylcholine system with the amygdala in the emotional control of health.

Pilot Project 3 - 2012 to 2013

Title: "Determinants of multipotency and neurogenesis from reactive astrocytes"
Investigators: Jeffrey L. Spees, Ph.D., Associate Professor Medicine and Director, Stem Cell Core


            Identifying cell-extrinsic and cell-intrinsic determinants that control self-renewal and differentiation has become a fundamental area of discovery for stem cell biology and regenerative medicine. Cell-extrinsic determinants include: growth factors and cytokines, hormones, extracellular matrix proteins, ions (e.g. Calcium), oxygen tension, mechanical forces (stretch), and neural (electrical) input. Cell-intrinsic determinants include transcription factors, microRNAs, and epigenetic modifiers that control chromatin status and gene promoter activity (e.g. DNA methylation, histone modifications). The field of cellular reprogramming has elaborated many cell-intrinsic regulators of embryonic and adult stem cells, and also intrinsic mechanisms that control the fate and phenotype of differentiated adult cell types. Improved understanding of mechanisms controlling stem cell self-renewal and cell fate decisions may provide powerful tools to manipulate the behavior of stem cells and improve tissue repair after injury. In circumstances where it may prove difficult to transplant cultured stem/progenitor cells or to mobilize endogenous reparative cells to improve healing, growing evidence indicates that we may be able to learn how to alter cellular phenotype in situ. For example, after CNS injuries such as stroke, it may be possible to repair neural tissue by converting proliferating reactive astrocytes of the peri-infarct area into multipotent neural stem/progenitor cells (NSCs/NPCs) that can then be directed to produce neurons. The ability to convert reactive astrocytes into NPCs/NSCs could potentially be very useful because there is no neuroblast migration from the subventricular zone (SVZ) niche toward the infarct core after focal cortical strokes that do not injure the striatum. In addition, larger-size strokes that do damage the striatum still do not elicit a sufficient response from SVZ-derived neuroblasts to replace the vast numbers of neurons destroyed by cerebral ischemia.
             Recently, with lineage-tracing based on expression of glial fibrillary acidic protein (GFAP), we identified proliferating cortical reactive astrocytes of the peri-infarct area as a potential source of multipotent NSCs after stroke. Similar to adult SVZ-NSCs, we found that reactive astrocyte-derived NSCs (Rad-NSCs) grew in culture as neural spheres, self-renewed, and differentiated into neurons, astrocytes, and oligodendrocytes. We found that signals from injured peri-infarct tissue were critical to generate Rad-NSCs, as we could not produce them from uninjured contralateral tissues. Notably, lineage-tracing in vivo for 1 month showed that proliferating reactive astrocytes did not generate neurons in the peri-infarct area after stroke. Therefore, signals may be required from both the peri-infarct area and the culture environment to induce multipotency and neurogenesis from reactive astrocytes of peri-infarct tissues. In studies with conditional knockout mice, we demonstrated that Notch1 signaling through Notch Intracellular Domain (NICD1) was required to produce Rad-NSCs from cortical peri-infarct tissues (see preliminary data). Because hypoxia in peri-infarct tissues increases levels of Hypoxia-Inducible Factor 1 alpha (HIF1 alpha), a transcription factor that stabilizes Notch1 signaling, HIF1 alpha is likely also to control the formation of Rad-NSCs from reactive astrocytes. Furthermore, we were able to generate multipotent Rad-NSCs from cortical peri-infarct tissues with NSC medium that contained only EGF. Therefore, initial Notch1/HIF1 alpha/EGFR signaling from the peri-infarct area combined with high dose EGFR signaling in culture may regulate Rad-NSC formation, self-renewal, and/or differentiation. Improved understanding of the mechanisms controlling Rad-NSCs and their formation from reactive astrocytes may identify ways to promote multipotency and neurogenesis in situ from reactive astrocytes of peri-infarct tissues after stroke.

Pilot Projects 4 - 2013 to 2014

Title: "Mitochondrial Dysfunction and Asynchronous Release in Diabetic Mice"
Investigator: John D. Tompkins, Ph.D., Research Associate, Department of Neurological Sciences


            Diabetes can profoundly disrupt autonomic coordination of visceral organ function. Urogenital, gastrointestinal and cardiovascular systems are compromised, producing erectile dysfunction, bladder dysfunction, gastroparesis, constipation, postural hypotension, arrhythmias and sudden cardiac death. In an effort to elucidate the cellular mechanisms underlying the loss of autonomic control, I recently determined that type 2 diabetes alters neurotransmitter release from parasympathetic nerve terminals of the major pelvic ganglion (MPG). This alteration was evident as a significant increase in asynchronous neurotransmitter release both during and after tetanic stimulation. The purpose of this application is to determine whether the increased asynchronous release is a measure of mitochondrial dysfunction with diabetes.
             Nerve terminal mitochondria play an important role in buffering the transient rise in [Ca2+]i during repetitive stimulation of motor nerves. Inhibition of mitochondrial function, at the motor endplate, causes greater asynchronous release. Both type 1 and type 2 diabetes are known to disrupt mitochondrial bioenergetics as a consequence of the over abundance of oxidative substrates. It has not yet been determined whether a loss of mitochondrial function with diabetes disrupts Ca2+ homeostasis at autonomic nerve terminals. The objective in this application is to determine if compromised mitochondrial function causes the increased asynchronous release observed in type 2 diabetic mice. My hypothesis is that type 2 diabetes decreases mitochondrial sequestration of nerve terminal Ca2+ during tetanic stimulation causing greater asynchronous neurotransmitter release. This hypothesis is formulated, in part, based on the existing literature and my preliminary data demonstrating that depolarization of the mitochondrial membrane potential (Δψm) with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) also increases asynchronous release at MPG preganglionic nerve terminals.