Carbon dioxide (CO2) inhalation lowers brain pH and induces anxiety, fear, and panic responses in humans. In mice, CO2 produces freezing and avoidance behavior that has been suggested to depend on the amygdala. However, a recent study in humans with bilateral amygdala lesions revealed that CO2 can trigger fear and panic even in the absence of amygdalae, suggesting and important role for extra-amygdalar brain structures. Because the bed nucleus of the stria terminalis (BNST) contributes to fear- and anxiety-related behaviors and expresses acid sensing ion channel-1A (ASIC1A), we hypothesized that the BNST plays an important role in CO2-evoked fear-related behaviors in mice. We found that BNST lesions decreased both CO2-evoked freezing and CO2-conditioned place avoidance. In addition, we found that CO2 inhalation caused BNST acidosis, and that acidosis was sufficient to depolarize BNST neurons and induce freezing behavior; both responses depended on ASIC1A. Finally, disrupting Asic1a specifically in the BNST reduced CO2-evoked freezing whereas viral vector mediated expression of ASIC1A in the BNST of Asic1a-/- and Asic1a+/+ mice increased CO2-evoked freezing. Together, these findings identify the BNST as an extra-amygdalar fear circuit structure important in CO2-evoked fear-related behavior. Genetic disruption of the acid-sensing ion channel-1A (ASIC1A) in mice results in deficits in several fear- and anxiety-related behaviors. These deficits have been largely attributed to the loss of ASIC1A in neurons. However, recent studies have identified ASIC1A in several types of non-neuronal cells, including glia. To test the hypothesis that it is the loss of ASIC1A in neurons that results in the behavioral deficits seen in Asic1a-/- mice, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. To validate these mice, we confirmed by PCR that the Asic1a floxed allele was disrupted in brain, but not tail DNA. We further detected a reduction in ASIC1A protein in the SynCre+Asic1aloxP/loxP mice by western blotting and ASIC1A immunohistochemsitry. Further characterization of cre expression with a Rosa26 cre reporter mouse revealed that cre expression did not occur in all neurons, but verified that cre expression was neuron-specific. This neuron-specific knockout of ASIC1A led to behavioral deficits in several models of fear and anxiety, including cued and context fear conditioning, predator odor-evoked freezing and CO2-evoked freezing. Together, these findings suggest that it is ASIC1A in neurons that mediates these fear- and anxiety-related behaviors. Trimethylthiazoline (TMT), a predator odor isolated from fox feces, elicits freezing and avoidance responses in rodents. This TMT-evoked freezing behavior depends on the bed nucleus of the stria terminalis (BNST), a brain region thought to contribute to anxiety in both humans and mice. Because the acid-sensing ion channel-1A (ASIC1A) is robustly expressed in the BNST and has been previously implicated in TMT-evoked freezing, we hypothesized that the BNST might be a site of ASIC1A action in the TMT-evoked freezing response. Consistent with previous studies, we found that TMT-evoked freezing depended both on the olfactory bulb and on ASIC1A. Viral-mediated disruption of ASIC1A in the BNST reduced TMT-evoked freezing, whereas, viral mediated expression of ASIC1A in the BNST of Asic1a-/- mice increased TMT-evoked freezing. We further observed that TMT exposure induces a modest acidosis, likely due to TMT-induced respiratory suppression. However, this respiratory suppression was not unique to odors that evoke freezing, suggesting that it does not drive the TMT-evoked freezing response. Together, these findings suggest that the BNST is a key site of ASIC1A action in TMT-evoked freezing. Regulation of cerebral blood flow (CBF) is critical to insure that the brain has adequate resources to maintain normal function. One of the strongest regulators of CBF is carbon dioxide (CO2). CO2 and acidosis are thought to induce vasodilation and increase CBF by initiating nitric oxide (NO) synthesis, though the mechanism by which this occurs is unknown. Recent unpublished studies have suggested that the acid-sensing ion channel-1A (ASIC1A) plays a role in hypercapnia-induced vasodilation. Therefore, we hypothesized that CO2-induced NO production would depend on ASIC1A. We found that CO2 induced robust NO production in Asic1a+/+ but not Asic1a-/- mice. To test the role of neuronal ASIC1A in CO2-induced NO production, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. We found that CO2 did not induce significant NO production in the SynCre+Asic1aloxP/loxP mice, suggesting that it is ASIC1A in neurons that mediates this response. Together, these studies suggest that ASIC1A may mediate neurovascular coupling and regulate CBF.

Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-β (Aβ) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥ 4.5 hr post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and Aβ, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or Aβ conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to Aβ25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of sigma-1 and sigma-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or Aβ results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate Aβ-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K+. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra. These comorbidities of ischemia and Aβ toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and Aβ influence cellular activity and survival within core and penumbral regions. Application of Aβ increased the [Ca2+]i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca2+]i overload induced by Aβ. Furthermore, afobazole (100 uM) abolished Aβ potentiation of ischemia + acidosis evoked [Ca2+]i overload, which may represent a therapeutic strategy for mitigating injury produced by Aβ and stroke.

It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.