D-Aspartate (D-Asp) is an endogenous compound found in the central nervous system (CNS) of a variety of organisms. Despite its prevalence, however, relatively little understood of its physiological role. The prevailing theory is that D-Asp is an alternate agonist of N-methyl-D-aspartate receptor (NMDAR) channels. The goal of this work was to characterize the currents activated by D-Asp in neurons Aplysia californica, focusing on cells of the buccal S cluster (BSC). First, a general electrophysiological characterization was carried out, examining ion permeability, agonist dose-response, and the kinetics of activation, inactivation, and desensitization. D-Asp activated non-specific cation currents characterized by permeability to Na+ and K+. D-Asp-induced currents shared similar current-voltage relationships and time courses of activation and inactivation with L-glutamate (L-Glu)-induced currents. D-Asp currents, however, were subject to prolonged desensitization. Additionally, D-Asp activated currents independently of L-Glu, the known agonist of NMDAR channels, suggesting a non-NMDAR-dependent role of D-Asp. Next, select antagonists were used in an effort to pharmacologically characterize D-Asp receptor channels. These experiments suggested that D-Asp whole cell currents may be characterized by activation of multiple receptor sites, including NMDARS, excitatory amino acid transporters (EAATs), and a putative non-L-Glu D-Asp receptor. Furthermore, bath-applied D-Asp attenuated L-Glu-activated currents. Finally, D-Asp currents were compared to those evoked by acetylcholine (ACh) and serotonin (5-HT) in BSC cells. Results suggested that D-Asp activated receptor channels independently of ACh and 5-HT. Ten minute bath application of 5-HT was found to potentiate D-Asp current responses, likely through activation of a protein kinase C (PKC)-dependent mechanism, suggesting that D-Asp induced currents may be subject to synaptic plasticity associated with learning. While the identity of the putative D-Asp receptor remains elusive, the current work has advanced our understanding of the role D-Asp may play in the nervous system. These results should provide the groundwork for future studies aimed at identifying this unknown receptor channel, as well as investigation of the potential relationship of D-Asp receptor modulation to learning and memory in Aplysia, which may have relevance in higher organisms.

Patch clamping is considered the gold standard in measuring the bioelectrical activity of a cell. It is used to detect and measure ion transport through ion channels located throughout a cell membrane. Ion movement is crucial to cell viability and cell-to-cell communication. Pharmaceutical companies increasingly target ion channels because of their significance in disease and to help design better targeted drugs. However, the traditional method of patch clamping is cumbersome and is being replaced by planar high throughput screening (HTS) systems. These systems are reaching their limits due to materials and cost of processing; cell handling methods and small varieties of applicable cell types are also issues to be addressed. In this work, the core components of a new kind of planar patch clamping device have been designed and developed, after analysis of currently available HTS systems. This design approaches patch clamping using polymers to overcome some of the limitations in current systems, specifically cell handling and positioning, by using a simple modification technique to provide distinct attractive areas for cell binding. This uniquely allows the culture of both single cells and cell networks to increase the range of cell types that can be measured and circumvents challenges from using suction to pull cells onto measurement holes. The components of the design are a 10 x 10 array of small holes drilled in a polymer then aligned modifications for precise cell placement are added and a planar electrode array for individual addressing of each cell. A study of methods to produce a leak-tight seal required between microfluidic chambers was done. Cell adhesion parameters for the modification techniques were established. The principle viability of this approach was confirmed using the modification technique to culture cells over holes and measure their resistance using a rig developed for this work.

Whole-cell patch clamp electrophysiology of neurons in vivo enables the recording of electrical events in cells with great precision, and supports a wide diversity of morphological and molecular analysis experiments important for the understanding of single-cell and network functions in the intact brain. However, high levels of skill are required in order to perform in vivo patching, and the process is time-consuming and painstaking. Robotic systems for in vivo patching would not only empower a great number of neuroscientists to perform such experiments, but would also open up fundamentally new kinds of experiment enabled by the resultant high throughput and scalability. We discovered that in vivo blind whole cell patch clamp electrophysiology could be implemented as a straightforward algorithm and developed an automated robotic system that was capable of performing this algorithm. We validated the performance of the robot in both the cortex and hippocampus of anesthetized mice. The robot achieves yields, cell recording qualities, and operational speeds that are comparable to, or exceed, those of experienced human investigators. Building upon this framework, we developed a multichannel version of “autopatcher” robot capable establishing whole cell patch clamp recordings from pairs and triplets of neurons in the cortex simultaneously. These algorithms can be generalized to control arbitrarily large number of electrodes and the high yield, throughput and automation of complex set of tasks results in a practical solution for conducting patch clamp recordings in potentially dozens of interconnected neurons in vivo.

Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+-dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+-induced Ca2+-release; mechanisms known to influence the AHP. We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin. These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.

The cardiac sodium-calcium exchanger (NCX) is an important membrane protein which regulates cellular calcium necessary for the optimal contractile function of the heart. NCX has become a focal point in ischemic heart disease (IHD) research as evidence suggests that reactive oxygen species (ROS) produced during IHD can cause NCX to malfunction resulting in an intracellular calcium overload leading to cardiac contractile abnormalities. Therefore, I hypothesized that NCX function is mediated by ROS increasing NCX1 activity during cardiac ischemia-reperfusion. To research this hypothesis, I investigated cellular mechanisms which may play a role in NCX dysfunction and also examined methods to correct NCX function. I found that reactive oxygen species directly and irreversibly modify NCX protein, increasing its activity, thereby worsening the calcium overload which is deleterious to cardiac function. I also elucidated the molecular means by which NCX protein modification occurs. Exploring pharmacological means by which to decrease NCX function to relieve the calcium overload and reduce the damage to the heart, I discovered that ranolazine (Ranexa), indicated for the treatment of angina pectoris inhibits NCX activity directly, thereby further reducing the calcium overload-induced injury to the heart. Furthermore, many IHD patients are also co-morbid for type-2 diabetes. These patients are prescribed sulfonylurea (SU) agents which act at the ATP sensitive K+ channel (KATP). One agent such as glibenclamide is known to have cardiotoxic side effects. Therefore, SUs devoid of any cardiac side effects would beneficial. Interestingly, patients possessing the genetic variant E23K-S1369A KATP channel have improved blood glucose levels with the use of the SU gliclazide. Therefore, I determined the functional mechanism by which gliclazide has increased inhibition at the KATP channel. These findings have implications for type-2 diabetes therapy, in which 20% of the type-2 diabetic population carries the KATP channel variant. In summary, the findings presented in this thesis have implications on treatment strategies in the clinical setting, as a NCX inhibitor can be beneficial in IHD and possibly type-2 diabetes. Moreover, a pharmacogenomic approach in treating type-2 diabetes may also provide a positive outcome when considering co-morbid cardiac complications such as atrial fibrillation and heart failure.

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Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

Extended periods of oxygen deprivation cause brain death in mammals but the western painted turtle overwinters in anoxic mud for months without damage. Neural protection is achieved through decreases in the whole cell currents of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) that are dependent on a mild increase in intracellular calcium from the mitochondria. The goal of this research was to determine if natural anoxic decreases in reactive oxidative species (ROS) serve as the signal to bring about these changes. Reductions in cellular ROS levels were demonstrated to have no effect on AMPAR currents or intracellular calcium and produced massive increases in NMDAR currents, indicating that ROS depression does not directly mediate anoxic alterations. Interestingly, mammalian neural tissue also experiences a similar increase in NMDAR whole cell current in response to reducing agents suggesting a possible conserved mechanism for normoxic receptor control.

Extended periods of oxygen deprivation cause brain death in mammals but the western painted turtle overwinters in anoxic mud for months without damage. Neural protection is achieved through decreases in the whole cell currents of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) that are dependent on a mild increase in intracellular calcium from the mitochondria. The goal of this research was to determine if natural anoxic decreases in reactive oxidative species (ROS) serve as the signal to bring about these changes. Reductions in cellular ROS levels were demonstrated to have no effect on AMPAR currents or intracellular calcium and produced massive increases in NMDAR currents, indicating that ROS depression does not directly mediate anoxic alterations. Interestingly, mammalian neural tissue also experiences a similar increase in NMDAR whole cell current in response to reducing agents suggesting a possible conserved mechanism for normoxic receptor control.