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Elucidating the process by which an animal can transduce a brief signal into a predictable set of behaviours has important implications for understanding brain function. I explored the transition from quiescence to repetitive activity in the bag cell neurons of Aplysia californica. Using both cultured neurons and intact ganglia, I demonstrated the involvement of cholinergic transmission in this marked change to excitability. The bag cell neurons are a group of 200-400 electrically-coupled neuroendocrine cells that initiate a set of prescribed reproductive behaviours, culminating in deposition of an egg mass. This fixed action pattern, lasting ~1 h, follows a brief (≤10-sec) stimulus from an afferent input to the bag cell neuron cluster, which causes these previously silent neurons to continuously fire for ~30 min. Central to the maintenance of this increased excitability, are the elevation of various second messenger pathways that modulate multiple ion channels. As such, the initiating stimulus for afterdischarge generation was thought to involve metabotropic receptors. However, I report that an acetylcholine-gated ionotropic current triggers the afterdischarge, as well as two, distinct nicotinic responses that participate in excitability: one associated with channel opening and the other through the inhibition of K+ currents. My data suggest that the interplay between inward Ca2+ and cation currents, and outward K+ channels, regulated by intracellular messengers protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP), respectively, set the baseline level of excitability prior to cholinergic activation. I also observed, distinct negative-feedback mechanisms on the acetylcholine ionotropic current. First, an increase in cAMP inhibits the cholinergic current shortly after the start of the afterdischarge, and once the afterdischarge is fully underway, dephosphorylation by a Src family tyrosine kinase further inhibits the channel. In addition, FMRFamide, an afterdischarge suppressor, appears to directly block the cholinergic channel. By exploring both canonical and non-canonical cholinergic roles in the afterdischarge, I have determined that complex signalling pathways can be reduced to a single variable, provided that the necessary precursors are in place. Furthermore, based on post-synaptic receptor composition and regulation, my work indicates the potential for profound diversity in cholinergic pathways. / Thesis (Ph.D, Physiology) -- Queen's University, 2013-06-28 17:51:30.733

"Retinal prostheses are becoming a viable therapy for inner retinal degeneration caused by age related macular degeneration (AMD) and retinitis pigmentosa (RP). The majority of ocular and periocular prosthetic devices employ photodiodes and a microelectrode interface to convert light into a nerve impulse within the retina. Problems with this design include the need of an external power source, the lack of biocompatibility of the microelectrode array, and the need for complicated surgical procedures. Self-assembled monolayer (SAM) technology offers an alternative strategy, where neurons can be stimulated by light activation of a single layer of a photovoltaic SAM. We have developed a SAM structure where the photoexcitable dye 2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-caboxymethylbenzothiazolium bromide (NK5962) was covalently immobilized to an indium tin oxide (ITO) and 3-(aminopropyl) trimethoxysilane (APTMS) surface. The NK562 derivatized surface was characterized through contact angle goniometry, electrochemical impedance spectroscopy, grazing angle infrared spectroscopy, and ultraviolet-visible absorption spectrophotometry. NG108-15 neurons were differentiated onto the surface and neural responses from electrical stimulation and photostimulation of the system were measured using whole-cell current and voltage clamp methodologies. We found an average 2.9 mV decrease in NG108-15 threshold potential for every 10 mV increase in ITO surface potential. Following photostimulation, there was a 1.8±0.2 fold increase (p < 0.05) in the sodium channel current amplitude and a 2.00 ± 0.22 fold increase (p < 0.05 ) in voltage amplitude of NG108-15 neurons on the ITO-APTMS-NK5962 surface due to transfer of energy from the excited dye surface to the attached neurons. The degree of photostimulation decreased upon using 344, 430, and 603 nm optical filters to block increasing amounts of the wavelengths of incident light capable of being absorbed by NK5962. The sodium current amplitude slightly increased at 50% transmittance of incident light relative to 100% transmittance, then sharply decreased at 12.5%, 6.25%, 3.13% transmittance. Upon addition of tetrodotoxin (TTX), sodium channel blockage was observed and portrayed by decreased sodium current and voltage response amplitudes, validating the voltage and current clamp results described above. Our findings indicate that the NK5962 photoelectric film shows promise as an implant for restoring light sensitivity to the retina. "

Object recognition memory (ORM) refers to both recognition of an object and the memory of having seen it before. In humans, ORM has been investigated using functional neuroimaging and electrophysiological techniques with tests of episodic memory retrieval involving recollection of previously studied items. Processes involved in the maintenance of a mental state adopted for the performance of a retrieval task (retrieval mode) appear to involve right frontal neural regions. More transient processes occurring at the time of item recollection (retrieval success) have shown scalp activity over parietal and right frontal regions. This activity is thought to originate in the medial temporal lobes and the underlying right frontal cortex respectively. The aforementioned findings have been derived mainly from studies using verbal stimuli. It is uncertain whether the same neural regions are involved in object recollection. It is also uncertain whether sustained modal and transient item-related activity involve the same or different right frontal regions. In this study, steady-state probe topography (SSPT) was used to investigate both sustained and transient processes involved in the retrieval of abstract pictorial objects from memory. The ability to vary the evaluation period of the steady-state visually evoked potential (SSVEP) allows investigation of cognitive processes occurring over different time scales. Neural regions involved in sustained modal processes were identified by examining the SSVEP values averaged over the duration of a memory retrieval task. Sustained SSVEP effects were observed over right fronto-temporal regions. Neural regions involved in transient retrieval success processes were identified by comparing the transient SSVEP responses to tasks with different memory loads. Comparison of a higher with a lower memory load condition showed SSVEP effects over parieto-temporal and right inferior frontal regions. Larger differences between memory loads gave effects that were larger and more right lateralized. Retrieval mode and retrieval success processes showed SSVEP effects over different right frontal regions. It was also found that, in contrast to the left lateralized parietal ERP response to recollected verbal stimuli, the SSVEP effects produced with abstract pictorial shapes showed a more bilateral pattern. This was considered to reflect the relatively non-verbalizable pictorial nature of the stimuli.

Novices and experts show distinct differences in the performance of many tasks. Experts may perform a task quickly and accurately with seemingly little attention or effort, whilst novices will perform the same task more slowly and with great effort. The transition from novice to expert performance occurs only after extended practice and has been conceptualized as a transition from controlled to automatic processing, and has been modeled as a reduction in attention or cognitive resources. Alternatively, based on findings relating to learning in the domain of number arithmetic, it has also been modeled as a transition from an algorithmic, or computationally-based process, to the use of memory retrieval. However, relatively few studies have investigated the changes in brain activity associated with such a transition. In this study, the Steady-State Probe Topography technique was used to investigate differences in the topography of the Steady-State Visual Evoked Potential (SSVEP) between an unpracticed and a well-practiced analogue of number arithmetic, Alphabet arithmetic. Subjects showed decreases in response time with practice that followed a power law and were suggestive of automatization. During initial, unpracticed performance of the task, processing of the Alphabet Arithmetic equations was characterised by increased SSVEP amplitude and decreased latency in frontal regions, whilst after extended practice, performance was characterised by reduced SSVEP amplitude and increased latency. It is suggested that the frontal activity during the initial, unpracticed stage of the task implicates a role for working memory, whilst the amplitude decrease and latency increase observed in the well-practiced task may reflect a reduction in excitation, consistent with ideas of an improvement in brain efficiency, and possibly an increase in inhibitory processes.

Insects detect specific chemicals in the environment with olfactory receptors (ORs), which represent a novel class of ligand-gated ion channel. Insect ORs are comprised of at least one common subunit (OR83b in Drosophila) and at least one odorant-binding subunit. However, the molecular details of insect OR architecture, such as how they bind odorants, are unknown. This lack of knowledge hinders the development of compounds that may modulate OR function and potentially control insects involved in disease propagation and agricultural damage. The intent of this project is to investigate the structure and function of insect ORs. To this end, the utility of the Xenopus oocyte heterologous expression system was explored. Assay optimization, accuracy, and investigations on functional requirements were first performed using the Drosophila OR (DmOR) 35a/83b. The utility of the assay system was also demonstrated by identification of the honey bee (Apis mellifera) OR 11/2 as a receptor for the queen pheromone, 9-oxo-2-decenoic acid. A series of DmORs was cloned and expressed in Xenopus oocytes and individual receptors were selected for further study. DmOR85a/83b was shown to possess an incredibly high degree of enantioselectivity for the odorant ethyl 3-hydroxybutyrate. The receptive range of DmOR67a/83b was explored and observations were made on potential features of the odorant-binding site and a ligand odorophore. DmORs were also used to investigate the contributions of individual subunits toward the odorant-binding site and pore structure. Also, evidence for receptor antagonism by odorants was revealed. DmORs were screened with methanethiosulfonate reagents and the substituted cysteine accessibility method to identify residues 146-150 of DmOR85b as functionally important in receptor activation. This region, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2, was shown to be physically adjacent to the odorant-binding site itself. Finally, residues within the extracellular half of TMS3 in DmOR85b were implicated in odorant-induced activation by screening DmOR85b mutants for altered ligand preferences. Therefore, this project provides the first identification of insect OR subunit components involved in odorant recognition, and represents an important starting point for detailed analysis of the molecular basis for insect OR activation by odorants.