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Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2012-10-02 17:59:20.589

Functional magnetic resonance imaging (fMRI) offers a number of opportunities to non-invasively study the properties of the human visual system. Advances in scanner technology, particularly the development of high-field scanners, allow improvements in fMRI such as higher resolution and higher signal to noise ratio (SNR). We aimed to examine what these advances in scanner technology, combined with novel analysis techniques, can tell us about the processing of motion stimuli in the human visual cortex. In Chapter 3 we investigated whether high-resolution fMRI allows us to directly study motion-selective responses in MT+. We used event-related and adaptation methods to examine selectivity for coherent motion and selectivity for direction of motion, and examined the potential limitations of these techniques. One particular analysis technique that has been developed in recent years uses multivariate methods to classify patterns of activity from visual cortex. In Chapter 4 we investigated these methods for classifying direction of motion, particularly whether successful classification responses are based on fine-scale information such as the arrangement of direction-selective columns, or a global signal at a coarser scale. In Chapter 5 we investigated multivariate classification of non-translational motion (e.g. rotation) to see how this compared to the classification of translational motion. The processing of such stimuli have been suggested to be free from the large-scale signals that may be involved in other stimuli, and therefore a more powerful tool for studying the neural architecture of visual cortex. Chapter 6 investigated the processing of plaid motion stimuli, specifically ’pattern’ motion selectivity in MT+ as opposed to ’component’ motion selectivity. These experiments highlight the usefulness of multivariate methods even if the scale of the signal is unknown.

Calcium ions play an important role in many signalling pathways involved in normal cell metabolism. Pertrebations of normal Ca++ signalling may also play a pivotal role in the initiation of cell death. In these studies I have examined the influx of 45Ca++ into the extensor tibiae muscle of the locust (Schistocerca gregaria ). 45Ca++ entry could be stimulated by the addition of glutamate receptor-agonists or by activation of voltage activated calcium channels. L-glutamate, L-quisqualate and NMDA stimulated the influx of 45Ca++ while L-aspartate had only a small effect. DL-ibotenate, kainate, AMPA and glycine had no effect on 45Ca++ uptake (all agonists were tested at concentrations up to (100μM). Glycine (1μM) enhanced the 45Ca++ entry induced by NMDA and L-glutamate. Only the glycine potentiation of L-glu-stimulated responses was abolished in the presence of Mg++ (2mM) or AP5 (10μM) whereas the NMDA-stimulated response was completely abolished by these agents. These finding suggests that in the presence of glycine, L-glutamate may activate NMDA receptors and that in the absence of glycine L-glu-stimulated 45Ca++ entry occurs via activation of the qGluR. Depolarisation of the extensor tibiae muscles (50mM KCl) stimulated 45Ca++ influx by activation of voltage-sensitive calcium channels. Philanthotoxin-343 (0.1μM) had no effect on depolarisation activated calcium entry, however, nifedipine (1μM) an L-type calcium channel antagonist inhibited this Ca++ influx. Nifedipine did not inhibit L-glu-stimulated Ca++ entry suggesting that in these muscles L-type Ca++ channels are not involved in the Ca++ influx pathway following G1uR activation. Philanthotoxin-433 (PhTX-433) and many of its synthetic analogues are potent inhibitors of locust GluR. In the future these analogues may prove as useful potential neuroprotective agents or as novel pesticides. Over 100 analogues of PhTX-433 have been synthesized with changes made in the four regions of the structure, the thermospermine moiety, the tyrosyl moiety, the butyryl moiety and the terminal amino moiety. The effects of different concentrations (10-4M to 10-14M) of synthetic analogues of PhTX-433 (PhTX-343, PhTX-343-Arg, PhTX-4) were investigated in the 45Ca++ influx assay using locust extensor tibiae muscle. PhTX-343-Arg was more potent (IC50= - 7x10-9) than PhTX- 343 (IC50= - 10-8M) or PhTX-4 in blocking 45Ca H uptake. These findings were further supported by electrophysiological studies. The interaction of these synthetic analogues of philanthotoxin with GluR of locust muscle were further investigated by examining the effect of these compounds on evoked excitatory post synaptic potentials. In recent years control of ticks have been very important issue because of the social and economical damage they cause. Neuromuscular transmission is a main target site for the chemical control of many pests. Philanthotoxin and its analogues block the glutamate receptors which are involved in arthropod neuromuscular transmission and thus may prove useful as novel pesticides. The action of synthetic analogues of philanthotoxin (C7PhTX-343, DNP12-, PhTX-343 and PhTX-343) were examined on evoked excitatory postsynaptic potential in tick coxal muscle. These compounds all antagonized the evoked EPSP. C7PhTX-343 and DNP12-PhTX-343 exhibited same potency (IC50 = 10-8M) and both were more potent than PhTX-343 (IC50 ='2X10-5M). In recent years Xenopus oocyte has taken over a new role as a test tube for the study of the biogenesis, functional architecture and modulation of plasma membrane protein. Attempts were made to express mRNA from embryonic tissue of tick and locust leg muscle in to Xenopus oocyte for pharmacological studies. Xenopus oocytes failed to translate RNA faithfully and efficiently from these sources. Rat brain RNA injected oocytes used as control, expressed routinely.

A ubiquitous feature of neural responses is their dependence on stimulus context. One prominent contextual effect is the reduction in neural response size with stimulus repetition, known as “adaptation”. As adaptation is often stimulus-specific, it has been used in visual neuroimaging studies to probe mechanisms of stimulus representation that would otherwise be hidden due to the limited spatial resolution of the available measurement techniques. However, work on the visual system has suggested that stimulus-specific adaptation may not only reflect stimulus representations, but may itself also modify representational information. The four studies described in this report examined the effects of stimulus context on auditory cortical responses using electroencephalography (EEG). The first study used adaptation to examine the neural representation of musical pitch in auditory cortex. Whilst pitch is often treated as a single dimension, namely, the repetition rate of the stimulus waveform, in music, pitch actually has two dimensions: pitch height (the octave in which a note resides) and pitch chroma (the position of the note within an octave). The current study provided evidence for an explicit representation of pitch chroma in an anterolateral region of non-primary auditory cortex. The second, third and fourth studies examined the auditory “mismatch response” (MMR). The MMR refers to the increase in response size to a stimulus when it is presented infrequently (as a “deviant”) compared to when it is presented frequently (as a “standard”). The second study found that the MMR could not be fully accounted for by a passive release from adaptation. Instead, the MMR seemed to reflect a sharpening of the neural representation of the adaptor stimulus with repeated presentation. This suggests that the MMR may be involved in perceptual learning. The third study examined the time courses of the contextual effects on neural responses. Both short- and longer-term effects were observed, with the effects differing between the different components of the auditory evoked response. Notably, the N1 component was influenced by complex effects that seemed to partially reflect the longer-term probabilities of certain short segments of the stimulus sequence, whereas the P2 was influenced by a strong suppressive effect with a remarkably short time course. The fourth study examined whether the contextual effects on auditory-evoked transient and sustained responses are sensitive to the absolute, or the relative, stimulus probabilities. For the transient N1 response, the most striking finding was that adaptation was broadly tuned for deviant stimuli, but sharply tuned for stimuli that were, in terms of their relative probabilities, standards. In contrast, the sustained response appeared to be influenced by a different effect, which facilitated responses to deviant stimuli. The current results suggest that contextual effects differ vastly between different deflections of the auditory-evoked responses, that they include effects that are both complex and long-lasting (of the order of ten seconds or longer), and that they involve not only suppressive, but also facilitatory effects.