Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

The nonlinear modeling capabilities of artificial neural networks (ANN’s) are renowned in the field of artificial intelligence (Al) for capturing knowledge that can be very difficult to understand otherwise. Their ability to be trained on representative data within a particular problem domain and generalise over a set of data make them efficient predictive models. One problem domain that contains complex data that would benefit from the predictive capabilities of ANN’s is that of optical emission spectra (OES). OES is an important diagnostic for monitoring plasma species within plasma processing. Normally, OES spectral interpretation requires significant prior expertise from a spectroscopist. One way of alleviating this intensive demand in order to quickly interpret OES spectra is to interpret the data using an intelligent pattern recognition technique like ANN’s. This thesis investigates and presents MLP ANN models that can successfully classify chemical species within OES spectral patterns. The primary contribution of the thesis is the creation of deployable ANN species models that can predict OES spectral line sizes directly from six controllable input process parameters; and the implementation of a novel rule extraction procedure to relate the real multi-output values of the spectral line sizes to individual input process parameters. Not only are the trained species models excellent in their predictive capability, but they also provide the foundation for extracting comprehensible rules. A secondary contribution made by this thesis is to present an adapted fuzzy rule extraction system that attaches a quantitative measure of confidence to individual rules. The most significant contribution to the field of Al that is generated from the work presented in the thesis is the fact that the rule extraction procedure utilises predictive ANN species models that employ real continuously valued multi-output data. This is an improvement on rule extraction from trained networks that normally focus on discrete binary outputs

This thesis examines the processing of accented speech in both infants and adults. Accents provide a natural and reasonably consistent form of inter-speaker variation in the speech signal, but it is not yet clear exactly what processes are used to normalise this form of variation, or when and how those processes develop. Two adult studies use ERP data to examine differences between the online processing of regional- and foreign-accented speech as compared to a baseline consisting of the listeners’ home accent. These studies demonstrate that the two types of accents recruit normalisation processes which are qualitatively, and not just quantitatively, different. This provided support for the hypothesis that foreign and regional accents require different mechanisms to normalise accent-based variation (Adank et al., 2009, Floccia et al., 2009), rather than for the hypothesis that different types of accents are normalised according to their perceptual distance from the listener’s own accent (Clarke & Garrett, 2004). They also provide support for the Abstract entry approach to lexical storage of variant forms, which suggests that variant forms undergo a process of prelexical normalisation, allowing access to a canonical lexical entry (Pallier et al., 2001), rather than for the Exemplar-based approach, which suggests that variant word-forms are individually represented in the lexicon (Johnson, 1997). Two further studies examined how infants segment words from continuous speech when presented with accented speakers. The first of these includes a set of behavioural experiments, which highlight some methodological issues in the existing literature and offer some potential explanations for conflicting evidence about the age at which infants are able to segment speech. The second uses ERP data to investigate segmentation within and across accents, and provides neurophysiological evidence that 11-month-olds are able to distinguish newly-segmented words at the auditory level even within a foreign accent, or across accents, but that they are more able to treat new word-forms as word-like in a familiar accent than a foreign accent.

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

No description available.

Dentate microcircuitry is thought to be involved in filtering, integrating, and relaying extrinsic hippocampal inputs to the hippocampus proper, which contributes to memory formation and retrieval. The axons of granule cells are called mossy fibres (MFs), and contain multiple terminal types that form characteristic synaptic connections with their postsynaptic targets. This diversity of presynaptic release sites that exists on the same MF provides an extremely interesting axonal type to study the organizing principles of presynaptic release regulation. A remarkable set of neurotransmitters and receptors present at the MF synaptic complex allow diverse computational modification of information from the dentate gyrus to the hippocampus. There are several types of glutamate receptors expressed at MF, such as group II/III mGluRs and kainate receptors (KARs). Presynaptic KARs modulate transmission at MF-CA3 pyramidal cell synapses; however, it is not known whether presynaptic KARs affect other synapses made by MFs. The aim of the first part of this thesis was to establish the principles of synapse-specific actions of presynaptic KARs in MFs. Combining electrophysiology and calcium imaging, this study provides compelling evidence that presynaptic KARs and Ca²⁺ stores can be activated by glutamate release from a single action potential in a single MF axon. This contributes to short-term, use-dependent facilitation of presynaptic Ca²⁺ entry and glutamate release exclusively at MF-CA3 pyramidal cell synaps, but not at other MF synapses, on hilar mossy cells or interneurons. Thus, our findings indicate that the presynaptic KARs, coupled with intracellular stores, exist in a synapse-specific autoreceptor mechanism. Activation of KARs strengthened MF-CA3 pyramidal cell synapses by increasing the Ca²⁺ influx at giant boutons, which might also contribute to the KAR-dependent hyper-excitability of the MF circuitry related to the mechanisms of temporal lobe epilepsy (TLE). This makes KARs good potential targets for therapies in CNS disorders such as epilepsy and other neurological and psychiatric disorders. The second part of this thesis was to explore the actions on the hippocampus of purified antibodies from a limbic encephalitis (LE) patient. LE is a CNS disease characterized by subacute onset of memory loss and temporal lobe seizures. The serum of these patients strongly labels MFs apparently co-localizing with the VGKC. The patients improve with immunotherapies that reduce the VGKC antibody levels in the serum, thus, strongly suggesting that these antibodies cause the condition. We found that LE serum IgGs enhance CA3 pyramidal cell excitability by blocking α-DTX sensitive VGKCs, which results in the increased release of glutamate. This, in turn, strengthens and desynchronizes MF and CA3 pyramidal cells synaptic transmission. However, these effects were occluded by α-DTX, a Kv1.1, Kv1.2 and Kv1.6 antagonist which, when applied alone, mimicked the action of the LE IgG, suggesting that they may share similar mechanisms of action. In contrast serum taken from healthy control patients had no significant effect under same recording conditions. Thus, this study provides the first evidence that the LE IgG functionally affects VGKC containing Kv1.1, Kv1.2 and/or Kv1.6 at both presynaptic MF axon terminals as well as the postsynaptic somatodendritic domain of CA3 pyramidal cells. Whatever defines the exact nature of LE IgG action, our results suggest that drugs acting specifically as openers of VGKC might help to protect the hippocampus from immune-mediated damage. In conclusion my data is consistent with the increasingly documented idea that MFs play a critical role in regulating the excitability of the hippocampal circuits and the dysfunction of MF transmission profoundly impairs hippocampal function.

The field of cardiac electrophysiological imaging has advanced tremendously in the past three decades with developments in fluorescent dyes, photodetectors, optical filters, illumination sources, computers and electronics. This thesis describes several scalable multi-parametric imaging systems and their application to cardiac tissue preparations at various levels of complexity. Using off-the-shelf components, single-camera multi-parametric optical mapping systems are described for various fluorescent dye combinations and single-element photodiode-based fibre-optic detection systems are described for drug-testing applications. The instruments described take advantage of modern voltage-sensitive dyes, multi-band optical filters and powerful light-emitting-diodes, from the ultraviolet to the red. The two electrophysiological parameters focused on were transmembrane voltage and the intracellular calcium concentration. Several voltage and calcium dye combinations were established, which produce no signal cross-talk. Furthermore, second- and third-generation voltage dyes were characterized in cardiac tissue, in vitro and in vivo. The developed systems were then applied to isolated Langendorff-perfused whole-hearts, in vivo whole-hearts, thin ventricular tissue-slices and human induced pluripotent stem cell-derived cardiac tissue. The interventions applied include accurately-timed electrical and mechanical local stimulation of the whole-heart to generate ectopic beats, cardiotoxic drugs and flash-photolysis of caged-compounds. With the high-throughput demands of drug discovery and testing, further development of scalable optical electrophysiological systems may prove critical in reducing attrition and costs. And for in vivo optical mapping, development of minimally-invasive and clinically-relevant optical systems will be essential in validating existing theories based on in vitro experiments and exploring cardiac function and behaviour with the heart intact in the organism.

Morphine is one of the most widely used drugs for the treatment of pain but its clinical efficacy is limited by adverse effects including persistent constipation and colonic inflammation. Morphine-induced colonic inflammation is facilitated by microbial dysbiosis and bacterial translocation. In this study, we demonstrate that secondary inflammation and persistent constipation are modulated by enteric glia. In chronic morphine treated mice (75 mg morphine pellet/5 days), ATP-induced currents were significantly enhanced in enteric glia isolated from the mouse colon myenteric plexus. Chronic morphine resulted in significant disruption of the colonic epithelium and increased Il-1β in the myenteric plexus. The increase in ATP-induced currents, IL-1β expression and ATP release were also observed in isolated glia treated with lipopolysaccharide (LPS) consistent with bacterial translocation as a potential mediator of chronic morphine-induced inflammation. These effects of LPS were reversed by carbenoxolone, a connexin43 hemichannel blocker. In-vivo treatment with carbenoxolone (25 mg/kg) prevented 1) ATP-induced currents in enteric glia, 2)the decrease in neuronal density, and 3) colonic inflammation in chronic morphine treated mice. Inhibition of connexin43 in enteric glia also reversed morphine mediated decrease in gastrointestinal transit. These findings indicate that bacterial translocation-induced enteric glial activation and inflammation is a significant modulator of morphine-related constipation.

Use-dependent long-term plasticity in synaptic connections represents the cellular substrate for learning and memory. The hippocampus is the most thoroughly investigated brain area for long-term synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) are both well characterized in glutamatergic excitatory connections between hippocampal principal cells in vitro and in vivo. An increasing number of studies based on acute brain slice preparations report LTP and LTD in excitatory synapses onto postsynaptic hippocampal GABAergic inhibitory interneurons. However, a systematic study of activity-induced long-term plasticity in excitatory synaptic connections to inhibitory GABAergic interneurons in vivo is missing. To determine whether LTP and LTD occur in excitatory synaptic connections to the hippocampal CA1 area GABAergic interneurons types in intact brain, I have used juxtacellular recording to measure synaptically evoked short-delay postsynaptic action potential probability in identified CA1 neurons in the urethane-anaesthetized rats. Plasticity in excitatory synaptic connections to CA1 cell types was measured as a change of afferent pathway stimulation-evoked postsynaptic spike probability and delay. In the study only experiments with monosynaptic-like short-delay (range 3-12 ms) postsynaptic spikes phase-locked to afferent stimulation were used. Afferent fibres were stimulated from the CA1 area of the hippocampus at the contralateral (left) side to avoid simultaneous monosynaptic activation of GABAergic fibres and to exclude antidromic spikes in recorded CA1 cells (in right hemisphere). Plasticity in pathways was tested using theta-burst high-frequency stimulation (TBS, 100 pulses), which is one of the most common synaptic plasticity induction protocols in acute brain slice studies. I discovered that TBS elicited permanent potentiation in single shock-evoked postsynaptic spike probability with shortening or no change in evoked spike latency in various postsynaptic neuron types including three identified pyramidal cells and parvalbumin-expressing (PV+) interneurons. Most fast-spiking PV+ cells showed LTP including an axo-axonic cell and one bistratified cell, whereas two identified basket cells exhibited LTD in similar experimental conditions. In addition, I discovered diverse plasticity in non-fast spiking interneurons, reporting LTP in an ivy cell, and LTD in three incompletely identified regular-spiking CA1 interneurons. I report that the underlying brain state, defined as theta oscillation (3-6 Hz) or non-theta in local field potential, failed to explain whether LTP, LTD or no plasticity was generated in interneurons. The results show that activity-induced potentiation and depression similar to LTP and LTD also occur in excitatory synaptic pathways to various CA1 interneurons types in vivo. I propose that long-term plasticity in excitatory connections to inhibitory interneurons may be take place in learning and memory processes in the hippocampus.

The activity of cortical neurons in awake brains changes dynamically as a function of the behavioural and attentional state. The primary motor cortex (M1) plays a central role in regulating complex motor behaviors. Despite a growing knowledge on its connectivity and spiking pattern, little is known about intra-cellular mechanism and rhythms underlying motor-command generation. In the last decade, whole-cell recordings in awake animals has become a powerful tool for characterising both sub-and supra-threshold activity during behaviour. Seminal in vivo studies have shown that changes in input structure and sub-threshold regime determine spike output during behaviour (input-output transformations). In this thesis I make use of computational and experimental techniques to better understand (i) how the brain regulates the sub-threshold activity of the neurons during movement and (ii) how this reflects in their input-output transformation properties. In the first part of this work I present a novel probabilistic technique to infer input statistics from in-vivo voltage-clamp traces. This approach, based on Bayesian belief networks, outperforms current methods and allows an estimation of synaptic input (i) kinetic properties, (ii) frequency, and (iii) weight distribution. I first validate the model on simulated data, thus I apply it to voltage-clamp recordings of cerebellar interneurons in awake mice. I found that synaptic weight distributions have long tails, which on average do not change during movement. Interestingly, the increase in synaptic current observed during movement is a consequence of the increase in input frequency only. In the second part, I study how the brain regulates the activity of pyramidal neurons in the M1 of awake mice during movement. I performed whole-cell recordings of pyramidal neurons in layer 5B (L5B), which represent one of the main descending output channels from motor cortex. I found that slow large-amplitude membrane potential fluctuations, typical of quiet periods, were suppressed in all L5B pyramidal neurons during movement, which by itself reduced membrane potential (Vm) variability, input sensitivity and output firing rates. However, a sub-population of L5B neurons ( 50%) concurrently experienced an increase in excitatory drive that depolarized mean Vm, enhanced input sensitivity and elevated firing rates. Thus, movement-related bidirectional modulation in L5B neurons is mediated by two opposing mechanisms: 1) a global reduction in network driven Vm variability and 2) a coincident, targeted increase in excitatory drive to a subpopulation of L5B neurons.