Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 88-97).

Maintaining the intrinsic excitability of neurons is crucial for stable brain activity. This can be achieved by the homeostatic regulation of membrane ion channel conductances, although it is not well understood how these processes influence broader aspects of neuron and network function. One of the many mechanisms which contribute towards this task is the modulation of potassium channel conductances by activity-dependent nitric oxide signalling. Here, we first investigate this mechanism in a conductance-based neuron model. By fitting the model to experimental data we find that nitric oxide signalling improves synaptic transmission fidelity at high firing rates, but that there is an increase in the metabolic cost of action potentials associated with this improvement. Although the improvement in function had been observed previously in experiment, the metabolic constraint was unknown. This additional constraint provides a plausible explanation for the selective activation of nitric oxide signalling only at high firing rates. In addition to mediating homeostatic control of intrinsic excitability, nitric oxide can diffuse freely across cell membranes, providing a unique mechanism for neurons to communicate within a network, independent of synaptic connectivity. We next conduct a theoretical investigation of the distinguishing roles of diffusive homeostasis mediated by nitric oxide in comparison with canonical non-diffusive homeostasis in cortical networks. We find that both forms of homeostasis robustly maintain stable activity. However, the resulting networks differ, with diffusive homeostasis maintaining substantial heterogeneity in activity levels of individual neurons, a feature disrupted in networks with non-diffusive homeostasis. This results in networks capable of representing input heterogeneity, and linearly responding over a broader range of inputs than those undergoing non-diffusive homeostasis. We further show that diffusive homeostasis interferes less than non-diffusive homeostasis in the synaptic weight dynamics of networks undergoing Hebbian plasticity. Overall, these results suggest a novel homeostatic mechanism for maintaining stable network activity while simultaneously minimising metabolic cost and conserving network functionality.

Dopaminergic systems regulate many brain functions and dysfunction of dopaminergic neurotransmission is thought to underlie numerous disorders, including schizophrenia, attention deficit hyperactivity disorder (ADHD), depression and Alzheimer’s disease. In the hippocampus, a dopaminergic projection from the ventral tegmental area (VTA) is proposed to be essential for controlling entry of sensory information into long-term memory through novelty and salience detection. However, the effects of the VTA-dopamine system on hippocampal synaptic transmission are largely under-explored and the underlying mechanisms are unclear. The goal of this project was to investigate mechanisms involved in dopaminergic modulation of hippocampal neurophysiology. Specifically, I (1) examined if dopamine modulates hippocampal synaptic transmission in a region- and input-specific manner, and (2) studied the signaling mechanisms underlying such modulation. In the first aim for the study, I tested whether SKF38393, a dopamine D1-like receptor agonist, differentially affects excitatory synaptic transmission in perforant path synapses onto dentate gyrus granule cells and whether such effects differ from those at area CA1 synapses. I found that SKF38393 produced a concentration-dependent increase in field excitatory postsynaptic potential (fEPSP) in both subregions, but that higher concentrations were needed in the dentate gyrus to produce comparable effects. This synaptic enhancement was long-lasting and largely irreversible which suggests it may be a form of long term enhancement (LTP). Also, the increase in synaptic transmission at medial perforant path synapses was larger than in the lateral perforant path. Importantly, effects in the dentate gyrus, unlike those in CA1, differed substantially along the dorsoventral axis, with effects being significantly larger at the dorsal compared to the ventral pole. In the second aim, various combinations of D1 and D2-like receptor agonists and antagonists as well as inhibitors of second messenger systems, demonstrated that differential mechanisms were required for initiation and maintenance of SKF38393-mediated early and late-phase enhancement and that a novel non-canonical phospholipase-C (PLC) dependent signaling pathway may be involved. Based on recent discoveries in other brain regions, we hypothesized that multiple subcellular signaling pathways may contribute to PLC activation which may include but are not limited to D1(5)-D2 heteromers and Gβγ complex. In conclusion, this work uncovers novel dopaminergic signaling pathways regulating hippocampal physiology, which will lead to development of better (functionally selective) therapeutic agents.

abstract: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein, E6-AP) in neurons. Currently, the mechanism on how loss-of-function of the enzyme influences the nervous system development remains unknown. We hypothesize that impaired metabolism of proteins, most likely those related to E6-AP substrates, may alter the developmental trajectory of neuronal structures including dendrites, spines and synaptic proteins, which leads to disrupted activity/experience-dependent synaptic plasticity and maturation. To test this hypothesis, we conducted a detailed investigation on neuronal morphology and electrophysiological properties in the prefrontal cortex (PFC) layer 5 (L5) corticostriatal pyramidal neurons (target neurons). We found smaller soma size in the maternal Ube3a deficient mice (m-/p+; 'AS' mice) at postnatal 17-19 (P17-19), P28-35 and older than 70 days (>P70), and decreased basal dendritic processes at P28-35. Surprisingly, both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) decreased on these neurons. These neurons also exhibited abnormalities in the local neural circuits, short-term synaptic plasticity and AMPA/NMDA ratio: the excitatory inputs from L2/3 and L5A, and inhibitory inputs from L5 significantly reduced in AS mice from P17-19; Both the release probability (Pr) and readily-releasable vesicle (RRV) pool replenishment of presynaptic neurons of the target neurons were disrupted at P17-19 and P28-35, and the change of RRV pool replenishment maintained through adulthood (>P70). The AMPA/NMDA ratio showed abnormality in the L5 corticostriatal neurons of PFC in AS mice older than P28-35, during which it decreased significantly compared to that of age-matched WT littermates. Western Blot analysis revealed that the expression level of a key regulator of the cytoskeleton system, Rho family small GTPase cell division control protein 42 homolog (cdc42), reduced significantly in the PFC of AS mice at P28-35.These impairments of synaptic transmission and short-term synaptic plasticity may account for the impaired neuronal morphology and synaptic deficits observed in the PFC target neurons, and contribute to the phenotypes in AS model mice. The present work reveals for the first time that the E6-AP deficiency influences brain function in both brain region-specific and age-dependent ways, demonstrates the functional impairment at the neural circuit level, and reveals that the presynaptic mechanisms are disrupted in AS model. These novel findings shed light on our understanding of the AS pathogenesis and inform potential novel therapeutic explorations. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2017

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions. / Medicine, Faculty of / Graduate

The effects of anaesthetic applications of isoflurane on 82 CA₁ neurons were studied in in vitro preparations (guinea pigs) using intracellular recording techniques. Various parameters of their excitability such as membrane electrical properties, action potentials and their afterhyperpolarizing potentials as well as synaptic transmission were determined during bath perfusion of clinically relevant concentrtaions of isoflurane. Concentrations of isoflurane were detected in the bath with ¹⁹fluorine nuclear magnetic resonance techniques, and were found to range between 0.02 and 0.3 mM. No consistent effects on the membrane properties were observed. When synaptic activity was blocked by tetrodotoxin, isoflurane induced a hyperpolarization (3-5 mV) without affecting input conductance which was computed from the voltage responses to injections of hyperpolarizing current pulses and the slopes of current-voltage relations for each cell. Responses to depolarizing pulses revealed that the threshold, amplitude and duration of the evoked spikes were not greatly altered, although repetitive spike firing was suppressed in a dose-dependent manner by isoflurane. Similarly, the amplitude and duration of the long-lasting hyperpolarizations following the elicitation of multiple (4 or 5) spikes were reduced in a reversible and dose-dependent manner. Reductions in amplitude and duration of excitatory and inhibitory postsynaptic potentials evoked by electrical stimulation of stratum radiatum were observed; these effects also were strictly dependent on the dose, as well as on duration of the application. These investigations have revealed that isoflurane interferes with synaptic transmission in the hippocampal slice preparation and suggest that presynaptic actions on transmitter release, in addition to postsynaptic effects / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

In the hippocampus, transient tetanic stimulations of inputs, or brief simultaneous pairings of conditioning intracellular postsynaptic depolarizations with activated presynaptic afferents at low stimulation frequencies, result in input specific long-term potentiation (LTP) of synaptic transmission. LTP lasts for hours in vitro, or weeks in vivo, and it is thought to be involved in memory and learning. Experimental evidence in the literature suggests that postsynaptic mechanisms mediate LTP induction, whereas presynaptic mechanisms are involved in its maintenance. Since LTP is thought to be generated by postsynaptic mechanisms and to be subsequently maintained by presynaptic processes, this suggests the presence of feedback interactions during LTP development, however, the experimental evidence for such interactions is presently not available. Consequently, the present studies were conducted to examine possible feedback interactions between postsynaptic and presynaptic elements in the hippocampus. Furthermore, the experiments tested the hypothesis that substances released during tetanic stimulations caused the release of endogenous substances that interacted with activated afferents resulting in alterations in presynaptic functions and LTP production. Experiments were conducted using transversely sectioned guinea pig hippocampal slices. Briefly, physiological medium containing 3.5 mNi Ba++ and 0.5 mM Ca (denoted as Ba medium) was used to induce the asynchronous release of transmitters, observed as evoked miniature EPSPs (minEPSPs) in CA1b neurons after stimulation of the stratum radiatum. During transient Ba++ applications, short bursts of evoked minEPSPs were observed following stimulations of the stratum radiatum or conditioning depolarizing current injections into CA1b neurons. Moreover, the frequencies of minEPSPs were significantly increased immediately after simultaneous stimulations of the stratum radiatum and conditioning depolarizing current injections into CA1b neurons. Significant increases in the frequencies of evoked minEPSPs were also observed during LTP induced by tetanic stimulations. The above increases in the frequencies of evoked minEPSPs were attributed, in part, to presynaptic changes resulting in increases in transmitters released. However, a thorough quanta! analysis is requirea to substantiate this conclusion. In order to determine whether any substances released during tetanic stimulations were involved in the mooulation of presynaptic functions and induction of LTP, samples were collected from guinea pig hippocampus and rabbit neocortex. It was found that samples that were collected during tetanic stimulations of the guinea pig hippocampus in vivo or rabbit neocortex in vivo produced LTP in the guinea pig hippocampal slice in vitro. Applications of these samples after heating and cooling failed to induce LTP. Subsequent studies demonstrated that PC-12 cells incubated in growth medium treated with samples collected during tetanic stimulations of the rabbit neocortex developed extensive neurite growths. In contrast, PC-12 cell cultures incubated in (1) heated and cooled samples, (2) samples collected in the absence of tetanic stimulations of the rabbit neocortex, or (3) plain growth medium, failed to develop neurite growths. In addition, PC-12 cell cultures that were incubatea in growth medium containing samples collected during tetanic stimulations plus saccharin (10 mM), a substance known to inhibit N6F-dependent neurite growth, failed to develop neurites. In separate experiments it was found that saccharin could block (1) the synaptic potentiating effects of the above collected and applied endogenous substances, and (2) LTP induced with tetanic stimulations, in the guinea pig hippocampus in vitro. The concentrations of saccharin used in these studies had insignificant effects on resting membrane potentials, input resistances, spontaneous or evoked responses of CA1b neurons. Furthermore, CA1b neuronal depolarizations induced by N-methyl-DL-aspartate (NMDA) or with tetanic stimulations of the stratum radiatum, were not altered by saccharin applications. In addition, saccharin had insignificant effects on paired-pulse facilitation, post-tetanic potentiations, minEPSP frequencies in CA1b neurons, and Schaffer collaterals terminal excitability. These results suggest that saccharin blocked LTP through mechanisms different from either non-specific alterations in CA1b cell properties or NMDA receptor activation. Perhaps the agent antagonized LTP at a step beyond NMDA receptor activation. That saccharin blocked LTP caused by the applied neocortical sample as well as by tetanic stimulation of the stratum radiatum, and that saccharin also blocked neurite growth in PC-12 cells induced by the neocortical samples, raises the prospect that growth related substances are involved in LTP generation. In other control experiments, it was found that the potentiating effects of the collected endogenous substances were not antagonised by atropine or dihydro-e-erythroidine. Heated and then cooled solutions of glutamate (a putative transmitter at the Schaffer col laterals-CA1b synapses) still maintained their actions on the CA1b population spike. While brief applications of 2.5 μg/ml exogenous NGF (from Vipera lebetina) during low frequency stimulations of the stratum radiatum did not consistently induce LTP, this peptide significantly facilitated the development of LTP when applied in association with tetanic stimulations of weak inputs in the CA1b area. These weak inputs could not support LTP if tetanized in the absence of the exogenous NGF. The results of the studies in this thesis suggested that postsynaptic depolarizations modulated presynaptic functions in the hippocampus. Tetanic stimulations in hippocampus and neocortex caused the release of diffusible substances, which were probably growth related macromolecules, that interacted with activated presynaptic afferents and/or subsynaptic dendritic elements resulting in LTP development. The precise locus of actions of these agents awaits further investigations. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Au cours des dernières années, la biologie et plus généralement la recherche médicale, a connu des avancées majeures grâce aux nombreux progrès de la microscopie, de la génétique, de la biologie moléculaire, de la protéomique, et du séquençage à haut débit. Il s’agit maintenant d’identifier à partir de toutes ces données gigantesques, les grandes lois de la biologie. Le principe de la biologie intégrative ou systémique est de poser les bases d'une véritable théorie de l'organisation fonctionnelle du vivant à partir des différents mécanismes découverts expérimentalement. De la même manière que l'on décrit la matière par les théories des mathématiques, de la physique, et de la chimie, on veut pouvoir comprendre, formaliser et modéliser le fonctionnement des mécanismes du vivant. Cette thèse a consisté à réaliser une bibliothèque de modèles fonctionnels des réactions chimiques qui prennent place dans les cellules nerveuses aussi appelés modèles élémentaires et de les assembler afin d’obtenir un système mimant le plus finement possible les mécanismes de la propagation du signal électrique au sein d’une synapse. Les travaux réalisés jusqu'alors ont permis de modéliser les mécanismes essentiels et de reconstituer le comportement d’une synapse glutamatergique. En particulier, l’utilisation de cette nouvelle méthode de recherche et de développement de médicaments a pour objectif de proposer des molécules innovantes, en optimisant leurs propriétés biochimiques. Cette technique permettra, dans un futur proche, l'avènement d'une nouvelle génération de pharmaceutiques, dit multi-cibles, c'est-à-dire permettant d'intervenir sur les différents mécanismes d'une pathologie. / In recent years, biology and medical research more generally, has seen major advances in microscopy, genetics, molecular biology, proteomics, and high-throughput sequencing. We now identify from these huge data, the great laws of biology. The principle of integrative biology or systemic is to defined the basis of the foundations for a theory of the functional organization of living from different mechanisms discovered experimentally. In the same way that matter is described by the theories of mathematics, physics, and chemistry, we want to understand, formalize and model the functioning of living mechanisms. This thesis has been to achieve a library of functional models of chemical reactions that take place in nerve cells also called elementary models and assemble them to obtain a system mimicking the finest possible mechanisms of electrical signal propagation within of a synapse. The work done so far allow us to model the essential mechanisms and reconstruct the behavior of a glutamatergic synapse. In particular, the use of this new method of research and drug development aims to offer innovative molecules by optimizing their biochemical properties. This technique will, in the near future, the advent of a new generation of pharmaceuticals, said multi-target, able to intervene on the different mechanisms of disease.

<p> Alterations in neural tissues associated with environmental variables have been studied for many years. Anatomical changes in the neocortex of rats in response to exposure to complex environments were observed and replicated in a number of studies both within and across species. These changes are not dependent on the age of the animal or on the duration of exposure, and have been demonstrated in structures outside of the cortex. Due to the undisputed involvement of both the neocortex and the hippocampus in learning and memory, researchers applied a widely used model system of a synaptic mechanism for learning, long-term potentiation (LTP), to the environmental enrichment paradigm and demonstrated significant enhancements in hippocampal field potentials in enriched rats. The present study examines whether the neocortex also showed evidence of plasticity in synaptic transmission. No effects for environmental enrichment were observed on the maximum amplitude of neocortical field responses evoked from the corpus callosum. To assess the plasticity of the chronic preparation used in the study, the animals were exposed to trains of pulses previously shown to induce electrical LTP in the cortex, but revealed only a slight, although significant, depression of the evoked response amplitude. An alteration in the stimulation parameters did not result in an enhanced response. Cortical depth measures suggested that the enriched environment was indeed sufficient to produce plastic changes in anatomy, if not in the efficacy of synaptic transmission. The importance of these findings in the neocortex leads us to question the validity of the LTP model of learning and memory.</p> / Thesis / Master of Science (MSc)