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This thesis investigated the contribution of the nucleus accumbens core (AcbC) and the hippocampus (H) to choice and learning involving reinforcement that was delayed or unlikely. Animals must frequently act to influence the world even when the reinforcing outcomes of their actions are delayed. Learning with action-outcome delays is a complex problem, and little is known of the neural mechanisms that bridge such delays. Impulsive choice, one aspect of impulsivity, is characterized by an abnormally high preference for small, immediate rewards over larger delayed rewards, and is a feature of attention-deficit/hyperactivity disorder (ADHD), addiction, mania, and certain personality disorders. Furthermore, when animals choose between alternative courses of action, seeking to maximize the benefit obtained, they must also evaluate the likelihood of the available outcomes. Little is known of the neural basis of this process, or what might predispose individuals to be overly conservative or to take risks excessively (avoiding or preferring uncertainty, respectively), but risk taking is another aspect of the personality trait of impulsivity and is a feature of a number of psychiatric disorders, including pathological gambling and some personality disorders. The AcbC, part of the ventral striatum, is required for normal preference for a large, delayed reward over a small, immediate reward (self-controlled choice) in rats, but the reason for this is unclear. Chapter 3 investigated the role of the AcbC in learning a free-operant instrumental response using delayed reinforcement, performance of a previously learned response for delayed reinforcement, and assessment of the relative magnitudes of two different rewards. Groups of rats with excitotoxic or sham lesions of the AcbC acquired an instrumental response with different delays (0, 10, or 20 s) between the lever-press response and reinforcer delivery. A second (inactive) lever was also present, but responding on it was never reinforced. The delays retarded learning in normal rats. AcbC lesions did not hinder learning in the absence of delays, but AcbC-lesioned rats were impaired in learning when there was a delay, relative to sham-operated controls. Rats were subsequently trained to discriminate reinforcers of different magnitudes. AcbC-lesioned rats were more sensitive to differences in reinforcer magnitude than sham-operated controls, suggesting that the deficit in self-controlled choice previously observed in such rats was a consequence of reduced preference for delayed rewards relative to immediate rewards, not of reduced preference for large rewards relative to small rewards. AcbC lesions also impaired the performance of a previously learned instrumental response in a delay-dependent fashion. These results demonstrate that the AcbC contributes to instrumental learning and performance by bridging delays between subjects' actions and the ensuing outcomes that reinforce behaviour. When outcomes are delayed, they may be attributed to the action that caused them, or mistakenly attributed to other stimuli, such as the environmental context. Consequently, animals that are poor at forming context-outcome associations might learn action-outcome associations better with delayed reinforcement than normal animals. The hippocampus contributes to the representation of environmental context, being required for aspects of contextual conditioning. It was therefore hypothesized that animals with H lesions would be better than normal animals at learning to act on the basis of delayed reinforcement. Chapter 4 tested the ability of H-lesioned rats to learn a free-operant instrumental response using delayed reinforcement, and their ability to exhibit self-controlled choice. Rats with sham or excitotoxic H lesions acquired an instrumental response with different delays (0, 10, or 20 s) between the response and reinforcer delivery. H-lesioned rats responded slightly less than sham-operated controls in the absence of delays, but they became better at learning (relative to shams) as the delays increased; delays impaired learning less in H-lesioned rats than in shams. In contrast, lesioned rats exhibited impulsive choice, preferring an immediate, small reward to a delayed, larger reward, even though they preferred the large reward when it was not delayed. These results support the view that the H hinders action-outcome learning with delayed outcomes, perhaps because it promotes the formation of context-outcome associations instead. However, although lesioned rats were better at learning with delayed reinforcement, they were worse at choosing it, suggesting that self-controlled choice and learning with delayed reinforcement tax different psychological processes. Chapter 5 examined the effects of excitotoxic lesions of the AcbC on probabilistic choice in rats. Rats chose between a single food pellet delivered with certainty (probability p = 1) and four food pellets delivered with varying degrees of uncertainty (p = 1, 0.5, 0.25, 0.125, and 0.0625) in a discrete-trial task, with the large-reinforcer probability decreasing or increasing across the session. Subjects were trained on this task and then received excitotoxic or sham lesions of the AcbC before being retested. After a transient period during which AcbC-lesioned rats exhibited relative indifference between the two alternatives compared to controls, AcbC-lesioned rats came to exhibit risk-averse choice, choosing the large reinforcer less often than controls when it was uncertain, to the extent that they obtained less food as a result. Rats behaved as if indifferent between a single certain pellet and four pellets at p = 0.32 (sham-operated) or at p = 0.70 (AcbC-lesioned) by the end of testing. When the probabilities did not vary across the session, AcbC-lesioned rats and controls strongly preferred the large reinforcer when it was certain, and strongly preferred the small reinforcer when the large reinforcer was very unlikely (p = 0.0625), with no differences between AcbC-lesioned and sham-operated groups. These results suggest that the AcbC contributes to action selection by promoting the choice of uncertain, as well as delayed, reward.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22252号 / 理博第4566号 / 新制||理||1656(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)准教授 上田 佳宏, 准教授 岩室 史英, 教授 長田 哲也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue. Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity. The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques. The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation. In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10. By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10. The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss. In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

(English) Phosphoinositides (PIs) are glycerophospholipids with a negative charge. As components of cell membranes, PIs are involved in membrane and cytoskeletal dynamics, cell movement and signalling, and the modulation of ion channels and transporters. Apart from the cytoplasm, phosphoinositides also localise to the cell nucleus. PIs play a role in crucial nuclear processes, such as DNA transcription, pre-rRNA and pre-mRNA processing, cell differentiation, DNA damage response, or apoptosis. Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are the most abundant phosphoinositides in the cell. However, their exact localisation and function in the nucleus are largely unknown. Here, we describe their localisation at super-resolution level and their involvement in some nuclear processes. PI(4)P is present in nuclear lamina, nuclear speckles and nucleoli, and it forms small foci in nucleoplasm. The majority of nuclear PI(4)P localises to the nucleoplasm, whereas almost 16 % is present in nuclear speckles. On the other hand, the majority of nuclear PI(4,5)P2 localises to nuclear speckles, almost 30 % localises to nucleoplasm and the lesser portion to nucleoli. In the nucleoplasm, PI(4,5)P2 forms small foci called nuclear lipid islets (NLIs). Their core is...

Drug-associated stimuli trigger craving and relapse in addiction. Murine morphine conditioned place preference (CPP) was used to model learning of opioid associations. We examined how morphine and learning interact to alter neuron morphology in the nucleus accumbens (NAc) core and shell after acquisition and extinction of CPP. Conditioning with morphine dose-dependently increased place preference compared to saline. In comparison to those from saline conditioned and morphine non-conditioned controls, neurons from the NAc core of morphine conditioned mice had increased dendritic complexity, as defined by increased dendritic length, number, and Sholl intersections. This effect is due to the combination of morphine and learning, which is different from effects of morphine or conditioning alone. Morphine administration without conditioning was associated with increased spine density in the core, which was reversed by CPP acquisition. Control conditioning with saline produced no morphology changes. Morphine CPP extinction was associated with decreased dendritic complexity, reversing the increased complexity seen after acquisition. Mice that extinguished CPP had similar dendritic complexity to saline conditioned mice, in terms of dendritic count and intersections, but less dendritic complexity than non-extinguished mice that retained CPP. Since dopamine release imbues salience to stimuli that coincide with drug use, and the dopamine D1 receptor mediates CPP acquisition, we tested the effect of SKF81297 D1 receptor agonist on CPP extinction and associated accumbal neuron morphology. SKF81297 (0.8 mg/kg) injected after each extinction training session impeded extinction, and produced increased dendritic complexity compared to controls. SKF81297 may have sustained conditioned associations, disrupted consolidation of extinction, and/or disrupted the decline in dopamine levels that may occur throughout extinction sessions. We hypothesize that changes occurred in the NAc core because this region mediates how stimuli and drug effects direct motor action. Since D1 receptors oppose extinction of drug-cue-induced behavior, they play a role in reinforcing opioid addiction. Acquisition and extinction may be opposite processes in the brain, as in behavior. Extinction may include some reversal of acquisition learning as well as being new learning with its own pathway. Interventions that target D1 receptors or that selectively reduce NAc core dendritic complexity may contribute to opioid addiction treatment.

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...

The lateral and basal nuclei of the amygdala receive dopaminergic projections from the ventral tegmental area and substantia nigra, and noradrenergic projections from the locus coeruleus (LC). Previously, we demonstrated postmortem indices of altered dopaminergic (amygdala) and noradrenergic (LC) neurochemistry in subjects with major depressive disorder (MDD). For example, decreased levels of dopamine transporter were observed in the amygdala in MDD, while concentrations of tyrosine hydroxylase (TH) were elevated in the LC in MDD. The present study investigated the quantitative distribution of TH in nuclei of the human amygdala from 5 control subjects, and measured amounts of TH in specific amygdaloid nuclei and the LC from 8-10 matched pairs of MDD and psychiatrically normal control subjects. Matched pairs included 3 females and 7 males (controls and MDD), average ages of 50±5 y (controls) and 51±5 y (MDD), average postmortem intervals of 16±2 h (controls) and 21±1 h (MDD), and average pH values of 6.58±0.08 (controls) and 6.59±0.09 (MDD). The lateral, basal, accessory basal, and central nuclei of the amygdala and the LC were punched from frozen sections of postmortem brain. TH-immunoreactivity was measured by quantitative Western blotting. In normal control subjects, TH levels in the LC were between 3000- and 4000-fold higher than TH levels in the nuclei of the amygdala. Within the amygdala, amounts of TH were highest in the basal and central nuclei, and lowest in the lateral nucleus. TH levels in the basal nucleus were highly variable across subjects. TH levels were significantly lower (-50%) in the lateral amygdaloid nucleus in MDD subjects as compared to control subjects. In contrast, TH levels in the LC were significantly higher (+75 %) in MDD subjects. This report is the first demonstration of altered TH levels in the human amygdala. The direction of change associated with MDD of TH in the lateral nucleus of the amygdala was opposite to that found in the LC. Whether abnormal amounts of TH in the amygdala are a result of altered dopaminergic or noradrenergic input to the amygdala requires further study.

The increase in extracellular dopamine (DA) following cocaine administration plays a major role in cocaine abuse. In vitro, cocaine binds to DA transporters (DAT) and blocks DA uptake. Moreover, cocaine can increase extracellular DA concentration as measured by in vivo neurochemical methods. The present study examined the effects of cocaine and other drugs on DA, NE and 5-HT uptake using an ex vivo assay. Rats were injected i.v. with saline or drug and sacrificed at various time points after injections. Brains were dissected for regional monoamine uptake studies ex vivo. In most brain regions, cocaine given in vivo blocked monoamine uptake as expected. [ H]DA uptake in nucleus accumbens was inhibited with an ED = 22.3 μmol/kg. Cocaine fully inhibited [ H]NE uptake (ED = 4.58 μmol/kg) in the occipital cortex and partially inhibited [ H]5-HT uptake (33% at 30 μmol/kg) in the midbrain. However, under the same conditions [ H]DA uptake in the striatum was not inhibited after injections of cocaine up to 56 μmol/kg. Although the mechanism for this discrepancy is unclear, DA binding and uptake sites may be distinct and/or there may be regional differences in DA transporters.

Philosophiae Doctor - PhD / Observations of galaxies at di↵erent wavelengths have shaped our understanding of their formation and evolution through time. The commonly derived parameters, such as stellar mass and star formation rate (SFR), rely on the assumption that the radiation received is exclusively generated by the stars within the galaxy. This assumption is true for pure star-forming (SF) galaxies, but not in the presence of an active galactic nucleus (AGN). AGNs are structures that also radiate in the full electromagnetic spectrum, inducing additional flux to that emitted by stars. Their small sizes in comparison to the host galaxy (⌧1 %) generally make them invisible in galaxy images. AGNs come in many variations making the most powerful (e.g., quasi-stellar objects) easily identifiable, whereas others with much weaker signatures can be hidden in the total emission from the host. Therefore it is imperative to find accurate methods to separate and study the properties of AGNs versus pure SF galaxies.