Neuronal nicotinic acetylcholine receptors (nAChRs) are key players in both cognitive and autonomic processes. In the cognitive domains of the brain, destruction of cholinergic inputs or disruption of nAChR function result in cognitive deficits as observed in Alzheimer's disease, schizophrenia, brain trauma and aging. By contrast, moderate activation of nAChRs supports neuroprotection and improves cognitive functions. In addition, neuronal nAChRs are also expressed in important autonomic centers such as the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV) that support autonomic visceral reflexes and homeostasis. In this study, the underlying mechanisms of nAChR activation and its pharmacology were investigated in the hippocampus and the NTS, critical brain regions supporting cognitive and autonomic functions, respectively. Specific Aim 1 of this study was to determine the capacity of physiological levels of choline to activate α7 nAChRs in hippocampal CA1 pyramidal neurons and interneurons. A weak persistent activation of α7 nAChRs can be neuroprotective. These levels of activation can be achieved by selective or non-selective α7 nAChR agonists or inhibitors of ACh esterase (AChEI). However, nicotinic agonists desensitize α7 nAChRs while AChEI produces side effects limiting their overall clinical and pre-clinical effectiveness. These limitations can be avoided by using a novel class of drugs; type-II positive allosteric modulators of α7 nAChRs (α7-PAMs) such as PNU-120596 (i.e., PNU). At physiological levels, choline alone is ineffective as an α7 agonist because of its low concentration in the cerebrospinal fluid (~10 µM) and low potency for α7 activation (EC50~1.5 mM). However, the results pertaining to Specific Aim 1 demonstrate that in the presence of PNU (1-5 µM) , 10 µM choline produces persistent α7 activation expressed on CA1 pyramidal and interneurons which may be fine-tuned to achieve optimal neuroprotection and cognitive benefits. Specific Aim 2 was to test the novel concept that PNU mediated changes in α7 receptor kinetics can alter the biophysical properties of α7 channel-drug interactions and thereby increase the probability and the apparent affinity of open channel block. The results of this study suggest that the compounds (e.g., Bicuculline) that do not potently interact with α7 ion channels in the absence of PNU begin to interact potently in its presence. These emergent properties of α7 channel-drug interactions in the presence of PNU need to be recognized in drug development as they may lead to unanticipated side effects and serious misinterpretation of data. Specific Aim 3 investigated the pharmacology and mechanisms of action of pre-synaptic non-α7 and α7 nAChRs in the caudal NTS neurons. Although, activation of nAChRs is known to enhance pre-synaptic release of glutamate in subsets of caudal NTS neurons, its mechanism of action has been elusive. However, the results from this study demonstrated that nicotine-mediated enhancement of glutamate release requires Ca2+ influx via nAChRs but does not require any contribution from voltage-gated Ca2+ ion channels (VGCCs) and presynaptic Ca2+ stores. Moreover, both functional α7 and non-α7 nAChRs were found to contribute to the presynaptic effects of nicotine in subsets of NTS neurons. However, co-expression of α7 and non-α7 nAChRs on the same glutamatergic presynaptic terminals was not detected. Collectively, these studies may help in developing new therapeutic strategies to selectively target nAChR-associated pathways that support cognitive and autonomic functions in health and disease.

OBJECTIVE: Previous studies have shown the significant contribution of sympathoinhibition in response to sodium loading to prevent increases in mean arterial blood pressure in salt resistant phenotypes. It has also been shown that brain Gαi2 protein gated signal transduction plays a major role in this pathway, however, the specific mechanisms through which this pathway is activated remain less well understood. The purpose of this study was to elucidate the relative contribution of increased sodium in either the plasma or the cerebrospinal fluid (CSF) to the regulation of mean arterial pressure and natriuresis. Additionally we explored the potential for using the novel CLARITY Imaging technique to identify the relative activity of neurons in areas of the brain thought to play a major role in body fluid homeostasis in response to salt. METHODS: Rats that were pre-treated with either scrambled or Gαi2 oligodeoxynucleotides (ODN), to selectively down regulate brain Gαi2 proteins, were challenged either peripherally or centrally with sodium. Upon sodium loading physiological parameters were measured for two hours after which the animal's brains were recovered for immunohistochemical (IHC) analysis of the paraventricular nucleus, a known regulatory center for body fluid homeostasis and blood pressure regulation. Additionally we adapted a version of the published CLARITY Imaging protocols for optically clearing tissue through application of electrophoretic tissue clearing (ETC) to a larger rat model. RESULTS: In scrambled ODN pre-treated rats we observed a temporary increase in MAP in response to both the peripheral and central sodium challenge. In the Gαi2 ODN pre-treated animals we saw some form of attenuation to this response in both studies, however, where in the peripheral challenge there was an increase in the amount of time that it took the rats to return to normotension with no alteration in natriuresis, in the central challenge there was a large attenuation in natriuresis with no differences in the time to return to baseline MAP. Our IHC analysis also showed a decrease in neuronal activation of paraventricular medial parvocellular neurons in Gαi2 pre-treated rats that were challenged peripherally vs their SCR pre-treated counterparts. No such difference was observed in either of the pre-treatment groups from the central sodium challenge study. In the CLARITY study we found that it is possible to adapt the method for optically clearing tissue to the larger model, however, we encountered several issues related to tissue swelling and peripheral tissue damage. CONCLUSION: Based on our current results it seems evident that there are at least two different mechanisms that activate the cardiovascular regulatory control centers in the brain that prevent long term increases in mean arterial pressure in response to increased salt. It also appears that these two different mechanisms are triggered either by increases in plasma or CSF salinity, though which of these two mechanisms may be directly responsible for the development of salt sensitive hypertension requires further investigation. While we had some success at optically clearing larger tissue volumes through ETC, problems we encountered with maintaining tissue integrity for investigations of intact neural networks prevented us from applying this technique, in its current form, to our investigation of salt sensitive hypertension.

The daily variation in background light intensity (irradiance) can entrain the endogenous clock in the suprachiasmatic nucleus (SCN) to the external environment. The only source of this photic information in mammals is the eye, which is primarily a visual organ. It is therefore highly specialised to detect high frequency spatiotemporal modulations. This together with the adaptation which occurs within the retina could be present difficulties when encoding global irradiance. This raises the question of whether spatial patterns, which are present in our everyday viewing, might affect the ability of the SCN to receive 'true irradiance' signals and entrain to the external environment. My first approach was to determine whether individual SCN cells might receive a 'true irradiance' signal. To this end I mapped and characterised the receptive field properties of SCN neurons using in vivo electrophysiology. Indeed a handful of neurons (full field cells) responded to light anywhere in the visual scene and thus may act as 'irradiance detectors'. However, the vast majority of cells only sampled local radiance from a limited area of the visual scene. Having mapped the receptive field properties it became clear that cells which sampled from a limited area of the visual scene would be sensitive to spatial contrast (patterns). To examine the effect of spatiotemporal contrast on the SCN I examined two SCN outputs: locomotor activity and neuronal firing rates. Although spatiotemporal modulation in light intensity could induce large amplitude oscillations in neuronal activity; the time averaged firing rate and locomotor activity, which are believed to be determined by irradiance, were largely unaffected by spatial patterns. This led to the conclusion that the SCN can multiplex photic information into information regarding irradiance, and spatial information by encoding them under different timescales. Melanopsin has been heralded as the key photopigment for encoding irradiance and entraining the SCN. However such experiments have been only performed using diffuse light stimuli. Here I investigated the role of melanopsin under natural viewing conditions which incorporated spatial patterns. Under such stimuli the SCN response can be almost entirely accounted for by the melanopic irradiance of the stimuli.

Age-related hearing loss, presbycusis, is a complex disorder involving the interaction of both peripheral and central neurological deficits. Central auditory dysfunction may contribute to poor temporal discrimination of complex sounds such as speech. This research is timely since our population over 60 years old is increasing rapidly due to advances in medicine and nutrition as well as the advancing age of baby boomers. This study was designed to provide a better understanding of age-related changes in dorsal cochlear nucleus (DCN) physiology. DCN was chosen because it receives direct input from the auditory nerve and much is known about its neuronal morphology, physiology and circuitry. In young animals, DCN output neurons, fusiform cells, receive excitatory inputs from the acoustic nerve, which is modulated and shaped by inhibitory glycinergic inputs from nearby vertical cells. A number of studies in rodents suggested an age-related impairment of glycinergic neurotransmission. To access the functional impact of reduced putative glycinergic input in the central auditory system, this study compared the physiological responses of DCN neurons from young adult and aged rats in response set of simple and more complex acoustic stimuli. Single-unit extracellular recordings were made from two groups of DCN neurons: fusiform cells and cartwheel cells. Fusiform cells reflect the culmination of DCN processing, therefore were good candidates for studying the effect of aging on one ascending auditory stream. Two specific aims were directed at fusiform cell: SA1) Examine the effects of aging on fusiform cell response properties to simple tone burst stimuli; SA2) Examine the effect of aging on DCN output neuron response to complex temporal stimuli. A third aim, SA3) Examine the impact of aging on the response properties of cartwheel cells, a DCN inhibitory interneuron. Fusiform cells recorded from aged rats displayed significantly higher maximum discharge rates to characteristic frequency (CF) tones, fewer nonmonotonic CF rate-level functions and more wide-chopper type post-stimulus time histograms (PSTHs) when compared to neurons from young adult rats. These findings were consistent with an age-related loss of inhibitory glycinergic input. To elucidate how loss of inhibition could lead to functional deficits in temporal processing, fusiform cells were challenged to encode sinusoidally amplitude modulated (SAM) tones. DCN output neurons were presented with SAM tones at three modulation depths at 30 dB above hearing level/response threshold with the carrier frequency set to each unit’s CF. Temporal synchronicity to the SAM envelope was measured using vector strength from temporal modulation transfer functions (tMTFs). Firing rate to SAM tones was also assessed in rate modulation transfer functions (rMTFs). DCN output neurons from aged rats showed no loss of rate response (rMTF) but displayed a selective loss of temporal precision to SAM tones with significant age-related changes in peak vector strength (best modulation frequency), and the shape and category of tMTF. Wide-chopper PSTH types had significantly lower vector strength values than buildup and pauser PSTHs in both young and aged fusiform cells. Since a significantly greater proportion of aged neurons exhibited wide-chopper responses, this could explain, in part, the loss of temporal processing. The age-related response changes in the present study mimicked results from earlier studies were glycine inhibition onto young adult fusiform cells was pharmacologically blocked. Cartwheel cells receive excitatory inputs from granule cell parallel fibers as well as somatosensory dorsal column nucleus and project glycinergic inputs onto DCN output neurons. They appear to play a role in the integration of auditory and somatosensory inputs such as sensing head position. Aged cartwheel neurons exhibited signs of disinhibition showing increased spontaneous activity, increased maximum discharge rates and altered rate-level functions. The observed age-related changes in cartwheel cells are consistent with deafferentation studies using acoustic trauma. Collectively, the changes in DCN output neurons and cartwheel cells reflect a potentially maladaptive age-related neuroplasticity in response to a loss of excitatory acoustic nerve input. These in vivo extracellular findings were consistent with a global downregulation of glycinergic input within the DCN of aged rats. This reduced inhibition may contribute to functional deficits, particularly in activities that require precise timing of events such as response to speech-like stimuli.

A biomarker is a measurable biological characteristic that can be evaluated as an indicator of normal (physiological) or abnormal (pathogenic) processes. In cancer research, there remains a need for the identification of new biomarkers that can be used to close the gaps in the current understanding of cancer development, progression and treatment. HLXB9 is a homeobox gene located at 7q36. It encodes a transcription factor important in embryonic development. Its accurate regulation is significant in the organogenesis of the endodermal germ layer particularly in the development of the pancreas. After development, its expression is downregulated in the majority of adult tissues. Recently, aberrant expression of HLXB9 has been found in certain cancers such as hepatocellular carcinoma, testicular cancer, pancreatic cancer and leukaemia. The location of genes and chromosomes in the nuclei of healthy human cells has been shown to be non-random, therefore understanding the mechanisms that regulate nuclear genome organisation is important in understanding of cancer biology in cases where genes are relocated. The nuclear localisation of genes as a biomarker of tumour development in cancer is a relatively new but promising field in cancer research. Previous research by our group found overexpression of HLXB9 corresponded to an altered positioning of this gene in the nucleus of paediatric leukaemia patients harbouring the translocation t(7;12)(q36;p13). In this project, HLXB9 was evaluated as a biomarker in cancer development. In the first study, a new dual colour probe for the detection of the t(7;12)(q36; p13) was validated by fluorescence in situ hybridisation (FISH) in leukaemia patient samples previously described as harbouring the translocation. The expression of HLXB9 was then analysed by RT-PCR in 48 patients diagnosed with various haematological disorders. 25% of patients analysed expressed HLXB9. Additionally, HLXB9 expression in leukaemia was found in patients with normal copies of chromosome 7 suggesting HLXB9 expression can occur independently of chromosome 7 abnormalities. An attempt was made to evaluate the link between HLXB9 expression and its nuclear localisation in these patients. Four online databases were interrogated to identify cancer types and subtypes that exhibit differential HLXB9 expression. HLXB9 expression was not altered in the majority of cancer cases investigated. However, aberrant HLXB9 expression was found in cancer types not previously reported as showing differential HLXB9 expression such as kidney cancer, lung cancer and endometrial cancer. The identification of aberrant expression of HLXB9 in these cancer types provides a new avenue for research into understanding cancer development and progression in these tumour types. Finally, the expression of HLXB9 was analysed in four breast cancer cell lines by quantitative RT-PCR and immunofluorescence. Additionally, the prognostic significance of HLXB9 expression was evaluated in publicly available breast cancer survival databases (Kaplan Meier plotter and BreastMark). Altogether, the findings emerging from this thesis work show that, although the potential for HLXB9 to be used as biomarker is appealing, further work is required to confirm the value of this biological parameter in the diagnosis, prognosis and progression of cancer.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-30T18:09:44Z No. of bitstreams: 2 Dissertação - Lucas Nojosa Oliveira - 2014.pdf: 3496210 bytes, checksum: ee39300fc3e6a76f167fcc74a33b646f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-30T18:10:02Z (GMT) No. of bitstreams: 2 Dissertação - Lucas Nojosa Oliveira - 2014.pdf: 3496210 bytes, checksum: ee39300fc3e6a76f167fcc74a33b646f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-01-30T18:10:02Z (GMT). No. of bitstreams: 2 Dissertação - Lucas Nojosa Oliveira - 2014.pdf: 3496210 bytes, checksum: ee39300fc3e6a76f167fcc74a33b646f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by fungi of the genus Paracoccidioides which is composed of two species P. lutzii e P. brasiliensis. The fungus grows as mycelium in saprophytic environment, and as yeast in host. Paracoccidioides spp present multinucleated yeast cells. The characterization of the nuclear proteome an important step to understand the biology of the fungus, and contributes to future studies of possible pharmacological targets. In this study we aimed to characterize the profile of the nuclear proteome of Paracoccidioides species in yeast form. A total of 570 proteins were identified and classified as nuclear, using a combination of method sample enrichment, proteomic of high accuracy technique, NanoUPLC-MS E , and bioinformatic filters. The results revealed important proteins related to DNA maintenance, regulation and gene expression, synthesis and processing of messenger and ribosomal RNA and nuclear-cytoplasmic traffic. Uncharacterized proteins and proteins commonly known as cytoplasmic were identified in fraction enriched nuclear and require further studies to demonstrate its nuclear roles. This is the first descriptive nuclear proteome of Paracoccidioides spp. and opens the way for new researches. / A paracoccidioidomicose (PCM) é uma doença endêmica da América Latina causada pelos fungos do gênero Paracoccidioides contituído de duas espécies P. lutzii e P.brasiliensis. O fungo cresce como micélio no ambiente saprofítico, e como levedura no hospdeiro. As células leveduriformes de Paracoccidioides spp. apresentam-se multinucleadas. Caracterizar o perfil protéico nuclear é um importante passo para compreender a biologia do fungo, e contribui para estudos futuros de possíveis alvos farmacológicos. Neste trabalho objetivamos caracterizar o perfil do proteoma nuclear das espécies de Paracoccidioides nas formas leveduriformes. Um total de 570 proteínas foram identificadas e classificadas como nucleares utilizando a combinação de um método de enriquecimento da amostra, uma técnica proteômica de alta acurácia, NanoUPLC-MS E , e a aplicação de filtros de bioinformática. Os resultados revelaram importantes proteínas relacionadas à manutenção do DNA, regulação e expressão gênica, síntese e processamento de RNA mensageiro e ribossomal e transporte núcleo-citoplasma. Proteínas não caracterizadas e proteínas comumente conhecidas como citoplasmáticas foram identificadas na fração enriquecida nuclear e necessitam de estudos adicionais para elucidar seus papéis nucleares. Esse é o primeiro trabalho proteômico descritivo do núcleo de Paracoccidioides spp., onde demonstra novas moléculas para estudos futuros.

Orientador: Cláudia Herrera Tambeli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T16:51:32Z (GMT). No. of bitstreams: 1 Miranda_Josiane_M.pdf: 1166629 bytes, checksum: b11fc8ed7e534b6c9265e384b6a80eef (MD5) Previous issue date: 2014 / Resumo: Neste estudo, foi testada a hipótese de que a transição da hiperalgesia aguda para a persistente e a hiperalgesia persistente reduzem a atividade e induzem alterações plásticas num circuito de analgesia endógena, o controle nociceptivo ascendente (CNA). Este circuito é de grande importância para mediar uma forma de analgesia endógena, conhecida como analgesia induzida por capsaicina, e é dependente de receptores µ-opióide no núcleo accumbens. Portanto, nós também investigamos se a transição da hiperalgesia mecânica aguda para a persistente e a hiperalgesia mecânica persistente altera a participação dos receptores µ-opióide intra-accumbens na analgesia induzida por capsaicina. No modelo animal de cronificação da dor utilizado, 14 dias consecutivos de injeções intraplantares de PGE2 na pata traseira de ratos (referido como o período de indução da hiperalgesia persistente), induz um estado permanente de sensibilização dos nociceptores (referido como o período de manutenção da hiperalgesia persistente), que se mantém por até 30 dias após a interrupção do tratamento com PGE2. A hipersensibilidade dos nociceptores foi medida pela diminuição do intervalo de tempo para o animal responder a uma leve estimulação mecânica na pata traseira. Para avaliar a expressão dos receptores µ-opióide no núcleo accumbens, foi utilizado o método de Western Blotting. Foi encontrada uma redução significativa na duração da analgesia induzida por capsaicina nos dias 7 e 14 do período de indução e nos dias 1, 7, 14 e 21 do período de manutenção da hiperalgesia mecânica persistente. A administração intra-accumbens do antagonista seletivo de receptor µ-opióide Cys2,Tyr3,Orn5,Pen7amide (CTOP), 10 minutos antes da injeção subcutânea de capsaicina na pata dianteira dos ratos, bloqueou a analgesia induzida por capsaicina. No entanto, não ocorreram alterações significativas na expressão dos receptores µ-opióide. Tomados em conjunto, estes resultados indicam que a transição da hiperalgesia aguda para a persistente e a hiperalgesia persistente reduzem a duração da analgesia induzida por capsaicina, sem afetar sua dependência de mecanismos mediados por receptores µ-opióide no núcleo accumbens. A atenuação da analgesia endógena durante a cronificação da dor e dor crônica sugerem que os circuitos endógenos de controle da dor desempenham um importante papel no desenvolvimento e manutenção da dor crônica / Abstract: In this study, we tested the hypothesis that the transition from acute to persistent hyperalgesia and persistent hyperalgesia reduces the activity and induces plastic changes in an endogenous analgesia circuit, the ascending nociceptive control (ANC). An important mechanism mediating this form of endogenous analgesia, referred as capsaicin-induced analgesia, is its dependence on nucleus accumbens µ-opioid receptor mechanisms. Therefore, we also investigated whether the transition from acute to persistent mechanical hyperalgesia and persistent mechanical hyperalgesia alters the requirement for nucleus accumbens µ-opioid receptor mechanisms in capsaicin-induced analgesia. We used an animal model of pain chronification in which daily intraplantar PGE2 injection into the rat's hind paw for 14 days, referred as the induction period of persistent hyperalgesia, induces a long lasting state of nociceptior sensitization referred as the maintenance period of persistent hyperalgesia, that lasts for at least 30 days following the cessation of the PGE2 treatment. The nociceptor hypersensitivity was measured by the shortening of the time interval for the animal to respond to a mechanical mild stimulation of the hind paw. Western blot analysis were used to evaluate the expression of µ-opioid receptors in nucleus accumbens. We found a significant reduction in the duration of capsaicin-induced analgesia at day 7 and 14th of the induction period and at days 1, 7, 14 and 21th of the maintenance period of persistent mechanical hyperalgesia. Intra-accumbens administration of the µ-receptor selective antagonist Cys2,Tyr3,Orn5,Pen7amide (CTOP) 10 min before the subcutaneous injection of capsaicin into the rat's fore paw blocked capsaicin-induced analgesia. However, no significant changes occurred in the expression of µ-opioid receptors. Taken together, these findings indicate that the transition from acute to persistent hyperalgesia and persistent hyperalgesia reduces the duration of capsaicin-induced analgesia, without affecting its dependence on nucleus accumbens µ-opioid receptor mechanisms. The attenuation of endogenous analgesia during pain chronification and chronic pain suggests that endogenous pain circuits play an important role in the development and maintenance of chronic pain / Mestrado / Fisiologia Oral / Mestra em Odontologia

Stroke is the third leading cause of death and the primary cause of adult long-term disability in Canada. Despite advances in rehabilitation research, stroke survivors experience an unusually high incidence of depressive symptoms which undermine recovery outcomes by reducing patient motivation levels. Human and animal studies have linked the incidence of post-stroke depression and the extent of prefrontal cortex (PFC) damage. The PFC and the lateral habenula (LHb) are limbic structures that are strongly connected to the serotonergic dorsal raphe nucleus (DRN), a key neuronal hub for mood regulation. We hypothesized that PFC stroke produces a depressive phenotype by triggering maladaptive reorganization in mood-related networks. We used viral and optogenetic strategies to functionally characterize PFC and LHb projections to DRN. Moreover, we found that PFC stroke causes a time-dependent remodeling of LHb inputs to DRN 5-HT neurons which results in altered postsynaptic glutamate receptor number and subunit composition. This remodeling likely reflects a homeostatic upregulation of LHb-DRN synapses in response to stroke-induced challenge to network activity. Since these synapses encode stress and aversion, potentiation of this pathway could contribute to depressive symptoms following stroke. However, more work will be needed to identify the behavioral and network-level consequences of altered LHb-DRN dynamics. Thus, a deeper understanding of circuit mechanisms implicated in post-stroke depression will provide insights into this disease and open new treatment avenues to improve recovery.

In order to anticipate the predictable changes in the environment associated with the earth’s rotation, most organisms possess intrinsic biological clocks. To be useful, such clocks require a reliable signal of ‘time’ from the external world. In mammals, light provides the principle source of such information; conveyed to the suprachiasmatic nucleus circadian pacemaker (SCN) either directly from the retina or indirectly via other visual structures such as the thalamic intergeniculate leaflet (IGL). Nonetheless, while the basic pathways supplying sensory information to the clock are well understood, the sensory signals they convey or how these are processed within the circadian system are not. One established view is that circadian entrainment relies on measuring the total amount of environmental illumination. In line with that view, the dense bilateral retinal input to the SCN allows for the possibility that individual neurons could average signals from across the whole visual scene. Here I test this possibility by examining responses to monocular and binocular visual stimuli in the SCN of anaesthetised mice. In fact, these experiments reveal that SCN cells provide information about (at most) irradiance within just one visual hemisphere. As a result, overall light-evoked activity across the SCN is substantially greater when light is distributed evenly across the visual scene when the same amount of light is non-uniformly distributed. Surprisingly then, acute electrophysiological responses of the SCN population do not reflect the total amount of environmental illumination. Another untested suggestion has been that the circadian system might use changes in the spectral composition of light to estimate time of day. Hence, during ‘twilight’, there is a relative enrichment of shortwavelength light, which is detectable as a change in colour to the dichromatic visual system of most mammals. Here I used a ‘silent substitution’ approach to selectively manipulate mouse cone photoreception, revealing a subset of SCN neurons that exhibit spectrally-opponent (blue-yellow) visual responses and are capable of reliably tracking sun position across the day-night transition. I then confirm the importance of this colour discrimination mechanism for circadian entrainment by demonstrating a reliable change in mouse body temperature rhythms when exposed to simulated natural photoperiods with and without simultaneous changes in colour. This identification of chromatic influences on circadian entrainment then raises important new questions such as which SCN cell types process colour signals and do these properties originate in the retina or arise via input from other visual regions? Advances in mouse genetics now offer powerful ways to address these questions. Our original method for studying colour discrimination required transgenic mice with red-shifted cone sensitivity – presenting a barrier to applying this approach alongside other genetic tools. To circumvent this issue I validated a modified approach for manipulating wildtype cone photoreception. Using this approach alongside optogenetic cell-identification I then demonstrate that the thalamic inputs to the SCN are unlikely to provide a major source of chromatic information. To further probe IGL-contributions to SCN visual responses, I next used electrical microstimulation to show that the thalamus provides inhibitory input to both colour and brightness sensitive SCN cells. Using local pharmacological inhibition I then show that thalamic inputs supress specific features of the SCN light response originating with the contralateral retina, including colour discrimination. These data thus provide new insight into the ways that arousal signals reaching the visual thalamus could modulate sensory processing in the SCN. Together then, the work described in this thesis provides important new insight into sensory control of the circadian system and the underlying neural mechanisms.

Doctor of Philosophy / Department of Psychological Sciences / Mary E. Cain / Drug addiction is a chronic cyclical disease characterized by periods of drug use and abstinence. Drug craving increases as a function of abstinence period, such that longer periods of abstinence result in greater feelings of craving. Longer periods of abstinence may render cues to become more powerful motivators of drug seeking behavior because of the greater craving response. Neurobiological evidence suggests that changes in glutamatergic transmission in the nucleus accumbens (NAc) plays a pivotal role in the incubation of craving and drug seeking motivation. Specifically, the upregulation of Ca²⁺ permeable AMPA receptors may increase drug seeking following the presentation of a drug cue. Environmental housing manipulations also change the expression of metabotropic glutamate receptors (mGlur) and psychostimulant self-administration. In the current experiments, Sprague-Dawley rats were reared in enriched (EC) or isolated (IC) conditions from PND 21-51. Then rats were implanted with indwelling jugular catheters and allowed to self-administer amphetamine (0.1 mg/kg/infusion) or saline paired with a cue light for 16 days for 1h. Then rats went through a forced abstinence period of 1 day and were then tested in a cue-induced seeking test. Immediately after the seeking test, half the rats were sacrificed and the NAc was dissected and prepared for western blot analyses. The other half of rats rested for 40 days and were tested again in the cue-induced seeking test. Immediately following the seeking test, rats were sacrificed and their NAc was dissected. Factorial ANOVA results indicate that rearing in the IC environment increased drug seeking when compared to EC rats after 1 day of abstinence and after 40 days of abstinence, but drug seeking did not increase after 40 days. Rats in the saline groups showed an increase in seeking after 40 days of abstinence, providing evidence of increased responding. Saline responding was significantly lower when compared to rats that responded for amphetamine. When rats self-administered saline, generally IC rats had more responding than EC rats. Western blot analyses indicated that expression of AMPA subunits GluA1, and GluA2, as well as metabotropic glutamate receptors 1 and 5 (mGlur1, and mGlur5) were not different across the experimental groups, suggesting another mechanism could be implicated in drug seeking after short and long abstinence periods. These results suggest that early life experience can have long lasting effects into adulthood and increase the vulnerability of drug abuse. Our results provide mixed results of incubated seeking. Positive early life experiences reduce drug seeking motivation after short and long abstinence periods, providing evidence for further research to examine how early life experience changes the reward seeking and subsequent structures in the mesocorticolimbic pathway.