Abstract The characteristics of humoral immunity have been proposed to reflect the bias between two T helper (Th) lymphocyte subsets: Th1 cells, which activate cell-mediated immunity, and Th2 cells, which mediate humoral immunity. The present study aimed to characterize the humoral immunity to beta-cell autoantigens insulin and glutamic acid decarboxylase (GAD65) in preclinical and clinical type 1 diabetes. Insulin antibodies were analyzed in pregnant women with or without type 1 diabetes and their newborn infants and in prediabetic children. Epitope or/and isotype-specific GAD65 antibodies (GAD65Abs) were analyzed in prediabetic children, in children and adolescents diagnosed with type 1 diabetes, and in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome. Antibodies were determined by radioligand immunoassays. The humoral immune response to insulin and GAD65 was observed to be a highly dynamic process, comprising mainly the IgG1 subclass and, less frequently, other IgG subclasses. GAD65Abs were directed primarily to the middle region and secondarily to the C-terminal region of GAD65 as a consequence of epitope spreading. Young children who progressed to overt type 1 diabetes were characterized by a broad initial isotype response to insulin and GAD65 and by a strong IgG1 and IgG3 response to insulin. Children who did not progress to clinical type 1 diabetes were characterized by an emerging IgG4 response to GAD65. Rising levels of GAD65Abs targeted to the middle region of GAD65 were associated with high titers of islet cell antibodies and a decreased requirement for exogenous insulin, probably reflecting a persistent residual beta-cell mass, in patients with manifest type 1 diabetes. Non-immunoglobulin insulin-binding activity was observed to be induced by pregnancy. APECED-associated humoral autoimmunity to GAD65 did not differ markedly from that observed in subjects with type 1 diabetes alone. In conclusion, isotype-specific GAD65 and especially insulin antibodies are valuable markers of the risk of progression to type 1 diabetes in young children. The appearance of an initial IgG3 subclass response and a strong IgG3 response to insulin in children who progressed to overt type 1 diabetes may reflect the role of cytotoxic Th1-biased immunity in the disease process leading to clinical presentation of type 1 diabetes.

Our previous results showed that oligodendrocyte development is regulated by both nociceptin and its G-protein coupled receptor, the nociceptin/orphanin FQ receptor (NOPR). The present in vitro and in vivo findings show that nociceptin plays a crucial conserved role in both human and rodent brain astrocytes, regulating the levels of the glutamate/aspartate transporter GLAST/EAAT1. This nociceptin-mediated response takes place during a critical developmental window that coincides with astrocyte maturation and synapse formation. GLAST/EAAT1 upregulation by nociceptin is mediated by NOPR and the downstream participation of a complex signaling cascade that involves the interaction of several kinase systems, including PI-3K/AKT, mTOR and JAK. Because GLAST is the main glutamate transporter during brain maturation, these novel findings suggest that nociceptin plays a crucial role in regulating the function of early astrocytes and their capacity to support glutamate homeostasis in the developing brain.

In order to process information, neurons must connect together to form a neuronal circuit. The formation of neuronal circuits is dependent on synaptic activity through glutamate receptors and downstream molecules within the post-synaptic density (PSD). The pathways downstream of glutamate receptors play an important role in maintaining appropriate synapses and forming neural circuits; mutations in genes that encode PSD proteins disrupt these pathways and are associated with many forms of intellectual disability in humans (Grant 2012). The development of neuronal circuits relies on two key developmental events; neurons must first send out axons locally and to disparate brain regions and then, neurons must form connections with the dendrites of target neurons. The rodent trigeminal system is a neuronal circuit that processes somatosensory information from whiskers on the facepad via nuclei in the brainstem to the thalamus and ultimately the cerebral cortex. Brain regions that comprise the trigeminal system are organised in a manner that topographically recapitulates the whisker pattern; each whisker on the rodent facepad corresponds to a physiological and anatomical unit in the primary somatosensensory cortex (S1) called a barrel. This topographical organisation creates a pattern consisting of thalamocortical axons (TCA) clustered into distinct whisker-related bundles and layer IV spiny stellate cells which segregate around the outside of TCA bundles. Three different anatomical patterns can be identified within the mouse S1 by labelling the cell soma, axons or the extracellular matrix. This strict organisation makes the rodent S1 an excellent model for discerning the proteins involved in neural circuit formation, and by screening genetic mutants for S1 patterning defects the molecular pathway involved in setting up neuronal circuits can be elucidated. Furthermore an understanding of these pathways may provide insight into how neuronal networks are disrupted in human intellectual disability. In the first data chapter, the expression profile of three neurofilament subunits were characterised in order to develop a method of identifying anatomical defects in barrel morphology. The precise organisation of the rodent S1 can also be used as a method to identify the cellular localisation of neurofilament subunits ex vivo. Neurofilaments are polymers formed from three subunits identified by their relative molecular weight. By using the unique patterning of S1, each neurofilament subunit shows a unique spatialtemporal expression pattern in the somatosensory cortex. Two neurofilaments subunits; the medium and the heavy neurofilament subunits can be used to identify TCA which can be used as an indicator of anatomical defects in barrel patterning. In chapter 4 neurofilament labelling was used in conjunction with other histological techniques to investigate S1 organisation in mice lacking synapse associated protein 102 (SAP102). SAP102 is a PSD scaffolding molecule that binds to both NMDA receptor subunits and SynGAP, a synaptic GTPase activating protein, furthermore it is associated with X-linked mental retardation in humans (Tarpey et al., 2004; Zanni et al., 2010). Mutant mice lacking functional NMDA receptors or PSD proteins such as SynGAP show defects in S1 pattern formation (Barnett et al., 2006; Iwasato et al., 2000; Wijetunge, Till, Gillingwater, Ingham, & Kind, 2008). SAP102 null mutants (SAP102-/y) have defects in synaptic plasticity and are slow to learn on behavioural tasks (Cuthbert et al., 2007), however it is unclear how the loss of SAP102 may disrupt neural networks. SAP102-/y were found to have a reduction in brain mass compared to wild-type littermates, but cortical thickness and patterning of S1 is unchanged. SAP102-/y have fewer TCA reaching the cortex compared to littermates; furthermore SAP102-/y mutants have layer specific defects in the density of dendritic spines. These data suggest that in the absence of SAP102 connectivity in the S1 is altered by layer specific changes in synapses number and fewer axons innervating cortical layer IV. In the final experimental chapter (chapter 5) the combined role of SAP102 and Postsynaptic density protein 95 (PSD95) in S1 patterning was investigated. SAP102 and PSD95 are the main members of the membrane-associated guanylate kinase (MAGUK) family expressed during early cortical development. These proteins share a similar protein structure, perform similar functions at the synapse (Elias & Nicoll, 2007) and have been shown to compensate for each other in vitro (Elias, Elias, Apostolides, Kriegstein, & Nicoll, 2008). Genetic mutants lacking both SAP102 and PSD95 are not viable and do not survive beyond birth (Cuthbert et al., 2007; Petrie, 2008). Therefore in order to investigate the combined role of these proteins a novel approach was developed that utilises X-inactivation to produce mosaic animals containing cells that lack both proteins. The distribution of cells containing the same X-chromosome was investigated and found to be evenly distributed throughout the cortex, demonstrating that this method could be used to investigate allosomal genes. In mosaic animals where approximately half the cells only lack PSD95 and the remaining cells lack both SAP102 and PSD95, double knockout cells are viable and are equally represented in S1. These double knockout cells contribute to normally barrel formation which suggests that SAP102 and PSD95 are not required for barrel formation.

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.

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.

Ketamine is a non-competitive NMDA antagonist that has been shown to have antidepressant properties both clinically as well as in preclinical studies when administered at a subanesthetic dose. In vivo electrophysiological recordings were carried in male Sprague Dawley rats 30 minutes following ketamine administration (10 mg/kg) to first assess its effects on monoaminergic firing. Whilst no change in the firing activity of serotonin (5-HT) neurons was observed in the dorsal raphe nucleus (DRN), an increase in the firing activity was observed for dopamine (DA) and noradrenergic (NE) neurons in the ventral tegmental area (VTA) and locus coeruleus (LC), respectively. The effect of ketamine on these electrophysiological parameters was prevented by pre-administration of the AMPA receptor antagonist NBQX 10 minutes prior to ketamine administration. In a second series of experiments, an increase in AMPA-evoked response was observed within 30 minutes in the CA3 layer of the hippocampus (HPC) following acute ketamine administration. These findings suggest that acute ketamine administration produces a prompt enhancement of AMPA transmission in the forebrain and also results in increased catecholaminergic activity. These effects may play a crucial role in the rapid antidepressant effects of ketamine observed shortly following its infusion in the clinic.

O nosso grupo está explorando como alternativa para a identificação de novas estratégias de tratamento o reposicionamento de drogas. Nesse contexto, nosso laboratório mostrou que memantina, um antagonista de receptores de glutamato NMDA, apresenta uma atividade tripanocida no ciclo de vida do T. cruzi, in vitro. Com base nessas informações, avaliamos o efeito da memantina em animais infectados com a cepa Y. O tratamento apresentou uma redução da parasitemia (40%) no 8º dpi e redução da carga parasitária no tecido cardíaco no 15º dpi. Uma vez que a cepa Y invade preferencialmente macrófagos, avaliamos o efeito de memantina em células RAW 264.7. Memantina diminuiu a produção de NO por células estimuladas com LPS, diminuiu o Ca2+i, e não houve uma redução de EROs. Memantina também interferiu no ciclo intracelular do parasita, reduzindo o número de células infectadas. Nossos resultados sugerem que o tratamento com memantina pode direcionar o sistema imune de modo a modular os receptores de tipo NMDA, através de um efeito direto ou indireto produzido pelo tratamento. / The search for new therapeutic targets has been of extreme importance for the specific treatment of Chagas disease. Our group is exploring as an alternative, a strategy for drug repurposing. In this context, our laboratory showed that memantine, an antagonistic of NMDA glutamate receptors, has a trypanocidal activity along the life cycle of T. cruzi, in vitro. Based on this information, we evaluated the effect of memantine in animals infected with Y strain. The treatment showed a reduction of parasitemia (40%) in 8th dpi and reduced parasitic load in cardiac tissues at 15th dpi. As the Y strain preferably invades macrophages, we evaluated the effect of memantine in RAW 264.7 cells. Memantine decreased NO production by cells stimulated with LPS, decreased Ca2+i, and did not cause a reduction in ROS. Memantine also interfere with the intracellular parasite cycle, reducing the number of infected cells. Our results suggest that treatment with memantine can target the immune system to modulate the NMDA receptor, through direct or indirect effect caused by the treatment.

Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 introns, similarly to the Arabidopsis IRE gene. MtIRE is a new member of the IRE family and it is a putative Ser/Thr protein kinase. MtIRE is a nodule- and flower-specific gene, suggesting that nodulation may have recruited it from other developmental processes. MtIRE is likely to be involved in the invasion process, or in the maturation of the symbiosome, or of the cells that contain rhizobia, rather than infection thread initiation and elongation or in nitrogen fixation. Nodule invasion precedes the onset of MtIRE expression and the expression pattern changes in time within the nodule. RNA interference results support MtIRE expression data and suggest a possible role in preventing extensive defense responses. Our study demonstrates the existence of an Arabidopsis IRE homolog in Medicago truncatula root nodules with an entirely new function and regulation.

The anterior cingulate cortex (ACC) is part of the cognitive and emotional brain circuitry that mediates social interaction. Imbalances between inhibitory, GABAergic neurons, and excitatory, glutamatergic neurons, in this region are essential to brain circuity during social responses and are thought to be involved with behaviors associated with autism spectrum disorder (ASD). Enriched cell populations of glutamatergic neurons, obtained through laser capture microdissection, were used for gene expression studies of GABAergic receptors (GABRA1, GABRA4, and GABBR1). Additionally, proteins that impact GABAergic synapses (Spinophilin, CPLX1, mTOR, IGF1R, PSD95, PARP1) were investigated using Western Blotting with punchdissected homogenate brain tissue from ACC and frontal cortical brain regions. No significant differences in gene expression nor protein were identified between ASD and control brain donors. Evidence of GABAergic synaptic pathology was not found; however, future studies of alternative GABAergic markers and increased study numbers are needed to confirm these findings in ASD human tissue.

N-methyl-D-aspartate receptors (NMDARs) are heterotetramers containing two obligatory glycine-binding (GluN1) and two glutamate/glycine-binding (GluN2/3) subunits. These receptors mediate excitatory synaptic transmission in the central nervous system and play a key role in high order neuronal processes as a learning and formation of memory. It has been shown that dysregulation of NMDARs is involved in the pathophysiology of neurological and psychiatric disorders. Each receptor is composed of four protomers exhibiting a conserved domain organization. The most distal part to the cell membrane is the amino-terminal domain that is linked to the ligand binding domain (LBD), which is connected to the pore-forming transmembrane domain (TMD) communicating with the intracellular carboxy-terminal domain. LBD and TMD are connected via three polypeptide chains - linkers. Channel opening is the key step in the NMDAR gating that allows the flux of ions across the membrane. The energy of agonist binding-evoked conformational changes is transferred via linkers to M3 helices forming an ion channel. The rearrangement of M3 helices in activated receptor makes the central cavity of the channel accessible. The details of energy transfer are not yet fully characterized, although accurate knowledge of the receptor gating...